CA2443770A1 - Proteins and nucleic acids encoding same - Google Patents
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- CA2443770A1 CA2443770A1 CA002443770A CA2443770A CA2443770A1 CA 2443770 A1 CA2443770 A1 CA 2443770A1 CA 002443770 A CA002443770 A CA 002443770A CA 2443770 A CA2443770 A CA 2443770A CA 2443770 A1 CA2443770 A1 CA 2443770A1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
Disclosed are polypeptides and nucleic acids encoding same. Also disclosed are vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using same.
Description
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
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NOTE POUR LE TOME / VOLUME NOTE:
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
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THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTTON
The invention relates to polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, and methods for using the same.
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded therefrom.
More specifically, the invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
Heart disease is the primary cause of death in most western societies. Death from heart disease is often induced by platelet-dependent ischemic syndromes which are initiated by atherosclerosis and arteriosclerosis and include, but are not limited to, acute myocardial infarction, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastornosis of vascular grafts, and chronic cardiovascular devices (e.g., in-dwelling catheters or shunts "extracorporeal circulating devices"). These syndromes represent a variety of stenotic and occlusive vascular disorders thought to be initiated by platelet activation either on vessel walls or within the lumen by blood-borne mediators but are manifested by platelet aggregates which form thrombi that restrict blood flow.
For example, Thrombospondin-1-like proteins associate with the extracellular matrix and inhibits angiogenesis in vivo. In vitro, Thrombospondin-like proteins block capillary-like tube formation and endothelial cell proliferation. The antiangiogenic activity is mediated by a region that contains 3 type 1 (properdin or thrombospondin) repeats.
In addition, Selectin-like proteins such as P-selectin, also called GMP-140, CD62, or selectin P, is a 140-kD adhesion molecule, expressed at the surface of activated cells, that mediates the interaction of activated endothelial cells or platelets with leukocytes. In endothelial cells, the protein is localized to the membranes of Weibel-Palade bodies, the intracellular storage granules for von Willebrand factor.
Many disease states are characterized by uncontrolled cell proliferation.
These diseases involve a variety of cell types and include disorders such as cancer, psoriasis, pulmonary fibrosis, glomeruloneplmitis, atherosclerosis and restenosis following angioplasty. Vital cellular functions such as cell proliferation and signal transduction are regulated in part by the balance between the activities of protein-tyrosine kinases (PTI~) and protein-tyrosine phosphatases (PTPase). Oncogenesis can result from an imbalance.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV1, NOV2, NOV3, NOV4, NOVS, NOV6, NOV7, NOVB, NOV9, NOVlOa, NOVlOb, NOV 11, NOV 12, NOV 13, NOV 14, NOV 1 S, and NOV 16 nucleic acids and polypeptides.
These nucleic acids and polypeptides, as well as variants, derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX"
nucleic acid or polypeptide sequences.
1 S In one aspect, the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ m NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, and 33. Tn some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ lD NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, and 33.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ m NOS:1, 3, S, 7, 9, 11, 13, 1 S, 17~ 19, 21, 23, 2S, 27, 29, 31, and 33) or a complement of said oligonucleotide.
Also included in the invention are substantially purified NOVX polypeptides (SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide mzder conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specif c cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX
nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX
polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., those described for the individual NOVX nucleotides and polypeptides herein, and/or other pathologies and disorders of the like.
The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX-specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed below and/or other pathologies and disorders of the like. The polypeptides can be used as irrnnunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX rnay be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The method includes contacting a test compound with a NOVX
polypeptide and determining if the test compound binds to said NOVX
polypeptide. Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above andlor other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX
polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
Tn a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subj ect (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
W yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby.
Included in the invention are the novel nucleic acid sequences and their polypeptides. The sequences are collectively referred to as "NOVX nucleic acids" or "NOVX
polynucleotides"
and the corresponding encoded polypeptides are referred to as "NOVX
polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers NOVX Internal SEQ SEQ gomolo ID ID gy Identification NO (nt)NO (aa) 1 CG93221-01 1 2 Paladin Plasma membrane ring finger 2 CG93210-01 3 4 protein Thrombospondin-1 domain 3 CG93275-01 5 6 containing protein Protocadherin alpha C2 short 4 CG93187-O1 7 8 form COR CG95083-01 9 10 Nuclear protein 6 COR CG94989-Ol 11 12 Secretory protein Transmission-blocking target 7 COR CG94978-O1 13 14 antigen 5230 precursor 8 COR CG94713-02 l5 16 Nuclear protein 9 COR CG94702-01 17 18 Hemicentin precursor 10a COR CG94661-01 19 20 Selectin lOb COR CG94661-02 21 22 Selectin 11 COR CG94325-01 23 24 Nuclear protein 12 COR CG94282-O1 25 26 Plasma membrane protein 13 COR CG94399-01 27 28 BHLH Factor MATH6 Putative protein-tyrosine 14 COR CG94366-01 29 30 phosphatase CG95387-02 31 32 LRR protein 16 CG95419-02 33 34 RhoGEF
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
FIELD OF THE INVENTTON
The invention relates to polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, and methods for using the same.
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded therefrom.
More specifically, the invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
Heart disease is the primary cause of death in most western societies. Death from heart disease is often induced by platelet-dependent ischemic syndromes which are initiated by atherosclerosis and arteriosclerosis and include, but are not limited to, acute myocardial infarction, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastornosis of vascular grafts, and chronic cardiovascular devices (e.g., in-dwelling catheters or shunts "extracorporeal circulating devices"). These syndromes represent a variety of stenotic and occlusive vascular disorders thought to be initiated by platelet activation either on vessel walls or within the lumen by blood-borne mediators but are manifested by platelet aggregates which form thrombi that restrict blood flow.
For example, Thrombospondin-1-like proteins associate with the extracellular matrix and inhibits angiogenesis in vivo. In vitro, Thrombospondin-like proteins block capillary-like tube formation and endothelial cell proliferation. The antiangiogenic activity is mediated by a region that contains 3 type 1 (properdin or thrombospondin) repeats.
In addition, Selectin-like proteins such as P-selectin, also called GMP-140, CD62, or selectin P, is a 140-kD adhesion molecule, expressed at the surface of activated cells, that mediates the interaction of activated endothelial cells or platelets with leukocytes. In endothelial cells, the protein is localized to the membranes of Weibel-Palade bodies, the intracellular storage granules for von Willebrand factor.
Many disease states are characterized by uncontrolled cell proliferation.
These diseases involve a variety of cell types and include disorders such as cancer, psoriasis, pulmonary fibrosis, glomeruloneplmitis, atherosclerosis and restenosis following angioplasty. Vital cellular functions such as cell proliferation and signal transduction are regulated in part by the balance between the activities of protein-tyrosine kinases (PTI~) and protein-tyrosine phosphatases (PTPase). Oncogenesis can result from an imbalance.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV1, NOV2, NOV3, NOV4, NOVS, NOV6, NOV7, NOVB, NOV9, NOVlOa, NOVlOb, NOV 11, NOV 12, NOV 13, NOV 14, NOV 1 S, and NOV 16 nucleic acids and polypeptides.
These nucleic acids and polypeptides, as well as variants, derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX"
nucleic acid or polypeptide sequences.
1 S In one aspect, the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ m NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, and 33. Tn some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ lD NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, and 33.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ m NOS:1, 3, S, 7, 9, 11, 13, 1 S, 17~ 19, 21, 23, 2S, 27, 29, 31, and 33) or a complement of said oligonucleotide.
Also included in the invention are substantially purified NOVX polypeptides (SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide mzder conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specif c cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX
nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX
polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., those described for the individual NOVX nucleotides and polypeptides herein, and/or other pathologies and disorders of the like.
The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX-specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed below and/or other pathologies and disorders of the like. The polypeptides can be used as irrnnunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX rnay be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The method includes contacting a test compound with a NOVX
polypeptide and determining if the test compound binds to said NOVX
polypeptide. Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above andlor other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX
polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
Tn a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subj ect (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
W yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby.
Included in the invention are the novel nucleic acid sequences and their polypeptides. The sequences are collectively referred to as "NOVX nucleic acids" or "NOVX
polynucleotides"
and the corresponding encoded polypeptides are referred to as "NOVX
polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers NOVX Internal SEQ SEQ gomolo ID ID gy Identification NO (nt)NO (aa) 1 CG93221-01 1 2 Paladin Plasma membrane ring finger 2 CG93210-01 3 4 protein Thrombospondin-1 domain 3 CG93275-01 5 6 containing protein Protocadherin alpha C2 short 4 CG93187-O1 7 8 form COR CG95083-01 9 10 Nuclear protein 6 COR CG94989-Ol 11 12 Secretory protein Transmission-blocking target 7 COR CG94978-O1 13 14 antigen 5230 precursor 8 COR CG94713-02 l5 16 Nuclear protein 9 COR CG94702-01 17 18 Hemicentin precursor 10a COR CG94661-01 19 20 Selectin lOb COR CG94661-02 21 22 Selectin 11 COR CG94325-01 23 24 Nuclear protein 12 COR CG94282-O1 25 26 Plasma membrane protein 13 COR CG94399-01 27 28 BHLH Factor MATH6 Putative protein-tyrosine 14 COR CG94366-01 29 30 phosphatase CG95387-02 31 32 LRR protein 16 CG95419-02 33 34 RhoGEF
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
10 nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological conditions can be diagnosed by determinng the amount of the new protein in a sample or by 15 determining the presence of mutations in the new genes. Specific uses are described for each of the sixteen genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cell growth, cell metabolism, cell differentiation, cell proliferation, andlor cell signaling.
In one embodiment of the present invention, NOVX or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NOVX. Examples of such disorders include, but are not limited to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, _;
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; neurological disorders such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Takob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; and disorders of vesicular transport such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking including acquired immunodeficiency syndrome (A)DS), allergic reactions, autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, osteoarthritis, scleroderma, Chediak-Higashi syndrome, Sjogren's syndrome, systemic lupus erythiematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helininthic, and protozoal infections, as well as additional indications listed for the individual NOVX clones.
The NOVX nucleic acids and proteins of the invention are useful in potential S diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diag~iostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
These also include potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotaxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), (v) an agent promoting tissue regeneration i~a vitro and in vivo, and (vi) a biological defense weapon.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 1 polypeptide has been identified as a Paladin-like protein (also referred to as CG93221-O1). The disclosed novel NOV1 nucleic acid (SECT m NO:l) of 2600 nucleotides is shown in Table 1A. The novel NOV1 nucleic acid sequences maps to the chromosome 10.
An ORF begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAG cadon at nucleotides 2583-2585. A putative untranslated region and/or downstream from the termination codon is underlined in Table IA, and the start and stop codons are in bold letters.
Table 1A. NOV1 Nucleotide Sequence (SEQ ID NO:1) GCTGCTGGCAGACTATGGGTACAACGGCCAGCACAGCCCAGCAGACGGTCTCGGCAGGCACCCCATT
TGAGGGCCTACAGGGCAGTGGCACGATGGACAGTCGGCACTCCGTCAGCATCCACTCCTTCCAGAGC
ACTAGCTTGCATAACAGCAAGGCCAAGTCCATCATCCCCAACAAGGTGGCCCCTGTTGTGATCACGT
ACAACTGCAAGGAGGAGTTCCAGATCCATGATGAGCTGCTCAAGGCTCATTACACGTTGGGCCGGCT
CTCGGACAACACCCCTGAGCACTACCTGGTGCAAGGCCGCTACTTCCTGGTGCGGGATGTCACTGAG
AAGATGGATGTGCTGGGCACCGTGGGAAGCTGTGGGGCCCCCAACTTCCGGCAGGTGCAGGGTGGGC
TCACTGTGTTCGGCATGGGACAGCCCAGCCTCTTAGGGTTCAGGCGGGTCCTCCAGAAACTCCAGAA
GGACGGACATAGGGAGTGTGTCATCTTCTGTGTGCGGGAGGAACCTGTGCTTTTCCTGCGTGCAGAT
GAGGACTTTGTGTCCTACACACCTCGAGACAAGCAGAACCTTCATGAGAACCTCCAGGGCCTTGGAC
CCGGGGTCCGGGTGGAGAGCCTGGAGCTGGCCATCCGGAAAGAGATCCACGACTTTGCCCAGCTGAG
CGAGAACACATACCATGTGTACCATAACACCGAGGACCTGTGGGGGGAGCCCCATGCTGTGGCCATC
CATGGTGAGGACGACTTGCATGTGACGGAGGAGGTGTACAAGCGGCCCCTCTTCCTGCAGCCCACCT
ACAGGTACCACCGCCTGCCCCTGCCCGAGCAAGGGAG'T'CCCCTGGAGGCCCAGTTGGACGCCTT'T'GT
CAGTGTTCTCCGGGAGACCCCCAGCCTGCTGCAGCTCCGTGATGCCCACGGGCCTCCCCCAGCCCTC
GTCTTCAGCTGCCAGATGGGCGTGGGCAGGACCAACC'T'GGGCATGGTCCTGGGCACCCTCATCCTGC
TTCACCGCAGTGGGACCACCTCCCAGCCAGAGGCTGCCCCCACGCAGGCCAAGCCCCTGCCTATGGA
GCAGTTCCAGGTGATCCAGAGCTTTCTCCGCATGGTGCCCCAGGGAAGGAGGATGGTGGAAGAGGTG
GACAGAGCCATCACTGCCTGTGCCGAGTTGCATGACCTGAAAGAAGTGGTCTTGGAAAACCAGAAGA
AGTTAGAAGGTATCCGACCGGAGAGCCCAGCCCAGGGAAGCGGCAGCCGACACAGCGTCTGGCAGAG
GGCGCTGTGGAGCCTGGAGCGATACTTCTACCTGATCCTGTTTAACTACTACCTTCATGAGCAGTAC
CCGCTGGCCTTTGCCCTCAGTTTCAGCCGCTGGCTGTGTGCCCACCCTGAGCTGTACCGCCTGCCCG
TGACGCTGAGCTCAGCAGGCCCTGTGGCTCCGAGGGACCTCATCGCCAGGGGCTCCCTACGGGAGGA
CGATCTGGTCTCCCCGGACGCGCTCAGCACTGTCAGAGAGATGGATGTGGCCAACTTCCGGCGGGTG
CCCCGCATGCCCATCTACGGCACGGCCCAGCCCAGCGCCAAGGCCCTGGGGAGCATCCTGGCCTACC
TGACGGACGCCAAGAGGAGGCTGCGGAAGGTTGTCTGGGTGAGCCTTCGGGAGGAGGCCGTGTTGGA
GTGTGACGGGCACACCTACAGCCTGCGGTGGCCTGGGCCCCCTGTGGCTCCTGACCAGCTGGAGACC
CTGGAGGCCCAGCTGAAGGCCCATCTAAGCGAGCCTCCCCCAGGCAAGGAGGGCCCCCTGACCTACA
GGTTCCAGACCTGCCTTACCATGCAGGAGGTCTTCAGCCAGCACCGCAGGGCCTGTCCTGGCCTCAC
CTACCACCGCATCCCCATGCCGGACTTCTGTGCCCCCCGAGAGGAGGACTTTGACCAGCTGCTGGAG
GCCCTGCGGGCCGCCCTCTCCAAGGACCCAGGCACTGGCTTCGTGTTCAGCTGCCTCAGCGGCCAGG
GCCGTACCACAACTGCGATGGTGGTGGCTGTCCTGGCCTTCTGGCACATCCAAGGCTTCCCCGAGGT
GGGTGAGGAGGAGCTCGTGAGTGTGCCTGATGCCAAGTTCACTAAGGGTGAATTTCAGGTAGTAATG
AAGGTGGTGCAGCTGCTACCCGATGGGCACCGTGTGAAGAAGGAGGTGGACGCAGCGCTGGACACTG
TCAGCGAGACCATGACGCCCATGCACTACCACCTGCGGGAGATCATCATCTGCACCTACCGCCAGGC
GAAGGCAGCGAAAGAGGCGCAGGAAATGCGGAGGCTGCAGCTGCGGAGCCTGCAGTACTTGGAGCGC
TATGTCTGCCTGATTCTCTTCAACGCGTACCTCCACCTGGAGAAGGCCGACTCCTGGCAGAGGCCCT
TCAGCACCTGGATGCAGGAGGTGGCATCGAAGGCTGGCATCTACGAGATCCTTAACGAGCTGGGCTT
CCCCGAGCTGGAGAGCGGGGAGGACCAGCCCTTCTCCAGGCTGCGCTACCGGTGGCAGGAGCAGAGC
TGCAGCCTCGAGCCCTCTGCCCCCGAGGACTTGCTGTAGGGGGCCTTACTCCCT
Variant sequences of NOV1 are included in Example 3, Table 18. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 1 protein (SEQ m N0:2) encoded by SEQ >D NO:1 is 856 amino acid residues in length and is presented using the one-letter amino acid code in Table 1B. Psort analysis predicts the NOV 1 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 1B. Encoded NOV1 protein sequence (SEQ ID NO:2) MGTTASTAQQTVSAGTPFEGLQGSGTMDSRHSVSIHSFQSTSLHNSKAKSIIPNKVAPWITYNC
KEEFQIHDELLKAHYTLGRLSDNTPEHYLVQGRYFLVRDVTEKMDVLGTVGSCGAPNFRQVQGGL
TVFGMGQPSLLGFRRVLQKLQKDGHRECVIFCVREEPVLFLRADEDFVSYTPRDKQNLHENLQGL
GPGVRVESLELAIRKEIHDFAQLSENTYHVYHNTEDLWGEPHAVAIHGEDDLHVTEEWKRPLFL
QPTYRYHRLPLPEQGSPLEAQLDAFVSVLRETPSLLQLRDAHGPPPALVFSCQMGVGRTNLGMVL
GTLILLHRSGTTSQPEAAPTQAKPLPMEQFQVIQSFLRMVPQGRRMVEEVDRAITACAELHDLKE
WLENQKKLEGIRPESPAQGSGSRHSWQRALWSLERYFYLILFNYYLHEQYPLAFALSFSRWLC
AHPELYRLPVTLSSAGPVAPRDLIARGSLREDDLVSPDALSTVREMDVANFRRVPRMPIYGTAQP
SAKALGSILAYLTDAKRRLRKWWVSLREEAVLECDGHTYSLRWPGPPVAPDQLETLEAQLKAHL
SEPPPGKEGPLTYRFQTCLTMQEVFSQHRRACPGLTYHRIPMPDFCAPREEDFDQLLEALRAALS
KDPGTGFVFSCLSGQGRTTTAMWAVLAFWHIQGFPEVGEEELVSVPDAKFTKGEFQWMKWQL
LPDGHRVKKEVDAALDTVSETMTPMHYHLREIIICTYRQAKAAKEAQEMRRLQLRSLQYLERYVC
LILFNAYLHLEKADSWQRPFSTWMQEVASKAGIYEILNELGFPELESGEDQPFSRLRYRWQEQSC
SLEPSAPEDLL
Tn all BLAST alignments described herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size.
It decreases exponentially with the Score (S) that is assigned to a match between two sequences.
Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001, with the filter to remove low complexity sequence turned off. In BLAST 2.0, the Expect value is also used instead of the P
value (probability) to report the significance of matches. For example, an E
value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance.
See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering of the query for low-complexity sequence that is performed to prevent artifactual hits The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., " ") or the letter "X" in protein sequences (e.g., "XXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571, (1996).
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 1 C.
Table 1C. Patp results for NOVl Smallest Sum eadingigh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:AAB41108Human ORFX ORF872 polypeptide +1 4187 0.0 >patp:AAB35276Murine dual specificity phosphatase+1 120 5.2e-06 >patp:AAB73211Murine phosphatase AA023073 +1 120 5.2e-06 m >patp:AAB73231Human phosphatase BAA91172 h +1 115 1.8e-05 >patp:AAG67455Amino acid sequence of a human +1 115 1.8e-0 polypeptide In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2063 of 2508 bases (82%) identical to a gb:GENBANK-m:MMPAL~acc:X99384.1 mRNA from Mus musculus (Paladin gene). The full amino acid sequence of the protein of the invention was found to have 695 of 859 amino acid residues (80%) identical to, and 754 of 859 amino acid residues (87%) similar to, the 859 amino acid residue ptnr:SPTREMBL-ACC:P70261 protein from Mus rnusculus (PALADIN
GENE). NOVl also has homology to the proteins shown in the BLASTP data in Table 1D.
Table 1D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~6331287~dbj~BAABKIAA1274 protein752 752/752 752/752 0.0 6588.1 (AB033100)[Homo Sapiens] (100%) (100%) gi~14738662~ref~XPKIAA protein 748 747/748 747/748 0.0 _ (similar to (99%) (99%) 046314.1 mouse (XM 046314) paladin) (Homo sapi ens]
gi~7305365~ref~NPpaladin [Mus 859 673/841 730/841 0.0 _ musculus] (80%) (86%) 38781.1 (NM 013753) gi~15228672~refINPputative protein1232 207/821 340/821 7e-45 _ [Arabidopsis (25%) (41%) 191760.1 (NM 116066) thaliana]
gi~12836455~dbj~BABdata source:SPTR,144 24/60 33/60 Ze-04 23663.1I(AK004912)source (40%) (55%) key:Q9NX48, evidence:ISS-.homo log to CDNA
FLJ20442 FIB, CLONE
KAT04828-putative [Mus musculus]
A multiple sequence alignment is given in Table 1E, with the NOV 1 protein being shown on line 1 in Table 1E in a ClustalW analysis, and comparing the NOV1 protein with the related protein sequences shown in Table 1D. This BLASTP data is displayed graphically in the ClustalW in Table 1E.
Table 1E. ClustalW Analysis of NOVl 1) > NOVl; SEQ ID NO:2 2) > gi~6331287/ KIAA1274 protein [Hofrao Sapiens]; SEQ ID N0:35 3) > gi~1473866/ KIAA protein (similar to paladin) [Homo Sapiens]; SEQ ID
N0:36 4) > gi~7305365/ paladin [Mus musculus]; SEQ ID N0:37 5) > gi[1522867/ putative protein [Arabidopsis thaliana]; SEQ ID N0:38 6) > gi~1283645/ data source: SPTR, source key: Q9NX48, evidence: IBS-homolog to cDNA FLJ20442 FIB clone KAT04828 putative [Mus muscudus]; SEQ ID N0:39 gi~6331287 -_________________________________________________ gi~1473866 __________________________________________________ gi~7305365 MGTTASTAQQTVSAGTSLEGLQGGSSSSMDSQHSLGGVQSFRATSLHNSK
gi~1522867 __________________________________________________ gi~1283645 __________________________________________________ gi16331287 __________________________________________________ gi11473866 __________________________________________________ 3O gi~7305365 AKSIIPNKVAPWITYNCKEEFQIHDELLKAHYRMGRLSDATPEHYLVQG
gi11522867 ----------------------------MSIPKEPEQVMKMRDGSVLGKK
gi~1283645 __________________________________________________ $ gi~6331287 -------V
gi~1473866 ________ gi~7305365 RYFLVRDI
gi11522867 TILKSDHF
i 1283645 __________________________________________________ g .~. .~.
NOV1 ~ ~ ~ ---HR -iL - ~ Q QG
71D ~
gi16331287 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~1473866 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~7305365 ~ ~T~ ---L I ' E ~ ~~ES RD
giI1522867 RHIGAHKDGKQVKVLWTSLVYINGRPF VLRDVEKPFT GIN
EYT
gi~1283645 _______-__________________________________________ gi~6331287 gi~1473866 2$ gi~7305365 gi,1522867 gi~1283645 _______________________________________________-__ Nov1 gi1 6331287 gi~ 1473866 gi~ 7305365 3$ gi~ 1522867 gi~ 1283645_______ ___________________________________________ NOV1 .Q'~.. P ~~1 ILL. ~-____ gi1 6331287 Q ~ P ~i ILL t-----' gi1 1473866 Q ~ P V ILL ~-----~
giI 7305365 P ~ L L F ~-----~S H
R
gi~ 1522867------- --IN TEII~R ' I F
TT T
SD~GFPRN
4$ gi~ ~.283645_______ _,___________________-_____________________ ...
Nov1 --------$0 gi~6331287 --------gi11473866 --------giI7305365 --------gi~1522867 NSFGRIF ~N' gi11283645 __________________________________________________ $$
gi16331287 gi~1473866 gi~7305365 gi~1522867 gi~1283645 __________________________________________________ .~. .I.
i-TE ~, G " '~ Q T ~~ L
TE ~ G " '~iQ T ~~ L
__ ~ G .. ~~-iQ T v~ S
TE ~I L " '~ R P ~~ L
PGCQNKR~M PQIE '~ Y~~ S- R H AI~TA I~~
... .~.
_____p ...TQ. . . ~ ~ ~ L~ i~~ R
_____p ...TQ. . . ~ ~ ~ L~Mi~~ R
_____p ...TQ. . ~ ~ ~ L~ i~~ R
-----L ~~SPL~ ' ~ ~ ~G IC ~~ K
KAGENITVNL~ SEEAIRRGEYAVVR~LI~I1LE EGKRQ
The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological conditions can be diagnosed by determinng the amount of the new protein in a sample or by 15 determining the presence of mutations in the new genes. Specific uses are described for each of the sixteen genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cell growth, cell metabolism, cell differentiation, cell proliferation, andlor cell signaling.
In one embodiment of the present invention, NOVX or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NOVX. Examples of such disorders include, but are not limited to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, _;
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; neurological disorders such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Takob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; and disorders of vesicular transport such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking including acquired immunodeficiency syndrome (A)DS), allergic reactions, autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, osteoarthritis, scleroderma, Chediak-Higashi syndrome, Sjogren's syndrome, systemic lupus erythiematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helininthic, and protozoal infections, as well as additional indications listed for the individual NOVX clones.
The NOVX nucleic acids and proteins of the invention are useful in potential S diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diag~iostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
These also include potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotaxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), (v) an agent promoting tissue regeneration i~a vitro and in vivo, and (vi) a biological defense weapon.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 1 polypeptide has been identified as a Paladin-like protein (also referred to as CG93221-O1). The disclosed novel NOV1 nucleic acid (SECT m NO:l) of 2600 nucleotides is shown in Table 1A. The novel NOV1 nucleic acid sequences maps to the chromosome 10.
An ORF begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAG cadon at nucleotides 2583-2585. A putative untranslated region and/or downstream from the termination codon is underlined in Table IA, and the start and stop codons are in bold letters.
Table 1A. NOV1 Nucleotide Sequence (SEQ ID NO:1) GCTGCTGGCAGACTATGGGTACAACGGCCAGCACAGCCCAGCAGACGGTCTCGGCAGGCACCCCATT
TGAGGGCCTACAGGGCAGTGGCACGATGGACAGTCGGCACTCCGTCAGCATCCACTCCTTCCAGAGC
ACTAGCTTGCATAACAGCAAGGCCAAGTCCATCATCCCCAACAAGGTGGCCCCTGTTGTGATCACGT
ACAACTGCAAGGAGGAGTTCCAGATCCATGATGAGCTGCTCAAGGCTCATTACACGTTGGGCCGGCT
CTCGGACAACACCCCTGAGCACTACCTGGTGCAAGGCCGCTACTTCCTGGTGCGGGATGTCACTGAG
AAGATGGATGTGCTGGGCACCGTGGGAAGCTGTGGGGCCCCCAACTTCCGGCAGGTGCAGGGTGGGC
TCACTGTGTTCGGCATGGGACAGCCCAGCCTCTTAGGGTTCAGGCGGGTCCTCCAGAAACTCCAGAA
GGACGGACATAGGGAGTGTGTCATCTTCTGTGTGCGGGAGGAACCTGTGCTTTTCCTGCGTGCAGAT
GAGGACTTTGTGTCCTACACACCTCGAGACAAGCAGAACCTTCATGAGAACCTCCAGGGCCTTGGAC
CCGGGGTCCGGGTGGAGAGCCTGGAGCTGGCCATCCGGAAAGAGATCCACGACTTTGCCCAGCTGAG
CGAGAACACATACCATGTGTACCATAACACCGAGGACCTGTGGGGGGAGCCCCATGCTGTGGCCATC
CATGGTGAGGACGACTTGCATGTGACGGAGGAGGTGTACAAGCGGCCCCTCTTCCTGCAGCCCACCT
ACAGGTACCACCGCCTGCCCCTGCCCGAGCAAGGGAG'T'CCCCTGGAGGCCCAGTTGGACGCCTT'T'GT
CAGTGTTCTCCGGGAGACCCCCAGCCTGCTGCAGCTCCGTGATGCCCACGGGCCTCCCCCAGCCCTC
GTCTTCAGCTGCCAGATGGGCGTGGGCAGGACCAACC'T'GGGCATGGTCCTGGGCACCCTCATCCTGC
TTCACCGCAGTGGGACCACCTCCCAGCCAGAGGCTGCCCCCACGCAGGCCAAGCCCCTGCCTATGGA
GCAGTTCCAGGTGATCCAGAGCTTTCTCCGCATGGTGCCCCAGGGAAGGAGGATGGTGGAAGAGGTG
GACAGAGCCATCACTGCCTGTGCCGAGTTGCATGACCTGAAAGAAGTGGTCTTGGAAAACCAGAAGA
AGTTAGAAGGTATCCGACCGGAGAGCCCAGCCCAGGGAAGCGGCAGCCGACACAGCGTCTGGCAGAG
GGCGCTGTGGAGCCTGGAGCGATACTTCTACCTGATCCTGTTTAACTACTACCTTCATGAGCAGTAC
CCGCTGGCCTTTGCCCTCAGTTTCAGCCGCTGGCTGTGTGCCCACCCTGAGCTGTACCGCCTGCCCG
TGACGCTGAGCTCAGCAGGCCCTGTGGCTCCGAGGGACCTCATCGCCAGGGGCTCCCTACGGGAGGA
CGATCTGGTCTCCCCGGACGCGCTCAGCACTGTCAGAGAGATGGATGTGGCCAACTTCCGGCGGGTG
CCCCGCATGCCCATCTACGGCACGGCCCAGCCCAGCGCCAAGGCCCTGGGGAGCATCCTGGCCTACC
TGACGGACGCCAAGAGGAGGCTGCGGAAGGTTGTCTGGGTGAGCCTTCGGGAGGAGGCCGTGTTGGA
GTGTGACGGGCACACCTACAGCCTGCGGTGGCCTGGGCCCCCTGTGGCTCCTGACCAGCTGGAGACC
CTGGAGGCCCAGCTGAAGGCCCATCTAAGCGAGCCTCCCCCAGGCAAGGAGGGCCCCCTGACCTACA
GGTTCCAGACCTGCCTTACCATGCAGGAGGTCTTCAGCCAGCACCGCAGGGCCTGTCCTGGCCTCAC
CTACCACCGCATCCCCATGCCGGACTTCTGTGCCCCCCGAGAGGAGGACTTTGACCAGCTGCTGGAG
GCCCTGCGGGCCGCCCTCTCCAAGGACCCAGGCACTGGCTTCGTGTTCAGCTGCCTCAGCGGCCAGG
GCCGTACCACAACTGCGATGGTGGTGGCTGTCCTGGCCTTCTGGCACATCCAAGGCTTCCCCGAGGT
GGGTGAGGAGGAGCTCGTGAGTGTGCCTGATGCCAAGTTCACTAAGGGTGAATTTCAGGTAGTAATG
AAGGTGGTGCAGCTGCTACCCGATGGGCACCGTGTGAAGAAGGAGGTGGACGCAGCGCTGGACACTG
TCAGCGAGACCATGACGCCCATGCACTACCACCTGCGGGAGATCATCATCTGCACCTACCGCCAGGC
GAAGGCAGCGAAAGAGGCGCAGGAAATGCGGAGGCTGCAGCTGCGGAGCCTGCAGTACTTGGAGCGC
TATGTCTGCCTGATTCTCTTCAACGCGTACCTCCACCTGGAGAAGGCCGACTCCTGGCAGAGGCCCT
TCAGCACCTGGATGCAGGAGGTGGCATCGAAGGCTGGCATCTACGAGATCCTTAACGAGCTGGGCTT
CCCCGAGCTGGAGAGCGGGGAGGACCAGCCCTTCTCCAGGCTGCGCTACCGGTGGCAGGAGCAGAGC
TGCAGCCTCGAGCCCTCTGCCCCCGAGGACTTGCTGTAGGGGGCCTTACTCCCT
Variant sequences of NOV1 are included in Example 3, Table 18. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 1 protein (SEQ m N0:2) encoded by SEQ >D NO:1 is 856 amino acid residues in length and is presented using the one-letter amino acid code in Table 1B. Psort analysis predicts the NOV 1 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 1B. Encoded NOV1 protein sequence (SEQ ID NO:2) MGTTASTAQQTVSAGTPFEGLQGSGTMDSRHSVSIHSFQSTSLHNSKAKSIIPNKVAPWITYNC
KEEFQIHDELLKAHYTLGRLSDNTPEHYLVQGRYFLVRDVTEKMDVLGTVGSCGAPNFRQVQGGL
TVFGMGQPSLLGFRRVLQKLQKDGHRECVIFCVREEPVLFLRADEDFVSYTPRDKQNLHENLQGL
GPGVRVESLELAIRKEIHDFAQLSENTYHVYHNTEDLWGEPHAVAIHGEDDLHVTEEWKRPLFL
QPTYRYHRLPLPEQGSPLEAQLDAFVSVLRETPSLLQLRDAHGPPPALVFSCQMGVGRTNLGMVL
GTLILLHRSGTTSQPEAAPTQAKPLPMEQFQVIQSFLRMVPQGRRMVEEVDRAITACAELHDLKE
WLENQKKLEGIRPESPAQGSGSRHSWQRALWSLERYFYLILFNYYLHEQYPLAFALSFSRWLC
AHPELYRLPVTLSSAGPVAPRDLIARGSLREDDLVSPDALSTVREMDVANFRRVPRMPIYGTAQP
SAKALGSILAYLTDAKRRLRKWWVSLREEAVLECDGHTYSLRWPGPPVAPDQLETLEAQLKAHL
SEPPPGKEGPLTYRFQTCLTMQEVFSQHRRACPGLTYHRIPMPDFCAPREEDFDQLLEALRAALS
KDPGTGFVFSCLSGQGRTTTAMWAVLAFWHIQGFPEVGEEELVSVPDAKFTKGEFQWMKWQL
LPDGHRVKKEVDAALDTVSETMTPMHYHLREIIICTYRQAKAAKEAQEMRRLQLRSLQYLERYVC
LILFNAYLHLEKADSWQRPFSTWMQEVASKAGIYEILNELGFPELESGEDQPFSRLRYRWQEQSC
SLEPSAPEDLL
Tn all BLAST alignments described herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size.
It decreases exponentially with the Score (S) that is assigned to a match between two sequences.
Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001, with the filter to remove low complexity sequence turned off. In BLAST 2.0, the Expect value is also used instead of the P
value (probability) to report the significance of matches. For example, an E
value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance.
See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering of the query for low-complexity sequence that is performed to prevent artifactual hits The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., " ") or the letter "X" in protein sequences (e.g., "XXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571, (1996).
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 1 C.
Table 1C. Patp results for NOVl Smallest Sum eadingigh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:AAB41108Human ORFX ORF872 polypeptide +1 4187 0.0 >patp:AAB35276Murine dual specificity phosphatase+1 120 5.2e-06 >patp:AAB73211Murine phosphatase AA023073 +1 120 5.2e-06 m >patp:AAB73231Human phosphatase BAA91172 h +1 115 1.8e-05 >patp:AAG67455Amino acid sequence of a human +1 115 1.8e-0 polypeptide In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2063 of 2508 bases (82%) identical to a gb:GENBANK-m:MMPAL~acc:X99384.1 mRNA from Mus musculus (Paladin gene). The full amino acid sequence of the protein of the invention was found to have 695 of 859 amino acid residues (80%) identical to, and 754 of 859 amino acid residues (87%) similar to, the 859 amino acid residue ptnr:SPTREMBL-ACC:P70261 protein from Mus rnusculus (PALADIN
GENE). NOVl also has homology to the proteins shown in the BLASTP data in Table 1D.
Table 1D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~6331287~dbj~BAABKIAA1274 protein752 752/752 752/752 0.0 6588.1 (AB033100)[Homo Sapiens] (100%) (100%) gi~14738662~ref~XPKIAA protein 748 747/748 747/748 0.0 _ (similar to (99%) (99%) 046314.1 mouse (XM 046314) paladin) (Homo sapi ens]
gi~7305365~ref~NPpaladin [Mus 859 673/841 730/841 0.0 _ musculus] (80%) (86%) 38781.1 (NM 013753) gi~15228672~refINPputative protein1232 207/821 340/821 7e-45 _ [Arabidopsis (25%) (41%) 191760.1 (NM 116066) thaliana]
gi~12836455~dbj~BABdata source:SPTR,144 24/60 33/60 Ze-04 23663.1I(AK004912)source (40%) (55%) key:Q9NX48, evidence:ISS-.homo log to CDNA
FLJ20442 FIB, CLONE
KAT04828-putative [Mus musculus]
A multiple sequence alignment is given in Table 1E, with the NOV 1 protein being shown on line 1 in Table 1E in a ClustalW analysis, and comparing the NOV1 protein with the related protein sequences shown in Table 1D. This BLASTP data is displayed graphically in the ClustalW in Table 1E.
Table 1E. ClustalW Analysis of NOVl 1) > NOVl; SEQ ID NO:2 2) > gi~6331287/ KIAA1274 protein [Hofrao Sapiens]; SEQ ID N0:35 3) > gi~1473866/ KIAA protein (similar to paladin) [Homo Sapiens]; SEQ ID
N0:36 4) > gi~7305365/ paladin [Mus musculus]; SEQ ID N0:37 5) > gi[1522867/ putative protein [Arabidopsis thaliana]; SEQ ID N0:38 6) > gi~1283645/ data source: SPTR, source key: Q9NX48, evidence: IBS-homolog to cDNA FLJ20442 FIB clone KAT04828 putative [Mus muscudus]; SEQ ID N0:39 gi~6331287 -_________________________________________________ gi~1473866 __________________________________________________ gi~7305365 MGTTASTAQQTVSAGTSLEGLQGGSSSSMDSQHSLGGVQSFRATSLHNSK
gi~1522867 __________________________________________________ gi~1283645 __________________________________________________ gi16331287 __________________________________________________ gi11473866 __________________________________________________ 3O gi~7305365 AKSIIPNKVAPWITYNCKEEFQIHDELLKAHYRMGRLSDATPEHYLVQG
gi11522867 ----------------------------MSIPKEPEQVMKMRDGSVLGKK
gi~1283645 __________________________________________________ $ gi~6331287 -------V
gi~1473866 ________ gi~7305365 RYFLVRDI
gi11522867 TILKSDHF
i 1283645 __________________________________________________ g .~. .~.
NOV1 ~ ~ ~ ---HR -iL - ~ Q QG
71D ~
gi16331287 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~1473866 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~7305365 ~ ~T~ ---L I ' E ~ ~~ES RD
giI1522867 RHIGAHKDGKQVKVLWTSLVYINGRPF VLRDVEKPFT GIN
EYT
gi~1283645 _______-__________________________________________ gi~6331287 gi~1473866 2$ gi~7305365 gi,1522867 gi~1283645 _______________________________________________-__ Nov1 gi1 6331287 gi~ 1473866 gi~ 7305365 3$ gi~ 1522867 gi~ 1283645_______ ___________________________________________ NOV1 .Q'~.. P ~~1 ILL. ~-____ gi1 6331287 Q ~ P ~i ILL t-----' gi1 1473866 Q ~ P V ILL ~-----~
giI 7305365 P ~ L L F ~-----~S H
R
gi~ 1522867------- --IN TEII~R ' I F
TT T
SD~GFPRN
4$ gi~ ~.283645_______ _,___________________-_____________________ ...
Nov1 --------$0 gi~6331287 --------gi11473866 --------giI7305365 --------gi~1522867 NSFGRIF ~N' gi11283645 __________________________________________________ $$
gi16331287 gi~1473866 gi~7305365 gi~1522867 gi~1283645 __________________________________________________ .~. .I.
i-TE ~, G " '~ Q T ~~ L
TE ~ G " '~iQ T ~~ L
__ ~ G .. ~~-iQ T v~ S
TE ~I L " '~ R P ~~ L
PGCQNKR~M PQIE '~ Y~~ S- R H AI~TA I~~
... .~.
_____p ...TQ. . . ~ ~ ~ L~ i~~ R
_____p ...TQ. . . ~ ~ ~ L~Mi~~ R
_____p ...TQ. . ~ ~ ~ L~ i~~ R
-----L ~~SPL~ ' ~ ~ ~G IC ~~ K
KAGENITVNL~ SEEAIRRGEYAVVR~LI~I1LE EGKRQ
gi~6331287 gi~1473866 gi~7305365 gi~1522867 gi~1283645 ________________,_________________________________ ... .I.
NOV1 ___________________ ~ W ~R ~Em pw -_ gi16331287 ~ ____________________ , . .E " p,. __ 1$ gi11473866 ~ ____________________ ~ , ~R ~E " P~~ __ gi~7305365 ~L-____________________ ~G, ~ E " L,~ __ gi~1522867 ~MGALGYAAMKPSLIKIAESTDG 'HEMSW ~SGAVLGSQTVLKSDH
gi~1283645 __________________________________________________ NOV1 _________ , . .. .-r~ .~~ . . . T,' .
gi~6331287 _________ , .,.. .~.~.r~, ii,. T,.
gi11473866 _________ , . .. ~ .~. . . . T,.
2$ giI7305365 ----_---- , . .~. . . . S,.
gi11522867 SPGCQILNLPE~ EGAP 'E ~GF~ ; ~TIDGIR IERVGSSR-gi~1283645 __________________________________________________ .~.
NOV1 R ' 'S ' ~ Y ~W' " --------- ~'~Q T
gi16331287 R ' S ' ~ y ~W~ .._________ ..,Q T
gi~1473866 R ~ S ' ~ Y 'W' ~~--------- ~~~Q T
giI7305365 'Q IF ' ~ WP' --------L~~E
3$ giI1522867 GG~P F HN~PV~ KPFVmEVE ~YKNMLEYTGIDR~R~GM
gi~1283645 ---NFS L -------- RLAG~LORL~---------AHYQF LDQG
NOV1 ~~. ~ 'P~GKE ~LT ~.~ ___,___________ , , gi16331287 ~~ ~ ~P'GKE ~LTY~ ~ _______________ v v gi~1473866 ~~ ~ ~P~GKE ~LT ' ~ _______________ gi~7305365 ~~ ~ ~ TKS~TAP~ ~K---------------- T~
4S gi~1283645 VRHLVS~T~EAKRYD IMVIHE-KDGQIFDLWENVDADSVQ_PL--YKP
50 gi~6331287 gi~1473866 gi~7305365 gi~1522867 gi11283645 .~....~... ,I.
NOV1 ~ ~ . . .F Q ___________F. .,. _ gi~6331287 ~ ~ ~ . .F Q ___________F. .,. _ gi~1473866 ~ ~ ~ ~ ~F Q -----------F~ ~
gi~7305365 ~ ~ ~ ~ ~C ----------C~ ~
gi~1522867 M R~GTVIiC~KLR YGRPIKVLYDVLT IVD SS GGEETG
gi~1283645 L F ~ G-_-__TMrr::rr~~CYLVK-________________~G_____ NOV1 _____________________ ~iiTiii~ ~~ R ~-i1~1 gi16331287 _____________________ ~ ~ ., R
gi~1473866 _____________________ ~ ~ ~~ R
gi~7305365 -____________________ ~
gi~1522867 SNNAEARPRNSGRRTEEEQG GMDDILLLW'~iTTR FDN~TESREAL~~
gi~1283645 ____________________________DATAETRR~R~G__________ Nov1 ~ .T _____________________________ .. .. .
gi16331287 ~ ~T ___________________________ , v gi~1473866 ~ ~T _____________________________ gi~7305365 ~ ~I _____________________________ gi11522867 VI~RC QNIREAVLQYRKVFNQQHVEPRVRSAALKRGAEYLERYF~L
gi~1283645 __________________________________________________ gi~6331287 C __________ gi~1473866 C __________ 2$ giI7305365 S ----------gi~1522867 AF LGSKAFDGFFVEi gi~1283645 S~E E-----------.~. .~. .~.
NOVl ~ ~ " Q ~S ~ --- E ~ G ~
gi~6331287 ~ ~~~ Q ~S ~ --- E G D~
gi~1473866 ~ ~~ Q ~S ~ --- E G ~
giI7305365 ~ R STS ~'~ ~T ~ ---- Q ~ I E~
3S gi11522867 IPEE RAQHES~HGDAVMESI ERS SVLSKGS KMYF ~GQRTSSRL
gi~1283645 _________-________________________________________ ~n NOV1 ~F~~ ~ ~.CSL.~S~,~PE~L -------------------------gi~6331287 ~F ~ ~ ~ CSL ~S~,~PE~L -------------------------gi~1473866 ~F ' ~ ~ CSL ~S~PE~L -________________________ gi~7305365 ~L ' ~ ~ RDP ~CDVG~F -------------------------gi11522867 QINGAPHVYKVDRYPVYSMiTPTISGAKKMLAYLGTKI~KEEGGGSTERIV
4$ gi~1283645 __________________________________________________ The NOV1 Clustal W alignment shown in Table 1E was modified to begin at amino residue 1050. The data in Table 1E includes all of the regions overlapping with the NOV 1 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (htip:www.ebi.ac.uk/interpro~. Table 1F lists the domain description from DOMAIN analysis results against NOV 1.
NOV1 ___________________ ~ W ~R ~Em pw -_ gi16331287 ~ ____________________ , . .E " p,. __ 1$ gi11473866 ~ ____________________ ~ , ~R ~E " P~~ __ gi~7305365 ~L-____________________ ~G, ~ E " L,~ __ gi~1522867 ~MGALGYAAMKPSLIKIAESTDG 'HEMSW ~SGAVLGSQTVLKSDH
gi~1283645 __________________________________________________ NOV1 _________ , . .. .-r~ .~~ . . . T,' .
gi~6331287 _________ , .,.. .~.~.r~, ii,. T,.
gi11473866 _________ , . .. ~ .~. . . . T,.
2$ giI7305365 ----_---- , . .~. . . . S,.
gi11522867 SPGCQILNLPE~ EGAP 'E ~GF~ ; ~TIDGIR IERVGSSR-gi~1283645 __________________________________________________ .~.
NOV1 R ' 'S ' ~ Y ~W' " --------- ~'~Q T
gi16331287 R ' S ' ~ y ~W~ .._________ ..,Q T
gi~1473866 R ~ S ' ~ Y 'W' ~~--------- ~~~Q T
giI7305365 'Q IF ' ~ WP' --------L~~E
3$ giI1522867 GG~P F HN~PV~ KPFVmEVE ~YKNMLEYTGIDR~R~GM
gi~1283645 ---NFS L -------- RLAG~LORL~---------AHYQF LDQG
NOV1 ~~. ~ 'P~GKE ~LT ~.~ ___,___________ , , gi16331287 ~~ ~ ~P'GKE ~LTY~ ~ _______________ v v gi~1473866 ~~ ~ ~P~GKE ~LT ' ~ _______________ gi~7305365 ~~ ~ ~ TKS~TAP~ ~K---------------- T~
4S gi~1283645 VRHLVS~T~EAKRYD IMVIHE-KDGQIFDLWENVDADSVQ_PL--YKP
50 gi~6331287 gi~1473866 gi~7305365 gi~1522867 gi11283645 .~....~... ,I.
NOV1 ~ ~ . . .F Q ___________F. .,. _ gi~6331287 ~ ~ ~ . .F Q ___________F. .,. _ gi~1473866 ~ ~ ~ ~ ~F Q -----------F~ ~
gi~7305365 ~ ~ ~ ~ ~C ----------C~ ~
gi~1522867 M R~GTVIiC~KLR YGRPIKVLYDVLT IVD SS GGEETG
gi~1283645 L F ~ G-_-__TMrr::rr~~CYLVK-________________~G_____ NOV1 _____________________ ~iiTiii~ ~~ R ~-i1~1 gi16331287 _____________________ ~ ~ ., R
gi~1473866 _____________________ ~ ~ ~~ R
gi~7305365 -____________________ ~
gi~1522867 SNNAEARPRNSGRRTEEEQG GMDDILLLW'~iTTR FDN~TESREAL~~
gi~1283645 ____________________________DATAETRR~R~G__________ Nov1 ~ .T _____________________________ .. .. .
gi16331287 ~ ~T ___________________________ , v gi~1473866 ~ ~T _____________________________ gi~7305365 ~ ~I _____________________________ gi11522867 VI~RC QNIREAVLQYRKVFNQQHVEPRVRSAALKRGAEYLERYF~L
gi~1283645 __________________________________________________ gi~6331287 C __________ gi~1473866 C __________ 2$ giI7305365 S ----------gi~1522867 AF LGSKAFDGFFVEi gi~1283645 S~E E-----------.~. .~. .~.
NOVl ~ ~ " Q ~S ~ --- E ~ G ~
gi~6331287 ~ ~~~ Q ~S ~ --- E G D~
gi~1473866 ~ ~~ Q ~S ~ --- E G ~
giI7305365 ~ R STS ~'~ ~T ~ ---- Q ~ I E~
3S gi11522867 IPEE RAQHES~HGDAVMESI ERS SVLSKGS KMYF ~GQRTSSRL
gi~1283645 _________-________________________________________ ~n NOV1 ~F~~ ~ ~.CSL.~S~,~PE~L -------------------------gi~6331287 ~F ~ ~ ~ CSL ~S~,~PE~L -------------------------gi~1473866 ~F ' ~ ~ CSL ~S~PE~L -________________________ gi~7305365 ~L ' ~ ~ RDP ~CDVG~F -------------------------gi11522867 QINGAPHVYKVDRYPVYSMiTPTISGAKKMLAYLGTKI~KEEGGGSTERIV
4$ gi~1283645 __________________________________________________ The NOV1 Clustal W alignment shown in Table 1E was modified to begin at amino residue 1050. The data in Table 1E includes all of the regions overlapping with the NOV 1 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (htip:www.ebi.ac.uk/interpro~. Table 1F lists the domain description from DOMAIN analysis results against NOV 1.
Table 1F Domain Analysis of NOVl Model Region of Score (bits) E value Homology #PD396342 19-118 431 4e-43 PALADIN GENE
#PD148197 119-399 1175 e-129 PALADIN GENE
#PD222597 119-231 119 6e-07 DOMAIN
OF UNKNOWN
#PD306865 354-454 132 2e-08 PALADIN GENE
#PD024454 356-445 84 0.007 PLASMID ORFS
#PD325716 400-604 800 6e-86 PALADIN GENE
#PD326847 458-594 97 2e-04 #PD222597 505-602 113 3e-06 bOMAIN
OF UNKNOWN
#PD277963 595-648 97 2e-04 HYDROLASE
CDNA
#PD148197 605-678 340 1e-32 PALADIN GENE
#PD306865 680-856 765 7e-82 PALADIN GENE
#PD325716 751-820 86 0.004 PALADIN GENE
Consistent with other known members of the Paladin-like family of proteins, has, for example, multiple Paladin gene signature sequences and homology to other members of the Paladin-like Protein Family. NOV 1 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 1 nucleic acids and polypeptides can be used to identify proteins that are members of the Paladin like family of proteins. The NOV 1 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 1 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism and signal transduction. These molecules can be used to treat, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atnial septal defect (ASD),atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, hyperthyroidism and hypothyroidism, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmmne disease, allergies, immunodeficiencies, transplantation, graft vesus host, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphedema, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, cancer, trauma, regeneration (in vitro and irz vivo), viral/bacteriallparasitic infections, as well as other diseases, disorders and conditions.
In addition, various NOV 1 nucleic acids and polypeptides according to the invention are useful, izzter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV1 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Paladin-Iike protein family.
Paladin proteins are a family of protein-tyrosine phosphatases. The protein phosphatases can be divided into 2 large families: the serinelthreonine phosphatases, which are metalloproteins, and the protein-tyrosine phosphatases, which proceed via a thiol-phosphate enzyme intermediate. The protein-tyrosine phosphatase family includes the VHl-like dual-specificity phosphatases. These phosphatases dephosphorylate phosphotyrosine-as well as phosphoserine- and phosphothreonine-containing substrates. Members of the dual-specificity phosphatase protein family inactivate mitogen-activated protein (MAP) kinase through dephosphorylation of critical threonine and tyrosine residues. Members of the MAP kinase family play a pivotal role in cellular signal transduction. Using a subtractive screen of mouse gastrulation, Pearce et al. (1996) identified a novel mouse gene, paladin, with similarity to the dual specificity protein phosphatase family.
The NOV1 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and endocrine physiology. As such, the NOV 1 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD),atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, hyperthyroidism and hypothyroidism, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft vesus host, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeflciencies, transplantation, graft versus host disease (GVHD), lymphedema, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, cancer, trauma, regeneration (ira vitro and iya vivo), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
The NOV1 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 1 nucleic acid is expressed in brown adipose, heart, aorta, Ve111, umbilical vein, adrenal gland/suprarenal gland, pancreas, thyroid, salivary glands, parotid salivary glands, stomach, liver, gall bladder, small intestine, colon, bone marrow, lymphoid tissue, spleen, lymph node, tonsils, thymus, cartilage, muscle, brain, thalamus, hypothalamus, pituitary gland, amygdala, substantia nigra, hippocampus, spinal chord, cervix, mammary gland/breast, ovary, placenta, uterus, vulva, prostate, testis, lung, lung pleura, kidney, retina, dermis.
Additional utilities for NOV 1 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV2 polypeptide has been identified as a Plasma Membrane Ring Finger-like protein (also referred to as CG93210-01). The disclosed novel NOV2 nucleic acid (SEQ m N0:3) of 1205 nucleotides is shown in Table 2A. The novel NOV2 nucleic acid sequences maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 17-19 and ends with a ATT codon at nucleotides 1149-1151. A putative untranslated region and/or downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
Table 2A. NOV2 Nucleotide Sequence (SEQ ID N0:3) CTCGCCGGGTCCGGCCATGGGCCCCGCCGCTCGCCCCGCGCTGAGATCGCCGCCGCCGCCTCCGCCG
CCGCCTCCGTCTCCGCTGCTGCTGCTGCTGCCCCTGCTGCCGCTGTGGCTGGGCCTGGCGGGGCCCG
GGGCCGCGGCGGACGGCAGCGAGCCGGCGGCCGGGGCGGGGCGGGGCGGAGCCCGCGCCGTGCGGGT
GGACGTGAGACTGCCGCGCCAGGACGCTCTGGTCCTGGAGGGCGTCAGGATCGGCTCCGAAGCCGAC
CCGGCGCCCCTGCTGGGCGGTCGTCTGCTGCTGATGGACATCGTGGATGCCGAGCAGGAGGCACCAG
TGGAAGGCTGGATTGCAGTGGCATACGTGGGCAAGGAGCAGGCGGCCCAGTTCCACCAGGAGAATAA
GGGCAGTGGCCCGCAGGCCTATCCCAAGGCCCTGGTCCAGCAGATGCGGCGGGCCCTCTTCCTGGGT
GCCTCTGCCCTGCTTCTTCTCATCCTGAACCACAACGTGGTCCGAGAGCTGGACATATCCCAGCTTC
TGCTCAGGCCAGTGATCGTCCTCCATTATTCCTCCAATGTCACCAAGCTGTTGGATGCATTGCTGCA
GAGGACCCAGGCCACGGCTGAGATCACCAGCGGAGAGTCCCTGTCTGCCAATATCGAGTGGAAGTTG
ACCTTGTGGACCACCTGTGGCCTCTCCAAGGATGGCTATGGAGGATGGCAGGACTTGGTCTGCCTTG
GAGGCAGTCGTGCCCAGGAGCAGAAACCCCTGCAGCAGCTGTGGAACGCCATCCTGCTGGTGGCCAT
GCTCCTGTGCACAGGCCTCGTGGTCCAGGCCCAGCGGCAGGCGTCGCGGCAGAGCCAGCGGGAGCTC
GGAGGCCAGGTGGACCTGTTTAAGCGCCGCGTGGTGCGGAGACTGGCATCCCTCAAGACACGGCGCT
GCCGGCTGAGCAGGGCAGCGCAGGGCCTCCCAGATCCGGGTGCTGAGACCTGTGCGGTGTGCCTGGA
CTACTTCTGCAACAAACAGTGGCTCCGGGTGCTGCCCTGTAAGCACGAGTTTCACCGAGACTGTGTG
GACCCCTGGCTGATGCTCCAGCAGACCTGCCCACTGTGCAAATTCAACGTCCTGGGTGAGCACCGCT
ACTCCGATGATTAGCTGCCCAGCTGGACTCTGCACATGGGGATGGACCCCTCCTGCCTGCACCCCG
The NOV2 protein (SEQ ID N0:4) encoded by SEQ ID NO:3 is 378 amino acid residues in length and is presented using the one-letter amino acid code in Table 2B. Psort analysis predicts the NOV2 protein of the invention to be localized at the plasma membrane with a certainty of 0.6400.
Table 2B. Encoded NOV2 protein sequence (SEQ ID N0:4) MGPAARPALRSPPPPPPPPPSPLLLLLPLLPLWLGLAGPGAAADGSEPAAGAGRGGARAVRVDVR
LPRQDALVLEGVRIGSEADPAPLLGGRLLLMDIVDAEQEAPVEGWIAVAYVGKEQAAQFHQENKG
SGPQAYPKALVQQMRRALFLGASALLLLILNHNVVRELDISQLLLRPVIVLHYSSNVTKLLDALL
QRTQATAEITSGESLSANIEWKLTLWTTCGLSKDGYGGWQDLVCLGGSRAQEQKPLQQLWNAILL
VAMLLCTGLVVQAQRQASRQSQRELGGQVDLFKRRWRRLASLKTRRCRLSRAAQGLPDPGAETC
AVCLDYFCNKQWLRVLPCKHEFHRDCVDPWLMLQQTCPLCKFNVLGEHRYSDD
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 2C.
Table 2C. Pat results for Smallest Sum eadingigh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAB42695HumanORFX ORF2459 polypeptide +1 1715 3.0e-276 >patp:AAM79288Humanprotein SEQ TD NO 1950 +1 612 2.3e-59 >patp:AAM80272Humanprotein SEQ TD NO 3918 +1 534 4.2e-51 >patp:AAU28202Novelhuman secretory protein +1 201 5.1e-13 >patp:ABB50251Humantranscription factor TRFX-102+1 148 5.5e-13 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 287 of 489 bases (58%) identical to a gb:GENBANK-1D:SSI132828~acc:AJ132828.1 mRNA from Spermatozopsis similis (mRNA
for p210 protein, partial). The full amino acid sequence of the protein of the invention was found to have 341 of 379 amino acid residues (89%) identical to, and 355 of 379 amino acid residues (93%) similar to, the 379 amino acid residue ptnr:SPTREMBL-ACC:Q9DCW1 protein from Mus musculus (0610009J22RIK PROTEIN).
NOV2 also has homology to the proteins shown in the BLASTP data in Table 2D.
Table 2D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~12832380~dbj~BABdata source:SPTR,379 340/380 354/380 e-173 22082.1 (AK002414)source (89%) (92%) key:Q9Y6U7, evidence:ISS~homo log to WUGSC:H DJ130H16.
PROTEIN(FRAGMENT) putative [Mus musculus]
gi~5441942~gb~AAD43supported by 347 336/336 336/336 e-148 187.1~AC004997 mouse EST (100%) (100%) (AC004997) AA538043 (NID:g2284036) [Homo Sapiens]
gi~17485136~refiXPsimilar to data272 271/283 272/283 e-146 _ source:SPTR, (95%) (95%) 066294.1 (XM 066294) source key:Q9Y6U7 evidence:ISS-homo log to WUGSC:H_DJ130H16.
(FRAGMENT)-putati ve [Homo Sapiens]
giI17861674~gb~AAL3GH20973p 461 26/57 42/57 9e-13 9314.1 (AY069169)[Drosophila (45%) (73%) melanogaster]
gi~18485962~ref~XPsimilar to 461 26/57 42/57 1e-12 - goliath (H. (45%) (73%) 080778.1 (XM 080778) Sapiens) [Drosophila melanogaster]
A multiple sequence alignment is given in Table 2E, with the NOV2 protein being shown on line 1 in Table 2E in a ClustalW analysis, and comparing the NOV2 protein with the related protein sequences shown in Table 2D. This BLASTP data is displayed graphically in the ClustalW in Table 2E.
Table 2E. ClustalW Analysis of NOV2 1) > NOV2; SEQ 1D N0:4 2) >gi~12832380~/ data source:SPTR, source key:Q9Y6U7, evidence:ISS~homolog to WITGSC:H DJ130HI6.6 PROTEIN(FRAGMENT)~putative [Mus musculus]; SEQ ID N0:40 S 3) >gi~5441942[/ supported by mouse EST AA538043 (NID:g2284036) [Homo sapierrs]; SEQ ID
N0:41 4) >gi~17485136~/ similar to data source:SPTR, source key:Q9Y6U7 evidence:ISS~homolog to WUGSC:H DJ130H16.6 PROTEIN (FRAGMENT)~putative [Homo Sapiens]; SEQ ID N0:42 S) >gi~17861674~/ GH20973p [D>"osophila rnelanogaster°]; SEQ ID
N0:43 6) >gi~18485962~/ similar to goliath (H. Sapiens) [Drosophila melarrogaster];
SEQ ID N0:44 1 S NOV2 1 -- GP ~~ P RSP--P ~ 'PPP PPLL ~:f'LPI#LPLWL ~ GPG ~~ . EP ' 52 gi~128323801 1 -- -GS~P RSPSLP " PPS PPLL ~LP~iLPLWL GPG ~EPAT E 54 gi I 5441942 I 1 -- --GP~ARP RSP--P "PPP PPLL T.iLPLPLWLG~AGPGAAAS,EP '.. 52 gi~17485136~ 1 ______~_____________________.____'___________________________ 1 gi ~ 17861674 ~ 1 MYIRKTLL~i1CLVL FGG--L"LTF ~,'TTT AAh?tSIANQD ERYFRPG THSFS
2O gi ~ 18485962 I 1 MYIRKTT~LL~CLVLBFGG--L~LTF~A'~'TTT~AAN~SIANQDBERYFRPG~'~HSF~ 58 p...
NOV2 53 GRGG~i =R----------------VL1't7RLP~.QDj~~I,LVL ------ ~ GS~EAT7PP 87 2S gi ~ 12832380 ~ 55 GRGGf,~~1,PL R=---------------V~T~KLP~.QD~~,LVL ------ ~
gi~5441942~ 53 GRGG~RA~R _______________~RLP~2QD~LVL ________G'EM3PP 87 gi 17485136 Z
gi~178616741 59 EDRI~i1VD YNYAFLNWSYVEHGNMLC~EFA~Q~~,RY KVLNVTGRLH T,ATDFD 118 gi~18485962~ 59 EDRI~~YNYAFLNWSYVEHGNMLC~EFAQFQ~RYG~KVLNVTGRL,~HBT~TDF~.S,D
30 ' gi~12832380~ 90 145 3S gi~5441942~ 88 143 gi~17485136~ 1 48 gi~I7861674~ 119 177 gi~184859621 119 177 gi~128323801 146 203 gi~54419421 144 201 4S gi~174851361 49 106 gi~17861674~ 178 237 gi~18485962~ 178 237 NOV2 202 ~ 1'I' ~ _____________ ______~ "___ 236 gi~12832380~ 204 ~ S ~ _____________ ______~ ~~-__ 238 gi~54419421 202 ~ T _____________ ______~ w-__ 236 gi~17485136~ 107 ~ ~ _______________ _______, w-__ 141 SS gi ~ 17861674 ~ 238 VS FIVL~III~L LFY~'~~~,'IQRFRYMQAKDQQSRNL StrT
~KAIMKIPTKT KE~S~EKD 297 gi ~ 18485962 I 238 VS SFTVL~IISL L~FYIQRFRYMQAKDQQSRNL S~tt'I' ~KAIMKIPTKT
IC~'S~EKD 297 gi~Z2832380~ 238 gi~5441942~ 236 287 gi117485136~ 141 192 gi~178616741 298 357 6S gi~18485962~ 298 357 ~y y ~ .I~ ~~~ ~I. ~~~ .y y y . ~~. y w y v~~w~~ aw ' ~w NOV2 288 sn-- ' '~~Q '~' CDY .KQWLRVLP 342 7~1 ~vV
gi~12832380~ 290 ~E~-- ~ ~~HS~~E''T CDY KQWLRVLP 344 gi~54419421 288 ~ ~-- ~~Q ~~~ C~iDY ------- 334 gi~17485136~ 193 ~ ~-- ' '~~Q ~~~ ~ LRVLP K-------- 239 .YY
gi ~ 17861674 ~ 358 SE~SILE~IYC~PDPP~L,V 'DESAD '~RDF ~FPRVFVLDSGCWGAREMLFPCR
gi~18485962~ 358 ~- --~XQTPSP~HTiP~AIEEVPV~UViVPH~~Q~LQPLQiSN~iSS~APSHYFQSSR
1~ 430 440 450 460 470 NOV2 343 CK---HEFHDC'VD LLsQC~ CF GEHRYS~_?~~D------------- 378 gi~12832380! 345 CK---HEFHR!DC~D L~IL~Q'~C~ CAF GHYSD~-------------- 379 gi~54419421 334 ________ K __~ ~ ApG ___ ____-_________ 347 15 gy 17485136 ~ 239 -----HEFH~DC~DQP1LL~QC~CF G1i.~T:~RYSD~~--------------gi~17861674! 418 IPERSQSSLSLRQARDW,~SLMN E~QQ RMRND MQQVIK-------- 461 gi~18485962~ 413 SP---SSSVQQLTYQPHPQQAASERGRRISAPATMPHAITASHQVTDV 461 20 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Iliterpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 2F lists the domain description from DOMAIN analysis results against NOV2.
Table 2F Domain sis of Anal NOV2 Model Region of Score E value (bits) Homology Ring Finger 325-365 49.3 4.0e-07 zf-C3HC4 325-365 34.7 2.2e-09 PHD 324-368 -10.4 1.1 Consistent with other known members of the Membrane Ring Finger-like family of proteins, NOV2 has, for example, a Ring Finger signature sequence and homology to other members of the Plasma Membrane Ring Finger-Iike Protein Family. NOV 2 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV2 nucleic acids and polypeptides can be used to identify proteins that are members of the Plasma Membrane Ring Finger-like Protein Family. The NOV2 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 1 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism and signal transduction.
These molecules can be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, immunodeficiencies, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cancer, trauma, i regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
In addition, various NOV2 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV2 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Plasma Membrane Ring Finger-like Protein Family .
The NOV2 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of immune and renal physiology. As such, the NOV2 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., anemia, ataxia-telangiectasia, autoimmume disease, immunodeficiencies, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cancer, trauma, regeneration (in vitro and in vdv0), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
The NOV2 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV2 nucleic acid is expressed in peripheral blood, and a pool of various mammalian tissues. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of CuraGen Acc. No. CG93210-Ol.The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-1D: gb:GENBANK-ID:SSI132828~acc:AJ132828.1) a closely related Spe~matozopsis similis mRNA for p210 protein, partial homolog in species Spe~n2atozopsis sirnilis :kidney.
Additional utilities for NOV2 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV3 polypeptide has been identified as a Thrombospondin type 1 (tsp_1) domain containing protein (also referred to as CG93275-OI). The disclosed novel NOV3 nucleic acid (SEQ ID NO:S) of 799 nucleotides is shown in Table 3A. The novel NOV3 nucleic acid sequences maps to the chromosome 16.
An ORF begins with an ATG initiation codon at nucleotides 51-53 and ends with a TGA codon at nucleotides 744-746. A putative untranslated region and/or downstream from the termination codon is underlined in Table 3A, and the start and stop codons are in bold letters.
Table 3A. NOV3 Nucleotide Sequence (SEQ ID NO:S) GAATATATTTAGTGTGTTGTTTTTTTTTTTAATGTGGCTACTGAAACCTAATGGGAATGCAAATAGA
ACTTTTTTGTCTTCTCAAGTGTTCCAAGACCTGTGGACGAGGGGTGAGGAAGCGTGAACTCCTCTGC
AAGGGCTCTGCCGCAGAAACCCTCCCCGAGAGCCAGTGTACCAGTCTCCCCAGACCTGAGCTGCAGG
AGGGCTGTGTGCTTGGACGATGCCCCAAGAACAGCCGGCTACAGTGGGTCGCTTCTTCGTGGAGCGA
GTGTTCTGCAACCTGTGGTTTGGGTGTGAGGAAGAGGGAGATGAAGTGCAGCGAGAAGGGCTTCCAG
GGAAAGCTGATAACTTTCCCAGAGCGAAGATGCCGTAATATTAAGAAACCAAATCTGGACTTGGAAG
AGACCTGCAACCGACGGGCTTGCCCAGCCCATCCAGTGTACAACATGGTAGCTGGATGGTATTCATT
GCCGTGGCAGCAGTGCACAGTCACCTGTGGGGGAGGGGTCCAGACCCGGTCAGTCCACTGTGTTCAG
CAAGGCCGGCCTTCCTCAAGTTGTCTGCTCCATCAGAAACCTCCGGTGCTACGAGCCTGTAATACAA
ACTTCTGTCCAGCTCCTGAAAAGAGAGAGGATCCATCCTGCGTAGATTTCTTCAACTGGTGTCACCT
AGTTCCTCAGCATGGTGTCTGCAACCACAAGTTTTACGGAAAACAATGCTGCAAGTCATGCACAAGG
AAGATCTGATCTTGGTGTCCTCCCCAGCCTTAGGGCCAGGGGCTTACCTTTCAACCTCTAGA
The NOV3 protein (SEQ ID N0:6) encoded by SEQ m N0:5 is 231 amino acid residues in length and is presented using the one-letter amino acid code in Table 3B. Psort analysis predicts the NOV3 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 3B. Encoded NOV3 protein sequence (SEQ ID N0:6) MGMQIELFCLLKCSKTCGRGVRKRELLCKGSAAETLPESQCTSLPRPELQEGCVLGRCPKNSRLQ
WVASSWSECSATCGLGVRKREMKCSEKGFQGKLITFPERRCRNIKKPNLDLEETCNRRACPAHPV
YNMVAGWYSLPWQQCTVTCGGGVQTRSVHCVQQGRPSSSCLLHQKPPVLRACNTNFCPAPEKRED
PSCVDFFNWCHLVPQHGVCNHKFYGKQCCKSCTRKI
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 3C.
Table 3C. Patp results for Smallest Sum eadingigh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAE09696Humangene 7 encoding protein +1 1248 9.2e-127 >patp:AAE09699Humangene 10 encoding protein +1 1245 1.9e-126 >patp:AAU72893Humanmetalloprotease partial +1 1204 4.2e-122 sequence #5 >patp:AAU72891Humanmetalloprotease partial +1 693 3.5e-70 sequence #$3 >patp:AAB21253Humanmetalloproteinase KIAA0605 +1 327 5.5e-28 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 392 of 396 bases (98%) identical to an EST
AA057409 mRNA from human). The full amino acid sequence of the protein of the invention was found to have 74 of 216 amino acid residues (34%) identical to, and 107 of 216 amino acid residues (49%) similar to, the 237 amino acid residue ptnr:SPTREMBL-ACC:Q9HBS6 protein from Homo Sapiens (HYPOTHETICAL 25.7 I~DA PROTEIN.
NOV3 also has homology to the proteins shown in the BLASTP data in Table 3D.
Table 3D. BLAST
results for Gene Index/ Protein/ OrganismLength Tdentity PositivesExpect Tdentifier (aa) (%) (%) gi~18598706~ref~XPhypothetical 1123 181/183 183/183 e-200 _ protein XP_091253 (98%) (99%) 091253.7.~(XM_091253 [Homo Sapiens]
gi~19171150~emb~CACADAMTS18 protein1081 61/62 62/62 4e-27 83612.1~(AJ311903)[Homo Sapiens] (98%) (99%) gi~7662202~ref~NP_0ItIAA0605 gene 951 79/216 99/216 9e-23 55509.11(NM 014694)product (36%) (45%) [Homo Sapiens]
gi~18561227IrefIXPhypothetical 1365 51/112 74/112 4e-21 _ protein XP_094442 (45%) (65%) 094442.1~(XM_094442 [Homo Sapiens]
gi~17432918Isp~Q9H3HUMAN ADAMTS-10223 74/223 104/223 5e-20 24IAT10' precursor (A (33% (46%) disintegrin and metalloproteinase with thrombospondin motifs 10) (ADAM-TS 10) (ADAM-TS10)(Fragment) A multiple sequence alignment is given in Table 3E, with the NOV3 protein being shown on Iine 1 in Table 3E in a ClustalW analysis, and comparing the NOV3 protein with the related protein sequences shown in Table 3D. This BLASTP data is displayed graphically in the ClustalW in Table 3E.
Table 3E. ClustalW Analysis of NOV3 1) > NOV3; SEQ ID N0:6 2) >gi~18598706~/ hypothetical protein XP_091253 [Homo Sapiens]; SEQ >D N0:45 3) >gi~19171150~/ ADAMTS18 protein [Homo Sapiens]; SEQ ID N0:46 4) >gi~7662202~/ I~IAA0605 gene product [Homo Sapiens]; SEQ ID N0:47 5) >gi~ 18561227/ hypothetical protein XP_094442 [Homo Sapiens]; SEQ ID N0:48 6) >gi~17432918~/ AT10_HCTMAN ADAMTS-10 precursor (A disintegrin and metalloproteinase with thrombospondin motifs 10) (ADAM-TS 10) (ADAM-TS10) (Fragment); SEQ IT3 N0:49 NOV3 1 _______________________________________________~__MQIE~iF~LL 11 gi1185987061 769 GGVRSAKVLSLEEWIKSETTL-ARKEQQQPSTGWMPGEQS<TQa~KA~-AeG'IGQQSKIQ~VQ 826 gi1191711501 872 ______________________,__________________________ gi176622021 686 -------------------------------PQWEMSE ~E TA GERSVVT~DSE 715 gi1185612271 986 QGGGCTPSPPADGLVQNQLRLGRHTQAQCLDQSCSVG S ~R.T =~GAQS~t,P~Q TR
S gi 117432918 ~ 807 ______________________________________ Ti ~,AQ
yn~~rGSQV,A'(?'E RN 828 .1....1....1....1....1....1....1....1.. .1. .1. .1.. .1 NOV3 12 ~_________________________________________~ Kip~~ E~',,j,~ G 30 1O gi 118598706 1 827 ~t"jIKPFQKEEAVLHSLCPVST~TQVI~A~NSHA~PPQ~SLGP~SQ Kf 'I~ELL~ G 886 gi1191711501 882 S-_______________________________________E ~ YINVKA~ LR 901 gi176622021 716 DEKLCDP---------NT ' GE~~ TGPP RQ TVSD GP GQ RTI'H'~Y 1T 766 gi1185612271 1045 ~1HYDSE-PVPASLCPQP SSR~ SQS PP SAGP 1'I_' ~ S 1103 gi1174329181 829 LDSSAVAPHYCS-AHSKL KRQTEP PPD G SL R6 'S S ,;, QR 887 .1....1. .1....1.. .1. .1. .1....1. .1. ..1. .1. .1 NOi3 1 31 ----AA'E.''T~ Q L E~.Q~G=~ LG..~KNSRL~ ~-~8S .E L ~ 85 ~ V n VW n V 1.
.1, V V/1 V
gi 18598706 887 ---AAT E Q ~L E~ G LG 'KNSRLiSS E L 941 20 gi1191711501 902 D----QNTQ"NS F SATCT.~ TEPKI AFS ~ ---A 'PGE T _K8 Q~S' v v - ~t c gi176622021 767 ;----DGR .~E Q QMT ~LAIHP- GD ---- ~QD E T ~ 817 gi ~ 17432918 1 8884 RVSAAEE~L ~~A P~~ ~~L~A- LGPT ~KPK P~vLD E 'I'PEP ~~ ' .1. ..1....1....1....1. .1. ..1....1. .1. ..1....1.. .1 NOV3 86 E~I~ S ~GFQ ~LI~FPER RNT' ~.LDL~E~'---- --AHPVYNMVA~ Y 138 gi1185987061 942 E~ S 'GFQ' LI,~~,FPER RNA ~VLDL E~----- --AHPVYNMVA I1Y 994 gi 119171150 1 954 ICS ~ ~KPFQ ~EEAVLHSL P STST~--TQU~?'A---- S -PQ------- S
30 gi176622021 818 L'~L LA- PQ~RSGPE GLA --P EST--- FE'P F---------- Y 858 gi1185612271 1163 FI~~ ~YVS ~YRELASK SHIP ~SLELBR~!~'PHLLLRI~P~GAAGLPHPGLREV P
gi1174329181 942 VjUL KS---ADHRADLPPAH SP°~ -PATMR-____~'"L.~_______p__ 3S ....1....1....1. .1....1. ~1..~...1. ..1....1....1. .1....1 NOV3 139 S--L~ QI,~ ------r;.~ G----- tot's .Q-----------QG.~~ S-- 170 v v gi1185987061 995 S--L~"QQ _____ G_____ ~T.8~ Q___________QG ~~ S-- 1026 gi1191711501 996 L-- ~ S~,'7 ---- V ~R----- ~ 'E~:i~KGSA--------AETL~E Q--gi176622021 859 TS--' S---- ~ ------ ' ~ YQG------------TDIVRG-- 890 gi1185612271 1223 LTRVL~"P PLIHLFRP SGSPCTVP VSYQHNKPIPRRREHPPREHLTQ~'SP 1282 gi1174329181 983 AG--E~G: --- AQ ---- Q~yQ~'7SVR~TS-----------HTGQ~H--E 1015 1.. ~~~~~1~.~ 1 .1. .1 ..1....1....1.. 1 1 ..1 4S NOV3 171 LLHQ ~PVL , ---- TF ~~PEKR--------------DPSC~DFF ~'~'''~~'CH- 206 gi1185987061 1027 LLHQ'~PVL . --------TF ~ PEKR~GEMQAELDSKLSGFQTIS~IWFESEG
gi1191711501 1031 TSLP ELQ~,~,. V --------LG ~KNSRLQ-------------- WVASSG~SE-gi176622021 891 DPLVVGR~,I --------LQP ~TEPPD-----------------SCQDQPGTN- 923 gi1185612271 1283 AKRKYGQKTDLDTLPIPLWAPLS~SPEPRG----PEICEQ----QGLD TECP LLL
SO gi1174329181 1016 TEAL~PTTf,~Q~EAK--- -CD,~PT~GDGPE--------------CKD~NKVACP- 1053 NOV3 206 - 1.~PQHGV~NHKFYGKQCC~I--.TRKI--I_---1----1- 231 SS gi1185987061 1078 NERL~PSFSLHLGGKNGIQYPKRLPBEQKENMALAAIKMLQSTF 1123 gi1191711501 1062 - 'V'WIRSH~RRLRPSWLTQ---------------------- 1081 gi176622021 923 - C ~IKVNL GHWYYSKACC~-- RPPHS------------- 951 gi1185612271 1335 AIG- IiPCQARDTESRPQGPVP~;--P GQDIEK------------ 1365 gi1174329181 1053 --- ,.LKFQF~SRAYFRQMCC'-- QGH--------------- 1077 The NOV3 Clustal W alignment shown in Table 3E was modified to begin at amino residue 1321. The data in Table 3E includes all of the regions overlapping with the NOV3 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (hrip:www.ebi.ac.uk/interpro/). Table 3F lists the domain description from DOMAIN analysis results against NOV3.
Table 3F Domain sis of Anal NOV3 Model Region of Score E value (bits) Homology tsp 1 12-58 -6.8 4.1 tsp 1 66-125 14.6 0.015 tsp 1 141-187 19.3 0.0041 Consistent with other known members of the Thrombospondin type 1 (tsp_1) family of proteins, NOV3 has, for example, three tsp_1 domain signature sequences and homology to other members of the tsp_1 Domain-containing Protein Family. NOV 3 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV3 nucleic acids and polypeptides can be used to identify proteins that are members of the tsp_1 Domain-containing Protein Family. The NOV3 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV3 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, tuberous sclerosis, fertility, hypogonadism, as well as other diseases, disorders and conditions.
In addition, various NOV3 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV3 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the tsp-1 Domain-containing Protein Family.
Thrombospondin type 1 domain (TSP1, IPR000884) is a repeat found in the thrombospondin protein where it is repeated 3 times. Likewise, the tsp_1 domain is repeated three times in the NOV3 polypeptide. Now a number of proteins involved in the complement pathway (properdin, C6, C7, CBA, C8B, C9) (Patthy,L., J. Mol. Biol. 202: 689-696 (1988)) as well as extracellular matrix protein like mindin, F-spondin (Okamoto, et al., Developnaeht 126:
3637-3648 (1999)), SCO-spondin and even the circumsporozoite surface protein 2 and TRAP
proteins of Plasmodium (Wengelnik, et al., EMBO J. 18: 5195-5204 (1999);
Rogers, et al., Mol. Bioclzefn. Parasitol. 53: 45-51 (1992)) contain one or more instance of this repeat. It has been involved in cell-cell interraction, inhibition of angiogenesis (Krutzsch, et al., Circulation 100: 1423-1431 (1999)), apoptosis [Krutzsch, et al., Cancer Res. 57: 1735-1742 (1997)).
The NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of urogenital physiology. As such, the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat reproductive and metabolic disorders, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, tuberous sclerosis, fertility, hypogonadism, as well as other diseases, disorders and conditions.
The NOV3 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV3 nucleic acid is expressed in eye and testis.
Additional utilities for NOV3 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV4 polypeptide has been identified as a Protocadherin Alpha C2 Short Form-like protein (also referred to as CG93187-O1). The disclosed novel NOV4 nucleic acid (SEQ 1D
NO:7) of 600 nucleotides is shown in Table 4A. The novel NOV4 nucleic acid sequences maps to the chromosome 11.
An ORF begins with an ATG initiation codon at nucleotides 41-43 and ends with a TAG codon at nucleotides 2546-2548. A putative untranslated region and/or downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
Table 4A. NOV4 Nucleotide Sequence (SEQ ID N0:7) CACCATAAAAGCTCAGAAAATAGACTTTTCCTCTGCCTCTATGGAGGGGCAGGCCAGATCTGGGGAA
GGGATGGGACAGCCTGGCATGAAGAGCCCCAGGCCCCACCTCCTGCTACCATTGCTGCTGCTGCTGC
TGCTGCTGCTGTCTTCGCCTCGCCGTGCACGCGTGCGCCTCCCAGAGGACCAGCCGCCTGGGCCCGC
GGCTGGCACGCTCCTAGCCCGCGACCCGCATCTGGGCGAGGCTGCACGCGTGTCCTATCGGCTGGCA
TCTGGCGGGGACGGCCACTTCCGGCTGCACTCAAGCACTGGAGCGCTGTCCGTGGTGCGGCCGTTGG
ACCGCGAACAACGAGCTGAGCACGTACTGACAGTGGTGGCCTCAGACCGAGCTCCCCGCCCGCGCTC
GGCCACGCAGGTCCTGACCGTCAGTGTCGCTGACGTCAACGACGAGGCGCCTACTTTCCAGCAGCAG
GAGTACAGCGTCCTCTTGCGTGAGAACAACCCTCCTGGCACATCTCTGCTCACCCTGCGAGCAACCG
ACCCCGACGTGGGGGCCAACGGGCAAGTGACTTATGGAGGCGTCTCTAGCGAAAGCTTTTCTCTGGA
TCCTGACACTGGTGTTCTCACGACTCTTCGGGCCCTGGATCGAGAGGAACAGGAGGAGATCAACCTG
ACAGTGTATGCCCAGGACAGGGGCTCACCTCCTCAGTTAACGCATGTCACTGTTCGAGTGGCTGTGG
AGGATGAGAATGACCATGCACCAACCTTTGGGAGTGCCCATCTCTCTCTGGAGGTGCCTGAGGGCCA
GGACCCCCAGACCCTTACCATGCTTCGGGCCTCTGATCCAGATGTGGGAGCCAATGGGCAGTTGCAG
TACCGCATCCTAGATGGGGACCCATCAGGAGCCTTTGTCCTAGACCTTGCTTCTGGAGAGTTTGGCA
CCATGCGGCCACTAGACAGAGAAGTGGAGCCAGCTTTCCAGCTGAGGATAGAGGCCCGGGATGGAGG
CCAGCCAGCTCTCAGTGCCACGCTGCTTTTGACAGTGACAGTGCTGGATGCCAATGACCATGCTCCA
GCCTTTCCTGTGCCTGCCTACTCGGTGGAGGTGCCGGAGGATGTGCCTGCAGGGACCCTGCTGCTGC
AGCTACAGGCTCATGACCCTGATGCTGGAGCTAATGGCCATGTGACCTACTACCTGGGCGCCGGTAC
AGCAGGAGCCTTCCTGCTGGAGCCCAGCTCTGGAGAACTGGTGTTGCTTGAACCTCTAGACTTTGAA
AGCCTGACACAGTACAATCTAACAGTGGCTGCAGCTGACCGTGGGCAGCCACCCCAAAGCTCAGTCG
TGCCAGTCACTGTCACTGTACTAGATGTCAATGACAACCCACCTGTCTTTACCCGAGCATCCTACCG
TGTGACAGTACCTGAGGACACACCTGTTGGAGCTGAGCTGCTGCATGTAGAGGCCTCTGACGCTGA.C
CCTGCCCTCATGGCCTCCTCAGGCGACCCATCAGGGCTCTTTGAGCTGGATGAGAGCTCAGGCACCT
TGCGACTGGCCCATGCCCTGGACTGTGAGACCCAGGCTCGACATCAGCTTGTAGTACAGGCTGCTGA
CCCTGCTGGTGCACACTTTGCTTTGGCACCAGTGACAATTGAGGTCCAGGATGTGAATGATCATGGC
CCAGCCTTCCCACTGAACTTACTCAGCACCAGCGTGGCCGAGAATCAGCCTCCAGGCACTCTCGTGA
CCACTCTGCATGCAATCGACGGGGATGCTGGGGCTTTTGGGAGGCTCCGTTACAGCCTGTTGGAGGC
TGGGCCAGGACCTGAGGGCCGTGAGGCATTTGCACTGAACAGCTCAACAGGGGAGTTGCGTGCGCGA
GTGCCCTTTGACTATGAGCACACAGAAAGCTTCCGGCTGCTGGTGGGTGCTGCTGATGCTGGGAATC
TCTCAGCCTCTGTCACTGTGTCGGTGCTAGTGACTGGAGAGGATGAGTATGACCCTGTATTTCTGGC
ACCAGCTTTCCACTTCCAAGTGCCCGAAGGTGCCCGGCGTGGCCACAGCTTGGGTCACGTGCAGGCC
ACAGATGAGGATGGGGGTGCCGATGGCCTGGTTCTGTATTCCCTTGCCACCTCTTCCCCCTATTTTG
GTATTAACCAGACTACAGGAGCCCTGTACCTGCGGGTGGACAGTCGGGCACCAGGCAGCGGAACAGC
CACCTCTGGGGGTGGGGGCCGGACCCGGCGGGAAGCACCACGGGAGCTGGGGCTCCACCTGGACTCT
TACCAGAGTCACTCCAAGTCCTGTCTCAGGCAGAATACTCAGATCTATTCCAAGCACCTTCCCTGGG
ATCTCAGGCGCATACTGAGAACCAGTGGGACAGGGTTGAGAGAGAGAGCCAACCGAGAATCTCAAAT
GAACCAAACTGAGAAAGATGCCCCTCAGTGGGGCTACAGACCGACACCCCACCATGGGGCAACAGAA
AAACCAAGACCCCCTCCCCAAAGGAATCAAACCAATCGGGAAAAGGAAGGAGGCGTTGGCCGTGCCT
AGGATAT
The NOV4 protein (SEQ 1D N0:8) encoded by SEQ ID N0:7 is 835 amino acid residues in length and is presented using the one-letter amino acid code in Table 4B. Psort analysis predicts the NOV4 protein of the invention to be localized at the plasma membrane with a certainty of 0.7900.
Table 4B. Encoded NOV4 protein sequence (SEQ ID N0:8) MEGQARSGEGMGQPGMKSPRPHLLLPLLLLLLLLLSSPRRARVRLPEDQPPGPAAGTLLARDPHL
GEAARVSYRLASGGDGHFRLHSSTGALSVVRPLDREQRAEHVLTWASDRAPRPRSATQVLTVSV
ADVNDEAPTFQQQEYSVLLRENNPPGTSLLTLRATDPDVGANGQVTYGGVSSESFSLDPDTGVLT
TLRALDREEQEEINLTWAQDRGSPPQLTHVTVRVAVEDENDHAPTFGSAHLSLEVPEGQDPQTL
TMLRASDPDVGANGQLQYRILDGDPSGAFVLDLASGEFGTMRPLDREVEPAFQLRIEARDGGQPA
LSATLLLTVTVLDANDHAPAFPVPAYSVEVPEDVPAGTLLLQLQAHDPDAGANGHVTYYLGAGTA
GAFLLEPSSGELVLLEPLDFESLTQYNLTVAAADRGQPPQSSWPVTVTVLDVNDNPPVFTRASY
RVTVPEDTPVGAELLHVEASDADPALMASSGDPSGLFELDESSGTLRLAHALDCETQARHQLWQ
AADPAGAHFALAPVTIEVQDVNDHGPAFPLNLLSTSVAENQPPGTLVTTLHAIDGDAGAFGRLRY
SLLEAGPGPEGREAFALNSSTGELRARVPFDYEHTESFRLLVGAADAGNLSASVTVSVLVTGEDE
YDPVFLAPAFHFQVPEGARRGHSLGHVQATDEDGGADGLVLYSLATSSPYFGINQTTGALYLRVD
SRAPGSGTATSGGGGRTRREAPRELGLHLDSYQSHSKSCLRQNTQIYSKHLPWDLRRILRTSGTG
LRERANRESQMNQTEKDAPQWGYRPTPHHGATEKPRPPPQRNQTNREKEGGVGRA
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 4C.
Table 4C. Patp results for Smallest Sum ReadingHigh Prob Sequences producing Frame Score P(N) High-scoring Segment Pairs:
>patp:AAU07054 Flamingo protein +1 968 1.8e-98 Human >patp:AAU07053 Flamingo polypeptide +1 968 2.0e-98 Human >patp:ABG21921 human diagnostic protein +l 642 3.1e-64 Novel #21912 >patp:ABG21921 human diagnostic protein +1 642 3.1e-64 Novel #21912 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 273 of 415 bases (65%) identical to a gb:GENBANI~-ID:AF061573~acc:AF061573.2 mRNA from Homo sapie~rs (protocadherin (PCDHB) mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 273 of 415 amino acid residues (65%) identical to, and 273 of 415 amino acid residues (65%) similar to, the 4076 amino acid residue gb:GENBANK-m:AF061573~acc:AF061573.2 protein from Homo Sapiens (protocadherin (PCDHB) mRNA, complete cds).
NOV4 also has homology to the proteins shown in the BLASTP data in Table 4D.
Table 4D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~17461472~ref~XPsimilar to 1415 459/682 503/682 0.0 _ protocadherin (67%) (73%) 052786.2I(XM 16 [Homo Sapiens]
gi~16933557iref~NPprotocadherin 443/676 490/676 0.0 _ precursor; (65%) (71%) 003728.1~(NM_003737 fibroblast cadherin FIB1;
cadherin 19;
fibroblast cadherin 1;
dachsous homologue [Homo Sapiens]
gi~6753408~ref~NPcadherin EGF 3034 48/693 358/693 1e-98 _ seven-pass G-type (35%) (50%) 34016.1~(NM_009886) receptor [Mus musculus]
gi~13325064~ref~NPcadherin EGF 2923 246/679 345/679 2e-98 LAG
_ seven-pass G-type (36%) (50%) 001399.1~(NM
~
receptor 2;
EGF-like-domain, multiple 2;
epidermal growth factor-like 2;
multiple epidermal growth factor-like domains 3;
cadherin, EGF
LAG
seven-pass G-type receptor 2, flamingo (Drosophila)homol og gi~10727655Igb~AAF5Stan gene product3606 241/700 361/700 3e-98 8763.2 (AE003828)[Drosophila (34% (51%) melanogaster]
A multiple sequence alignment is given in Table 4E, with the NOV4 protein being shown on line 1 in Table 4E in a ClustalW analysis, and comparing the NOV4 protein with the related protein sequences shown in Table 4D. This BLASTP data is displayed graphically in the ClustalW in Table 4E.
Table 4E. ClustalW Analysis of NOV4 1) > NOV4; SEQ >D N0:8 2) >gi~ 17461472/ similar to protocadherin 16 (H, Sapiens) [Homo Sapiens]; SEQ
)D N0:50 3) >gi~16933557~/ protocadherin 16 precursor; fibroblast cadherin FIB1;
cadherin 19; fibroblast cadherin 1; dachsous homologue [Homo sapieras] ; SEQ >D N0:51 4) >gi~6753408~/ cadherin EGF LAG seven-pass G-type receptor [Mus rnusculus] ;
SEQ >D N0:52 5) >gi~13325064~/ cadherin EGF LAG seven-pass G-type receptor 2; EGF-like-domain, multiple 2;
epidermal growth factor-like 2; multiple epidermal growth factor-like domains 3; cadherin, EGF LAG
seven-pass G-type receptor 2, flamingo (Drosoplaila) homolog; ; SEQ )D N0:53 6) >gi~10727655~gb~AAF58763.2~(AE003828) Stan gene product [Dr~osophila rnelanogaster] ; SEQ >D
N0:54 rrov4 1 3 gi~ 17461472~81 ga.~ 16933557~1153 1211 g3.~ 67534081955 1014 gi~
gi~ 10727655~1055 NOV4 4 QARSGE-GMGQ-_______________pGMKS~___________________________ 19 gi1174614721 140 L~ASGAAGGG P ;Q PAR ~P 'I'L~uTTI:iQ~i ~'~ EI GT L T'T~T- PGS
gi I 16933557 I 1212 L~,ASGAAGGG'L~t'Pt2.~Q PIJR ~P TL~TT,LiQ~ ~' ~ E GT L TAT-gi~6753408~ 1015 EKDE--- ~'E~F E~NS~ S IRi ~'~ P Q I Q~~E P Q LL 1068 gi ~ 13325064 ~ 922 Ej~DE--- FDV~F~E~NS~LA~AR~Ti,~~~,T~ ~ ~ T Q Q~VE IP _ Q IF
gi ~ 10727655 ~ 1114 ASDK--- ~CTI~Y PNS SV GE'IHi ~ ~ ~ VUH SI~I DSN S TRP
NOV4 19 __________________ .HL~ _P-________ ___LL~,tL~sL~:,.SS-__ _______ 37 gi~174614721 199 --~ LTAAP IRA-E~~H T~S~HDQGSP~RSSLQ LVQ PSARLAP~~D 253 15 gi~16933557~ 1271 --LTAAP IRA-E~~H TS~,~HDQGSP~RS~SLQ~L. PSARLAFSP~~D 1325 gi I 6753408 ~ 1069 ---~7 RALVE FE- 'RD Qi~i,TS-- ~LVRAT4V~i~~, QN-- ~7 ~ ~E P
gi ~ 13325064 ~ 976 -- TALVD YE-D' ~E Q~TS-- ~LVRAT~'HU7~,~r~DRN-- 't~ ~ G
gi I 10727655 ~ 1168 SERA LTMTE YEST KR~'E u.~R~AS---P ~LRNDAIiE~L'S~TDVN---~3N ~ R 1221 NOV4 37 ____________________________________________________________ 37 gi ~ 17461472 ~ 254 k~RDPAAPVPWLT GL ~GSL S~~P~ GVG' T ~L~~ ;PEGTF ~ 313 gi~169335571 1326 RDPAAPVPWLT EGL ~GSL S, ~P~~ GVG T TL~ PEGTF Afi1385 gi~67534081 1118 FQILFNN----Y KSNSF'SGV,~ ~~P~~,H~ LSD~~a 'PFQ BLS-LLL ~P,A'~
gi I 13325064 ~ 1025 NFEILFNN----YRSSSF'GGA'I R~t P~iH>~~7' ~ ISDS T SFE ~ BLS-LVL G'i' 1079 gi~10727655~ 1222 ~.IFQVIFN--------NFRDHF~SGEiI P~FADVSD RIS ~AN-LL 51272 NOV4 37 ___________________________________________________-_____p__ 38 gi 17461472 314 373 gi~16933557~ 1386 1445 giI6753408~ 1173 1231 giI13325064~ 1080 1138 gi~10727655~ 1273 NOV4 38 ____________________________________________________________ 38 gi117461472~ 374 RTRSPAQRCTLSARRTPTAP TATCATACCARSRPCRASPGRAHRGVSSARPGPRD---gi~16933557~ 1446 PE-NPEPGAALYTF~SDADG'-GPNSDVRY.LLRQEPPVP R'~.iDARTGALSAPRG---gi~6753408~ 1232 EKFLSPLLSLFVEG ~~TVLSTTKDDIFVFNI~NDTDVSS-NI ~ TFSALLPGGTRG--R
giI13325064~ 1139 ERFLSPLLGLFIQAVTLAT~PDHWVFNV~RDTDAPGGHI YSLSVGQPPGPGGGPP
gi~10727655~ 1332 EAFLSPLLNFFLDGL~IIPC KEHIFVFSItDDTDVSS-RI j SFSARRPDVSHE--E
NOV4 38 -_________________ _ __ _ ___ __ R~~a~R~=~~RLP~T'JQP~ 52 gi~17461472~ 431 HSRAAAAGGSHRPA~QRQPPSCFSA;~TSR~QR~TRLS,---P~RLP~QP~ 485 gi ~ 16933557 ~ 1501 LDRETTPALL'~L~1EATDRPANARAARVSARVFVTDEN~3NAPVASPS
~=RLP1QP ~ 1560 gi ~ 6753408 ~ 1289 FFPSEDLQEQ~Y' ~ 'TLLTTI.~~ LP",DD C EPCE'~CV~LRF~SS ~ 1344 gi~133250641 1199 FLPSEDLQERIar9 'SLLTAIAQ LP DN~Cxr EPCERCVS=LRFSS ~ 1254 ' ~ ~ i..
gi~10727655~ 1389 FYTPQYLQER~3Y' ILARLTVEVLP~DN~C,'EPCL~'EECLT~LKFGASE 1444 55 ~ ,.
1630 7.640 1650 1660 1670 1680 . .,I. .~~ ~~~~ .~. ..y y ...I. y . .I.. .y ~V
NOV4 52 GP, P~LGE ~ YRLAS.~ ------~GHF ~S TGAL ' 'P~.iDv -- 101 gi~17461472~ 486 GP~ ~DP LGE YRLAS ------~GHF S TGAL~ ~PhD~ -- 535 gi~16933557~ 1561 GP~~LH ~DPDLGE _YRLAS ------~GHF S TGAL~,~ 'PI~D~ -- 1610 gi~6753408~ 1345 FIST F'PI PITGL~CRCPPGFT ----DYCTEID CY PC ' CRS' GGY 1400 gi~13325064~ 1255 FI~SSF'PI~PVGGL'CRCPPGFT ----DYCTEVD CY RPCGPHGyyCRS~ GGY
gi~10727655~ 1445 FIND F'PI'Y~PVNTFAC~CPEGFT SKEHYLCdTEVD CY DPCQ~1GGTC'tt ' NOV4 101 ____I....~.. .~. .'....~ .._.i....~ I"~ I,, I ~ I,~ ~' --Q ~ L DB=--~pRP ATQ T'.t~~~~SV~TD' FQ~QQY'VL . ~ E 151 gi ~ 17461472 ~ 535 ---_---Q L --~PRP~~ATQ T~S~'~' V~17:: ~ .F~~QQ Y~sVL ~E
gi I 16933557 I 1610 -------Q L DH---GSPP32u~ATQ TVS~ 'TF~,bQf~~Ys.~'r, VL 'E
70 gi I 6753408 ~ 1401 TCECFEDFT CQVNV G~-CAGVC~GGTC4~tN,~L~I,GG~VC
~PG~"YHPYCE~TST 1459 gi ~ 133250641 1311 TCLCRDGYTG~CEVSAR~ -CTPGVCGGTCVNT.~L~GGFKCC~GDFPYCQ~STTT
gi 1107276551 1505 TCVCPSTHTG~IStCE~G~GHrLyPCPETCEGGLSC~fiTYP- ~
QPPPYTA~I'CE~~yA 1558 gi1174614721 586 645 gi1169335571 1661 1720 gi167534081 1460 1518 gi1133250641 1370 1428 gi1107276551 1559 1617 gi1174614721 646 705 gi1169335571 1721 1780 gi16753408~ 1519 1578 gi1133250641 1429 1488 gi1107276551 1618 1677 1.. .~....1....~....1....1....1. .1. ..1. 1....~.. .1 NOV4 272 ~i7GQ ....~;_________-____________-R__-_~.~ ~GAFVLD~' G~ G-Tv P 303 gi1174614721 706 ~iGQ ~1'~' ----------------------R-- D~GAFVLD GG-Tv P 737 gi1169335571 1781 iINGQ ~'----------------------R----~ ~~GAFVLD ' GG-T ~P 1812 gi167534081 1579 JAVA ~._GSYVGNYSCAAQGTQSGSKKSLDLTGPLL ~ LPEDFPH~RQ GC ~ 1638 gi1133250641 1489 TGV 'GSVLGNYSCAAQGTQGGSKKSLDLTGPL DLPESFP'9'RMRQ GC ' 1548 gi1107276551 1678 NRT~LDKRCSLLTETCH-------RFLDLTGPLQT?'?'G~VL~'RIPAHFP~TRGCSD
gi1174614721 738 794 gi1169335571 1813 1869 gi167534081 1639 1698 gi1133250641 1549 1608 giI107276551 1731 1790 .1....1. .1. ..1....1....1 1....1. ..~. ~.~..1....1 NOV4 360 ---------- ~.1'L ~QLQ ~PDAGANG~1V~'GAGT~,~~I,, ~ PSS LVLLEPLD 409 e'E
gi 117461472 1 794 ------- '~L QLQAH~PDAGANGHV'~Y~GAGT~,~, ' ~ PAS ~LR'.AAALD
gi 116933557 1 1869 ------- -~. TL QLQ ~PDAGANG~3"~'Y GAGTi ~ PAS LItAAALD
gi167534081 1699 CEQAMPHPQRFT ES''V~t(LWSDL~ITISVPW'x7~GLMFRTRKEDG ~, gi1133250641 1609 CAQEMANPQHFL ~SAWHGLSLPISQPW~h~L_MFRTRQiDG
S~AI'~RGRS'~.'ITLQL 1668 gi1107276551 1791 CQDNIPAPWRFGS SFNPLLRPIQLPWTT'~5~RTRQKE~ IQI~NSAAVCL 1850 . ..1. ~_ ..1... 1 ..1....1....~ .1. ..1....1.. ~ m..1 NOV4 410 F.SLTQ. L~~ ~--- RG-QP-PQSSWP~Tj~TVLDV~NP------- PV3.:TRA 453 gi1174614721 844 QCPS TFVSA ~--- G,~-~aAGPLSTTVS T TVT~D HA------- PT~PTS 888 gi1169335571 1919 ' QCPS TFSA ~--- G3~-AGPLSTTVS~T T~I2,D~---------PTPTS 1963 gi 1 67534081 1759 LNSYI1;EVYGPS~ ASMQLKRITDGGWHHiL
E~.,~SAKGKDIKYLAVMTLD~'~','GMD 1818 gi1133250641 1669 GHVMLSVEGTGLQASSLRLEPGRANDGDWHHAQ~GASGGPG----HAILSFD~~GQQ
gi110727655~ 1851 '~QGVLY~IFDGEP-----MYLG~SFLSDGEWHRVE~RWS~QGIH------FS-VD'YaGQR 1898 1 ...1.. 1 . ~ ..1.,...1....1....1. 1 ...1....1. ..1 CO NOV4 454 SYR~~J"T~~~~PEDT~- '~GAEL2aHVEASDADP--------ALN~AS~,.'rGD--------'SGL 491 gi 1174614721 889 PLRT~.~RPRP ~SFSTPT~iAI~AT~R.~.A~;bRDAGAN-----ASILY LF~ ------ 'PPG 935 gi1169335571 1964 PLRRI~PRP ~SFSTPT~TRDAGAN-----ASILY'_ --------'PPG 2010 gi167534081 1819 QST~QGNQL~GLKMRTVTGG~VT~KVSVRHG--FRGCMQG ' GE STNIATLNMNDA
gi1133250641 1725 RAEGN~I~GPRLHGLHLSN~T GG~PGPAGGVARG--FRGCLQG ~wD
PEGVNSLD~SHG 1782 65 gi1107276551 1899 SGS~P1~SQKVQGLYVGKT7GSPDGSIGAVPEASPFEGCIQD~ Z~GAG----1.. .1....1....1....1....1. ..1 ...1....1. 1 ...1....1 NOV4 492 FE~'pESS~TLRL~~,HAL------DCETQARHQ. Q ~PAG-AHFALAPVT~!EVQDVN-- 542 70 gi1174614721 936 TT~~SYT~EIRV~RSP------V~LGPRDR IV~T~L~RPARSATG~II'VGLQGEA--32 ~~ _~
gi1169335571 2011 TTU[.1~SYT EIRViRSP------VLGPRDRV~~IVT~L
RPARSATG~iII'S~GLQGEA-- 2062 gi~67534081 1877 LRVKD CDVEDPCASSPCPPHRPCRDTWDS'~SCIC~ YFGKKCVDACL~iNPCKHVAA
gi1133250641 1783 ESIt~VEQ CSLPDPCDSNPCPANYCSNDWDSSCC~P YYGDNCTNVCD~iNPCEHQSV
gi I 107276551 1955 IRE;E~VED CESRi~IQCP-DHCPNHSCQSSWD~STCEC~S
YVGTDCAPCT:StRPCASG-V 2012 .~.
.I.. .1....1....1....1. ..I....I....I....I....~..f..," I....p, NOV4 542 -DH ~ F~LNLLSTSVAE---NQPP ~L~ITTLHAIDGDAG-AF RLRYLF.~AGPGPEG597 gi1174614721 987 -E~ ~RF SEATIRE---NAPP P~~ S~RAVH- G-T ~ITYS~LS --- 1037 1o gi1169335571 2062 -E' ~RF SEATIRE---NAPP P VS~RAVH- 'G-TV ~ITY~TLS V - , 217.2 gi167534081 1937 C ~S~ T GYCECGPG-HYGQYCEN d7L~CPKGWW PVC ~CHC~51'S~ F~PDC
giI107276551 2013 CRANTSL HGYDCECNSSSRHGDYCE~~QQ~CPGGWW ERVC ~CRCD~.tA, YHPDC
.1....I.. .1....I... I ...I I....I....I. ..I.. I . .I
NOV4 598 EAFALNSS GEL,: ~'.AR------VPFD'~'EHTESF LVGAADAGNLSAS~TVSVLVTGEIE651 ga. 11174614721 1038 G"~'FSIQPS GAITVRSA----EGLDF'EVSPRL'~VLQAESGGAFAFT
LiTLTLQDA13N3 1092 giI169335571 2113 G'.~'FSIQPS GAITVRSA----EGLD.~.~EVSPRL'~iVLQAESGGAFAFT~L~TLTLQDA~N 2167 2O gi167534081 1996 TNGQCQCKENYYPPAQDACLPCDCPHGSHS~ CDMDTGQCACKP ~GRQCNRCP2055 gi~133250641 1902 °'GECHCKENHY PPGSPTCLLCDC3~PTGSLS sCDPEDGQCPCKP
~GRQCDRCi~P' 1962 gi110727655~ 2073 TGQCYCKQNHYPPNETACLSCDC~'SIGSFSGACNPLTGQCECREGuGRRCDSCS~P2132 25 ....~....I....I....I....I....I....I ...I. .I. ..1....1....I
NOV4 652 DPVF PAFHFQVPEGARRGH----SLGHaj'QATDEk~GGAD L~7LYATSSP-------- 699 gi1174614721 1093 ~,~~1,PRF RPHY LPES~PL G--- LL~?EADDL~QGS QISY~AASQP ----gi1169335571 2168 ~~,PRF RPHY LPES~PL G----~LLt~EADDL~7QGS 'Q,~SY~cAASQP ----gi~67534081 2056 ~iE=uTSLGCE I'~NGCP~ F GIWW QTFGQPAAVPCP 'S~ GN~,RHCSGE~ -3~ gi1133250641 1962 iiIEuiTTNGCE YDSCP~ I GIWW RTFGLPAAAPCP SFGT,,'RHCDE -gi1107276551 2133 iEuT~SGCE~~DACP~SFAGGWW~RTPiGGVAI!GCPPPARGKGQRSCDVQS SWNTP
.I.. I . .I... 1....I. ..1....I ...1....I. ..~....I....I
3S NOV4 700 YFGINQi~TG - ---- ------ .YLRVDS~ P ---G GT ------------- 725 gi1174614721 1144 LFHVDP~~TGT~TTTAIL ~EIW ~? LMA'D~GP G~ATL ------------ 1190 gi116933557~ 2219 LFHVDP~TGTTTTAILREIW T LMAD~GP GATL ------------ 2265 gi167534081 2115 ELFNCTSGSF~DLKAL LN T GN L L RN=TQGNS------------- 2161 gi~133250641 2021 NLFNCTTTFSELKGFF~~LQ ~ SGF~Q'L~LL RN~~~,TQH~A-------'-----gi110727655~ 2193 DMYNCT~EPFITELRRQLSLEKL LGE~TSFVAIEQ RCEAVDRRGASKDQKISGN
.'..I....I....I....I....I....I....I....I....I....I....I....I
NOV4 725 ______________________________________________gGGG-GRTRREAPR 738 45 gi1174614721 1190 ____________________________________________DTNDNRPTIPQPW
gi1169335571 2265 ____________________________________________~7jOTT7DNRPTIPQPW 2281 gi167534081 2161 ----------------------------------------TLFGNDC~RTAYQLLARILQH
gi1133250641 2067 ----------------------------------------GYFGSDKVAYQLATRLLAH
gi1107276551 2253 GRPNRRYKMESSFLLSNGGNVWSHELEMDYLSDELKFTHDRLYGADLVTEGLLQELINY
.I....I....I....I....I....I....I....I....I....I.'.'I....I
NOV4 739 ~GLHLDSYQS-----------------------HS----------------------KS 753 gi1174614721 1207 RVSED LG~IAQVTG~~DS P YVLSPGP~ DPFSVGRY RVS TGPL~FEQ 1266 gi ~ 16933557 1 2282 RVSED LGI~IAQVTGI~(1'~'DS P YVLSPGP~DPFSVGRY RVS TGPLFEQ
w gi167534081 2182 SRQQGFD REANFHEI~~HT S LAPATE~3SWQIQRSE-- Q LRHFAYF 2239 gi1133250641 2088 STQRGF SA'QDVHFTL'~""iR. S LDTANKRHW_LIQQTE-- TAW LQHYAYA
gi1107276551 2313 QSGL SH~C.~DKYFI~L~AASVLDRKYEEWRRATELIQ- PDD DAFNKYL 2371 .I.. L~. I ...I....I....p....l....l....l....l....~....1 NOV4 754 CLRQNT~IYKHL Ia~_iRRILRTSGTG~iRRA~3RESQ----------------------- 790 gi1174614721 1267 CDRY~LvLLA;E~D ~GRANLT~ Em n APAFSQSI~Y'VML~~~EHTP~GS--------ga.11693355i ~ 2342 CDRYL~LLA'~iD ~GRANLT E~ ~ APAFSQS~.aYVMLEHTP~GS--------gi~6753408 2240 SNVAN~T XL F~TANM~ WF~KL~FTGAQVPFED'~~QEEL~RE-----LES 2294 gi I 133250641 2146 S NMR.I-~TYLS FT. ;
~VTPNI~3~;SWRL~KGFAGAK'~.tPYEA~iRGEQ~PD-----LET 2200 gi ~ 10727655 I 2372 W~S~ID~~'1'TS
~F~ZTQPNMA~GL~~VTTE~LFGYEPE~LSEYHRSKYLICPNAFTTES 2431 ....I....I....I....I....I.'..1....1....1....1....1....1....1 NOV4 790 - -MNQ EIi;D PQW YR-________-_____-______pT~H TE ~RPPPQRN823 gi1174614721 1319 A~LSVS~p G~ ~~~~~,SYH------------------LAS~ FSVD~ GTLF~'~'I
giI16933557~ 2394 A'~Z.tSVS~,D G~~SYH--------------'---LAS~ FSVD~ GTLFTI 2434 gi~6753408~ 2295 StT;SFPAD FPPEKKE~P~VRLTNR--------------RTTPLTAQPEPRAERETSS~~2340 gi~13325064~ 2201 TjVLPE'~UF~;----ETPPRP-----------------------A~ePGEAQEPEELAR~'t 2233 gi~10727655~ 2432 V;VV11PDTGFLQHARQRPISFPKYNNYILDRRKFDQHTKVLV~LEMLGIT~PESDEI~,t~ 2491 l~ NOV4 824 TN~'yIEKEGG Ri-_______________________________________________ 835 gi~17461472~ 1360 VGTVALGHD SGAVD ~~ KHETTG------------------------------------gi~I6933557~ 2435 VGTVALGHD SGAVD LEARDHGAPGRAAR.ATVH-------------------VQLQDQ
giI6753408~ 2341 R ~HPDEPGQFiiIVAL ;;,IYRTLGQLLPEHYDP--------------------------gi~13325064~ 2234 Q HPELSQ~eE~iAS IYRTLAGLLPHNYDP----------------------------1S gi~10727655~ 2492 SG'RGSSHDHRiIVAYAQYKDVGQLLPDLYDETITRRWGVDVELATPILSLQILVPSMER 2551 The NOV4 Clustal W alignment shown in Table 4E was modified to begin at amino residue 1201 and end at amino acid residue 2760. The data in Table 1E includes all of the 20 regions overlapping with the NOV4 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 4F lists the domain description from 25 DOMAIN analysis results against NOV4.
Table 4F Domain Anal sis of NOV4 Model Region of Score (bits) E value Homology cadherin 41-131 97.7 2.4e-25 T25P N 16-223 ~ -I17.2 1.3 cadherin 145-233 104.1 2.7e-27 cadherin 247-337 78.1 1.8e-19 cadherin 351-441 112.9 6e-30 cadherin 455-539 64.7 2e-15 cadherin 553-646 77.5 2.8e-19 cadherin 660-745 15.4 0.036 Consistent with other known members of the Protocadherin Alpha C2 Short Form Protein-like family of proteins, NOV4 has, for example, seven Cadherin domain signature 30 sequences and homology to other members of the Protocadherin Alpha C2 Short Form Protein-Like Protein Family. NOV4 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV4 nucleic acids and polypeptides can be used to identify proteins that are members of the Protocadherin Alpha C2 Short Form Protein-like Protein Family. The NOV4 nucleic acids and polypeptides 35 can also be used to screen for molecules, which inhibit or enhance NOV4 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, fertility, endometriosis, hypogonadism, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimrnune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphaedema, as well as other diseases, disorders and conditions.
In addition, various NOV4 nucleic acids and polypeptides according to the invention are useful, i~atef° alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV4 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Protocadherin Alpha C2 Short Form Protein-like Protein Family.
Cadherins (Takeichi, Arahu. Rev. Biochem. 59: 237-252 (1990); Takeichi Trends Genet. 3: 213-217 (1987)), first discovered in mouse teratocarcinoma cells (Liaw, EMBO.I. 9:
2701-2708 (1990)), are a family of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Cadherins preferentially interact with themselves in a homophilic manner in connecting cells; thus acting as both receptor and ligand. There are a number of different isoforms distributed in a tissue-specific manner in a wide variety of organisms. Cells containing different cadherins tend to segregate ira vitro, while those that contain the same cadherins tend to preferentially aggregate together. This observation is linked to the finding that cadherin expression causes morphological changes involving the positional segregation of cells into layers, suggesting they may play an important role in the sorting of different cell types during morphogenesis, histogenesis and regeneration. They may also be involved in the regulation of tight and gap junctions, and in the control of intercellular spacing. Cadherins are evolutionary related to the desmogleins which are component of intercellular desmosome junctions involved in the interaction of plaque proteins.
Structurally, cadherins comprise a number of domains: these include a signal sequence;
a propeptide of around 130 residues; an extracellular domain of around 600 residues; a single transmembrane domain; and a well-conserved C-terminal cytoplasmic domain of about 150 residues. The extracellular domain can be subdivided into 5 parts, 4 of which are repeats of about 110 residues, and the fifth contains 4 conserved cysteines. The calcium-binding region of cadherins is thought to be located in the extracellular domain. This indicates that the sequence of the invention has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Maniatis et al. has identified 52 novel human cadherin-like genes organized into three closely linked clusters (Wu and Maniatis, Cell 97(6):779-90 (1999).) Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.
The NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of urogenital, nerve, and endocrine physiology. As such, the NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat reproductive and nervous system disorders, e.g., Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, fertility, endometriosis, hypogonadism, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphaedema, as well as other diseases, disorders and conditions.
The NOV4 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV4 nucleic acid is expressed in Heart, Aorta, Umbilical Vein, Thyroid, Colon, Peripheral Blood, Spleen, Lymph node, Bone, Cartilage, Brain, Left cerebellum, Right Cerebellum, Parietal Lobe, Temporal Lobe, Cerebral Medulla/Cerebral white matter, Hippocampus, Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Testis, Lung, and Retina.
Additional utilities for NOV4 nucleic acids and polypeptides according to the invention are disclosed herein.
NOVS
A NOVS polypeptide has been identified as a Nuclear protein-like protein (also referred to as CG95083-O1). The disclosed novel NOVS nucleic acid (SEQ ID
N0:9) of 2322 nucleotides is shot~cni in Table SA.
An ORF begins with an ATG initiation codon at nucleotides 70-72 and ends with a TAA codon at nucleotides 2320-2322. A putative untranslated region and/or downstream from the termination codon is underlined in Table SA, and the start and stop codons are in bold letters.
Table SA. NOVS Nucleotide Sequence (SEQ ID N0:9) GTGGAAGGACAGTCCAGAGCCCTTGTCATCGCACAGGAACTGCTATCTTCAGAGAAAGCATACGTGG
A_GATGCTCCAGCACTTAAATCTGTTCCTGGCAGAGCAGGCTATCAGCAGGAGAGGCCAGGGCTCCAA
AGCCCCAGGGGAAATCTGCCAAGGAGGACTTGTGCTCAGTCCTATCAACCTGTGGGTAACAGACCTT
TTGGTGTTTCAGGATTTCCATGGAGCTGTCATGAGGGCCTTGGATGACATGGACCATGAAGGCAGAG
ACACATTGGCCCGGGAGGAGCTGAGGCAGGGCCTGAGTGAACTCCCAGCCATCCACGACCTTCATCA
AGGCATCCTGGAGGAGCTGGAGGAAAGGCTGTCAAATTGGGAGAGCCAGCAGAAGGTAGCTGACGTC
TTCCTTGCCCGGGAGCAGGGGTTTGATCACCACGCCACTCACATCCTGCAGTTCGACAGGTACCTAG
GTCTGCTCAGTGAGAATTGCCTCCACTCTCCCCGGCTGGCAGCTGCTGTCCGTGAATTTGAGCAGAG
TGTACAAGGAGGCAGCCAGACTGCGAAGCATCGGCTGCTGCGGGTGGTTCAACGCCTCTTCCAGTAC
CAAGTGCTCCTCACAGACTATTTAAACAACCTTTGTCCGGACTCCGCCGAGTACGACAACACACAGG
GTGCACTGAGCCTCATCTCCAAAGTCACAGACCGTGCCAACGACAGCATGGAGCAAGGGGAAAACCT
GCAGAAGCTGGTCCACATTGAGCACAGCGTCCGGGGCCAAGGGGATCTCCTCCAGCCAGGAAGGGAG
TTTCTGAAGGAAGGGACGCTGATGAAAGTAACAGGGAAAAACAGACGGCCCCGGCACCTATTTCTGA
TGAACGATGTGCTCCTGTACACCTATCCCCAGAAGGATGGGAAGTACCGGCTGAAGAACACATTGGC
TGTGGCCAACATGAAGGCTCTTTACCATGGGGAAGGGGAAGGAGGAAGCACCTTTCTCAGCATGGAG
GTTTGTTCCCTTTTGGAACCAAAGGCTCCACCGAGGAGCCTGTTAGAAAAAGGCATGGGAGACGTGG
TCACTGGCAGGTACTTGTCCAACATGACAGTGCACCTGGGGTTGCCCGGGCTGGGCCCTGAGCATGA
CGCTCTGCAGCCTTCCCAGCGGTGGGTCAGCCGCCCTGTGATGGAGAAAGTGCCCTACGCTCTAAAG
ATTGAGACTTCCGAGTCCTGCCTGATGCTGTCTGCGAGGCTGCAGGTCAGGAAGTCCAAGGTCAAGG
CACTGACTGATTCGGTGTCTGCAGCCCTGGGAGTTAGGGGAATATCATTATTCCAGTGTAAGAAGAA
ACAGACCCAAGGACAGCTAATGGACCAGTGGTCTGCTCGTAAACCTAGTCTGGCAGGTGATCTCTTC
TTTGCTGGTGGTTCTGGGCAGTGTGAGAGGTGCAGGCTCAAGGGGCATCTGAGTGAGAACCTCATCC
ATGCCGAGATGGAGGCCCATGCCCGCAGCTCCTGTGCAGAGAGGGACGAGTGGTATGGCTGTCTGAG
CAGAGCCCTCCCTGAGGACTACAAGGCCCAGGCGCTGGCTGCATTCCACCATAGCGTGGAGATACGA
GAGAGGCTGGGGGTTAGCCTTGGGGAGAGGCCCCCCACCCTGGTGCCTGTCACACACGTCATGATGT
GCATGAACTGCGGCTGCGACTTCTCCCTCACCCTGCGGCGTCATCACTGTCACGCCTGTGGCAAGCA
GATCGTGTGCCGGAACTGTTCGCGGAACAAGTACCCGCTGAAGTACCTGAAGGACAGGATGGCCAAG
GTCTGCGACGGCTGCTTCGGGGAGCTGAAGAAGCGGGGCAGGGCTGTCCCGGGCCTGATGAGAGTTA
CAGAGCGGCCTGTGAGCATGAGCTTCCCGCTGTCTTCACCCCGCTTCTCGGGCAGTGCCTTTTCATC
CGTCTTCCAGAGCATTAACCCCTCGACCTTCAAGAAGCAGAAGAAAGTCCCTTCAGCCCTGACAGAG
GTAGCTGCCTCTGGAGAGGGCTCTGCCATCAGTGGCTATCTCAGCCGGTGTAAGAGGGGCAAGCGGC
ACTGGAAGAAGCTCTGGTTTGTCATCAAAGGCAAAGTTCTCTACACCTACATGGCCAGTGAGGACAA
AGTGGCCTTGGAGAGTATGCCTCTGCTAGGCTTCACCATTGCTCCAGAAAAGGAAGAGGGCAGCAGT
GAAGTAGGACCTATTTTTCACCTTTACCACAAGAAAACCCTATTTTATAGCTTCAAAGCAGAAGATA
CCAATTCATGGATCGAGGCCATGGAAGATGCGAGTGTGTTATAG
Variant sequences of NOVS are included in Example 3, Table 19. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOVS protein (SEQ ID NO:10) encoded by SEQ ID N0:9 is 750 amino acid residues in length and is presented using the one-letter amino acid code in Table SB. Psort analysis predicts the NOVS protein of the invention to be localized in the nucleus with a certainty of 0.3000.
Table 5B. Encoded NOVS protein sequence (SEQ ID N0:10) MLQHLNLFLAEQAISRRGQGSKAPGEICQGGLVLSPINLWVTDLLVFQDFHGAVMRALDDMDHEG
RDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHATHILQFD
RYLGLLSENCLHSPRLAAAVREFEQSVQGGSQTAKHRLLRWQRLFQYQVLLTDYLNNLCPDSAE
YDNTQGALSLISKVTDRANDSMEQGENLQKLVHIEHSVRGQGDLLQPGREFLKEGTLMKVTGKNR
RPRHLFLMNDVLLYTYPQKDGKYRLKNTLAVANMKALYHGEGEGGSTFLSMEVCSLLEPKAPPRS
LLEKGMGDWTGRYLSNMTVHLGLPGLGPEHDALQPSQRWVSRPVMEKVPYALKIETSESCLMLS
ARLQVRKSKVKALTDSVSAALGVRGISLFQCKKKQTQGQLMDQWSARKPSLAGDLFFAGGSGQCE
RCRLKGHLSENLIHAEMEAHARSSCAERDEWYGCLSRALPEDYKAQALAAFHHSVEIRERLGVSL
GERPPTLVPVTHVMMCMNCGCDFSLTLRRHHCHACGKQIVCRNCSRNKYPLKYLKDRMAKVCDGC
FGELKKRGRAVPGLMRVTERPVSMSFPLSSPRFSGSAFSSVFQSINPSTFKKQKKVPSALTEVAA
SGEGSAISGYLSRCKRGKRHWKKLWFVIKGKVLYTYMASEDKVALESMPLLGFTIAPEKEEGSSE
VGPIFHLYHKKTLFYSFKAEDTNSWIEAMEDASVL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table SC.
Table SC. Patp results for NOVS
Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAB93568Human protein sequence SEQ ID +1 577 1.7e-95 N0:12972 >patp:AAY51248Rat actin-binding protein frabin+1 312 1.9e-41 >patp:AAU21630Novel human neoplastic disease +l 256 1.6e-38 polypeptide >patp:AAU27818Human full-length polypeptide +1 300 2.6e-29 #143 >patp:ABG00573Novel human diagnostic protein +1 261 1.8e-26 #564 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 443 of 754 bases (58%) identical to a gb:GENBANI~-ID:AB037783~acc:AB037783.1 mRNA from H~~zo Sapiens (mRNA for KIAA1362 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 114 of 263 amino acid residues (43%) identical to, and 173 of 263 amino acid residues (65%) similar to, the 699 amino acid residue ptnr:SPTREMBL-ACC:Q9P2I5 protein from Ho»ao Sapiens (KIAA1362 PROTElI~.
NOVS also has homology to the proteins shown in the BLASTP data in Table SD.
Table 5D. BLAST
results for NOVS
Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~8922921~ref~NPhypothetical 432 135/284 169/284 5e-57 _ protein FLJ11183 (47%) (58%) 60821.1~(NM_018351) [Homo Sapiens]
gi~16716345~ref~NPethanol decreased431 131/284 171/284 2e-55 _ 4 [Mus musculus] (46%) (60%) 444302.1~(NM
gi~7243105~dbj~BAA9KIAA1362 protein699 111/251 166/251 2e-54 2600.1~(AB037783)[Homo Sapiens] (44%) (65%) gi~13648298~refIXPhypothetical 204 115/222 141/222 1e-49 - protein FLJ11183 (51%) (62%), 012133.21 (XM 012133) [Homo Sapiens]
gi(15426438~gb~AAH1Similar to 376 103/221 129/221 4e-40 3319.1~AAH13319 hypothetical (46%) (57%) (BC013319) protein FLJ11183 [Homo Sapiens]
A multiple sequence alignment is given in Table 5E, with the NOVS protein being shown on line 1 in Table SE in a ClustalW analysis, and comparing the NOVS
protein with the related protein sequences shown in Table SD. This BLASTP data is displayed graphically in the ClustalW in Table SE.
Table 5E. ClustalW Analysis of NOVS
1) > NOVS; SEQ ID NO:10 2) >giJ8922921J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ >D NO:55 3) >giJ16716345J/ ethanol decreased 4 [Mus musculus]; SEQ ID NO:56 4) >giJ7243105J/ KIAA1362 protein [Homo Sapiens]; SEQ ID N0:57 5) >giJ13648298J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ ID N0:58 6) >giJ 15426438J/ Similar to hypothetical protein FLJ11183 [Homo Sapiens];
SEQ m N0:59 ..
NOVS 1 ____________________________________________________________ 1 gi~8922921~ 1 ____________________________________________________________ 1 gi~16716345~ 1 ____________________________________________________________ 1 giI72431051 1 GIESDWQGLLVGEEKRSKPIKAYSTENYSLESQKKRKKSRGQTSAANGLRAESLDDQMLS 60 gi~7.3648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 ..
gi~89229211 1 ____________________________________________________________ 1 gi~16716345~ 1 ________________________________________,______________,____ 1 3O gi~7243105~ 61 RESSSQAPYKSVTSLCAPEYENIRHYEEIPEYENLPFIMATRKTQELEWQNSSSMEDADA
gi~13648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 ....~....J...
gi~8922921J 1 ____________________________________________________________ 1 g1(16716345( l __________________________________________________________ gi~72431051 121 NVYEVEEPYEAPDGQLQLGPRHQHSSSGASQEEQNDLGLGDLPSDEEEIINSSDEDDVSS
gi~13648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 J
J
J
J
NOV5 36 .... 64 ....
....J.,..J....J....
....J....J....J....J....J....
-----------------------INLWVTDLLVFQDFHGAVMRAL-----DDMDHE----gi J8922921J1 -___________________________________________________________ gi J16716345J1 -___________________________________________________________ gi J7243105J181ESSKGEPDPLEDKQDEDNGMKSKVHHIAKEIMSSEKVFVDVLKLLHIDFRDAVAHASRQL
giJ13648298J1 ____________________________________________________________ giJ15426438J1 ____________________________________________________________ J
J
J
....
NOV5 65 ....J 124 ....J....J....J....
....J....J....J....J....J....
GRDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHAT
giJ8922921J1 ____________________________________________________________ giJ16716345J1 ____________________________________________________________ IS giJ7243105J241GKPVIEDRILNQILYYLPQLYELNRDLLKELEERMLHWTEQQRIADIFVKKGPYLKMYST
gi~13648298J1 ____________________________________________________________ giJ15426438J1 ____________________________________________________________ 2.0 ....J....J....~....~....~....~.
.J.
.~.
~
.
...J
.
..J
.~
L
~
.
.
NOVS 125HILQFDRYLGLLSENCLHSPRLAAAVREFEQ "~GG,QTA
. r 184 .
.
..
.
._ .
~V
r ~;tFr Q~T
giJ892292111 _____________________________ . . r'.r r 30 .
giJ16716345J1 _____________________________ . v r 30 ~ v giJ7243105J301YIKEFDKNIALLDEQCKKNPGFAAVVREFEi C ~
r 360 r 25 giJ13648298J1 -___________________________________________________________ giJ15426438J1 -_____________________________~
.
.
r.
.r .
r 30 NOVS l85 244 giJ8922921J31 90 giJ16716345J31 90 giJ7243105J361 420 giJ13648298J1 ____________________________________________________________ 35 gi 1542643831 ~I~ilr 90 J J -, r r r r ' r v r v v r r .J. . .J....J. ...J. .~ . .J. . ..J,.. ~J...'....v..W ....J....J
NOV5 245E . T G~LR' E' . r ~~.... I E T YHGEGEG 304 Y ~ A~ i 'HL. QD
40 giJ8922921J91 I ~.. r ~ ~,t,~ 141 ~~_________ giJ16716345J91 T ~ r ~ ~ -------- 141 giJ7243105J421 I t' r ~ -------- 471 giJ13648298J1 ____________________________________________________________ giJ15426438J91 ~I 141 r r r --------J
....J....J....J
NOV5 305....J....J....J....J....J....J....J....J....
GSTFLSMEVCSLLEPKAPPRSLLEKGMGDVVTGRYLSNMTVHLGLPGLGPEHDALQPSQR
giJ8922921Jl41____________________________________________________________ 50 giJ16716345J141___________________________________-____,___________________ giJ7243105J471________________________________________-__________________ giI13648298Jl ____________________________________________________________ giJ15426438J141_____________________________________________ ____________ J
..J.. .J....~....J ....J
NOV5 365W.S~ . ~S SCL~ ....J....J....J....J....J....
RLQVRKSKVKALTDSVSAALGVRGISLFQCKKK
KVPY
giJ8922921J141- ~ ._______________________,_________ ~
giJ16716345J141- ~ ._________________________________ ~
60 giJ7243105J471- r ._________________________________ r giJ13648298J1 ____________________________________________________________ giJ15426438J141- 167 ~
~
._________________________________ 65 ....J....J....J....J....J....J....J....J....J....J..
.~.
.J
..
C r 484 giI8922921J167____________________________________________________ ~T r 174 giJ16716345J167____________________________________________________ r 174 giJ7243105J497____________________________________________________ T r 504 70 giI13648298J1 ____________________________________________________________ gi1154264381 167__,__________________________________________________~T~
.1. ._...~....~'i, ..~.E.P~ ~. 540 S NOV5 485..~....~....~ VF~IR'~RLG- L_ 'G
. 1.
YGCL ..
' -~
T.~P
DY
~ALAAFHH
gi1 89229211175 ~ ~ i ~ .. .~ 234 ..
gi1 167163451175~ S ~ -~ ~ 233 gi1 7243105)505~ ' ~ ~ S ' ~ 564 ' gi1 1364829811 _________________________ _____,______ ____.___________ 6 -154264381175' ~ S 234 gi1 ~ "
~
..
gi1 286 gi1 167163451234 -- ~ 285 gi1 72431051565 --gi1 1364829817 --gi1 154264381235 --..,.~. .~....~. ..~. .~. .~...
NOV5 601 RVTERPSMSFPLS ~RFS ~F FQ PS 660 gi189229211 287 ~ - ~ ~ 341 gi1167163451 286 ~L ~ - ~ 340 gi172431051 617 ~
gi1136482981 59 t - ~ 113 gi1154264381 287 ~ - ~
' ..1....1.,....1.,.. ,1....1 I 'I Y KVY
NOV5 661 ~ S ~ C ERG ~ ~H '1KL ~ ~ ~ :APE E . SEVGP~ 720 gi ~ 89229211 342 ~ ? ~ v . ,.I ~ 3'.. . Q ~ IQ ESK--~. 399 gi1167163451 341 ~ ~ L ~ ~~ ~ ~ Q~~TL ESK--~ 398 gi172431051 672 v v CCK-_-V~_____________________'__ 699 gi 1 13648298 ~ 114 ~ ~ ~ v ~ Q~FIQV~ytl.7~ESK--'~ l71 gi1154264381 342 ~ ~~ R,E~------- -- ------------ 376 NOV5 721 ~ ~ I~KT. ~ 15..:,~:T.. 1 .S~ ~..~. ... 1w~.-; ~A. 750 gi189229211 400 Q L ~ ~E~ .SAQ ~~F~G'.I'~ 432 gi 1167163451 399 Q~L~N~~ r, STQ D~F~G~ 431 gi172431051 699 -_ __________ ___,____ '_ __ 699 gi 1136482981 172 Q~L~mlM~ESAQIt~EiFGT~ 204 gi~15426438~ 376 _________________________________ 376 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table SF lists the domain description from DOMAIN ailalysis results against NOVS.
Table SF Domain Analysis of NOVS
Model Region of Score (bits) E value Homology RhoGEF 33-215 -1.9 1.2e-05 FYVE Ring 525-591 55.6 6.4e-14 Finger Plekstrin 657-748 49.3 8.0e-7 (PH) Consistent with other known members of the Nuclear Protein-like family of proteins, NOVS has, for example, an RhoGEF signature sequence and a FYVE Zinc Finger signature sequence, aw well as homology to other members of the Nuclear Protein-like Protein Family.
NOVS nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOVS nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOVS nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVS activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
In addition, various NOVS nucleic acids and polypeptides according to the invention are useful, ihteY alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOVS
nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Nuclear Protein-like Protein Family.
The NOVS nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and nerve physiology. As such, the NOVS nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
The NOVS nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOVS nucleic acid is expressed in Brown adipose, Vein, Umbilical Vein, Adrenal Gland/Suprarenal gland, Gall Bladder, Small Intestine, Colon, Lymphoid tissue, Spleen, Lymph node, Thymus, Brain, Temporal Lobe, Basal Ganglia/Cerebral nuclei, Substantia Nigra, Spinal Chord, Cervix, Ovary, Uterus, Testis, Lung, Lung Pleura, Larynx, Urinary Bladder, Kidney.
Additional utilities for NOVS nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV6 polypeptide has been identified as a Secretory Protein-like protein (also referred to as CG949~9-O1). The disclosed novel NOV6 nucleic acid (SEQ m NO:l 1) of 2372 nucleotides is shown in Table 6A. The novel NOV6 nucleic acid sequences maps to the chromosome 17.
An ORF begins with an ATG initiation codon at nucleotides 99-101 and ends with a TAA codon at nucleotides 1710-1712. A putative untranslated region and/or downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO:11) CCGGCAAGGATGACGCCTCCGGAGGCCCTGGCCTCACTCCCACCTGGGCGCTAGGAGCCATCCCGGG
GCTCCAGCCAGGAGCCCTGCTGCCCAGGGGCATGGCCAAACCTTTCTTCCGACTCCAGAAGTTTCTC
CGCCGAACACAGTTCCTGCTGTTCTTCCTCACGGCTGCCTACCTGATGACCGGCAGCCTGCTGCTGC
TGCAGCGGGTCCGCGTGGCTCTCCCACAGGGCCCCCGGGCACCCGGCCCCCTGCAGACCTTGCCAGT
GGCCGCCGTGGCGCTGGGCGTGGGCTTGCTGGACAGCAGAGCCCTGCACGACCCTCGAGTCAGCCCA
GAGCTGCTGCTGGGTGTGGACATGCTGCAGAGCCCCCTGACCCGGCCCCGGCCCGGCCCCCGCTGGC
TCCGGAGCCGCAACTCGGAGCTGCGTCAGTTGCGTCGCCGCTGGTTCCACCACTTCATGAGTNGACT
CCCAGGGACCGCCCGCCCTGGGCCCCGAGGCTGCCAGGCCCGCCATCCACAGCCGAGGTCCTATGTC
TACGCCGGCTTGGAGGCCGGGGCGGAGTGTTACTGCGGGAACCGGCTGCCAGCGGTGAGCGTGGGGC
TGGAAGAGTGTAACCATGAGTGCAAAGGCGAGAAGGGCTCTGTGTGCGGGGCTGTGGACCGGCTCTC
CGTGTACCGTGTGGACGAGCTGCAGCCGGGCTCCAGGAAGCGGCGGACCGCCACCTACCGCGGATGC
TTCCGACTGCCAGAGAACATCACACATGCCTTCCCCAGCTCCCTGATACAGGCCAATGTGACCGTGG
GGACTTGCTCGGGCTTTTGTTCCCAGAAAGAGTTCCCCTTGGCCATTCTCAGGGGCTGGGAATGCTA
CTGTGCTTACCCTACCCCCCGGTTCAACCTGCGGGATGCCATGGACAGCTCAGTATGTGGCCAGGAC
CCTGAGGCACAGAGGCTGGCAGAATACTGTGAGGTCTACCAGACACCTGTGCAAGACACTCGTTGTA
CAGACAGGAGGTTCCTGCCTAACAAATCCAAAGTGTTTGTGGCTTTGTCAAGCTTCCCAGGAGCCGG
GAACACGTGGGCACGGCACCTCATTGAGCATGCCACTGGCTTCTATACAGGGAGCTACTACTTTGAT
GGAACCCTCTACAACAAAGGGTTCAAGGGCGAAAAGGACCACTGGCGGAGCCGACGCACCATCTGTG
TCAAAACCCACGAGAGTGGCAGGAGGGAGATTGAGATGTTTGATTCAGCCATCCTGCTAATCCGGAA
CCCATACAGGTCCCTGGTGGCAGAATTCAACAGAAAATGTGCCGGGCACCTGGGATATGCAGCTGAC
CGCAACTGGAAGAGCAAAGAGTGGCCGGACTTTGTCAACAGCTACGCCTCGTGGTGGTCCTCGCACG
TCCTGGACTGGCTCAAGTACGGGAAGCGGCTGCTGGTGGTGCACTACGAGGAGCTGCGGCGCAGCCT
GGTGCCCACGTTACGGGAGATGGTGGCCTTCCTCAACGTGTCTGTGAGCGAGGAGCGGCTGCTCTGC
GTGGAGAACAACAAGGAGGGCAGCTTCCGGCGGCGCGGCCGGCGCTCCCACGACCCTGAGCCCTTCA
CCCCGGAGATGAAAGACTTGATCAATGGCTACATCCGGACGGTGGACCAAGCCCTGCGTGACCACAA
CTGGACGGGGCTGCCCAGGGAGTATGTGCCCAGATGATAGGCCTGGCCCACGCCGCCGCCCCCGCTG
AGTGACGCAATCGCACCACGGGGCTGCGCTCCCCACTCTGATGCTCAGGCCCGTGGCCTCACTGGGA
CGAACGGTGGGTGGGGGGCTCACCCTGGTGCTGCCTCCCGCACAAGGAGACCTGGACACAACAGACA
CACATCACAAGGCGAACACAAATGGACACACATACCTGGCCACGAACCCACACCTCCTCAGACACTC
AGACACCACTCCAGGCTCATAGCCCCGTCTTGATGCAGAGAAGCCACCCACGTGGGGTGTGCCAGGC
ACCCCCAGCTACAAATGCAGCCACGCACAGACGTAACACACAGGTGCCAGGCCGTGTGCTCCTGGAG
GCTGGCTGGCTGTCTCTCTCACACAGATACACGTGCGCTCCCTGGGATCCGGGAGGCCCTGGGCTTC
CTGTGTGTAGCCCTGGCATAGACTTGCTCGTCAGGGTGTTTGACTCTGGGATGCTGGGCCGGGCAGA
CATTTATGCTCTGAGCAGCAAGGACCATTGGGATGGAGGTGGGCACAAAGACTGCTGCTTCCAGGGT
GTGCGGCCCTGGCCGTGTGTCTGACATCCCATAAATGTGTGTGTGGTGTGACTACGGGCACCACAAA
CTCCGC
The NOV6 protein (SEQ m NO: I2) encoded by SEQ m NO:I 1 is 537 amino acid residues in length and is presented using the one-letter amino acid code in Table 6B. Psort analysis predicts the NOV6 protein of the invention to be localized outside the cell with a certainty of 0.6997.
Table 6B. Encoded NOV6 protein sequence (SEQ ID N0:12) MAKPFFRLQKFLRRTQFLLFFLTAAYLMTGSLLLLQRVRVALPQGPRAPGPLQTLPVAAVALGVG
LLDSRALHDPRVSPELLLGVDMLQSPLTRPRPGPRWLRSRNSELRQLRRRWFHHFMSXLPGTARP
GPRGCQARHPQPRSYVYAGLEAGAECYCGNRLPAVSVGLEECNHECKGEKGSVCGAVDRLSVYRV
DELQPGSRKRRTATYRGCFRLPENITHAFPSSLIQANVTVGTCSGFCSQKEFPLAILRGWECYCA
YPTPRFNLRDAMDSSVCGQDPEAQRLAEYCEVYQTPVQDTRCTDRRFLPNKSKVFVALSSFPGAG
NTWARHLIEHATGFYTGSYYFDGTLYNKGFKGEKDHWRSRRTICVKTHESGRREIEMFDSAILLI
RNPYRSLVAEFNRKCAGHLGYAADRNWKSKEWPDFVNSYASWWSSHVLDWLKYGKRLLVVHYEEL
RRSLVPTLREMVAFLNVSVSEERLLCVENNKEGSFRRRGRRSHDPEPFTPEMKDLINGYIRTVDQ
ALRDHNWTGLPREYVPR
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 6C.
Table 6C. Patp results for NOV6 Smallest Sum eading igh Prob Sequences Score P(N) producing High-scoring Segment Pairs: Frame >patp:ABB15485Human nervous system related polypeptide+1 92 0.0036 >patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042 protein >patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042 protein >patp:AAU18674Renal and cardiovascular-associated+1 79 0.085 protein >patp:AAB95341Human protein sequence SEQ ID +1 99 0.17 N0:17621 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2188 of 2189 bases (99%) identical to a gb:GENBANK-ID:AK000243~acc:AK000243.1 mRNA from Horrao sapiehs (cDNA
FLJ20236 fis, clone COLF5810, highly similar to ABOl 1095 Homo Sapiens mRNA
for KIAA.0523 protein). The full amino acid sequence of the protein of the invention was found to have 395 of 395 amino acid residues (100%) identical to, and 395 of 395 amino acid residues (100%) similar to, the 468 amino acid residue ptnr:SPTREMBL-ACC:060276 protein from Homo Sapiens (KIAA0523 PROTEIN)(Fig. 3B).
NOV6 also has homology to the proteins shown in the BLASTP data in Table 6D.
Table 6D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~14602977~gb~AAHOSimilar to 575 523/575 524/575 0.0 9975.1IAAH09975 KIAA0789 gene (90%) (90%) (BC009975) product [Homo Sapiens]
gi~3043570~dbj~BAA2KIAA0523 protein468 417/468 417/468 0.0 5449.1 (AB011095)[Homo Sapiens] (89%) (89%) gi~18489296~ref~XPCG9164 317 76/206 15/206 3e-28 _ [Drosophila (36%) (54%) 082751.1I(XM
melanogaster]
gi~16944644~emb~CADhypothetical 2117 43/131 62/131 5e-08 11404.11(AL513445)protein (32%) (46%) [Neurospora crassa]
gi~11359357~pir~~T4beta-1,3 1032 40/128 55/128 2e-05 3257 exoglucanase (31%) (42%) (EC
3.2.1.-) precursor -fungus (Trichoderma harzianum) A multiple sequence alignment is given in Table 6E, with the NOV6 protein being shown on line 1 in Table 6E in a ClustalW analysis, and comparing the NOV6 protein with the related protein sequences shown in Table 6D. This BLASTP data is displayed graphically in the ClustalW in Table 6E.
Table 6E. ClustalW Analysis of NOV6 1) > NOV6; SEQ ID NO:12 2) >gi~14602977~/ Similar to KIAA0789 gene product [Homo sapiens]; SEQ ID
N0:60 3) >gi~3043570~/ KIAA0523 protein [Homo Sapiens]; SEQ ID N0:61 4) >gi~18489296~/ CG9164 [Drosoplaila melanogaster]; SEQ 117 N0:62 5) >gi~ 16944644/ hypothetical protein [Neurospora cf assa]; SEQ ID N0:63 6) >gi~ 11359357/ beta-1,3 exoglucanase (EC 3.2.1.-) precursor - fungus [Trichodertna harzianum];
SEQ 1D N0:64 p....
NOV6 1 ------------------------MAKPFFR2QKFItRRTQFLLFFLTAAYt'C.yMT~-=--SL 32 gi1146029771 1 ------------------------KPFFRIiQKF~RRTQFLLFFLTAAY~.,MT - --SL
gi130435701 1 ________________________-___________________________________ 1 gi 18489296 1 ____________________________________________________________ 1 gi ~ 16944644 I 841 GTSPGLTTTEVTIRFTNKGGSDLNLI~DKSKPP~'IGSV~iGAQNPSSDLFEGMV2'KP KSESAT 900 gi1113593571 235 TFNGGLIG--------------AA~GNQQYT~RNL~TFNN--CAQPLSAASxaGSB----....~....~....~....~.. .~....~.. .,1....1....1....1..~.1....1 NOV6 33 LLLQRVRV~PQGPRAPGPLQT~.iiPVAAVAL~G~LDSR-ALHDPRVSPE~LLGVDMLQSP 91 gi~14602977~ 33 LLLQRVRV PQGPRAPGPLQTPVAAVAL G~LDSR-ALHDPRVSPE LLGVDMLQSP 91 gi130435701 1 ____________________________________________________________ 1 gi1184892961 1 ____________________________________________________________ 1 IS gi1169446441 901 LFFTPGAA~ADPIVYSGAWTLTFG~Fi7NFIGTLRATKVGPT~PDGSARFKYL 960 gi~11359357~ 275 RAISINNC.','!~lGIDMTAAESITLkr~. SSISGTP G~KTSFRRNQSPATSNS
NOV6 92 LTRPRPGPRWLRSRNS~LR---------------------------------------X7112 giI14602977~ 92 LTRPRPGPRWLRSRNSLR---------------------------------------',#~' gi130435701 1 ________________LR-______________________________________~5 gi1184892961 1 ___________________________________________________________- 1 gi116944644~ 961 GCYRDSSANRLETTQAFPSDNDNGKCQQYAITNKAAFAGTQYTYECWVGRSIPPASLiF
gi1113593571 335 PVAIQSSSGSTILAGGTTT----------------------------------------NOV6 113 RRRWF ~'i~X------------------------------L~GT .~G--PR------- 133 3o gi~146029771 113 RRRWF~~'MT7SQGP--===____ _____= PA'I~ ~E IHRG--=T~I 146 gi 1 30435701 6 RRRWFE., FMSDSQGP-- PAIi ~E ~IHRG-- Trr~~I 39 gi1184892961 1 __ _____________________________________ _________ 1 gi~16944644~ 1021 DDYLCTj~IzCPGDKSQFCGGVGSYMMMWYDTTGYFPENGT'~FRP~ASIC~VVGDW~
gi~11359357~ 355 AAWGQG~QYj~"PNGPTTFQG---------------- SItT SRPSLLGSN T 392 35 _.
.~....~....~....~....~....~....~. _ l~ ~I.
NOV6 134 ~QA--_______R_______________HpQp__ ~~y . ' ~ PA~~ 167 gi1146029771 147 FSDDGHERTLKGAVFYDLRKMTV-SHCQDAC~~'1,E Y~~' ~ PA205 gi~3043570~ 40 FSDDGHERTLKGAVFYDLRKMTV-SHCQDACiIE YV~ ' PA 98 gi~184892961 1 ________________________________~L~G~IR~'g S~TIII I -____'G__ gi1169446441 1081 ~RTDNSASPATRALNDRIVGQSSTNTIESCAQ~iCAG~S'F r .. PG~T 1140 gi~11359357~ 393 RSKPQYETLPVSSFRSVRSAGATGN-AVTDDTA~L~ATAC QI FDAG~ R'~~?S 451 .1. .1. .1. .1. .1.
NOV6 168 .E - E ~ 'E'EKGS~, VF.W . Z7DELQ~ _____________________ 202 gi~14602977~ 206 -- !E KGSi E L VDELQ~ _______________________ 240 gi~3043570! 99 ~ -- _E EKGS~Z~_ I VDELQ~ -____________________ 333 O gy 184892961 21 _ __________ __ __ __ V~~LSMNNI~
gi~16944644~ 1141 VA~KT~YV~ 1~PTEGSG~'yI~TCQKGTVI PSTGVSSSSGTASGTASATAS 1200 gi111359357i 452 T~S------IPP~ -AK~EYP~2IMSSGSFFND----------------------Q 479 . .1...~1. ~~~ .1...y y .~ . ~~. ~ ~~.. y , c - v _ r v-NOV6 203 R j' "I'~~TY~ C~RL~. . ----- !i! ' ~ ~5~~~~ ~ - '~!':2'C~GT S. -Q ~E
gi ~ 146029771 241 R ~TiiITY~ C RL~ _=---- TH~ ~ 5~~~ - --'i~T~TGT ;, -Q ~E
gi ~ 304357Q ~ 134 R WiTY~ C RL E ---- ~'H' ~ S~Ir~~ -- --,~T"~"sIGT ,~ -Q ~E
gi1184892961 34 HP4'PRIE~__ ________ -~ . __-_____ _ ___._ _p_ 53 gi 1 169446441 1201 STSSiIA~TPGN~Q'S'~GQYSSLGCY',~.",Di RSL~GKNTQSNVMSIiDD~T
!GY~yIY 1260 gi~11359357~ 480 SNP~PVVQ TPGQTG-------- QVE~SDM~TVSTQG- -----TQAGAVLIEWNL 520 .1. .1....1....1.. .1.~...~...1.~~ .1. ..1....1....1....1....1 NOV6 247 .PLAIL~~ E YCA ~T'P~tFNL~'~ n SS~ Gy P________________________ 282 gi114602977~ 285 PLAIL E YCAY~'T~P~R,FN~~W ySa GyP3~"'s -______-______________ gi~30435701 178 PLAIL E YCAY~'~P~FN~rw W9~G~',~,?rP~'." ______________________ gi1184892961 53 ____gp________ ~~gTI~ CR~LKYI ~- ________________________ 75 gi116944644~ 1261 ~GTEYSAECF~GNDLLNGAAP1~T~GRC~G QQQICGGSNGLSMYQLNPNGTSSSVT
gi1113593571 521 ATSGTPSe~vIWDVHTRIGGFKGSNLQ'~'~1QCPVTAS~----------------------4~ _:
..J.. .~....~.,...~ ..L ...~....~....~....~....~
NOV6 282 _______ ~QR(~..[~~ E~Q P~'ST;QD RC'DR~~2.F ~ K-___________________ gi~14602977~ 320 ------- Q ~ E EVY'Q PVQD RC DR~F ~ K-------------------- 349 gi~30435701 213 ------- fi~Q E EV~Q P~QD RC~DR~2.~' ~ K--------------------gi 18489296 75 ___________ZPIKSD~'Y ;'PSDVSAAL~S ~ _____________________ 102 gi~16944644) 1321 ASGSATQSATA~GTASGTASSSS ' T TS~AVPT ~ VSVKCPDNNNGTYLSLNGKTF
gi I 11359357 ~ 554 ------- TTt'V.NTA~IGAYMS ,:, I~ASASNL~'~'NIEI~-------------NOV6 311 _____________________________ ~~ . ~ _______________ 325 w gi~14602977~ 349 ______________________________. ~r _______________ 363 w 15 gi~30435701 242 ______________________________r~' S _______________ 256 gi~184892961 102 __-___________________________ pLT. ~ ________________ 114 gi~16944644~ 1381 LLECFTDHEGGDLALAYVDSYALCAEKCSTTDI,rC~ F~ GTGIQAPCYMKKSVGRGF
gi~113593571 582 ______________________________~T~DHDIDDS--__________N___ 597 1450 1460 7.470 1480 1490 1500 ....
NOV6 325 --------------------- F~ K 359 v gi~14602977~ 363 _____________________ y F~ K IC 397 v gi~30435701 256 --------------------- F~ K K 290 gi118489296~ 114 _____________________ I ~Y LKT P 4 148 gi~16944644~ 1441 SAAATMTSAASAMGSNSWGPS TGS TGSATDSTT 1500 gi~11359357~ 598 ------------------FWF ~..'AVEHH QYQ~ANT,638 gi~146029771 398 457 gi~3043570~ 291 350 gi1184892961 148 206 gi~16944644~ 1501 1560 gi~11359357~ 639 697 4o NOV6 420 ----I----I----I 458 gi~14602977~ 458 --------------- 496 gi~3043570~ 351 _,_____________ 389 gi~18489296~ 207 _______________ 249 g3~16944644~ 1561 IAPSENTTPSASVAP 1619 gi~11359357~ 697 --------------- 729 NOV6 458 --------L PT. E1~3't~~ 5~- ERW ~ I$G---------------- 494 giI14602977f 496 -_______L PT ~ES ER,~,~ 'j ' ~G-_______________ 532 gi ~ 3043570 ~ 389 --------L PT ~E~'1'C7~ ~ g~,~,, yG---------------- 425 gi~18489296~ 249 _-______TERE S ~D QFPQLt: IMy" I ~ ~_________________ 284 gi~16944644~ 1620 SNSWPSDS~APSAS~I~iPSA~~ yS~ASV~PSTSIAHS SESVAPAESIAPSASVSS
gi~11359357~ 729 --------N~FD~EGTTN~yNLGTVG,i~VI~IT~ LATS------S--------- 766 NOV6 494 --_________________________-_________-______________________ 494 gi~14602977~ 532 _-_________________________-________________________________ gi~3043570~ 425 ____________________________________________________________ gi118489296~ 284 ____________________________________________________________ gi~16944644~ 1680 GSNTGVAPTNSASVTPTNSASVATTISVSVAPTASDAPTTSITLSVAPGSSSSTTAPAW
gi~11359357~ 766 ____________________________________________________________ NOV6 494 _______________________, ~~~~ PE.: L~~GYIv Q RDHNW G 529 gi~146029771 532 -_____________-_________ ~~E~F PE"IDL~~TGYI~ Q~ RDHNW G 567 gi~3043570~ 425 _________________________ . ~.E. PE ~CDI~Ii~GYI~ ' Q; RDHNW G
gi~18489296~ 284 -------------------------;_~LLSF~1~ ES ~AE~QNR ~I YGL GRQEP--47 _ _ gi~169446441 1740 STTLSAPTVTSVPPAAGTSTTTAAAVT~TTTTTTATSTT TPVLFTTSTT~T 1799 gi~11359357~ 766 -------------------------SNVFADVIALFLASGSGGV~PPP'S~STTKAQT
NOV6 530 LPREY~7'PR-___________,____-__________________________________ 537 giI14602977~ 568 LPRE'IV'PR-___________,____-__________________________________ 575 gi13043570~ 461 LPREYPR-___________,___________________________-___________ gi118489296~ 317 ________________________________________________-___________ 1O gi~16944644~ 1800 gi~11359357~ 802 TFSTI';TSSPPKQTG-------------------------WNFLGCYSDNVNGRTLANQV
The NOV6 Clustal W alignment shown in Table 6E was modified to begin at amino residue 841 and end at amino acid residue 1860. The data in Table 6E includes all of the regions overlapping with the NOV6 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 6F lists the domain description from DOMAIN analysis results against NOV6.
Table 6F Domain Analysis of Model Region of Score (bits) E value Homology Disintegrin 151-159 5.8 0.73 WSC domain 120-186 36.8 5e-07 Peptidase family346-354 -0.2 8.4 Sulfotransferase288-528 -143.3 0.14 proteins Consistent with other known members of the Secretory Protein-like family of proteins, NOV6 has, for example, has homology to other members of the Secretory Protein-like Protein Family. NOV6 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV6 nucleic acids and polypeptides can be used to identify proteins that are members of the Secretory Protein-like Protein Family. The NOV6 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV6 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other diseases, disorders and conditions.
In addition, various NOV6 nucleic acids and polypeptides according to the invention are useful, intes~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV6 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Secretory Protein-like Protein Family.
The NOV6 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac physiology. As such, the NOV6 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiac and vascular system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other diseases, disorders and conditions The NOV6 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV6 nucleic acid is expressed in Aorta.
Additional utilities for NOV6 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV7 polypeptide has been identified as a Transmission Blocking Target Antigen 5230 Precursor-like protein (also referred to as CG9497~-O1). The disclosed novel NOV7 nucleic acid (SEQ )D N0:13) of 1629 nucleotides is shown in Table 7A. The novel NOV7 nucleic acid sequences maps to the chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TGA
codon at nucleotides 1627-1629. A putative untranslated region and/or downstream from the termination codon is underlined in Table 7A, and the start and stop codons are in bold letters.
Table 7A. NOV7 Nucleotide Sequence (SEQ ID N0:13) ATGGCGGTGCCCGGCGAGGCGGAGGAGGAGGCGACAGTTTACCTGGTAGTGAGCGGTATCCCCTCCG
TGTTGCGCTCGGCCCATTTACGGAGCTATTTTAGCCAGTTCCGAGAAGAGCGCGGCGGTGGCTTCCT
CTGTTTCCACTACCGGCATCGGCCTGAGCGGGCCCCTCCGCAGGCCGCTCCTAACTCTGCCCTAATT
CCTACCGACCCAGCCGCTGAGGGCCAGCTTCTCTCTCAGACTTCGGCCACCGATGTCCGGCCTCTCT
CCACTCGAGACTCTACTCCAATCCAGACCCGCACCTGCTGCTGCGTCATCTCGGTAAGGGGGTTGGC
TCAAGCTCAGAGGCTTATTCGCATGTACTCGGGCCGCCGGTGGCTGGATTCTCACGGGACTTGGCTA
CCGGGTCGCTGTCTCATCCGCAGACTTCGGCTACCTACGGAGGCATCAGGTCTGGGCTCCTTTCCCT
TCAAGACCCGGAAGGAACTGCAGAGTTGGAAGGCAGAGAATGAAGCCTTCACCCTGGCTGACCTGAA
GCAACTGCCGGAGCTGAACCCACCAGTGCTGATGCCCAGAGGGAATGTGGGGACTCCCCTGCGGGTC
TTTTTGGAGTTGATCCGGGCCTGCCGCCTACCCCCTCGGATCATCACCCAGCTGCAGCTCCAGTTCC
CCAAGACAGGTTCCTCCCGGCGCTACGGCAATGTGCCTTTTGAGTATGAGGACTCAGAGACTGTGGA
GCAGGAAGAGCTTGTGTATACAGCAGAGGGTGAAGAAATACCCCAAGGAACCTACCTGGCAGATATA
CCAGCCAGCCCCTGTGGAGAGCCTGAGGAAGAAGTGGGGAAGGAAGAGGAAGAAGAGTCTCACTCAG
ATGAGCTGTTCGGGTGTGCTGTGGTCATCCTCCCTGCGCACCTACAGCCGCAGACCGCCGGTGGGGG
GCGGGGGATGCCGGGCTGCCGCATCAGCGCCTGCGGCCCGGGGGCCCAGGAGGGGACGGCAGAGCAG
AGGTCGCCGCCGCCGCCCTGGGATCCCATGCCGTCCTCTCAGCCCCCGCCCCCAACTCCGACCTTGA
CTCCTACCCCGACCCCGGGTCAGTCCCCGCCGCTGCCGGACGCAGCTGGGGCTTCAGCAGGCGCGGC
CGAGGACCAGGAGCTGCAGCGCTGGCGCCAGGGCGCTAGCGGGATCGCGGGGCTCGCCGGCCCCGGA
GGGGGCTCTGGCGCGGCTGCGGGGGCGGGGGGCCGCGCGCTGGAGCTGGCCGAAGCACGGCGGCGGC
TGCTGGAGGTGGAGGGCCGCCGGCGCCTGGTGTCGGAGCTGGAGAGCCGCGTGCTGCAGCTGCACCG
CGTTTTCTTGGCGGCCGAGCTGCGCCTGGCGCACCGCGCGGAGAGCCTGAGCCGCCTGAGCGGCGGC
GTGGCGCAGGCCGAGCTCTACCTGGCGGCTCACGGGTCGCGCCTCAAGAAGGGCCCGCGCCGCGGCC
GCCGCGGCCGACCCCCCGCGCTGCTGGCCTCGGCGCTGGGCCTGGGCGGCTGCGTGCCCTGGGGTGC
CGGGCGACTGCGGCGCGGCCACGGCCCCGAGCCCGACTCGCCCTTCCGCCGCAGCCCGCCCCGCGGC
CCCGCCTCCCCGCAGCGCTGA
The NOV7 protein (SEQ m N0:14) encoded by SEQ m N0:13 is 542 amino acid residues in length and is presented using the one-letter amino acid code in Table 7B. Psort analysis predicts the NOV7 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 7S. Encoded NOV7 protein sequence (SEQ ID N0:14) MAVPGEAEEEATVYLVVSGIPSVLRSAHLRSYFSQFREERGGGFLCFHYRHRPERAPPQAAPNSA
LIPTDPAAEGQLLSQTSATDVRPLSTRDSTPIQTRTCCCVISVRGLAQAQRLIRMYSGRRWLDSH
GTWLPGRCLIRRLRLPTEASGLGSFPFKTRKELQSWKAENEAFTLADLKQLPELNPPVLMPRGNV
GTPLRVFLELIRACRLPPRIITQLQLQFPKTGSSRRYGNVPFEYEDSETVEQEELVYTAEGEEIP
QGTYLADIPASPCGEPEEEVGKEEEEESHSDELFGCAVVILPAHLQPQTAGGGRGMPGCRISACG
PGAQEGTAEQRSPPPPWDPMPSSQPPPPTPTLTPTPTPGQSPPLPDAAGASAGAAEDQELQRWRQ
GASGIAGLAGPGGGSGAAAGAGGRALELAEARRRLLEVEGRRRLVSELESRVLQLHRVFLAAELR
LAHRAESLSRLSGGVAQAELYLAAHGSRLKKGPRRGRRGRPPALLASALGLGGCVPWGAGRLRRG
HGPEPDSPFRRSPPRGPASPQR
A search against the Patp database, a proprietary database that'contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 7C.
Table 7C. Patp results for Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAU33166Novelhuman secreted protein #3657+1 1533 5.8e-157 >patp:AAE04880Humanprotease protein-7 (PRTS-7) +1 1533 5.8e-l57 >patp:AAB94023Humanprotein sequence SEQ ID N0:14157+1 1519 1.8e-155 >patp:AAU33124Novelhuman secreted protein #3615+1 390 7.7e-36 >patp:AAG02700Humansecreted protein, SEQ ID +1 268 3.5e-22 NO: 6781 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 874 of 876 bases (99%) identical to a gb:GENBANK-ID:AK022517~acc:AK022517.1 mRNA from Homo Sapiens (cDNA
FLJ12455 fis, clone NT2RM1000563, weakly similar to TRANSMISSION-BLOCKING
TARGET ANTIGEN 5230 PRECURSOR). The full amino acid sequence of the protein of the invention was found to have 290 of 292 amino acid residues (99%) identical to, and 290 of 292 amino acid residues (99%) similar to, the 525 amino acid residue pW
r:SPTREMBL-ACC:Q9H9Z3 protein from Homo Sapiens (CDNA FLJ12455 FIS, CLONE NT2RM1000563, PRECURSOR).
NOV7 also has homology to the proteins shown in the BLASTP data in Table 7D~
Table 7D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) (%) gi~18545154~ref~XPhypothetical 525 274/274 274/274 e-147 _ protein FLJ12455 (100%) (100%) 084046.1~(XM_084046 [Homo Sapiens]
gi~11545793IrefINPhypothetical 525 272/274 272/274 e-145 _ protein FLJ12455 (99%) (99%) 071361.1I(NM
[Homo Sapiens]
gi~18545156~ref~XPsimilar to 107 83/84 84/84 6e-37 _ hypothetical (98%) (99%) 086159.1~(XM
_ protein FLJ12455 [Homo Sapiens]
giI18545158~ref~XPhypothetical 141 135/137 136/137 2e-33 _ protein XP_097448 (98%) (98%) 097448.1~(XM_097448 [Homo Sapiens]
gi~~7.7562286IrefINPK07B1.7b.p 487 76/237 119/237 3e-30 _ [Caenorhabditis (32%) (50%) 505420.1~(NM_073019 e1 egans]
A multiple sequence alignment is given in Table 7E, with the NOV7 protein being shown on line 1 in Table 7E in a ClustalW analysis, and comparing the NOV7 protein with the select related protein sequences shown in Table 7D. This BLASTP data is displayed graphically in the ClustalW in Table 7E.
Table 7E. ClustalW Analysis of NOV7 1) > NOV7; SEQ m N0:14 2) > gig 18545154/ hypothetical protein FLJ12455 [Horrao Sapiens); SEQ m N0:65 3) > gi~11545793~/ hypothetical protein FLJ12455 [Homo Sapiens]; SEQ ID N0:66 NOV7 l 60 gi1185451541 1 60 gi~11545793~ 1 60 y . ~~~.~.I~ .~~ ~I~ ~I. y NOV7 61 ~~~ ~ ,~~~ ,. ~ ~ . .~ ~ . ~~w ~, 120 TRANSMISSION-FIS
#PD229850 55-90 179 4e-14 BLOCKING
TRANSMISSION-FIS
#PD138963 91-258 872 2e-94 BLOCKING
TRANSMISSION-FIS
Consistent with other known members of the Transmission Blocking Target Antigen 5230 Precursor-like family of proteins, NOV7 has, for example, three Blocking NT2RM1000563 Transmission-FIS Antigen Weakly Precursor Peptidase A2 signature sequences and homology to other members of the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family. NOV7 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV7 nucleic acids and polypeptides can be used to identify proteins that are members of the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family. The I O nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV7 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets fox the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection In addition, various NOV7 nucleic acids and polypeptides according to the invention are useful, irzte~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV7 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family.
The NOV7 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and nerve physiology. As such, the NOV7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection The NOV7 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV7 nucleic acid is expressed in Adipose, Heart, Aorta, Coronary Artery, Umbilical Vein, Pancreas, Liver, Gall Bladder, Colon, Bone Marrow, Thymus, Bone, Cartilage, Synovium/Synovial membrane, Skeletal Muscle, Brain, Left cerebellum, Right Cerebellum, Thalamus, Hypothalamus, Pituitary Gland, Frontal Lobe, Parietal Lobe, Cerebral Medulla/Cerebral white matter, Basal Ganglia/Cerebral nuclei, Substantia Nigra, Hippocampus, Cervix, Mammary gland/Breast, Uterus, Oviduct/LTterine Tube/Fallopian tube, Prostate, Testis, Lung, Bronchus, Larynx, Kidney, Retina, Skin, Epidermis.
Additional utilities for NOV7 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV8 polypeptide has been identified as a Nuclear Protein-like protein (also referred to as CG94713-O1). The disclosed novel NOV8 nucleic acid (SEQ ZD
NO:15) of 3807 nucleotides is shown in Table 8A. The novel NOV8 nucleic acid sequences maps to the chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 16-18 and ends with a TGA codon at nucleotides 3793-3795. A putative untranslated region and/or downstream from the termination codon is underlined in Table 8A, and the start and stop codons are in bold letters.
Table 8A. NOV8 Nucleotide Sequence (SEQ ID NO:15) ATGATAAAATAGAAGATGAATTGCAAACCTTCTTTACCAGTGATAAAGATGGAAATTACACATGCAT
ACAACCCGAAATCACCACCTACACAAAACTCTTCAGCCAGCAGTGTGAACTGGAATTCTGCCAACCC
AGATGACATGGTGGTTGATTATGAAACTGACCCTGCTGTAGTTACTGGTGAAAATATTTCTTTAAGC
CTTCAGGGTGTTGAAGTATTTGGTCATGAAAAGTCTTCTAGTGATTTCATTAGTAAGCAGGTGTTAG
ATATGCATAAAGATTCTATTTGTCAGTGTCCTGCACTTGTAGGTACTGAGAAGCCCAAATATCTGCA
ACACAGTTGTCATTCCCTAGAAGCAGTTGAGGGCCAGAGTGTTGAGCCATCTTTGCCTTTTGTGTGG
AAGCCTAATGACAATTTGAACTGTGCAGGCTACTGTGATGCCTTGGAGCTGAACCAAACATTTGACA
TGACAGTGGATAAAGTTAACTGCACCTTTATATCACATCATGCCATCGGAAAGAGTCAGTCCTTCCA
TACTGCTGGAAGCCTGCCACCAACTGGTAGGAGAAGTGGAAGTACATCTTCTTTATCCTATTCCACT
TGGACATCTTCCCATTCTGATAAGACGCATGCAAGAGAAACTACTTATGATAGAGAAAGCTTTGAAA
ACCCTCAAGTCACACCATCAGAAGCCCAAGACATGACTTACACAGCATTTTCTGATGTGGTGATGCA
AAGTGAGGTTTTTGTTTCAGATATTGGAAATCAGTGTGCATGTTCTTCAGGAAAGGTCACCAGTGAG
TACACAGATGGATCACAACAAAGACTAGTTGGAGAAAAAGAGACACAAGCACTAACACCAGTTTCTG
ATGGCATGGAAGTCCCCAATGATTCTGCATTACAAGAGTTCTTTTGTTTATCCCATGATGAATCCAA
TAGCGAACCACATTCACAGAGCTCATACAGGCACAAGGAAATGGGCCAAAATCTGAGAGAGACAGTG
TCCTATTGTCTTATTGATGATGAATGCCCTTTAATGGTGCCAGCTTTTGATAAGAGCGAAGCTCAAG
TGCTGAACCCAGAGCATAAAGTCACTGAGACTGAAGACACACAAATGGTCTCCAAAGGAAAGGATTT
GGGAACCCAAAATCATACCTCAGAATTGATTCTAAGTAGCCCGCCAGGACAAAAGGTGGGCTCGTCA
TTTGGACTGACTTGGGATGCAAATGATATGGTCATTAGCACAGACAAAACGATGTGCATGTCAACAC
CAGTCCTAGAACCCACAAAAGTAACCTTTTCTGTTTCACCGATTGAAGCGACGGAGAAATGTAAGAA
AGTGGAGAAGGGTAATCGAGGGCTTAAAAACATACCAGACTCGAAGGAGGCACCTGTGAACCTGTGT
AAACCCAGTTTAGGAAAATCAACAATCAAAACGAATACCCCAATAGGCTGCAAAGTTAGAAAAACTG
AAATTATAAGTTACCCAAGACCAAACTTCAAGAATGTCAAAGCAAAAGTTATGTCTAGAGCAGTGTT
GCAGCCCAAAGATGCTGCTTTATCAAAGGTCACGCCCAGACCTCAGCAGACCAGTGCCTCATCACCC
TCATCAGTGAATTCAAGACAACAAACAGTCTTGAGCAGAACACCGAGATCTGACTTGAATGCAGACA
AAAAAGCAGAAATTCTAATTAACAAGACACATAAGCAGCAGTTTAATAAACTCATTACTAGCCAGGC
TGTGCATGTTACAACTCATTCTAAAAATGCTTCACACAGGGTTCCAAGAACAACATCTGCCGTGAAA
TCGAATCAGGAAGATGTTGACAAAGCCAGTTCTTCTAACTCAGCATGCGAGACCGGGTCCGTTTCTG
CGTTGTTTCAGAAGATCAAAGGCATACTCCCTGTTAAAATGGAAAGTGCAGAATGTTTGGAAATGAC
CTATGTTCCCAACATTGATAGGATTAGCCCTGAAAAGAAGGGTGAAAAAGAAAATGGGACATCTATG
GAAAAACAAGAGCTGAAACAAGAGATTATGAATGAGACTTTTGAATATGGTTCTCTGTTTTTGGGCT
CTGCTTCAAAAACAACGACCACCTCAGGTAGGAATATATCCAAGCCTGACTCCTGCGGTTTGAGGCA
AATAGCTGCTCCAAAAGCCAAAGTGGGGCCCCCTGTTTCCTGTTTGAGGCGGAACAGTGACAATAGA
AATCCCAGTGCTGATCGAGCCGTATCTCCTCAGAGGATCAGGCGTGTGTCCAGTTCTGGAAAGCCTA
CATCCTTGAAAACTGCACAGTCGTCATGGGTGAATTTGCCTAGACCACTTCCTAAATCCAAAGCATC
TTTGAAAAGTCCTGCGCTGCGGAGGACAGGAAGCACCCCCTCAATAGCCAGCACCCACAGTGAGCTG
AGCACTTACAGCAACAATTCTGGTAATGCCGCTGTCATCAAATATGAGGAGAAACCTCCAAAACCAG
CATTTCAGAATGGTTCCTCAGGATCCTTTTATTTGAAGCCTTTGGTATCCAGGGCTCATGTTCACTT
GATGAAAACTCCTCCAAAAGGTCCTTCGAGAAAAAATTTATTTACAGCTCTTAATGCAGTTGAAAAG
AGCAGGCAAAAGAATCCTCGAAGCTTATGTATCCAGCCACAGACAGCTCCCGATGCGCTGCCCCCTG
AGAAAACACTTGAATTGACGCAATATAAAACAAAATGTGAAAACCAAAGTGGATTTATCCTGCAGCT
CAAGCAGCTTCTTGCCTGTGGTAATACCAAGTTTGAGGCATTGACAGTTGTGATTCAGCACCTGCTG
TCTGAGCGGGAGGAAGCACTGAAACAACACAAAACCCTATCTCAAGAACTTGTTAACCTCCGGGGAG
AGCTAGTCACTGCTTCAACCACCTGTGAGAAATTAGAAAAAGCCAGGAATGAGTTACAAACAGTGTA
TGAAGCATTCGTCCAGCAGCACCAGGCTGAAAAAACAGAACGAGAGAATCGGCTTAAAGAGTTTTAC
ACCAGGGAGTATGAAAAGCTTCGGGACACTTACATTGAAGAAGCAGAGAAGTACAAAATGCAATTGC
AAGAGCAGTTTGACAACTTAAATGCTGCGCATGAA.ACCTCTAAGTTGGAAATTGAAGCTAGCCACTC
AGAGAAACTTGAATTGCTAAAGAAGGCCTATGAAGCCTCCCTTTCAGAAATTAAGAAAGGCCATGAA
ATAGAAAAGAAATCGCTTGAAGATTTACTTTCTGAGAAGCAGGAATCGCTAGAGAAGCAAATCAATG
ATCTGAAGAGTGAAAATGATGCTTTAAATGAAAAATTGAAATCAGAAGAACAAAAAAGAAGAGCAAG
AGAAAAAGCAAATTTGAAAAATCCTCAGATCATGTATCTAGAACAGGAGTTAGAAAGCCTGAAAGCT
GTGTTAGAGATCAAGAATGAGAAACTGCATCAACAGGACATCAAGTTAATGAAAATGGAGAAACTGG
TGGACAACAACACAGCATTGGTTGACAAATTGAAGCGTTTCCAGCAGGAGAATGAAGAATTGAAAGC
TCGGATGGACAAGCACATGGCAATCTCAAGGCAGCTTTCCACGGAGCAGGCTGTTCTGCAAGAGTCG
CTGGAGAAGGAGTCGAAAGTCAACAAGCGACTCTCTATGGAAAACGAGGAGCTTCTGTGGAAACTGC
ACAATGGGGACCTGTGTAGCCCCAAGAGATCCCCCACATCCTCCGCCATCCCTTTGCAGTCACCAAG
GAATTCGGGCTCCTTCCCTAGCCCCAGCATTTCACCCAGATGACACCTCCCCAAA
Variant sequences of NOVB are included in Example 3, Table 20. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV8 protein (SEQ m N0:16) encoded by SEQ m NO:15 is 1259 amino acid residues in length and is presented using the one-letter amino acid code in Table 8B. Psort analysis predicts the NOV8 protein of the invention to be localized in the nucleus with a certainty of 0.7600.
Table 8B. Encoded NOV8 protein sequence (SEQ ID N0:16) MNCKPSLPVIKMETTHAYNPKSPPTQNSSASSVNWNSANPDDMVVDYETDPAWTGENISLSLQG
VEVFGHEKSSSDFTSKQVLDMHKDSICQCPALVGTEKPKYLQHSCHSLEAVEGQSVEPSLPFWK
PNDNLNCAGYCDALELNQTFDMTVDKVNCTFISHHAIGKSQSFHTAGSLPPTGRRSGSTSSLSYS
TWTSSHSDKTHARETTYDRESFENPQVTPSEAQDMTYTAFSDWMQSEVFVSDIGNQCACSSGKV
TSEYTDGSQQRLVGEKETQALTPVSDGMEVPNDSALQEFFCLSHDESNSEPHSQSSYRHKEMGQN
LRETVSYCLIDDECPLMVPAFDKSEAQVLNPEHKVTETEDTQMVSKGKDLGTQNHTSELILSSPP
GQKVGSSFGLTWDANDMVISTDKTMCMSTPVLEPTKVTFSVSPIEATEKCKKVEKGNRGLKNIPD
SKEAPVNLCKPSLGKSTIKTNTPIGCKVRKTEIISYPRPNFKNVKAKVMSRAVLQPKDAALSKVT
PRPQQTSASSPSSVNSRQQTVLSRTPRSDLNADKKAEILINKTHKQQFNKLITSQAVHVTTHSKN
ASHRVPRTTSAVKSNQEDVDKASSSNSACETGSVSALFQKIKGILPVKMESAECLEMTYVPNIDR
ISPEKKGEKENGTSMEKQELKQEIMNETFEYGSLFLGSASKTTTTSGRNISKPDSCGLRQIAAPK
AKVGPPVSCLRRNSDNRNPSADRAVSPQRIRRVSSSGKPTSLKTAQSSWVNLPRPLPKSKASLKS
PALRRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPAFQNGSSGSFYLKPLVSRAHVHLM
KTPPKGPSRKNLFTALNAVEKSRQKNPRSLCIQPQTAPDALPPEKTLELTQYKTKCENQSGFILQ
LKQLLACGNTKFEALTWIQHLLSEREEALKQHKTLSQELVNLRGELVTASTTCEKLEKARNELQ
TVYEAFVQQHQAEKTERENRLKEFYTREYEKLRDTYIEEAEKYKMQLQEQFDNLNAAHETSKLEI
EASHSEKLELLKKAYEASLSEIKKGHEIEKKSLEDLLSEKQESLEKQINDLKSENDALNEKLKSE
EQKRRAREKANLKNPQIMYLEQELESLKAVLEIKNEKLHQQDIKLMKMEKLVDNNTALVDKLKRF
QQENEELKARMDKHMAISRQLSTEQAVLQESLEKESKVNKRLSMENEELLWKLHNGDLCSPKRSP
TSSAIPLQSPRNSGSFPSPSISPR
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 8C.
Table 8C. results for Patp NOV8 Smallest Sum eading igh Prob equences Pairs: Frame ScoreP(N) producing High-scoring Segment >patp:AAG63542Aminoacidsequence human ATIP +1 6389 0.0 of a isoform >patp:AAG63529Aminoacidsequence human ATIP +1 6233 0.0 of a isoform >patp:AAG63541Aminoacidsequence human ATIP +1 3928 0.0 of a isoform >patp:AAG63537Aminoacidsequence ATIP isoform +l 3279 0.0 of a >patp:AAG63530Aminoacidsequence human ATIP +1 2954 1.5e-307 of a isoform In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 3751 of 3751 bases (100%) identical to a gb:GENBANI~-m:AB033114~acc:AB033114.1 mRNA from Homo Sapiens (mRNA for KIAA1288 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 1245 of 1245 amino acid residues (100%) identical to, and 1245 of 1245 amino acid residues (100%) similar to, the 1245 amino acid residue ptnr:SPTREMBL-ACC:Q9IJLD2 protein from Horno Sapiens (I~IAA1288 PROTEIN).
NOV8 also has homology to the proteins shown in the BLASTP data in Table 8D.
Table 8D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi163314071dbjIBAABKIAA1288 protein1245 1245/12451245/12450.0 6602.11(AB033114)[Homo Sapiens] (100%) (100%) gi1178656321refINPAT2 receptor- 436 404/436 409/436 0.0 065800.11(NM interacting (92%) (93%) protein 1 [Homo Sapiens]
gi1204367221dbjIBABunnamed protein240 239/240 239/240 e-107 14894.11 (AK024357)product (99%) (99%) [Homo Sapiens]
gi138822691dbjIBAA3KIAA0774 protein1163 135/366 224/366 9e-49 4494.11(AB018317)[Homo Sapiens] (36%) (60%) gi1174756301refIXPKIAA0774 protein901 135/366 224/366 3e-48 ' [Homo Sapiens] (36%) (60%) 029364.31 (XM 029364) A multiple sequence alignment is given in Table 8E, with the NOV8 protein being shown on line 1 in Table 8E in a ClustalW analysis, and comparing the NOVB
protein with the related protein sequences shown in Table 8D. This BLASTP data is displayed graphically in the ClustalW in Table 8E.
Table 8E. ClustalW Analysis of NOVB
1) > NOVB; SEQ m N0:16 2) > gi~6331407~/ KIAA1288 protein [Homo Sapiens]; SEQ ID N0:67 3) > gi~17865632~/ AT2 receptor-interacting protein 1 [Homo Sapiens]; SEQ m NO:68 4) > gi~10436722~/ unnamed protein product [Homo Sapiens]; SEQ m N0:69 5) > gi~3$82269~/ I~IAA0774 protein [Homo Sapiens]; SEQ ID N0:70 6) > gig 17475630/ KIAA0774 protein [Homo Sapiens]; SEQ m N0:71 ....1....1....1....1....1....1....1....1....1....1....1....1 gi163314071 1 ---------------THAYNPKSPPTQNSSASSVNWNSANPDDMWDYETDPAWTG--- 42 gi1178656321 1 ____________________________________________________________ 1 gi1104367221 1 ____________________________________________________________ 1 gi138822691 1 RGQIPGGGEGPQKTLPDHAVPAAFPATDSTSEGKSVRHPKPSTSESKQSTPSETQTVGAH 60 gi1174756301 1 ____________________________________________________________ 1 ....I....I....I....I....I....I....I....I....I....I....I....I
S gi I63314071 42 ---~---ENISLSLQGVEVFGHEKSSSDFISKQVLDMHKDSICQCPALVGTEKPKYLQHSC
gi ~17865632~ 1 _____________________________-______________________________ 1 gi ~10436722~ 1 ____________________________________________________________ gi 13882269~ 61 VLQVCSEHTSHSAHPEPALNLTLASKEIPSKLEAQLGQGKGEAKLDLKYVPPRRVEQEGK
gi ~17475630~ 1 ____________________________________________________________ gi I63314071 97 HSLEAVEGQSVEPSLPFVWKPNDN----LNCAGYCDALELNQTFDMTVDKVNCTFISHHA
15gi 1178656321 1 ____________________________________________________________ gi ~104367221 1 ____________________________________________________________ gi ~3882269~ 121AAQEGYLGCHKEENLSALEGRDPCGEAHPEATDALGHLLNSDLHHLGVGRGNCEEKRGVN
gi I174756301 1 ____________________________________________________________ gi I6331407~ 153IGKSQSFHT---AGSLPPTGRRSGSTSSLSYSTWTSSHSDKTHARETTYDRESFENPQVT
gi ~17865632~ 1 ____________________________________________________________ gi I104367221 1 ____________________________________________________________ gi ~3882269~ 181PGEQDSLHTTPKQGSASLGGADNQPTGKISPCAGEKLGERTSSSFSPGDSHVAFIPNNLT
gi ~174756301 1 ____________________________________________________________ ..
NOV8 224PSEAQDMTYTAFSDVVMQSEVFV'SD'~~ QCACSGKVTSYT~GSQ
~ B ~ LVGEETAL
B
B
giI 6331407~ 210PSEAQDMTYTAFSDVVMQSEVFSD~G QCACSGKVTSYTGSQ
Q
gi~ 17865632~ 1 _________________________.__________________________._______ gi~ 10436722~ 1 ___________________ __ ____ ____________________ ____ 3Sgi1 38822691 241DSKPLDVIEEERRLGSGNKDSVVL~?F PSVGE PL
SEAR~SKVT S 300 KTEVP PQS
gi 174756301 1 ~ ~ ~ ~ S 38 ~ ------------------- ~ PL~pQS ~
VLV'F,,~~,, PSVGE SEARSK~'VT
~KTEVP
g 284~SDGMEU'PN~7~AL Q~FFCLS~
ESNE~HSQSSj~'RHKEGQNL'RE~SYCLID ~ECPL~ 343 i 270SIaGMEU'PN~:?~AL Q ESN HSQSS'Y'RHKE ~ECPL
I SYCLID
i ' __._______.______ gi~ 17865632~ 1 ___________________________________________ i 10436722 1 ____________________________________________________________ B
gi 3882269 301~~.''NRN~~.~iLENADK I~STSAR SVL~TI~--APLPETTiVNMTYP~T
PSSSFQ~S===~ 355 I I ~ ~ s i 1747 I 5 39 NRN I SVLI --APLPETTNMTY PSSSFQ~ 93 g 1 LEN~K STS P T S
~
NOV8 344PAF~KSEAQ~ EHKVTETE QMVSKLGTQNHTSEILS PPGQKVGSSFGLTWD
.. C'~
S0gi~ 63314071 330PAF~KSEAQ~~ EHKVTETE ~QMVSKLGTQNHTSEILS
~PPGQKVGSSFGLTWD 389 gi~ 17865632~ 1 _____________________'_______________________________________ giI 10436722~ 1 ____________________________________________________________ gi1 3882269~ 356FG PTDSARLL SPKVPD KDTPSSQEGMENYQV
~ TCPSGIPKP'T;FTH
GSPL
P
g1~ 17475630~ 94 FGGSPL~P~ PTDSARLL ~SPKVPD~TCPSGIPKP~FTH
~KDTPSSQEGMENYQV 153 SS
NOVS 404ANDMVITDITjNjC 463 I LEPT
i TFSSPIEATEKCKKVEKGN
KNIDKE
L
T
~
A
~
EP~~
~
~
~
~
~
g NDMVITD T S DK 449 I !ICITFSSPIEATEKCKKVEKGNR L
VLEPT
KNI
gi~ 17865632~ 1 ____________________________________________________._______ gi~ 10436722~ 1 __________________________________________________________ gi 3882269 416EKTEER4LI~!:ET ~PI'IP KHVRPIITYRRNPQALGQVDASLVP
~ I ~ ~ PY
B P~''i'CT
~PH
~
~
~
~
gi 17475630 154EKTEERET P~'IPIKHVRP~PH 213 ~ ~ I<ITY~C!RRNPQALGQVDASLVPV
PYA
P~CTl ~
.~....~...
.~..
.~.
_ giI 6331407~ 450C'~P~LKSTIK'hNTPIGCKVRKTIISYPRPNFKNV
PICD Si P 509 SRAVL~
gi~ 178656321 1 _____ 1 _________________~_____________ _____ __'_ _ _ 70gi~ 104367221 1 ____________________________________________________________ gi~174756301 214 EK~GDLKP~ANLYEKFKPDLQKPRVFSSGLMVSGI~PPGHPFS~ S~KF~Q~DH~ 273 .
NOV8 524 Qi~TSAS SSVI~SRQQTVLS AD~CKAEIL'INTCTHKQQFN
gi 510 Q~TSAS ~SSVSRQQTVLS T ~ 569 ~ VHV'~TH,,H., 6331407 'TPR3S~'S'iD
~ ~TPRD~TAD~KAEIL2NT~THKQFNF~T~VHV~~TH
gi~1786563211 ___________'.'________,_________._____________________________ gi ~10436722~1 _ 1 _____ _____________________ _____ __ _________ __ _ IQ gi ~3882269i536 GEEFC PYAYEVPPTF
E --S
LKP~LGLGA~tRLPSA'ECSRT
~
RSAS
MS
~
YB
I
~
~
~
gi~17475630~274 G QLGLGA 329 EFC RLPSATtSRT
A
~
-RSAS
PYA
YEVPPTF
--S
LKP
, I NOV8 584 KIt~ASHRrV~~PRT
S gi~6331407~ 570 . 628 V
QE
~
ShCETGS
.
FQK-IKG~I'LP
E'AEC
EM
ICASHRPRT~AVQEy~
~S~CETGSV~A
FQK-IKGLPV'~EAECEM
gi~1786563211 _=__________________________________________________________ gi~10436722~1 _.___________________________________-______________________ ~ ~ ~
~
~
gi 17475630330 I LYSPS 389 ~ I PPGPTTA y KSNLPI
G
RPPGYSR
P
FGFRS
~S gi~63314071 629 Y~P~T~DRISPEKKG~ITSME~QELKQ
BIMNETFEYGS~ FLGS T~TTSGRNK 688 gi~1786563211 _______________.________.___________________________________ gi~10436722~1 ____________________________________________________________ gi 3882269 652 S~S~Cl'SSTQSGDSAP~ Q~RPATSTFG~EQ--====P~
KASLP~D~PKGAGR'V.A!P 705 I ~ ' ~
' ' gi 17475630390 ,~S Q RPAT~ t~. 443 ~ I SF , KASLP
~SSTQSGDSA~P ,STFG tD
EQ-- PKGAGR
P P
NOV8 703 ~ D~CGLRQI~P~K~GP~CLRRNSDNRNPSADRAV~
~QRI~~SSG ~ 74 6331407 689 D ~
i GLR L
A
I ' ' LRR
DN
S
A
V
#~
g QI KT
( ~ ~
I GP QRI
NS
RNP
ADR
C
P~
C
V
35 gi~17865632~ 1 -------MLLSPFS--------------------LTTHI~LTAKGLLRLR----giI10436722~ 1 ____________._________________________.______________.______ gi 706 S ------------ S 748 ~ -SVTiIPR~;.SLT.iP ~ TSiI ~
3882269 ~ ~ GTR
~ ~ T~PKDD
KPAV
~
~
~~
gi 444 S--SVT 'I-------------- S 486 ~ PR~SLPA TS
17475630 QKD~QD
~ KPAV
GT
Sl0 NOV8 763 SWVNLP ~PI~P~KAS ~
~R~t~,TGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 822 gi~63314071 749 SWVNLP~ P~.iP~KAS ' RtRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 808 gi~178656321 26 ____Lp____________SGFg,~'-_______ST___________________________ gi~10436722~ 1 ______________________=~_____________________________________ gi~3882269~ 748 ___ H GYP_-___________________________________ p ~
TT
gi~174756301 486 ____p~_TTI~ I-I~GYP_-___________________________________ 504 'QKN P'LCTQ'Q TA882 giI6331407~ 809 FQNGSSGSFYLKPLVSRAHVHLMKTPPKGPSRKNLFTALNAVEKS
'QKN P' TA868 LCIQ'~
gi~178656321 35 ____________________________________wFHTVEKS
'QKN P' TA59 LCIQ' giI10436722~ 1 _______________________________________-_______________-____ 1 gi~3882269~ 766 __,________________________________________ T ~PD781 Q -GFP
I
gi~17475630~ 504 ___________________ ~TF~~_GFP~I ~PD519 ______________________Q
gi~6331407~ 869 928 gi~17865632~ 60 gi~104367221 1 _______ 1 ____ _-__ _ _______________________ _____ gi~38822691 782 Q~1RE Q R E QyRQ~GVAGE KRAICT ~FF S
B ~ L ~ ~ ~ ~~ ~~
~ ~ ~
~
gi 520 Q Q V R E Q KRAICGA~AT FF S 578 ( RE L T'RQ7~ {R~
17475630 GVA~GE LV I~
I
70 gi~63314071 929 HST Q~ .' 3.L~VTATT~. E ~~ .'TV . F~~____~H~, KT. .'~ 984 gi I 17865632120 H~yT~Q~~GQI;VTATT~ETV~AFV~----~W
I KT~R~ICE 175 gi~10436722~1 _______ _-__ ___ _______ ________________________ __-__ 1 gi ~ 17475630579 E~E~I~~'~D~VAFHAIQ~EE~~RR~~DE~ L E' QL
~ I~RLGGV~Q~ LQ 638 ~
NOV8 999 F~';T E T~ II~~AEK v ~ ... , 1058 ' v v giI63314071985 F'~T YE 1~TIEAEKY~ ~ ~ '' 1044 ~
gi I 17865632176 F~'~T YE~i~T.I~~I~AEKY~ ~ 235 ~ ~ ~
gi~10436722~1 __________=_'~_______v ~ .. . . 39 v ~
gi ~ 3882269901 Q E QE G t~LLSI~;C~H~~ QDD~DTiK
~ ~ S .7AL TVA'~T~ 960 ~ ~ ,f"D T
gi ~ 17475630639 QLLSIC~ ~ r , QDD~iI7HK
f Qy~ , 698 "',E~Q~E~G vV,Ep T~S AL r a . ~VAT~
gi~6331407~1045 1202 gi~17865632~236 293 gi~10436722~40 97 gi~3882269,961 1020 gi~17475630~699 758 gi~6331407~1103 1162 gi1178656321294 353 giI10436722~98 157 gi~3882269~1021 1080 gi~174756301759 818 gi~633140711163 1222 gi~17865632~354 413 gi~10436722~158 217 gi13882269~1081 1140 gi~17475630~819 878 gi~ 633140711223 1245 gi~ 17865632~414 43b gi~ 104367221218 240 gi~ 3882269~1141 1163 gi~ 17475630~879 901 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the 55 Interpro website (http:www.ebi.ac.uk/interpro~. Table 8F lists the domain description from DOMAIN analysis results against NOVB.
Table 8F Domain Anal sis of NOV8 Model Region of Score (bits) E value Homology RNA polymerase1008-1094 -12.8 5.7 omega subunit Intermediate 967-1193 48.9 2.0e-06 filament Consistent with other known members of the Nuclear Protein-like family of proteins, NOV8 has, for example, an RNA polymerase omega subunit signature sequence and homology to other members of the Nuclear Protein-like Protein Family. NOV8 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV8 nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOVB
nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVB activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Hyperparathyroidism, Hypoparathyroidism as well as other diseases, disorders and conditions.
In addition, various NOV8 nucleic acids and polypeptides according to the invention are useful, ihtef° alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV8 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Nuclear Protein-like Protein Family.
The NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac or endocrine physiology. As such, the NOVB nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat infection, cardiovascular system, immune system, and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Hyperparathyroidism, Hypoparathyroidism as well as other diseases, disorders and conditions.
The NOV8 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV8 nucleic acid is expressed in Heart, Aorta, Coronary Artery, Vein, Umbilical Vein, Adrenal GlandlSuprarenal gland, Pancreas, Islets of Langerhans, Parathyroid Gland, Thyroid, Pineal Gland, Tongue, Salivary Glands, Stomach, Liver, Small Intestine, Colon, Ascending Colon, Lymphoid tissue, Spleen, Brain, Thalamus, Hypothalamus, Temporal Lobe, Amygdala, Cerebral Medulla/Cerebral white matter, Basal GangliaJCerebral nuclei, Substantia Nigra, Hippocampus, Spinal Chord, Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Prostate, Testis, Lung, Nasoepithelium, Larynx, Urinary Bladder, Kidney, Kidney Cortex, Retina, Skin, Foreskin, Epidermis, Dermis.
Additional utilities for NOV8 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV9 polypeptide has been identified as a Hemicentin precursor-like protein (also referred to as CG94702-OI). The disclosed novel NOV9 nucleic acid (SEQ ID
N0:17) of 11796 nucleotides is shown in Table 9A. The novel NOV9 nucleic acid sequences maps to the chromosome 9.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TAA
codon at nucleotides 11794-11796. A putative untranslated region and/or downstream from the termination codon is underlined in Table 9A, and the start and stop codons are in bold letters.
Table 9A. NOV9 Nucleotide Sequence (SEQ ID N0:17) ATGTCTGCCTTATTTGCAGCTGTGTACCAGATGCTAAA.ACCACGCCTGGTCCATAACAGCCCACATC
CGGTGACCTATCAAATTGAGGCAAGTTTAAAGCCAGAGCAGCCTGGTGTCACGCTGGTGTCCATCCC
AGTCTTCCTGGCACCTTCCTGGCACAAAGCCTCAGAGCTGATCCCGACCCAGTCCTTCCGAGCACAG
GGGGCAGGGAAGCAGCTCCTCGGCTCTCCTTGCCCCCAAGTGCCCCCCAGCATCCGGGAGGACGGGC
GCAAGGCCAACGTGTCGGGTATGGCCGGGCAGTCCCTGACGCTGGAGTGTGACGCGAACGGCTTTCC
AGTCCCTGAGATCGTGTGGCTGAAGGACGCGCAGCTGATTCCTAAGGTGGGCGGCCACCGCCTCCTG
GACGAGGGCCAGTCCCTCCACTTCCCCAGGATCCAGGAGGGTGATTCTGGGCTCTACTCCTGCCGGG
CAGAGAACCAGGCTGGCACCGCCCAGAGGGACTTCCATCTCCTTGTGCTCACCCCTCCTTCCGTGCT
TGGAGCCGGGGCCGCTCAGGAGGTGCTAGGATTGGCCGGTGCAGACGTGGAGCTGCAGTGTTGGACC
TCAGGGGTCCCCACGCCCCAGGTGGAGTGGACCAAGGACAGGCAGCCTGTCCTTCCGGGAGGCCCTC
ACCTGCAGGTCCAGGAGGATGGCCAGGTTCTCAGGATCACCGGCAGTCACGTGGGGGATGAGGGACG
ATACCAGTGCGTGGCCTTCAGCCCAGCTGGTCAGCAGGCCAGGGACTTCCAGCTCCGAGTTCATGCG
CCCCCCACTATCTGGGGCTCCAACGAGACAGGCGAGGTGGCCGTCATGGAGGACCACCTAGTGCAGC
TCCTGTGTGAGGCTCGAGGAGTGCCCACCCCAAACATCACCTGGTTCAAGGACGGGGCCCTGCTCCC
CACCAGCACCAAGGTGGTCTACACTAGGGGCGGTCGGCAGTTGCAGCTGGGGAGGGCCCAGAGCTCC
GATGCCGGCGTCTACACCTGCAAGGCCAGCAATGCTGTGGGGGCCGCAGAGAAGGCCACCAGGCTGG
ATGTTTATGTCCCACCTACCATCGAGGGCGCCGGTGGAAGACCATACGTGGTGAAGGCTGTGGCTGG
GAGGCCTGTGGCGCTGGAGTGCGTGGCCAGAGGCCACCCGTCCCCCACCCTCTCCTGGCACCACGAG
GGGCTGCCCGTGGCAGAGAGCAACGAGTCGCGGCTGGAGACAGACGGGAGTGTGCTGAGGCTGGAGA
GCCCGGGGGAGGCATCCAGTGGCCTGTACAGCTGTGTGGCCAGCAGTCCTGCCGGGGAAGCCGTCCT
GCAGTACTCCGTGGAGGTTCAGGTGCCCCCACAGCTCCTGGTGGCTGAAGGCTTGGGACAGGTGACC
ACCATCGTGGGACAGCCCCTGGAACTTCCCTGCCAGGCCTCAGGCTCCCCAGTACCCACTATCCAGT
GGCTGCAGAATGGCCGCCCAGCCGAGGAGCTGGCTGGGGTGCAGGTGGCCTCGCAGGGGACCACACT
GCACATTGACCATGTGGAGCTGGACCACTCAGGCCTCTTCGCCTGCCAGGCCACCAATGAGGCGGGC
ACTGCCGGGGCCGAGGTGGAGGTGTCTGTGCATGAGTTCCCATCGGTCAGTATCATTGGGGGTGAGA
ACATCACAGCTCCTTTCCTGCAGCCTGTGACCCTCCAGTGCATAGGGGATGGGGTGCCCACCCCAAG
CCTCCGTTGGTGGAAGGATGGTGTAGCCCTGGCAGCCTTTGGGGGGAACCTACAGATTGAGAAGGTG
GACCTGAGGGACGAGGGCATCTACACTTGTGCTGCTACCAACCTGGCTGGGGAGAGCAAGAGGGAAG
TGGCGCTGAAAGTTTTGGTGCCCCCCAACATCGAGCCAGGCCCAGTCAACAAGGCAGTGCTGGAAAA
TGCCTCAGTGACCTTGGAGTGTCTGGCTTCGGGCGTGCCCCCTCCTGATGTCTCCTGGTTCAAGGGC
CACCAACCTGTCTCTTCATGGATGGGAGTGACAGTATCAGTGGATGGGAGAGTTCTCCGCATTGAGC
AAGCCCAGCTTTCTGATGCTGGGAGCTACCGCTGTGTGGCATCCAATGTGGCAGGTAGCACAGAGCT
GCGGTATGGCCTACGGGTCAATGTGCCCCCTCGAATCACACTGCCACCCAGCCTGCCAGGCCCTGTG
TTGGTCAACACCCCTGTCCGGCTGACCTGCAATGCCACCGGTGCCCCCAGCCCCACACTGATGTGGC
TGAAGGATGGAAACCCTGTGTCCCCTGCAGGGACCCCTGGCCTGCAGGTCTTCCCTGGGGGCCGGGT
CCTCACCTTGGCTAGTGCCCGGGCCTCCGACTCTGGGAGGTACTCCTGCGTGGCTGTGAGCGCGGTG
GGCGAGGACCGCCAGGATGTTGTCCTGCAAGTCCACATGCCCCCGAGTATCCTTGGAGAAGAGCTGA
ATGTGTCCGTTGTGGCCAATGAGTCAGTGGCCCTGGAGTGCCAGAGCCACGCCATGCCCCCTCCTGT
GCTGAGCTGGTGGAAGGACGGGCGGCCCCTGGAACCACGGCCTGGAGTCCACCTCTCCGCAGACAAA
GCCTTGCTGCAGGTGGACAGAGCCGATGTGTGGGATGCGGGCCATTACACCTGTGAGGCACTGAACC
AGGCCGGCCACTCAGAGAAACACTACAATCTGAACGTCTGGGGTCAACCCCTCCCCGGGGAGGGGGC
TGGCCTCCAGCACGTGTCGGCTGTGGGGAGGCTGTTGTACCTGGGACAGGCCCAGCTGGCTCAGGAA
GGAACATACACCTGTGAATGCAGCAACGTGGTGGGGAACAGCAGCCAGGACCTGCAGCTGGAGGTGC
ACGTTCCCCCTCAGATTGCCGGTCCCCGGGAGCCTCCCACACAAGTCTCTGTGGTCCAGGATGGAGT
GGCCACTCTGGAGTGCAACGCCACAGGGAAACCCCCTCCGACAGTGACATGGGAGCGGGACGGCCAG
CCCGTGGGGGCTGAACTGGGCCTGCAGCTGCAGAACCAGGGTCAGAGCCTGCATGTGGAGCGGGCCC
AGGCTGCCCACACTGGACGCTACAGCTGTGTGGCCGAGAACCTGGCTGGGAGGGCAGAGAGGAAGTT
TGAGCTCTCCGTACTGGTGCCCCCAGAGCTCATTGGAGACTTGGACCCGCTGACCAACATCACTGCT
GCCTTGCACAGCCCCTTAACTCTGCTCTGTGAAGCCATGGGGATCCCACCTCCAGCCATCCGCTGGT
TCCGAGGGGAGGAGCCTGTCAGCCCCGGGGAGGACACCTACCTGCTGGCAGGTGGCTGGATGCTGAA
GATGACTCAGACACAGGAGCAAGACAGTGGCCTCTACTCATGCCTGGCAAGCAACGAGGCTGGGGAG
GCACGGAGGAACTTCAGTGTGGAGGTGCTGGTTCCTCCCAGTATTGAGAACGAGGACTTGGAGGAGG
TGATCAAGGTCCTTGATGGACAGACTGCCCATCTTATGTGCAACGTCACAGGCCACCCACAGCCCAA
GCTCACATGGTTCAAAGATGGCCGGCCTCTGGCTAGGGGAGATGCTCACCACATCTCCCCAGACGGA
GTCCTCCTGCAGGTCCTCCAGGCAAACCTGTCCAGTGCTGGCCACTACTCCTGCATTGCAGCCAACG
CTGTTGGGGAGAAGACCAAACACTTCCAGCTCAGTGTCCTGTTGGCTCCCACCATCCTGGGAGGGGC
CGAGGACAGTGCAGATGAGGAGGTGACCGTGACTGTCAACAACCCCATCTCTCTGATCTGCGAGGCC
CTGGCCTTCCCTTCCCCCAACATCACCTGGATGAAGGACGGGGCCCCGTTTGAGGCCTCCAGGAACA
TCCAGCTGCTCCCAGGTACCCACGGGCTGCAGATCCTGAATGCCCAGAAGGAAGATGCTGGCCAGTA
CACCTGCGTGGTCACCAATGAGCTCGGGGAGGCCGTGAAAAACTACCATGTGGAAGTGCTCATCCCC
CCTTCCATCTCCAAAGACGACCCCTTGGCGGAGGTCGGCGTGAAGGAGGTGAAGACCAAGGTCAACA
GCACCTTGACCTTGGAGTGTGAGAGCTGGGCTGTGCCCCCGCCCACCATCCGCTGGTACAAGGATGG
ACAGCCCGTGACCCCCAGCTCGCGGCTGCAGGTCCTGGGTGAAGGGCGACTGCTCCAGATCCAGCCC
ACACAGGTCTCAGACTCGGGGCGGTACCTGTGTGTGGCCACCAATGTGGCTGGCGAGGACGACCAGG
ACTTCAACGTGCTCATCCAGGTGCCCCCCATGTTCCAGAAGGTGGGTGATTTCAGTGCAGCCTTCGA
GATCCTGTCCCGGGAGGAGGAGGCCCGGGGCGGAGTCACGGAATACAGGGAGATCGTGGAGAACAAC
CCAGCCTACCTGTACTGCGACACCAACGCGATCCCACCCCCGGACCTCACCTGGTACAGAGAGGATC
AGCCCCTCTCGGCCGGGGATGAGGTGTCTGTGCTGCAAGGAGGCCGGGTCCTGCAGATCCCCCTGGT
GCGGGCAGAGAACGCCGGGAGGTACTCGTGCAAGGCCTCCAACGAGGTGGGCGAGGACTGGCTGCAC
TACGAGCTGCTGGTGCTGACCCCACCTGTGATCCTGGGTGACACAGAGGAGCTGGTGGAAGAGGTGA
CAGTCAATGCCAGCAGCACCGTCAGCCTGCAGTGCCCGGCCCTGGGAAACCCCGTGCCCACCATCTC
ATGGCTCCAGAATGGGCTGCCTTTCTCCCCGAGCCCACGGCTGCAGGTCCTGGAGGACGGGCAAGTC
TTGCAGGTTTCCACGGCAGAGGTGGCCGACGCCGCCAGCTACATGTGTGTGGCCGAGAACCAGGCGG
GCTCCGCTGAGAAGCTCTTCACCCTCAGGGTTCAAGGCCTGGACTTGGAGCAGGTCACTGCCATCCT
CAACAGCAGCGTCTCCCTCCCTTGCGACGTCCACGCTCACCCAAACCCCGAGGTCACGTGGTACAAG
GACAGCCAGGCCCTCTCCCTGGGTGAAGAGGTCTTCCTCCTGCCTGGCACCCACACGCTGCAGCTGG
GGAGAGCACGGCTGTCGGACTCCGGGATGTACACATGCGAAGCCCTCAATGCTGCCGGCCGAGACCA
GAAGCTGGTGCAGCTCAGTGTTCTGGTTCCCCCGGCCTTCAGGCAGGCTCCCAGAGGTCCCCAGGAT
GCGGTCCTGGTGAGGGTCGGGGACAAAGCTGTCCTGAGCTGCGAGACAGATGCGCTCCCTGAGCCAA
CTGTGACCTGGTACAAGGATGGGCAGCCCCTGGTCCTGGCACAGCGGACCCAGGCTCTGCGGGGTGG
GCAGAGGCTGGAGATCCAGGAAGCCCAGGTATCGGATAAAGGTTTATACAGCTGTAAAGTCAGCAAC
GTGGCTGGGGAGGCCGTGCGGACCTTCACCCTCACCGTCCAGGTGCCCCCAACATTTGAGAACCCCA
AGACAGAGACAGTGAGCCAGGTGGCTGGGAGCCCCCTGGTCCTGACCTGTGATGTGTCCGGGGTCCC
TGCACCCACGGTCACTTGGCTGAAGGACAGGATGCCTGTGGAGAGCAGCGCGGTGCACGGTGTGGTC
TCCCGGGGGGGCCGCCTCCAGCTGAGCCGCCTGCAACCGGCCCAGGCGGGCACCTACACGTGCGTGG
CTGAGAACACCCAGGCTGAGGCCCGCAAGGACTTCGTGGTAGCAGTGCTGGTGGCCCCCCGGATCCG
GAGCTCGGGCGTGGCGCGGGAGCACCATGTCTTGGAAGGGCAGGAGGTGCGGCTGGACTGTGAGGCC
GATGGGCAGCCGCCGCCGGACGTGGCCTGGCTGAAGGACGGCAGCCCGCTGGGCCAGGACATGGGCC
CCCACCTCCGGTTCTACCTGGACGGCGGCTCCCTGGTGCTAAAAGGCCTGAGGGCCTCGGACGCGGG
TGCCTACACCTGCGTGGCCCACAACCCAGCCGGGGAGGACGCCAGGCTGCACACGGTGAATGTGCTG
GTTCCTCCCACCATCAAGCAGGGAGCAGACGGCTCGGGGACCCTGGTGAGCAGGCCTGGGGAGCTGG
TGACCATGGTGTGCCCTGTGCGGGGCTCCCCGCCCATCCACGTGAGCTGGCTCAAGGACGGCCTGCC
CCTCCCGCTCTCCCAGCGCACCCTCCTCCACGGCTCTGGCCACACCCTCAGGATTTCCAAGGTGCAA
TTGGCAGACGCTGGCATCTTCACCTGTGTGGCCGCAAGCCCAGCTGGCGTGGCGGACAGGAACTTCA
CCTTGCAGGTGCAGGTGCCCCCTGTCCTGGAGCCGGTGGAGTTCCAGAATGACGTGGTGGTGGTTCG
TGGCTCCCTGGTGGAACTCCCGTGCGAGGCCCGGGGCGTTCCCCTGCCTCTCGTGTCGTGGATGAAG
GATGGGGAACCCTTGTTGTCCCAGAGCCTCGAGCAGGGGCCCAGCCTGCAGCTGGAGGCAGTGGGAG
CTGGTGACTCGGGGACCTACTCCTGTGTGGCCGTGAGCGAGGCGGGGGAAGCCAGGAGGCATTTCCA
GCTGACCGTCATGGAGCCCCCTCACATTGAGGACTCAGGCCAGCCTACAGAGCTGTCGCTGACCCCC
GGCGCCCCCATGGAGCTCCTCTGTGATGCCCAGGGCACCCCCCAGCCCAACATCACCTGGCATAAGG
ACGGGCAGGCCCTGACCAGGCTGGAGAACAACAGCAGAGCCACACGGGTGCTCCGGGTGGAGAATGT
GCAGGTTAGGGATGCTGGGCTGTACACTTGTCTGGCTGAAAGCCCTGCAGGTGCAATTGAGAAGAGC
TTCCGGGTCAGGGTTCAAGCCCCTCCAAACATTGTTGGGCCCCGAGGCCCCCGCTTTGTGGTCGGCC
TGGCCCCAGGGCAGCTGGTCCTGGAGTGTTCGGTGGAGGCAGAGCCAGCGCCCAAGATCACGTGGCA
CCGAGACGGCATTGTGCTGCAGGAGGACGCCCACACACAATTCCCGGAGCGGGGCAGGTTCCTCCAG
CTGCAGGCCCTGAGCACGGCTGACAGCGGCGACTACAGCTGCACAGCCCGCAACGCCGCAGGCAGCA
CTAGTGTCGCCTTCCGCGTGGAGATCCACACGGTGCCCACCATCCGGTCAGGACCACCTGCAGTGAA
CGTCTCAGTGAACCAGACAGCCCTGCTGCCTTGCCAGGCCGACGGCGTGCCCGCACCCCTCGTGAGC
TGGCGGAAGGACAGGGTCCCCCTGGATCCCAGGAGCCCCAGGGCAACCCCCATCCATTCTAGGTTTG
AAATTCTGCCTGAGGGTTCCCTGAGAATCCAGCCAGTCCTTGCCCAGGACGCCGGCCACTACCTCTG
CCTGGCATCCAACTCTGCTGGCTCCGATCGTCAAGGCCGTGACCTACGGGTCTTGGAGCCTCCAGCC
ATCGCCCCCAGCCCCTCCAACCTGACCCTGACCGCCCACACCCCAGCCTTGCTGCCCTGCGAGGCCA
GCGGCTCCCCTAAGCCCCTGGTGGTCTGGTGGAAGGACGGACAGAAGCTGGACTTCCGCCTGCAGCA
GGGCGCCTACCGGCTCCTGCCCTCCAACGCCCTGCTCCTCACGGCCCCCGGCCCCCAGGACTCAGCC
CAGTTTGAATGCGTGGTGAGCAATGAGGTGGGCGAGGCCCACAGGCTCTACCAGGTGACCGTCCATG
TGCCTCCCACCATTGCCGATGACCAGACAGACTTCACCGTGACCATGATGGCACCTGTGGTCCTCAC
ATGTCACAGCACGGGTATACCAGCTCCGACCGTGTCCTGGAGCAAGGCAGGCGCCCAGCTAGGAGCT
CGGGGGAGTGGCTATCGTGTCTCACCATCGGGCGCCCTGGAGATCGGGCAGGCCCTCCCCATCCACG
CAGGCCGCTACACCTGCTCAGCCCGCAACTCTGCCGGCGTAGCCCACAAGCACGTCTTCCTCACTGT
GCAAGCCTCCCCGGTGGTGAAGCCGCTGCCCAGCGTGGTTCGGGCAGTGGCAGAGGAGGAGGTGCTG
CTGCCCTGCGAGGCCTCAGGCATCCCCCGGCCGACCATCACCTGGCAGAAGGAAGGGCTCAACGTCG
CTACTGGAGTGAGTACCCAGGTCCTACCAGGCGGACAGCTGCGGATTGCCCATGCCAGCCCAGAGGA
TGCTGGAAACTATCTCTGCATCGCTAAGAACAGTGCGGGCAGTGCCATGGGGAAGACGCGGCTGGTG
GTGCAAGTCCCACCAGTGATCGAGAATGGCCTCCCAGACCTGTCCACCACCGAAGGCTCCCACGCCT
TCTTGCCTTGCAAGGCGAGGGGCAGTCCTGAGCCCAACATCACCTGGGACAAAGATGGCCAGCCTGT
GTCGGGCGCCGAGGGGAAGTTCACCATCCAGCCTTCTGGGGAGTTGCTGGTGAAGAACTTGGAGGGC
CAGGACGCAGGCACCTATACCTGTACCGCTGAGAACGCCGTGGGCCGGGCCCGCCGCCGCGTGCACC
TCACCATCCTGGTACTGCCTGTGTTCACCACCCTGCCTGGGGACCGCAGCCTGCGCCTTGGGGACAG
GCTGTGGCTTCGCTGTGCAGCCCGGGGCAGCCCCACCCCTCGCATTGGCTGGACTGTCAACGACCGG
CCAGTCACAGAAGGGGTGTCTGAGCAGGATGGAGGCAGCACGCTGCAGCGGGCCGCTGTCTCCAGAG
AAGACAGCGGGACCTATGTCTGCTGGGCGGAGAACAGAGTGGGCCGCACGCAGGCGGTCAGCTTCGT
CCACGTGAAGGAGGCTCCTGTCCTACAAGGGGAGGCTTTCTCCTACCTGGTGGAACCTGTAGGAGGC
AGCATTCAGCTAGACTGTGTGGTGCGTGGAGACCCAGTGCCGGACATCCACTGGATCAAAGATGGCC
TTCCACTGCGGGGCAGCCACCTCCGGCACCAGCTGCAGAATGGCTCGCTGACCATCCGCAGGACTGA
GGCAAGGCGGGGCCTGGCACCTTGGAGGGACGATGCGGGACGGTACCAGTGCCTGGCAGAGAATGAG
ATGGGCGTGGCGAAGAAAGTGGTGATCCTCGTCCTGCAGACCAGGATGGTGCCAGCAGAGCCCCACT
TGAAGCGCCAACTCCCACCGATCCCCAGCAATAATGAGGCACCCTCCCTGTTCCCGGGTGTCCATGG
AGGCCACGTGGGGAACCCGGACTTCCACTCTCATCTAGCAGAAGTTCTCGCCGTTCAGTTGCTGGCT
GGGTCCCTGCTCTTCTCAGCCAGGGCCATGCCGCAGGCCAGCACAGCAGCCATTTCCCTTTTGGCTC
CTACCAGTTTTGCCCCTTTTCCTGATGATATTTCTCAGGGCATACTTTCATCCTCTACTGCACATCA
AGGCAGCCCCCAGGGGTGGCAAAAGCTGCTGTTTTTCACAGCCATCCCTAATAAAACCACTGTGATG
GTCACGGTGGAGCCCCAGGACATGACAGTGAGATCTGGGGATGACGTGGCCCTGCGGTGCCAGGCCA
CTGGAGAGCCCACACCCACCATTGAATGGCTACAGGCGGGTCAACCCTTGCGGGCCAGCCGGCGGCT
CCGGACCCTGCCCGATGGGAGCCTGTGGCTGGAGAACGTGGAGACTGGGGATGCAGGCACCTACGAC
TGCGTCGCTCACAACCTCCTGGGCTCTGCCACAGCCCGGGCGTTCCTGGTCTGTGCCAGCCACGCCA
TCGTGGGCTCCCGGCATTTCAGAGACCCACAGGTCTTCTGTGAGTTTGTGGTCCCGCCTCCTCATTT
TACAGGGGAGCCCCAGGGGAGCTGGGGCAGCATGACTGGGGTGATAAATGGCCGGAAATTTGGCGTG
GCCACACTCAACACCAGCGTGATGCAGGAGGCACACTCCGGGGTCAGCAGCATCCACAGCAGCATCC
GCCATGTCCCAGCAAACGTGGGGCCTCTGATGCGGGTGCTCGTGGTCACCATCGCCCCCATCTACTG
GGCCCTGGCCAGAGAGAGTGGGGAAGCCCTGAATGGCCACTCTCTGACTGGGGGCAGGTTCCGGCAG
GAGTCACACGTGGAGTTTGCTACAGGGGAGCTGCTCACGATGACCCAGGTGGCCCGGGGTCTGGATC
CCGATGGCCTCCTGCTCCTCGACGTGGTGGTCAATGGCGTTGTCCCCGAGAGCCTGGCTGACGCAGA
TCTTCAAGTGCAGGACTTTGAGGAGCACTACGTGCAAACAGGGCCTGGCCAGCTGTTCGTGGGCTCC
ACACAGCGCTTCTTCCAGGGCGGCCTCCCCTCGTTCCTACGCTGCAACCACAGCATCCAGTACAACG
CGGCCCGGGGCCCCCAGCCCCAGCTGGTGCAGCACCTGCGGGCCTCAGCTATCAGCTCGGCCTTTGA
TCCAGAGGCCGAGGCCCTGCGCTTCCAGCTCGCTACAGCCCTGCAGGCGGAGGAGAACGAGGTCGGC
TGCCCCGAGGGCTTTGAGCTGGACTCCCAGGGAGCGTTTTGTGTGGACAGGGACGAGTGCTCAGGAG
GCCCTAGCCCCTGCTCCCATGCCTGCCTTAATGCACCCGGCCGCTTCTCCTGCACCTGCCCCACTGG
CTTCGCCCTGGCCTGGGATGACAGGAACTGCAGAGATGTGGACGAGTGTGCGTGGGATGCTCACCTC
TGCCGAGAGGGACAGCGCTGTGTGAACCTGCTCGGGTCCTACCGCTGCCTCCCCGACTGTGGGCCTG
GCTTCCGGGTGGCTGATGGGGCCGGCTGTGAAGATGTGGACGAATGCCTGGAGGGGTTGGACGACTG
TCACTACAACCAGCTCTGCGAGAACACCCCAGGCGGTCACCGCTGCAGCTGCCCCAGGGGTTACCGG
ATGCAGGGCCCCAGCCTGCCCTGCCTAGATGTCAATGAGTGCCTGCAGCTGCCCAAGGCCTGCGCCT
ACCAGTGCCACAACCTCCAGGGCAGCTACCGCTGCCTGTGCCCCCCAGGCCAGACCCTCCTTCGCGA
CGGCAAGGCCTGCACCTCACTGGAGCGGAATGGACAAAATGTGACCACCGTCAGCCACCGAGGCCCT
CTATTGCCCTGGCTGCGGCCCTGGGCCTCGATCCCCGGTACCTCCTACCACGCCTGGGTCTCTCTCC
GTCCGGGTCCCATGGCCCTGAGCAGTGTGGGCCGGGCCTGGTGCCCTCCTGGTTTCATCAGGCAGAA
CGGAGTCTGCACAGACCTTGACGAGTGCCGCGTGAGGAACCTGTGTCAGCACGCCTGCCGCAACACT
GAGGGCAGCTACCAGTGCCTGTGCCCCGCCGGCTACCGTCTGCTCCCCAGCGGGAAGAACTGCCAGG
ACATCAACGAGTGCGAGGAGGAGAGCATCGAGTGTGGACCCGGCCAGATGTGCTTCAACACCCGTGG
CAGCTACCAGTGTGTGGACACACCCTGTCCTGCCACCTACCGGCAGGGCCCCAGCCCTGGGACGTGC
TTCCGGCGCTGCTCGCAGGACTGCGGCACGGGCGGCCCCTCTACGCTGCAGTACCGGCTGCTGCCGC
TGCCCCTGGGCGTGCGCGCCCACCACGACGTGGCCCGCCTCACCGCCTTCTCCGAGGTCGGCGTCCC
CGCCAACCGCACCGAGCTCAGCATGCTGGAGCCCGACCCCCGCAGCCCCTTCGCGCTGCGTCCGCTG
CGCGCGGGCCTTGGCGCGGTCTACACCCGTCGCGCGCTCACCCGCGCCGGCCTCTACCGGCTCACCG
TGCGTGCTGCGGCACCGCGCCACCAAAGCGTCTTCGTCTTGCTCATCGCCGTGTCCCCCTACCCCTA
CTAA
Variant sequences of NOV9 are included in Example 3, Table 21. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV9 protein (SEQ m N0:18) encoded by SEQ 1D N0:17 is 3931 amino acid residues in length and is presented using the one-letter amino acid code in Table 9B. Psort analysis predicts the NOV9 protein of the invention to be localized at the plasma membrane IO with a certainty of 0.7300.
Table 9B. Encoded NOV9 protein sequence (SEQ ID N0:18) MSALFAAVYQMLKPRLVHNSPHPVTYQIEASLKPEQPGVTLVSIPVFLAPSWHKASELIPTQSFR
AQGAGKQLLGSPCPQVPPSIREDGRKANVSGMAGQSLTLECDANGFPVPETVWLKDAQLIPKVGG
HRLLDEGQSLHFPRIQEGDSGLYSCRAENQAGTAQRDFHLLVLTPPSVLGAGAAQEVLGLAGADV
ELQCWTSGVPTPQVEWTKDRQPVLPGGPHLQVQEDGQVLRITGSHVGDEGRYQCVAFSPAGQQAR
DFQLRVHAPPTIWGSNETGEVAVMEDHLVQLLCEARGVPTPNITWFKDGALLPTSTKVVYTRGGR
QLQLGRAQSSDAGVYTCKASNAVGAAEKATRLDVYVPPTIEGAGGRPYWKAVAGRPVALECVAR
GHPSPTLSWHHEGLPVAESNESRLETDGSVLRLESPGEASSGLYSCVASSPAGEAVLQYSVEVQV
PPQLLVAEGLGQVTTIVGQPLELPCQASGSPVPTIQWLQNGRPAEELAGVQVASQGTTLHIDHVE
LDHSGLFACQATNEAGTAGAEVEVSVHEFPSVSIIGGENITAPFLQPVTLQCIGDGVPTPSLRWW
KDGVALAAFGGNLQIEKVDLRDEGIYTCAATNLAGESKREVALKVLVPPNIEPGPVNKAVLENAS
VTLECLASGVPPPDVSWFKGHQPVSSWMGVTVSVDGRVLRIEQAQLSDAGSYRCVASNVAGSTEL
RYGLRVNVPPRITLPPSLPGPVLVNTPVRLTCNATGAPSPTLMWLKDGNPVSPAGTPGLQVFPGG
RVLTLASARASDSGRYSCVAVSAVGEDRQDWLQVHMPPSILGEELNVSWANESVALECQSHAM
PPPVLSWWKDGRPLEPRPGVHLSADKALLQVDRADWDAGHYTCEALNQAGHSEKHYNLNVWGQP
LPGEGAGLQHVSAVGRLLYLGQAQLAQEGTYTCECSNWGNSSQDLQLEVHVPPQIAGPREPPTQ
VSWQDGVATLECNATGKPPPTVTWERDGQPVGAELGLQLQNQGQSLHVERAQAAHTGRYSCVAE
NLAGRAERKFELSVLVPPELIGDLDPLTNITAALHSPLTLLCEAMGIPPPAIRWFRGEEPVSPGE
DTYLLAGGWMLKMTQTQEQDSGLYSCLASNEAGEARRNFSVEVLVPPSIENEDLEEVIKVLDGQT
AHLMCNVTGHPQPKLTWFKDGRPLARGDAHHISPDGVLLQVLQANLSSAGHYSCIAANAVGEKTK
HFQLSVLLAPTILGGAEDSADEEVTVTVNNPISLICEALAFPSPNITWMKDGAPFEASRNIQLLP
GTHGLQILNAQKEDAGQYTCWTNELGEAVKNYHVEVLIPPSISKDDPLAEVGVKEVKTKVNSTL
TLECESWAVPPPTIRWYKDGQPVTPSSRLQVLGEGRLLQIQPTQVSDSGRYLCVATNVAGEDDQD
FNVLIQVPPMFQKVGDFSAAFEILSREEEARGGVTEYREIVENNPAYLYCDTNAIPPPDLTWYRE
DQPLSAGDEVSVLQGGRVLQIPLVRAENAGRYSCKASNEVGEDWLHYELLVLTPPVILGDTEELV
EEVTVNASSTVSLQCPALGNPVPTISWLQNGLPFSPSPRLQVLEDGQVLQVSTAEVADAASYMCV
AENQAGSAEKLFTLRVQGLDLEQVTAILNSSVSLPCDVHAHPNPEVTWYKDSQALSLGEEVFLLP
GTHTLQLGRARLSDSGMYTCEALNAAGRDQKLVQLSVLVPPAFRQAPRGPQDAVLVRVGDKAVLS
CETDALPEPTVTWYKDGQPLVLAQRTQALRGGQRLEIQEAQVSDKGLYSCKVSNVAGEAVRTFTL
TVQVPPTFENPKTETVSQVAGSPLVLTCDVSGVPAPTVTWLKDRMPVESSAVHGWSRGGRLQLS
RLQPAQAGTYTCVAENTQAEARKDFWAVLVAPRIRSSGVAREHHVLEGQEVRLDCEADGQPPPD
VAWLKDGSPLGQDMGPHLRFYLDGGSLVLKGLRASDAGAYTCVAHNPAGEDARLHTVNVLVPPTI
KQGADGSGTLVSRPGELVTMVCPVRGSPPIHVSWLKDGLPLPLSQRTLLHGSGHTLRISKVQLAD
AGIFTCVAASPAGVADRNFTLQVQVPPVLEPVEFQNDWWRGSLVELPCEARGVPLPLVSWMKD
GEPLLSQSLEQGPSLQLEAVGAGDSGTYSCVAVSEAGEARRHFQLTVMEPPHIEDSGQPTELSLT
PGAPMELLCDAQGTPQPNITWHKDGQALTRLENNSRATRVLRVENVQVRDAGLYTCLAESPAGAI
EKSFRVRVQAPPNIVGPRGPRFWGLAPGQLVLECSVEAEPAPKITWHRDGIVLQEDAHTQFPER
GRFLQLQALSTADSGDYSCTARNAAGSTSVAFRVEIHTVPTIRSGPPAVNVSVNQTALLPCQADG
VPAPLVSWRKDRVPLDPRSPRATPIHSRFEILPEGSLRIQPVLAQDAGHYLCLASNSAGSDRQGR
DLRVLEPPAIAPSPSNLTLTAHTPALLPCEASGSPKPLWWWKDGQKLDFRLQQGAYRLLPSNAL
LLTAPGPQDSAQFECWSNEVGEAHRLYQVTVHVPPTIADDQTDFTVTMMAPWLTCHSTGIPAP
TVSWSKAGAQLGARGSGYRVSPSGALEIGQALPIHAGRYTCSARNSAGVAHKHVFLTVQASPWK
PLPSWRAVAEEEVLLPCEASGIPRPTITWQKEGLNVATGVSTQVLPGGQLRIAHASPEDAGNYL
CIAKNSAGSAMGKTRLWQVPPVIENGLPDLSTTEGSHAFLPCKARGSPEPNITWDKDGQPVSGA
EGKFTIQPSGELLVKNLEGQDAGTYTCTAENAVGRARRRVHLTILVLPVFTTLPGDRSLRLGDRL
WLRCAARGSPTPRIGWTVNDRPVTEGVSEQDGGSTLQRAAVSREDSGTYVCWAENRVGRTQAVSF
VHVKEAPVLQGEAFSYLVEPVGGSIQLDCWRGDPVPDIHWIKDGLPLRGSHLRHQLQNGSLTIR
RTEARRGLAPWRDDAGRYQCLAENEMGVAKKWILVLQTRMVPAEPHLKRQLPPIPSNNEAPSLF
PGVHGGHVGNPDFHSHLAEVLAVQLLAGSLLFSARAMPQASTAAISLLAPTSFAPFPDDISQGIL
SSSTAHQGSPQGWQKLLFFTAIPNKTTVMVTVEPQDMTVRSGDDVALRCQATGEPTPTIEWLQAG
QPLRASRRLRTLPDGSLWLENVETGDAGTYDCVAHNLLGSATARAFLVCASHAIVGSRHFRDPQV
FCEFWPPPHFTGEPQGSWGSMTGVINGRKFGVATLNTSVMQEAHSGVSSIHSSIRHVPANVGPL
MRVLWTIAPIYWALARESGEALNGHSLTGGRFRQESHVEFATGELLTMTQVARGLDPDGLLLLD
WVNGWPESLADADLQVQDFEEHYVQTGPGQLFVGSTQRFFQGGLPSFLRCNHSIQYNAARGPQ
PQLVQHLRASAISSAFDPEAEALRFQLATALQAEENEVGCPEGFELDSQGAFCVDRDECSGGPSP
CSHACLNAPGRFSCTCPTGFALAWDDRNCRDVDECAWDAHLCREGQRCVNLLGSYRCLPDCGPGF
RVADGAGCEDVDECLEGLDDCHYNQLCENTPGGHRCSCPRGYRMQGPSLPCLDVNECLQLPKACA
YQCHNLQGSYRCLCPPGQTLLRDGKACTSLERNGQNVTTVSHRGPLLPWLRPWASIPGTSYHAWV
SLRPGPMALSSVGRAWCPPGFIRQNGVCTDLDECRVRNLCQHACRNTEGSYQCLCPAGYRLLPSG
KNCQDTNECEEESIECGPGQMCFNTRGSYQCVDTPCPATYRQGPSPGTCFRRCSQDCGTGGPSTL
QYRLLPLPLGVRAHHDVARLTAFSEVGVPANRTELSMLEPDPRSPFALRPLRAGLGAWTRRALT
RAGLYRLTVRAAAPRHQSVFVLLIAVSPYPY
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 9C.
Table 9C. Patp results for NOV9 Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAY53667Sequence gi/3328186 +1 1529 6.5e-244 >patp:AAY87206Human secreted protein sequence +1 2235 6.4e-230 ID N0:245 >patp:AAE06183Human gene 57 encoded secreted +1 2235 6.4e-230 protein >patp:AAY87120Human secreted protein sequence +1 2235 6.4e-230 SEQ ID:159 >patp:AAE06097Human gene 57 secreted protein +1 2235 6.4e-230 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 625 of 1067 bases (58%) identical to a gb:GENBANK-ID:HSLTGFBP4~acc:Y13622.1 mRNA from Homo sapieszs (mRNA for latent transforming growth factor-beta binding protein-4). The full amino acid sequence of the protein of the invention was found to have 502 of 1665 amino acid residues (30%) identical to, and 767 of 1665 amino acid residues (46%) similar to, the 5198 amino acid residue ptnr:SPTREMBL-ACC:076518 protein from Cae~orhabditis elegaf~.s (HEMICENTIN
PRECURSOR).
NOV9 also has homology to the proteins shown in the BLASTP data in Table 9D.
Table 9D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~14575679~gb~AAK6hemicentin 5636 1230/30171785/30170.0 8690.1~AF156100 [Homo Sapiens] (40%) (58%) (AF156100) gi118547943~reflXP_hemicentrin 3645 979/2379 1413/23790.0 053531.3~(XM [Homo Sapiens] (4l%) (59%) gi~17568539~refINP_Ig superfamily 5175 857/3077 1348/30770.0 509636.1~(NM_077235repeats (I-type) (27%) (42%) [Caenorhabditis e1 egans]
gi~17568541IrefINP_IG 5198 857/3077 1348/30770.0 509635.1I(NM (immunoglobulin) (27%) (42%) superfamily (47 domains) [Caenorhabditis e1 egans]
gi~13872813~emb~CACfibulin-6 2673 552/1399 796/1399 0.0 37630.1~(AJ306906)[Homo Sapiens] (39%) (56%) A multiple sequence alignment is given ix~ Table 9E, with the NOV9 protein being shown on line 1 in Table 9E in a ClustalW analysis, and comparing the NOV9 protein with the related protein sequences shown in Table 9D. This BLASTP data is displayed graphically in the ClustalW in Table 9E.
Table 9E. ClustalW Analysis of NOV9 1) > NOV9; SEQ ID N0:18 2) > gig 14575679~J hemicentin [Homo Sapiens]; SEQ 1D N0:72 3) > gi~18547943~/ hemicentrin [Homo Sapiens]; SEQ ID N0:73 4) > gig 17568539/ Ig superfamily repeats (I-type) [Caeraorlaabditfs elegaras]; SEQ ID N0:74 5) > gig 17568541 ~/ IG (inununoglobulin) superfamily (47 domains) [Caenorlaabditis elegans]; SEQ ID
N0:75 6) > gi~13872813~/ fibulin-6 [Homo Sapiens]; SEQ ID N0:76 ..J....J....J....J....J.. ,.J....J. .
NOV9 2602 _________________________ _________ ____ giJ14575679J3929 _________________________ _________ ____ giJ18547943J1938 _________________________ _________ ____ giJ17568539J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
giJ17568541J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
giJ13872813J966 _________________________ _________ ____ ....J....J....J....J....J....J....J....J.. .J., J....J . .J
NOV9 2621---_______________________________________p . GQ~,PI . 2639 ..~
giJ14575679J3948-___,_____________________________________S
.~ TEL 3966 giJ18547943J1957-_________________________________________S
~ TvL ' giJ17568539J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
giJ17568541J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
giJ13872813J985 __________________________________________S
TvL 1003 ~.I
giJ14575679J 3967 4025 giJ18547943J 1976 giJ17568539J 3368 3427 giJ17568541J 3368 3427 giJ13872813J 1004 1062 giJ14575679J 4026 4085 giJ18547943J 2035 2094 giJ17568539J 3428 3486 giJ17568541J 3428 3486 giJ13872813J 1063 1122 ....~....J....J....J,...J....J....J....J...,J....J....J....J
giJ14575679J 4086 4143 ga.J18547943~ 2095 2152 giJ17568539J 3487 3546 giJ17568541J 3487 3546 giJ13872813J 1123 1180 .J....J,...J....J:..,J....J....J....J. .J.. .J....J.. .J
NOV9 2759 __________________ _______ ___ DLTT~~ ~1F ~ ~ S~E~ 2783 H' I v VI r t ~ Vm gi J 14575679 J 4144 T ~ ~ S STST Ti~H ~ RS'I!--E Y , v Q~iI ~ 'D ~T ~ v 4201 gi J 18547943 J 2153 T ' S STST T~~Ii ' RS,~--E ~Y_' QiI ~ iD ~T~ 2210 gi J 17568539 J 3547 I R~iS~E ~ T DIDLI ILL ' DIt'~'NTI PLAIVARTIY E PIS ~Q
~D'S~ 3606 giJ17568541J 3547 ' I RfiS EI~rT DIDLI ~L~~~ DKNTI PLAIVARTIY E PIS ~Q~ 3606 gi J 13872813 ~ 1181 H T ~ ~ S STST TtlIi ~ ~ RS!~--E YiT'VN~Qi~',T ~ iD ~T ~
.J....J....J . . .. .. J....J....J...
NOV9 2784 T~D~GQP~IS,GAE~F'~IQ~S~-QLVK~LEGQ~T~TyE ~ ' ~RARRR~fi~I,L~ 2842 gi~14575679~ 4202 4260 gi1185479431 2211 2269 gi1175685391 3607 3666 gi1175685411 3607 gi1138728131 1239 3666 NOV9 2843 ~...T.:. I..RL1. .1. .~.~S~~____T ~I.~1 'RP~'TEGVSE-I--Q~I 2892 gi1145756791 4261 'T E ' r - ~Q S T I~-----~~KL F I PAHFDS---- 4310 gi1185479431 2270 ~T E ' r - ~Q~ S I'T I~---- 'KL, F~~I~PAHFDS--- 2319 gi ~ 17568539 ~ 3667 ~E DIHGTQP 'KEE ;T~T,T T PIKLAEDIADQ~; . D~t7~
KDRAI7GDLTDNVDIS~J17 3726 gi ~ 27568541 ~ 3667 ~E IHGTQPi~~.KRE TAT T PIKLAEDIADQ'~~'ID;tTS
K'~,~RA~iDGDLTDNVDIS~77D 3726 gi~13872813~ 1298 ~T E ' r 4~ DQfl S It~~lTneI~-____~,. gIpp,HFDS-___~ 1347 a gi1145756791 4311 4369 gi1185479431 2320 2378 gi1175685391 3727 3786 gi1175685411 3727 3786 gi1138728131 1348 1406 gi1145756791 4370 4420 gi1185479431 2379 2429 30 gi~17568539~ 3787 3838 gi1175685411 3787 3838 gi1138728131 1407 1457 gi1145756791 4421 4480 gi1185479431 2430 2489 gi1175685391 3839 3898 gi1175685411 3839 3898 gi1138728131 1458 1517 .'....,....1,...1....1....1....1....1. .1....1,.. .1....1 NOV9 3063 t~GNPr-------------------------------FH- LA'S~QL_' ------ 3083 gi1145756791 4481 SWDrR --S Y A~ ~ErTSF'E ~ L ~P~I Q GF----- 4533 1 7 v r' gi ~ 18547943 ~ 2490 SWDrR ' Y 'yV--S Y ~A ErTS~.~'E V.~',' L V V P ~Q ~l GF---gi1175685391 3899 FDSPDGAR,'~ LKG PHL ~Tr GI?Y'T Q~L 'SEAS S~~L PPEINR17GI
gi 113872813 ~ 1518 SWD~RGLKG-A~ PHL[',J ;E~TS~E!E Q~LI L SEAS ~
P==~~''~~~''Q~~GFNRDGI 1570 .1. ..1. ..1....1....1....1. .1....1. ..1. I . .~.. .1 NOV9 3083 -- LLFj" .PQAST SLL~PTSF F~~IS~GILS~STAHQGSP ------ - 3132 gi I 14575679 ~ 4533 -- WSiiIW~ ~ C~'T GK ~IxCNQ L'L~ GG PCQ DIsEMR'~CQNPCP -D'S 4589 gi ~ 18547943 I 2542 - ~WS~i~ 'CSV'T GK QR~CNQ L~ GC~PCQ D~;EM~CQNPCP -D S
giI175685391 3959 DMSPLP~iQ L'~T.Q LAQG~PVPQ1~RWTLNGTALTHSTP ITASDGTFII SLSD
g7. 175685411 3959 DMSP ~LP~iQ~~v"'L~'IiQ LAQGT~PVPQR.WTLNGTALTHSTP
IT~,ASDixTFI"~3I 'L, SLSD 4018 gi 113872813 ~ 1570 ---~5~'~" C~S"''~T GKGQ~KRR~iCNQ~LGG~T~'PCQ
~iEM'k'i.2~CQNIfPCP -D~S 1626 ~. _..~....~....~....~....~....~
NOV9 3132 - --~QLLCLLFFTAIP~~ --------------- ----- T: TUE'---Q----- 3155 gi 114575679 ~ 4590 WSE~fL E ~CTRSCGRGi Q RTRTCNNPSVQH GPCEGNA'VE~~ ' ~C~ GA---gi118547943~ 2599 WSE~IL CTRSCGRG~QBRTRTCNNPSVQ GPCEGNAiSTE~ ,11 ~C~ GA---gi1175685391 4019 KGV~TC~I,~, AGSDNLMYNVDWQAPVISN T'KQVIEGE E L~EGY' PQVSWL
gi1175685411 4019 KGVY','~CAGSDNLMYNVDWQAPVISN T~QVIEGE~ E L~EGY~ PQVSWL 4078 gi 113872813 1 1627 WSETL~ CTRSCGRG~f~RTRTCNNPSVQH~eG ,PCEGNA3 ,ELI 3 !R~C~ GA-NOV9 3155 ----------------D-------M'VRS DVAL~CATGE~-------------T~TI 3179 gi~14575679~ 4646 ------------ W~'v'AWQPWGTCESC KGTQT' r~LC~ ------------P~ F
gi~18547943~ 2655 ------------ WAW~PWGTCESC KGTQT' _LC ~-------------P~ F 2687 gi~175685391 4079 RNGNRVETGVQGVRYVi!xDG~~MLTIIEARSLDSGIYLCSAT
EAGSAQQAYTLEVLVS~KI 4138 gi~17568541~ 4079 RNGNRVETGVQGVRYV"~'DGMLTIIEARSLDSGIYLCSATGSAQQAYTLEVLVS~KI
gi~13872813~ 1683 ------------ WAWPWGTCESC~KGTQT~LC -----------P~ F 1715 IO
NOV9 3179 -----------------------------E-~LQAGQPLRAS~~L'.LPDGSLWLN--- 3206 gi~14575679~ 4679 G YCDGAETQMQVCNE P~~H KWATW S,,,~ACSVSCG 'Q~ CSDPV~~_~P~YGGR
gi~185479431 2688 G YCDGAETQMQVCNE ' P;IFi KWATW ~~,,,AJCSVSCG _'Q~
CSDP~,PYGGR 2747 gi~17568539~ 4139 IT TPGVLTPSSGSKFSLP '~ YPDPII '15LNGNDIKD ENG I GT~HIEKAE
IS gi~175685411 4139 IT TPGVLTPSSGSKFSLP ~YPDPII 1,,:?~LNGNDIKD ENGH,I
GT~~'.rHIEKAE 4198 gi~13872813~ 1716 G YCDGAETQMQVCNE P~ KWATW ~ACSVSCG 'Q' CSDP~PYGGR 1775 NOV9 3206 - ----- '~GDAGYD . -----------LLGSAT ' L ------ 3236 V ~~;
gi ~ 14575679 I 4739 ---C G~sD~DFC~~P ~~ ~ ~ SGWGTCSRTCNGGQ ~RRT ~P ----- 4788 gi ~ 18547943 ~ 2748 ~----C G~~~~'D1(QDFC~TDP T G ~ ~ SGWGTCSRTCNGGQ ~R'~.'RT
, P------ 2797 gi~17568539~ 4199 e'RHLIY CAKNDAGA~~"'LEF~ QTIU ~KISTSGNRYINGSEGTETVI
°IESESSEF 4258 gi ~ 17568541 ~ 4199 ~RHLI C~'I'AI~NDAGA~.'?~'LEF ~ QTIV KISTSGNRYINGSEGTETVI
gi~13872813~ 1776 ----C G~D~~DFC~IDP ~T~G~W,~~WSGWGTCSRTCNGGQM~'yIR~RT~P------NOV9 3236 -HAI S'HFRD ___________ _________ __________ -VF ~. ~ 3257 r ~ ,~, r r 3~ gi ~ 14575679 ~ 4788 -PPS CG ~SQIQRCNTD1~CP~D~GSWGSWHS~ QCiTS,C GEKT~ IiP ~
gi~18547943~ 2797 -PPS ' CG ~SQIQRCNTD CP~GSWGSWHSQC~~C GEKT ~ 'HP ~ 2856 gi~17568539~ 4259 SWS PLLPSNLIFSEDYKL3~KI~t,(STRLSDQGE~, CTATQT ~GV ~ 4318 gi~17568541~ 4259 SWS PLLPSN~LIFSEDYKL~KISTRLSDQGE'~ CTS ~ ATQ~T GV 4318 gi ~ 13872813 ~ 1825 -PPS~G~ACGG~SQIQRCNTDCP~ST,DGSWGSWHS~J~QC~~~EKT;HP~ 1884 giI14575679~ 4848 gi~7.8547943~ 2857 4900 gi~17568539~ 4319 4378 gi~17568541~ 4319 4378 gi~13872813~ 1885 1937 .,..I
gi~145756791 4901 ----- 4953 gi~18547943~ 2910 ----- 2962 gi~17568539~ 4379 TGIPE 4438 gi~17568541~ 4379 TGIPE 4438 gi~13872813~ 1938 ----- 1990 gi~14575679~ 4953 5008 gi~18547943~ 2962 3017 gi~17568539~ 4439 4498 gi~17568541~ 4439 4498 gi~13872813~ 1990 2045 NOV9 3400 ------- F'~E ' yV ~1 FQ . L(PSFLRCi S~:Q ~ PQ ~Q . Q 3452 a r ...-gi ~ 14575679 ~ 5008 ------- Y'T D Iv ~ ~A ~L I ~ IISIPYT TUF L3Qi ~F E<T 5060 giI185479431 3017 _______ ,~X':T D ~~ ~ Y'AY 'L I~ I',SIPYT T'~F yQil ~,Q~ ~F T~ 3069 giI17568539~ 4499 PSDRPAPI:; CDE~ICG ~KI~TEYMID~GD P~DNPQLLP
KDVEDSSLNGSIAYRC~PGP 4558 gi~17568541~ 4499 PSDRPAPI,, CDE~CG KI~TEYMID'GD P~ NPQLLP KDVEDSSLNGSIAYRC.
gi113872813~ 2045 _______ yT ~~~ ~ v ~;AY 'L I~ I~SIPYT 'T''()F~_Qi~RMOFmE~T~
L, ~ ~ L,. . . I . I . I . I . . . I . . . . I . . . I . . . I . . I . . . I.
NOV9 3453 . ~ TS ~'~PE ~ . Q~.,iAT,~QAENEV . 'E ------ . E n Q n- n S 3505 gi I 14575679 ~ 5061 H~ S'ClIE Y~VQI IHAI~~SKG1~RSNQ 'S ----- ~ P ~-y ' 5113 gi~185479431 3070 H~ SUE D~QI~~T~ ~~HA~"~SKG~1RSNQ 'S ----- ~ P ~-W ~ 3122 gi1175685391 4559 R~ RTVLL~ P~FI~KP TT IGAIVEL S~1AGPPHPTI AKD KLIE~S~F I,~~, gi 1175685411 4559 R~ RTVLL~P~FI'U'KP TT~IGATVEL3 Sn~p,'AGPPHPTI AKD
KLIE~SIfiF Ii1 4618 gi 113872813 I 2098 H~ S~E~DY.~TQI~T~ ~I'HA~~'SKGRSNQ~S~e----- ~ P ~-~ ' 2150 gi1145756791 5114 5172 gi1185479431 3123 3181 gi1175685391 4619 4677 gi1175685411 4619 4677 gi1138728131 2151 2209 .I. .~. ..I....I....I....I....I....I....I....I....I....I
NOV9 3566 '~xiPD .P _______________-________________________________ 3576 gi1145756791 5173 ~ S ~RTS ~i---------------------- Cf~~IECQESPCHQ~CF1~ 5209 gi~18547943~ 3182 ~ S ~RTS~ T~----------------------- C~~IECQESPCHQ'CF3218 gi1175685391 4678 ERNQAYS LTWE ~ ~PMPKNLAGIHFMNNGSLVILDTS L~GLELY(~'CKV 'R 3 gi117568541~ 4678 ERNQAYS LTWE ~ PMPKNLAGIHFMNNGSLVILDTS L~G~LELY'CKV 'RF~
V V ~
gi I 13872813 ~ 2210 ~t~(1R~S 'RTS-----------------------CE~7~IECQESFSPCHQ~'yCF2246 30 ....I....I....I....I....I....1....1....1....1....1....1....1 NOV9 3576 -___________________________________________________________ 3576 gi1145756791 5210 5262 gi~185479431 3219 3271 gi~17568539~ 4738 4797 35 gi1175685411 4738 4797 gi1138728131 2247 2299 .I.. .I... I . .I. .I. .I ...I. ..I. .1....1....I...
NOV9 3577 ADGAG- E~'n L.LDD~. ~.'. PGGv '.S ~'~------------------ 3616 gi114575679i 5263 TKAEG ~W ICC7 T f1 vT RSS,' ~~ __________________ 5304 gii185479431 3272 ~TKAE'GT yI~KT~Q ~~ RGSG,..' ----------------- 3313 gi1175685391 4798 ~LPNNLVL~:~YDANSIGKAFDDTLNVYG------------------- 4838 gi1175685411 4798 ~tLPNNLVL~ICYD~ V~~ ANSIGKAFDDTLNVYE FLPLTGFEGSGINIDDS 4857 gi 113872813 I 2300 TKAE~tGT~T~~ .-, T,~IR~I~RGS----------------- 2341 ....I.
50 gi1145756791 5304 ------ 5353 gi~185479431 3313 ------ 3362 gi1175685391 4838 ----GS 4894 gi1175685411 4858 SNAGGS 4917 gi1138728131 2341 ------ 2390 gi1145756791 5354 5413 gi118547943~ 3363 3422 gi117568539~ 4895 4952 gi117568541~ 4918 4975 gi113872813~ 2391 2450 .. .I.. .1....I., .~. .~. ..~L. .I. .I. .I. .~. .I. .I
NOV9 3718 -- ' W ~P FIR--QG Tn n RVRL.~ E .~~ L.' ~~LP~u.' 3773 gi1145756791 5414 RTI'KT ~ SEA--S~DT ~I~ ENT~3 t F ~ I 'P' Q THE ~ ~T 5471 i 18547943 3423 RTI'KT ' SEA SDT ~I~ ENT v F ~ I P Q TH ~T 3480 gi1175685391 4953 TG-E FAMNPHTRI En_L~AFYQP F I Y G._,~ 'L~E ~Ee~-- 5009 gi~17568541~ 4976TG-E
FAMNPHTRI
E~
~
FYQP
F
I
YD
G
~
L
E
--r gi 7.3872813 2451RTI~'yKT L ~F ~ ~THT~S
~ ~ Z 2508 SEA P~Q
S~IDT~2 ~
ENT~)A
~
gi~14575679~ 5472 5531 gi~18547943~ 3481 3540 gi~17568539~ 5010 5059 gi117568541~ 5033 5082 gi~13872813) 2509 2568 gi~14575679~ 5531 5582 gi~18547943~ 3540 3591 gi~17568539) 5060 5118 gi117568541~ 5083 5141 gi~13872813~ 2568 2619 L, . . ~ ~...v., ~ . . I . . . . I . . . I .
~~ ~ 7 ~ i -NOV9 3883 .~PLRAGL ~T ~GL1~LT " P------- .~~~ .,ltL ~ .P ~ 3931 gi~14575679~ 5583 E-NL T'P RE~ T ' '~~ YS~=-GTIE'~~ T ~~Y ' 5636 V~ i1 gi~185479431 3592 ~ E-NL T~P RE~'T ' ~~S YS,~, -GTIE ~ T IyY ~ 3645 gi1175685391 5119 ' ~AVKRGHAQ G H ~~H DH~,~,TNELHAPK~L GQ ~~ 5175 gi~17568541~ 5142 T~AVKRGHAQ ' GH~K~~H DH~TNELHAPIt~ ~L GQ ~5198 gi ~ 13872813 ~ 2620. ~ E NLI~eV~T P RE~TS~YS ' -GTIEiY~T~~1~Y ' 2673 The NOV9 Clustal W alignment shown in Table 9E was modified to begin at amino residue 4080. The data in Table 9E includes all of the regions overlapping with the NOV9 35 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 9F lists the domain description from 40 DOMAIN analysis results against NOV9.
Table 9F Domain Anal sis of NOV9 Model Region of Score (bits) E value Homology ig 99-157 46.50 6.1e-10 ig 192-251 38.00 2.1e-07 ig 286-344 41.90 1.4e-08 ig 380-438 32,10 1.3e-05 ig 473-531 45.90 8.9e-10 ig 565-615 35.50 1.2e-06 ig 648-706 43.80 4e-09 ig 740-800 39.10 1e-07 ig 833-891 27.10 0.00041 ig 981-1039 42,90 7.1e-09 ig 1075-1133 28.90 0.00012 ig 1168-1226 30.80 3.2e-05 ig 1264-1322 39.60 7.2e-08 ig 1362-1420 43.40 5.2e-09 ig 1473-1531 29.40 8.2e-05 ig 1568-1626 35.70 1e-06 ig 1654-1712 _ 1.5e-09 45.20 ig 1749-1807 43.80 3.8e-09 ig 1841-1899 43.00 6.8e-09 ig 1934-1994 41.40 2e-OS
ig 2030-2088 49.90 5.8e-11 ig 2123-2177 49.60 6.8e-11 ig 2212-2268 37.50 3e-07 ig 2303-2361 28.60 0.00014 ig 2394-2459 28.20 0.00019 ig 2492-2552 32.90 7.2e-06 ig 2585-2643 22.80 0.0081 ig 2676-2733 37.50 3.1e-07 ig 2766-2824 43.10 6.3e-09 ig 2857-2913 44.10 3e-09 ig 2947-3012 18.00 0.22 ig 3162-3219 34.70 2.1e-06 EGF 3504-3539 38.30 1.8e-07 EGF 3589-3626 13.80 1.3 EGF 3632-3667 33.30 5,4e-06 EGF 3739-3773 38.40 1.6e-07 Consistent with other known members of the Hemicentin Precursor-like family of proteins, NOV9 has, for example, thirty-three immunoglobulin (ig) signature sequences and four epidermal growth factor (EGF) signature sequences, as well as homology to other members of the Hemicentin Precursor-like Protein Family. NOV9 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV9 nucleic acids and polypeptides can be used to identify proteins that are members of the Hemicentin Precursor-like Protein Family. The NOV9 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV9 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular differentiation, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Hyperparathyroidism, Hypoparathyroidism, Lymphedema, Allergies as well as other diseases, disorders and conditions..
In addition, various NOV9 nucleic acids and polypeptides according to the invention are useful, ifater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV9 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Hemicentin Precursor-like Protein Family.
Hemicentrin is an extracellular matrix protein with a modular sturcture. Like NOV9, the hemicentrin structure includes many immunoglobulin domains flanked by EGF
domains.
The protein is likely involved in cellular differentiation of epithelial tissue.
The NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune and endocrine physiology. As such, the NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and endocrine disorders, e.g., Cardiovascular diseases, Hyperparathyroidism, Hypoparathyroidism, Lymphedema, Allergies as well as other diseases, disorders and conditions.
The NOV9 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV9 nucleic acid is expressed in Adipose, Thyroid, Colon, Lymph node, Bone, Myometrium, Prostate, Testis, Aorta, Vein.
Additional utilities for NOV9 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV10 polypeptide has been identified as a Selectin-like protein. The novel nucleic acid sequences maps to the chromosome 9. Two alternative novel NOV10, NOVlOa and NOVlOb, nucleic acids and encoded polypeptides are provided.
NOVlOa A NOV10 variant is NOVlOa (alternatively referred to herein as CG94661-Ol), which includes the 1268 nucleotide sequence (SEQ m N0:19) shown in Table 10A. A NOV
1 Oa ORF begins with a ATG initiation codon at nucleotides 145-147 and ends with a TGA codon at nucleotides 871-873. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 10A, and the start and stop codons are in bold letters.
Table 10A. NOVlOa Nucleotide Sequence (SEQ ID N0:19) GCGGCCGCCACCCTCCGTGGCAAGGCGAGGCCCCGGGGGCGGGCCGGGGTCACCACGCCTGTCCCAG
GGAACCGCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAG
ATGAAGTGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCACGTGC
GCTAAGCTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGA
CCGTGCTCATGTTCCGCTGCCCCTCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTG
GAAGGGGAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACC
TTTGGCTTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGG
CCTTCCTCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGC
CCAGCTGTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAG
CACTTCAACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATG
GTGAGAGCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGC
TCTAAGCCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGA
CAGCCCCTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGT
GACCACGCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCA
GCTACAACTCCACATCAACTCCACATGCGCCCAGCTCGAGACTGATGAGTGGAATCAGCTTCCAGGT
GTAGGGACCCCTTGAGGGGCCGAGCTGACATCCAAGGCTGAGGACCCCAGTGGGGAGTGTTCTGTTC
CGGCATATCCTGGCCGTAACGATTTTTATAGTTATGGACTACTTGAAACCACTACTGAGGGTAATTT
ACTAGCTGTGGCCTCCCACTAACTAGCATTCCTTTAAAGAGACTGGGAAATGTTTTAAGCAAATCTA
GTTTTGTATAATAAAATAAGAAAATAGCAATAAACTTCTTTTCAGCAACTACF,~~e~AAAAAAA
The NOV 10a polypeptide (SEQ ID N0:20) encoded by SEQ ID N0:19 is 242 amino acid residues in length and is presented using the one-letter amino acid code in Table l OB. The Psort profile for the NOVlOa predicts that this peptide is likely to be localized at the plasma membrane with a certainty of 0.7000.
Table 10B. NOVlOa protein sequence (SEQ ID N0:20) MKDTIGLVMEWEIPEIICTCAKLRLPPQATFQVLRGNGASVGTVLMFRCPSNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOVlOb Alternatively, a NOV 10 variant is the novel NOV l Ob (alternatively referred to herein as CG94661-02), which includes the 887 nucleotide sequence (SEQ TD N0:21) shown in Table l OC. NOV l Ob was created by polymerase chain reaction (PCR) using the primers detailed in Example 1, Table 17. Primers were designed based on ih silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clone 143260::COR100348691 extn.698976.C20.
The NOVlOb ORF begins with a Kozak consensus ATG initiation colon at nucleotides 72-74 and ends with a TGA colon at nucleotides 1958-1960. Putative untranslated regions upstream from the initiation colon and downstream from the termination colon are underlined in Table l OC, and the start and stop colons are in bold letters.
Table IOC. NOVlOb Nucleotide Sequence (SEQ ID N0:21) GCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAGATGAAG
TGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCATGTGCGCTAAG
CTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGACCGTGC
TCATGTTCCGCTGCCCCCCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTGGAAGGG
GAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACCTTTGGC
TTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGGCCTTCC
TCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGCCCAGCT
GTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAGCACTTC
AACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATGGTGAGA
GCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGCTCTAAG
CCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGACAGCCC
CTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGTGACCAC
GCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCAGCTACA
ACTCCACATCAACTCC
Variant sequences of NOVlOb are included in Example 3, Table 22. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP
originates as a cDNA.
The NOV l Ob protein (SEQ ID N0:22) encoded by SEQ ID NO:21 is 242 amino acid residues in length and is presented using the one-letter code in Table l OD.
The Psort profile for NOV l Ob predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.7000.
Table 10D. NOVlOb protein sequence (SEQ ID N0:22) MKNIGLVMEWEIPEIICMCAKLRLPPQATFQVLRGNGASVGTVLMFRCPPNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOV10 Clones Unless specifically addressed as NOVlOa or NOVlOb, any reference to NOV10 is assumed to encompass all variants. NOVlOa differs from NOVlOb at amino acid position 18 (T>M) and amino acid position 50 (S>P) as shown in Tables 10B and l OD.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 10E.
Table 10E. Pat results for NOV10 Smallest Sum eading igh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAM93054Human digestive system antigen +1 210 7.2e-17 >patp:AAR05494Endothelial leukocyte adhesion +1 113 0.0016 molecule-1 >patp:AAR08116Endothelial leucocyte adhesion +1 l13 0.0016 molecule-1 >patp:AAW18839E-selectin +1 113 0.0016 >patp:AAW46733Endothelial leukocyte adhesion +1 113 0.0016 molecule-1 hi a BLAST search of public sequence databases, it was found, for example, that the NOVlOa nucleic acid sequence of this invention has 438 of 447 bases (97%) identical to a gb:GENBANI~-m:HSM802384~acc:AL137623.1 mRNA from Homo sapieras (cDNA
DKFZp434J1812 (from clone DKFZp434J1812)). The full amino acid sequence of the protein of the invention was found to have 110 of 139 amino acid residues (79%) identical to, and 123 of 139 amino acid residues (88%) similar to, the 269 amino acid residue ptnr:SPTREMBL-ACC:Q9D176 protein from Mus musculus (1700017I11R1K PROTEIN).
Similarly, it was found, for example, the NOV l Ob nucleic acid sequence of this invention has 438 of 447 bases (97%) identical to a gb:GENBANK-ID:HSM802384~acc:AL137623.1 mRNA from Homo Sapiens (cDNA DKFZp434J1812 (from clone DKFZp434J1812)). The full amino acid sequence of the protein of the invention was found to have 108 of 138 amino acid residues (78%) identical to, and 121 of 138 amino acid residues (87%) similar to, the 269 amino acid residue ptnr:SPTREMBL-ACC:Q9D176 protein from Mus musculus (1700017I11RIK PROTEIN.
Additional BLAST results are shown in Table 10F.
Table 10F. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier as >gi~15779059~gb~ASimilar to RIKEN255 192/225 192/225 e-101 AH14601.1~AAH1460cDNA 1700017I11 (85%) (85%) 1(BC014601) gene [Homo Sapiens]
>gi~12834785~dbj~Sushi domain 269 130/246 142/246 8e-56 BAB23043.1~(AK003(SCR repeat) (52%) (56%) 860) containing protein~data source:Pfam, source key:PF00084, evidence:ISS-put ative [Mus musculus]
>gi~128505441dbjlSushi domain 170 71/102 77/102 6e-35 BAB28764.1I(AK013(SCR repeat) (69%) (74%) 276) containing protein-.data source:Pfam, source key:PF00084, evidence:ISS-put ative [Mus musculus]
>gi~128389761dbjlSushi domain 149 55/73 61/73 2e-26 ' BAB24394.1~(AK006(SCR repeat) (75%) (83%) 068) containing protein-data source:Pfam, source key:PF00084, evidence:ISS-.put ative [Mus musculus]
>gi~7494498~pir~~scavenger 2043 30/87 42/87 2e-04 T18524 receptor (34%) (47%) cysteine-rich protein homolog srcrm2 - Geodia cydonium A multiple sequence alignment is given in Table 1 OG, with the NOV 10 protein of the invention being shown on line 1, in a ClustalW analysis comparing NOV10 with related protein sequences disclosed in Table IOF.
Table 10G. Information for the ClustalW proteins:
1) > NOVlOa; SEQ ID N0:20 2) > NOV l Ob; SEQ ID N0:22 3) > gi~1577905/ similar to RTKFN cDNA 1700017I11 gene [Homo Sapiens]; SEQ ID
N0:77 4) > gig 1283478/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:78 5) > gig 1285054/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:79 6) > gi~1283897/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084: ISS-putative [Mus nausculus]; SEQ ID N0:80 7) > gi~7494498/ scavenger receptor cysteine-rich protein homolog srcrm2-Geodia cydohiu~ra; SEQ ll~
NO:81 NOVlOa ___ IGLVMEWEIP--_____________________EIIC
RL
~ v NOVlOb -- NIGLVMEWEIP-----------------------EIIC
RL
K
.
gi~ 1577905--- WAAATLRGKARPRGRA--------- GNRT
RL
R TT
P
gi~ 1283478--- RTSATLRGRARPRWRA--------- VNQT Q
R TT P
P
V
V
~S gi~ 1285054--- RTSATLRGRARPRWRA--------- VNQT
' Q
R TT P
P
gi~ 1283897--- RTSATLRGRAR.PRWRA--------- VNQT
Q
IR TT P
P
gi1 7494498GRYTQDTGWIACEDGYQPTEGAADVLCTED PACSVS
P
TWSRT QV
NOVlOa ~ F~ -__________________________________________ NOVlOb v F~ -__________________________________________ gi~1577905 'v Fv -_____________________________-____________ gi~1283478 ~~v.~L~ ___________________________________________ giI1285054 ~ 7 -__________________________________________ ~~ L~
gi~1283897 ~~,.'~~1IL~ -__________________________________________ gi~7494498 SLSSPDTIPGLWSLVCDKGYTYDASSDGDFSWCGLDGEWNSTLG
NOVlOa _________,___________ L. .
NOVlOb _____________________ L. .p gi~ 1577905--___________________ L.
gi1 1283478--__________________ T L I
v n g7.I 1285054--__________________ T L I 1 gi1 1283897--___________________ T L I ' gi1 7494498TCKLVLCPAYSFNVTTNLRVSLTQ S TT T S 'FH SVI
S
.
.
NOVlOa T IAE ~L
' NOVlOb T IAE ~L ' . ' ' gi~ 1577905T IAE ~L
gi~ 1283478 TVD ' ' gi~1285054 TVD ' gi~1283897 TVD ~ --VPSSC~CPW
gi17494498 S S ---T GTV'H SRE " TKCPTLTISDHVTAS SETTINTVVS~
.) NOVlOa ------ S ' 'S ~SAQ .SQLKDED -ET ~~~Y G
NOVlOb ------ S ' 'S 'SAQ SQLKDED -ETi~~ y G
giI1577905 ------ S ' 'S 'SAQ SQLKDED -ET ~' Y G
1O gi~1283478 ------ Q~ E " TAQ YQLRGED -ET s~ Y G
giI1285054 ------ Q~ E " ~-------YSG-- -MSF~KLC
gi~1283897 PSS-__________p~SSSVC'KMSG--_______G_________ T
gi~7494498 TCDNGYFLKGDKI~E~LSTGVWNGTAPTCS~PNSCPSLI~SDH~TiSSTD
NOVlOa LKHFN-------------------KPVSGP ~ NHSF TDHGESTSKL
NOVlOb LKHFN-------------------KPVSGP ~ DNHSF TDHGESTSKL
g1~1577905 LKHFN-------------------KPVSGP t DNHSF TDHGESTSKL
2O gi~1283478 LKGHI~NSSSVGGGNGGPSGGGGKPGIQH ~ DNHSF TDPGD-IREQ
gi~1285054 VR-__-__-________-_______LLITSC~ PWG--____________ gi~1283897 GR-________________________ GTS-___-__-________ gi~7494498 TRINAVVTFTCD--DDRYTLNGNKIIACQSTGVWNGTAP~CKEIPTCPEL
NOVlOa ASVT KD~GIPR----------------------------ALSLSGS
NOVlOb ASVT DKD~GIPR----------------------------ALSLSGS
gi~1577905 ASVT TLD'GIPR----------------------------ALSLSGS
3O gi11283478 AGVTH DKD' TFR--------------------------MGTPGPGGC
giI1285054 ____- ___________________________________________ gi~1283897 -______________________-________________,_________ gi~7494498 TPSSHVIPST~DNSVGAEVSFQCEDGYTLQGEKKITCLPTQKWSANPPSC
NOVlOa S S~QAQ PRQPLP---AS T PQQPAAYALG---------NOVlOb S S'QAQ PRQPLP---AS T PQQPAAYALG---------gi'1577905 S S'QAQ PRQPLP---AS T PQQPAAYALG-________ 40 gi11283478 S S'GTYVMVHAL ---------S P PGRPKVYLPG--------gi~1285054 _____-___-_______-________________________________ gi~1283897 ___________,______________________________________ g1~7494498 G~TSQPLSNND~GSGTKVGPIVG~IG~ILVIVLIIVATAILFWKLSS
The NOV 10 Clustal W alignment shown in Table l OF was modified to begin at amino residue 1600 and end at amino acid residue 2000. The data in Table 1 OF
includes all of the regions overlapping with the NOV 10 protein sequences.
The NOV 10 Clustal W alignment shown in Table l OG was modified to begin at amino residue 1601. The data in Table l OG includes all of the regions overlapping with the NOV 10 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uklinterpro~. Table lOH lists the domain description from DOMAIN analysis results against NOV10.
Table lOH
Domain Anal sis of NOV10 Model Region of Score (bits) E value Homology Sushi domain 19-78 15.8 0.0075 (SCR repeat) Consistent with other known members of the Selectin-like family of proteins, has, for example, a Sushi domain (SCR repeat) signature sequences and homology to other members of the Selectin-like Protein Family. NOV 10 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
For example, NOV10 nucleic acids and polypeptides can be used to identify proteins that are members of the Selectin-like Protein Family. The NOV 10 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 10 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular adhesion and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous I5 sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Diabetes, Autoimmune disease, Renal artery stenosis, lilterstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Systemic lupus erythematosus, Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
In addition, various NOV 10 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 10 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Selectin-like Protein Family.
The NOV 10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and immune physiology. As such, the NOV10 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and immune disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic ~0 stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Diabetes, Autoixnmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
The NOV 10 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 10 nucleic acid is expressed in Heart, Thyroid, Parotid Salivaxy glands, Liver, Colon, Ascending Colon, Bone Marrow, Peripheral Blood, Lymphoid tissue, Spleen, Lymph node, Tonsils, Thymus, Cerebellum, Spinal Chord, Cervix, Mammary glandBreast, Ovary, Placenta, Uterus, Oviduct/LTterine Tube/Fallopian tube, Vulva, Prostate, Testis, Lung, Kidney, Kidney Cortex, Retina, Skin.
Additional utilities for NOV 10 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 11 polypeptide has been identified as a Nucleax Protein-like protein (also referred to as CG94325-O1). The disclosed novel NOV11 nucleic acid (SEQ ID
NO:23) of 8670 nucleotides is shown in Table 11A. The novel NOV 11 nucleic acid sequences maps to the chromosome 15.
An ORF begins with an ATG initiation codon at nucleotides 204-206 and ends with a TAA codon at nucleotides 7152-7154. A putative untranslated region and/or downstream from the termination codon is underlined in Table 11A, and the start and stop codons are in bold letters.
Table 11A. NOVll Nucleotide Sequence (SEQ ID N0:23) ACGCGTAGAGCCGCTTTGCGCGTGCGCATCACCTAGGCGGTTAGATTTGAATACTTCACTGAGGCGA
GCCGGGCGTTGTGAGCGGACTGCTAGAGGCGGCTGTCTGTTTCCGCTCTAAGGAAACTCAGAGCGTG
TGGACCCCAAACAAGTCTGCGCAAAATTTGTCGAGGAGGTTTGCCGCGGCAGAAAAGTTTTCTTCAA
AAATGGATGGGGTGTCTTCAGAGGCTAATGAAGAAAATGACAATATAGAGAGACCTGTTAGAAGACG
GCATTCTTCAATATTGAAACCCCCAAGGAGTCCTCTTCAGGACCTCAGAGGTGGGAATGAAAGAGTT
CAGGAATCCAATGCTTTGAGAAATAAGAAAAACTCTCGTCGAGTCAGCTTTGCAGATACTATAAAGG
TATTCCAGACGGAGTCTCATATGAAAATAGTGAGAAAGTCAGAAATGGAAGAAACAGAAACAGGAGA
AAATCTTCTTTTGATACAGAATAAGAAATTAGAAGATAATTACTGTGAAATTACTGGGATGAACACA
TTGCTTTCTGCTCCCATTCATACCCAGATGCAACAGAAGGAGTTTTCAATTATAGAACATACCCGTG
AAAGGAAACATGCAAATGACCAGACAGTCATTTTTTCAGATGAAAACCAGATGGACCTGACATCAAG
$1 TCACACTGTAATGATTACCAAAGGCCTTTTAGATAATCCCATAAGTGAAAAGTCCACCAAGATAGAT
ACCACATCATTTCTAGCTAATTTAAAGCTTCACACCGAGGACTCAAGAATGAAAAAAGAAGTAAATT
TTTCCGTGGATCAAAACACTTCTTCAGAAAATAAAATAGATTTCAATGACTTCATAAAAAGATTGAA
AACAGGAAAATGTAGTGCTTTTCCTGATGTGCCTGATAAAGAAAATTTTGAGATACCTATTTATTCC
AAGGAACCGAACAGTGCCTCTTCTACACATCAAATGCATGTATCTCTTAAGGAAGATGAAAATAACA
GTAATATTACTAGGCTCTTTAGAGAAAAAGATGATGGGATGAATTTCACCCAGTGTCATACAGCCAA
TATTCAGACATTGATTCCCACATCCAGTGAGACCAACTCACGGGAATCTAAAGGTAATGATATTACA
ATTTATGGCAATGACTTTATGGACTTGACATTTAACCACACTTTGCAGATCTTACCTGCAACAGGTA
ATTTTTCTGAAATAGAAAATCAAACTCAGAATGCCATGGATGTAACAACAGGTTATGGAACTAAAGC
TTCAGGAAATAAAACAGTTTTTAAGAGTAAACAAAATACTGCTTTTCAAGACCTTTCCATAAACTCT
GCAGACAAAATACATATTACCAGAAGTCATATTATGGGGGCAGAAACTCACATAGTCTCACAGACTT
GTAATCAGGATGCCAGAATATTAGCCATGACCCCAGAATCTATATATTCTAATCCATCTATTCAAGG
TTGTAAGACTGTTTTCTATTCTAGTTGTAATGATGCCATGGAAATGACCAAATGTCTCTCAAATATG
AGAGAGGAGAAAAATTTGCTAAAGCATGACAGTAATTATTCTAAAATGTATTGCAATCCAGATGCTA
TGTCTTCTCTCACAGAGAAAACTATTTATTCCGGAGAGGAGAACATGGACATTACCAAGAGTCATAC
AGTTGCAATAGATAATCAAATTTTTAAACAAGATCAATCAAATGTGCAAATAGCAGCTGCACCAACA
CCCGAAAAAGAAATGATGCTCCAAAATCTTATGACCACATCAGAAGATGGGAAAATGAATGTAAATT
GTAACTCAGTTCCTCATGTATCTAAGGAAAGAATACAGCAGAGCCTGTCAAATCCTTTGTCTATTTC
ATTGACTGATAGAAAGACTGAACTCTTATCAGGTGAAAATACGGATTTGACTGAAAGTCACACAAGT
AACTTAGGAAGTCAGGTTCCTCTTGCAGCTTATAATCTAGCACCGGAGAGTACCAGTGAATCTCACT
CTCAGAGCAAAAGCTCTTCAGATGAATGTGAAGAAATTACCAAAAGTCGTAATGAACCATTTCAGCG
ATCAGACATAATAGCCAAAAACAGCTTAACCGACACCTGGAACAAAGACAAAGATTGGGTTTTGAAG
ATTTTGCCCTACCTTGATAAAGATTCTCCTCAGTCAGCTGATTGTAATCAGGAGATAGCAACAAGCC
ATAATATAGTCTACTGTGGTGGAGTTCTTGATAAACAAATAACTAATAGAAATACAGTATCATGGGA
ACAATCTTTGTTTTCTACCACAAAGCCATTATTTTCATCAGGACAGTTCTCTATGAAAAATCATGAT
ACTGCTATAAGTAGTCATACAGTGAAATCTGTACTAGGCCAGAATTCTAAACTGGCTGAGCCACTGA
GGAAAAGTTTAAGCAATCCCACACCTGACTATTGCCATGACAAGATGATTATATGTTCAGAGGAAGA
GCAAAATATGGATCTAACAAAGAGCCACACTGTCGTCATTGGATTTGGTCCTTCTGAACTACAAGAA
CTTGGTAAAACTAATTTAGAACACACTACTGGCCAGCTAACAACAATGAACAGACAGATAGCTGTAA
AAGTTGAAAAATGTGGTAAAAGTCCCATAGAAAAAAGTGGAGTGCTTAAATCTAACTGTATTATGGA
TGTGTTAGAGGACGAAAGTGTACAGAAACCTAAATTTCCAAAGGAAAAGCAAAATGTCAAAATTTGG
GGAAGGAAAAGTGTTGGTGGACCAAAAATTGATAAGACTATTGTATTTTCAGAAGACGATAAGAATG
ATATGGATATCACTAAGAGTTATACAATAGAAATAAACCATAGACCTTTATTAGAGAAACGTGATTG
TCATTTGGTGCCATTGGCAGGAACTTCTGAAACTATTTTATATACATGTGGGCAGGATGACATGGAG
ATCACTAGAAGTCACACAACTGCCTTAGAATGTAAAACTGTCTCACCAGATGAAATAACTACTAGGC
CTATGGACAAAACTGTAGTGTTTGTAGATAATCATGTTGAACTAGAAATGACAGAGTCCCATACTGT
TTTCATTGACTACCAAGAAAAGGAAAGAACAGACAGACCTAACTTTGAACTATCCCAAAGGAAAAGC
CTAGGAACACCAACAGTGATATGTACTCCTACTGAGGAGAGTGTTTTCTTTCCAGGAAATGGTGAAA
GTGACCGTCTAGTAGCAAATGACAGCCAGCTAACCCCTCTGGAGGAATGGTCTAATAATAGGGGCCC
TGTAGAGGTAGCTGATAACATGGAATTGTCTAAATCAGCCACTTGCAAAAACATCAAAGATGTACAA
AGTCCTGGATTTCTGAATGAACCTCTATCAAGCAAAAGTCAGAGAAGAAAAAGCCTTAAGCTAAAAA
ATGACAAGACCATTGTATTTTCAGAGAATCATAAAAATGATATGGATATTACCCAGAGTTGTATGGT
GGAAATAGATAACGAAAGTGCCCTGGAGGATAAAGAGGACTTCCATTTGGCAGGGGCTTCTAAAACT
ATTTTGTATTCATGTGGGCAGGATGACATGGAGATCACTAGGAGTCACACAACTGCCTTAGAATGTA
AAACTCTCCTGCCAAACGAAATAGCTATTAGGCCCATGGACAAAACCGTATTGTTCACAGATAATTA
CAGTGATCTGGAAGTCACCGATTCCCATACTGTTTTCATTGACTGTCAAGCCACAGAGAAAATACTT
GAAGAAAACCCTAAATTTGGAATAGGAAAAGGAAAAAACTTGGGTGTTTCCTTTCCTAAGGATAATA
GCTGTGTTCAAGAAATCGCTGAAAAACAAGCACTGGCTGTAGGAAACAAAATAGTTCTTCACACCGA
GCAAAAGCAACAACTCTTTGCTGCTACTAATAGAACTACTAATGAAATCATCAAATTTCATAGTGCT
GCTATGGATGAAAAGGTCATAGGGAAAGTTGTAGACCAGGCCTGTACATTGGAAAAAGCGCAAGTTG
AAAGCTGTCAGTTAAATAATAGAGATAGAAGAAATGTGGACTTTACAAGTAGTCATGCAACTGCTGT
TTGTGGATCCAGTGATAATTATTCCTGTTTACCAAATGTTATTTCCTGTACTGATAATTTGGAGGGT
AGTGCCATGCTCTTATGTGATAAAGATGAGGAAAAAGCCAATTATTGCCCAGTGCAAAATGATCTTG
CTTATGCAAATGATTTTGCCAGTGAATATTACTTGGAATCTGAGGGACAGCCTCTCTCTGCTCCTTG
TCCTTTGTTAGAGAAGGAAGAAGTTATTCAAACCAGTACCAAAGGACAGTTAGACTGTGTTATAACA
CTGCACAAAGATCAAGATCTGATTAAGGATCCACGAAATCTATTGGCTAATCAAACTTTAGTATATA
GTCAAGATCTGGGGGAGATGACTAAACTTAATTCAAAGCGAGTATCTTTTAAGCTTCCAAAGGATCA
AATGAAAGTCTATGTTGATGACATTTATGTTATTCCTCAGCCTCATTTCTCAACCGACCAACCTCCA
TTACCTAAAAAAGGACAGAGTAGTATCAATAAAGAAGAAGTAATACTGTCTAAAGCTGGAAATAAGA
GTTTAAATATTATAGAAAATTCCTCTGCACCCATATGTGAAAACAAGCCCAAAATACTCAATAGTGA
GGAATGGTT'T'GCTGCAGCCTGTAAAAAAGAACTGAAGGAAAATATTCAAACAACTAACTATAATACA
GCTCTAGATTTCCACAGTAACTCAGACGTAACTAAGCAAGTCATTCAAACTCATGTCAATGCTGGAG
AAGCACCAGATCCTGTAATTACATCTAATGTTCCATGTTTTCATAGTATCAAACCAAATCTGAATAA
TTTGAATGGAAAAACTGGAGAGTTTTTAGCCTTTCAAACTGTTCATCTACCACCCCTTCCAGAGCAA
TTACTTGAATTAGGAAATAAGGCACACAATGATATGCATATAGTGCAAGCTACAGAAATACATAATA
TTAACATAATCTCCAGCAATGCTAAAGATAGTAGAGATGAGGAAAATAAAAAGTCTCATAATGGAGC
TGAAACCACCTCTCTACCGCCAAAGACAGTTTTTAAAGATAAAGTAAGGAGATGTTCTTTGGGAATC
TTTTTGCCTAGATTGCCCAACAAGAGAAATTGTAGTGTCACTGGTATTGATGACCTGGAACAGATTC
CAGCAGACACAACTGATATAAATCACTTAGAAACTCAGCCGGTCTCTAGCAAAGATTCAGGCATTGG
ATCTGTTGCAGGTAAACTGAACCTAAGTCCTTCTCAATATATAAATGAGGAAAATCTTCCTGTATAT
CCTGATGAGATCAATTCTTCAGACTCTATTAACATAGAAACTGAGGAAAAGGCCTTGATTGAGACAT
ACCAAAAAGAGATTTCACCATATGAAAATAAAATGGGAAAAACTTGCAATAGCCAAAAAAGAACGTG
GGTACAAGAAGAAGAAGATATTCATAAGGAGAAAAAAATCAGAAAAAATGAGATTAAGTTTAGTGAT
ACGACACAAGATCGGGAGATTTTTGATCACCATACTGAAGAGGATATAGATAAAAGTGCTAACAGTG
TATTGATAAAAAACCTGAGCAGGACCCCATCTAGTTGCAGCAGCTCTCTGGATTCAATCAAGGCTGA
TGGGACCTCTCTGGACTTCAGCACTTACCGCAGTAGTCAAATGGAATCACAGTTTCTCAGAGATACT
ATTTGTGAAGAGAGCTTGAGGGAGAAACTCCAAGATGGGAGAATAACAATAAGGGAGTTCTTTATAC
TTCTCCAGGTCCACATCTTGATACAGAAACCCCGACAGAGCAATCTCCCAGGCAATTTTACTGTAAA
CACACCACCTACTCCAGAAGACCTGATGTTAAGTCAATATGTTTACCGACCCAAGATACAGATTTAT
AGAGAAGATTGTGAGGCTCGTCGCCAAAAGATTGAAGAATTAAAGCTTTCTGCATCGAACCAAGATA
AGCTGTTGGTTGATATAAATAAGAACCTGTGGGAAAAAATGAGACACTGCTCTGACAAAGAGCTGAA
GGCCTTTGGAATTTATCTTAACAAAATAAAGTCATGTTTTACCAAGATGACTAAAGTCTTCACTCAC
CAAGGAAAAGTGGCTCTGTATGGCAAGCTGGTGCAGTCAGCTCAGAATGAGAGGGAGAAACTTCAAA
TAAAGATAGATGAGATGGATAAAATACTTAAGAAGATCGATAACTGCCTCACTGAGATGGAAACAGA
AACTAAGAATTTGGAGGATGAAGAGAAAAACAATCCTGTGGAAGAATGGGATTCTGAAATGAGAGCT
GCAGAAAAAGAATTGGAACAGCTGAAAACTGAAGAAGAGGAGCTTCAAAGAAATCTCTTAGAACTGG
AGGTACAAAAAGAGCAGACCCTTGCTCAAATAGACTTTATGCAAAAACAAAGAAATAGAACTGAAGA
GCTACTGGATCAGTTGAGCTTGTCTGAGTGGGATGTCGTTGAGTGGAGTGATGATCAAGCTGTATTC
ACCTTTGTTTATGACACGATACAACTCACCATCACCTTTGAAGAGTCAGTTGTTGGTTTCCCTTTCC
TGGACAAGCGTTATAGGAAGATTGTTGATGTCAATTTTCAATCTCTGTTAGATGAGGATCAAGCTCC
TCCTTCCTCCCTTTTAGTTCATAAGCTTATTTTCCAGTACGTTGAAGAAAAGGAATCCTGGAAGAAG
ACATGTACAACCCAGCATCAGTTACCCAAGATGCTTGAAGAATTCTCACTGGTAGTGCACCATTGCA
GACTCCTTGGAGAGGAGATTGAGTATTTAAAGAGATGGGGACCAAATTATAACCTAATGAACATAGA
TATTAATAAT.AATGAATTGAGACTTTTATTCTCTAGCTCCGCAGCATTTGCAAAGTTTGAAATAACT
TTGTTTCTCTCAGCCTATTATCCATCTGTACCATTACCTTCCACCATTCAGAATCACGTTGGGAACA
CTAGCCAAGATGATATTGCTACCATTCTATCTAAAGTGCCACTGGAGAACAACTACCTGAAGAATGT
AGTCAAGCAAATTTACCAAGATCTGTTTCAGGACTGCCATTTCTACCACTAGACCCTTGGACCACCA
TTGGAACAACCAAGCAGAATGTACTTGATATTATTTCAGGGTCCCATTGCTGTTCAGCCTTTGTTTT
TACGTCATTACAAGCTGAGTAAAATTCCTTCTGATGATGTTATAGTTAATCTGTATGTTTTTTATAT
CTCTGCAGAATGATGGTGATGAAGTCTGGATGGTAGGCCTCATAGCCTACTATCAACTTACTCATCT
TTGTACCAAAGGTTTAAGTAATAGGACACTTAGGAAAAATGTCTCCTAACTAAACTAGTGCTTTCTG
CTTTAGTACAAGCCCTAAGGATTAACTTAAGTATAAGAAGTGTTATCACTGACAAGAACATTAGCCA
TTTTCCCATAACTAGATAGAGCTATGATTTTTTAGGTTGCCTGGCTTCTGCCTAGCAGATATTTCTG
GAGTAGAAATGTATCTGTCTACAAACTATTATCCTTTTTCTCCGTTACTAAAATGCTATTAAGAGAA
AGTAGGGCTGGGTGTGAGCCACCACACCCAGCAATGTTTTCTTAATAAGTATAGTTTTTCTAGGGAA
AGTTAATTCATTTTTGTCTAGTACATATATGTAAATATATTAATGTTGTTTTTGTGTTTGTGATGTA
GTAAGGAGATGTACATAGAAATTCATTGAGGTATATAGATACTCATCTGTCTAGGCAGTTCCCAATT
TTCTGAAGAATGTTTTACAGCAAAATTTTCTATTTTCTTTTATTAAATAGTGACACGTCAAACAATG
TCACATCCAAAACACTAGTTTCATCAATTTCTAGCAGTAATAATAGACTTGCTGTAAGTATTGTTTT
CTGATGCCATACCCTTGTCATACATATTATTAAATGACCAATATTATGTATGAAGTAGACAAAAAAA
TTTACTCAAACTTCATTCAAATCCTAATTGTGATAATTTTTGTTTTATATTTAATTATAAACCAAAA
TACATTTGCATTTTTAAGCTAATTTGTCTCAAAATTTTGCTTTATATTTTTGGATCAGGTTAAAGTC
CTGTGGATCCCCTGAATGTTATTGTCCCTCTTGATTGGTTTTTACTTCTGAGCTATACGTCAAAAGA
CACATAAGCTTCAAAAGTCAAGACAAACCTCATTTGCCATAAAAATCAAGATATAGATGTTCTGTTC
CGTAAACTCCTTGAAAAACATTTTAAAGTCATCAATATGATCTGTTTCCCATGAAACTTAAGTTAGC
TTTCTTATTGGAGTTATTTCTTTTCTGTAAGTCTGAAAAGTAGAGATTTTGTTTTACGCATTTTAGT
AACCTGCAACAACCAACTCTAAAAA.AGATTTGGCTTGTAATGACGGTCTCTGCTTTTTTGGGTTTGG
AGTACACAATTGTAATATTTACTTAGTTATTTGTGTTTTTCTTTGTTCAAGGTATTGACTAGTTTCA
TAAATTTTTTGCAAGTTTTTCTTTCATTGGTTGGAAAGCAGATTACATTTTGCACTATTAAAATAAG
TTTATTACTTTP,AAAP~AAGTCGACG
Variant sequences of NOV11 are included in Example 3, Table 23. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 11 protein (SEQ m N0:24) encoded by SEQ m N0:23 is 2316 amino acid residues in length and is presented using the one-letter amino acid code in Table 11B. Psort analysis predicts the NOV 11 protein of the invention to be localized at the nucleus with a certainty of 0.8800.
Table 11B. Encoded NOVll protein sequence (SEQ ID N0:24) MDGVSSEANEENDNIERPVRRRHSSILKPPRSPLQDLRGGNERVQESNALRNKKNSRRVSFADTI
KVFQTESHMKIVRKSEMEETETGENLLLIQNKKLEDNYCEITGMNTLLSAPIHTQMQQKEFSIIE
HTRERKHANDQTVIFSDENQMDLTSSHTVMITKGLLDNPISEKSTKIDTTSFLANLKLHTEDSRM
KKEVNFSVDQNTSSENKIDFNDFIKRLKTGKCSAFPDVPDKENFEIPIYSKEPNSASSTHQMHVS
LKEDENNSNITRLFREKDDGMNFTQCHTANIQTLIPTSSETNSRESKGNDITIYGNDFMDLTFNH
TLQILPATGNFSEIENQTQNAMDVTTGYGTKASGNKTVFKSKQNTAFQDLSINSADKIHITRSHI
MGAETHIVSQTCNQDARILAMTPESIYSNPSIQGCKTVFYSSCNDAMEMTKCLSNMREEKNLLKH
DSNYSKMYCNPDAMSSLTEKTIYSGEENMDITKSHTVAIDNQIFKQDQSNVQIAAAPTPEKEMML
QNLMTTSEDGKMNVNCNSVPHVSKERIQQSLSNPLSISLTDRKTELLSGENTDLTESHTSNLGSQ
VPLAAYNLAPESTSESHSQSKSSSDECEEITKSRNEPFQRSDIIAKNSLTDTWNKDKDWVLKILP
YLDKDSPQSADCNQEIATSHNIVYCGGVLDKQITNRNTVSWEQSLFSTTKPLFSSGQFSMKNHDT
AISSHTVKSVLGQNSKLAEPLRKSLSNPTPDYCHDKMIICSEEEQNMDLTKSHTWIGFGPSELQ
ELGKTNLEHTTGQLTTMNRQIAVKVEKCGKSPIEKSGVLKSNCIMDVLEDESVQKPKFPKEKQNV
KIWGRKSVGGPKIDKTIVFSEDDKNDMDITKSYTIEINHRPLLEKRDCHLVPLAGTSETILYTCG
QDDMEITRSHTTALECKTVSPDEITTRPMDKTVVFVDNHVELEMTESHTVFIDYQEKERTDRPNF
ELSQRKSLGTPTVICTPTEESVFFPGNGESDRLVANDSQLTPLEEWSNNRGPVEVADNMELSKSA
TCKNIKDVQSPGFLNEPLSSKSQRRKSLKLKNDKTIVFSENHKNDMDITQSCMVEIDNESALEDK
EDFHLAGASKTILYSCGQDDMEITRSHTTALECKTLLPNEIAIRPMDKTVLFTDNYSDLEVTDSH
TVFIDCQATEKILEENPKFGIGKGKNLGVSFPKDNSCVQEIAEKQALAVGNKIVLHTEQKQQLFA' ATNRTTNEIIKFHSAAMDEKVIGKVVDQACTLEKAQVESCQLNNRDRRNVDFTSSHATAVCGSSD
NYSCLPNVISCTDNLEGSAMLLCDKDEEKANYCPVQNDLAYANDFASEYYLESEGQPLSAPCPLL
EKEEVIQTSTKGQLDCVITLHKDQDLIKDPRNLLANQTLVYSQDLGEMTKLNSKRVSFKLPKDQM
KVYVDDIYVIPQPHFSTDQPPLPKKGQSSINKEEVILSKAGNKSLNIIENSSAPICENKPKILNS
EEWFAAACKKELKENIQTTNYNTALDFHSNSDVTKQVIQTHVNAGEAPDPVITSNVPCFHSIKPN
LNNLNGKTGEFLAFQTVHLPPLPEQLLELGNKAHNDMHIVQATEIHNINIISSNAKDSRDEENKK
SHNGAETTSLPPKTVFKDKVRRCSLGIFLPRLPNKRNCSVTGIDDLEQIPADTTDINHLETQPVS
SKDSGIGSVAGKLNLSPSQYINEENLPVYPDEINSSDSINIETEEKALIETYQKEISPYENKMGK
TCNSQKRTWVQEEEDIHKEKKIRKNEIKFSDTTQDREIFDHHTEEDIDKSANSVLIKNLSRTPSS
CSSSLDSIKADGTSLDFSTYRSSQMESQFLRDTICEESLREKLQDGRITIREFFILLQVHILIQK
PRQSNLPGNFTVNTPPTPEDLMLSQYVYRPKIQIYREDCEARRQKIEELKLSASNQDKLLVDINK
NLWEKMRHCSDKELKAFGIYLNKIKSCFTKMTKVFTHQGKVALYGKLVQSAQNEREKLQIKIDEM
DKILKKIDNCLTEMETETKNLEDEEKNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQK
EQTLAQIDFMQKQRNR'T'EELLDQLSLSEWDVVEWSDDQAVFTFVYDTIQLTITFEESVVGFPFLD
KRYRKIVDVNFQSLLDEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLVVHHC
RLLGEEIEYLKRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHV
GNTSQDDIATILSKVPLENNYLKN~TVKQIYQDLFQDCHFYH
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 11 C.
Table 11C. results Pat fox NOVll Smallest Sum eading igh Prob equences igh-scoringegment Frame ScoreP(N) producing Pairs:
>patp:AAW8839.8Humantestis +1 2444 1.7e-253 secreted protein dol5 4 >patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253 >patp:AAU71961Humanbone marrowtissuepolypeptide#39+1 2444 1.7e-253 >patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253 >patp:AAU71961Humanbone marrowtissuepolypeptide#39+l 2444 1.7e-253 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 5584 of 5584 bases (100%) identical to a gb:GENBANK-m:AB046790~acc:AB046790.1 mRNA from Homo Sapiens (mRNA for KTA_A_1570 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 1790 of 1793 amino acid residues (99%) identical to, and 1792 of 1793 amino acid residues (99%) similar to, the 1833 amino acid residue ptnr:SPTREMBL-ACC:Q9NR92 protein from Horno sapieras (AF15Q14 PROTEIN).
NOV11 also has homology to the proteins shown in the BLASTP data in Table 11D.
Table 11D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) giI18308012~gb~AAL6AF15q14 isoform2316 2316/23162316/23160.0 7803.1~AF461041 [Homo Sapiens] (100%) (100%) 1(A
F461041) gi~9966807~refINPAF15q14 protein1833 1790/17931792/17930.0 _ [Homo Sapiens] (99%) (99%) 65113.1~(NM_020380) gi~14749154Iref~XP_AF15q14 protein1833 1789/17931791/17930.0 031524.1~(XM [Homo Sapiens] (99%) (99%) giI10047205~dbj~BABKIAA1570 protein1360 1360/13601360/13600.0 13396.1~(AB046790)[Homo Sapiens] (100%) (100%) gi~14749150~ref~XPsimilar to 915 900/900 900/900 0.0 _ KIAA1570 protein (100%) (100%) 012461.3 (XM 012461) [Homo Sapiens]
A multiple sequence alignment is given in Table 11E, with the NOV11 protein being shown on line 1 in Table 11E in a ClustalW analysis, and comparing the NOV11 protein with the related protein sequences shown in Table 11D. This BLASTP data is displayed graphically in the ClustalW in Table 11E.
Table 11E. ClustalW Analysis of NOVll 1) > NOVl 1; SEQ JD N0:24 2) > gi~18308012~/ AF15q14 isoform 2 [Homo Sapiens]; SEQ ID N0:82 3) > gi~9966807~/ AF15q14 protein [Homo Sapiens]; SEQ ID N0:83 4) > gi~14749154~/ AF15q14 protein [Homo Sapiens]; SEQ >D N0:84 5) > gi~10047205~/ KIAA1570 protein [Homo Sapiens]; SEQ >D N0:85 6) > gi~14749150~/ similar to KIAA1570 protein [Hoyno Sapiens]; SEQ >D N0:~6 .(. .( . .(..(. .~. .(..( .(.
NOV11 1 1 r 1 ~ 60 gi(18308012(1 1 1 1 ~ 60 gi(9966807(1 1 1 1 ~ 60 gi(14749154(1 1 1 1 ' ~ 60 gi(10047205(1 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ gi(14749150(1 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ ( ( ( ( ( ( ( ( ( ' ( ( gi(18308012(61 120 gi(9966807(6l gi(14749154(61 gi(1004720511 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ gi(14749150~1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ ( ( ( . (. .I. .( I. y .( ( ( .~ .
gi~183080121121 180 gi~9966807~121 gi(14749154(121 180 gi~10047205(1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ gi(14749150~1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( ( ( ( ( ( ( ( ( ( ( ( gi(18308012(181 240 gi~9966807(181 240 gi(14749154(181 240 gi(10047205(1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ gi(14749150(1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( . ( .( y .(.I ~ .( y y .
gi(183080121 241 300 gi(9966807( 241 45 gi(14749154( 241 300 giI10047205( 1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ gi(147491501 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ 50 ( gi(18308012( 301 360 gi(9966807( 301 gi(14749154( 301 360 gi(10047205~ 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ gi(14749150( 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( .( .( ( .(.. ( gi(18308012(361 gi(9966807(361 gi(14749154(361 g1(10047205(1 1 6$ _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ giI14749150(1 1 ( ( ( ( ( NOV11 421 .( . . ~.. . . .(. .(..(. . . .(..
.(. . 1 . . r 1 . . .. 480 8~ i1' ~6 1 v v 1 I v . y~ 11 1 1I'Il1 / '1 1 1 1 1 1 1 ! 1 1 1 r o v I Ir r 1 a 1 ~ -11 1 1 1 r 1 ,~ I 11 1 1 1 9~ I 11 1 1 11 ~ I ~ 1W 1 1 ~1 a 1 r 1 11 y 1 v 1 11 I v v .(. .(.. .(. (. . .(.
.(. . .( .(. .(..
.~.
.( r 1 1 v 1. v IvI 1 1 1 1 1 . 1 , 1 W 11 1 y1.
~
1 11 1 v ~~i1 gi(18308012( 421 m 1 1 SI '1' 480 gi(9966807( 42l v . 1 1 1 1 SI 1 480 gi (14749154( 421 . 1 1 ~ 1 1 1 1 480 gi(10047205( 1 ______ _____ ___________ ____ ________ ____ _____ _____ ____ ____ 1 _____ gi(14749150( 1 ______ _____ ___________ ____ ________ ________ _____ _____ ____ ____ 1 gi(18308012( 481 540 gi (9966807( 481 540 gi(14749154( 481 540 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ( (. .( .( (. .(. .(. . ( .( NOV11 541 Iv . 600 T
gi(18308012( 541 I TI i . 600 gi(9966807( 541 1 TI
gi(14749154( 541 1 ' ' 600 gi(10047205( 1 -_____ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 (..( ~ ~. .( . ~ ~..( . ( gi(18308012~ 601 660 gi(9966807( 601 660 30 gi(14749154( 601 660 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ _ gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(18308012( 661 720 gi(9966807( 661 720 gi(14749154( 661 720 40 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ___ gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 (. .( .(. ( .( ( (. ( .(. .
.(. . .( giI18308012( 721 780 gi~9966807( 721 780 gi(14749154( 721 780 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ( ( .~ .(. ( (. ( ( ~. .( .~.
5$ gi(18308012( 781 840 gi(9966807( 781 840 gi(14749154( 781 840 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ~ ( ( ( ( NOV11 841 .. .(. .( . .~. .(. . . . . . . . .(.. 900 .( . . . .
gi(18308012( 841 900 65 gi(9966807( 841 900 gi(14749154( 841 900 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 g7 r ~ 1 1 1 1 1 ~I' v v ~ 0 r .(. .(. . . (.. . .(..
.~ .(. . .(. .(. .( .(.
.( 1 v v 1 ~ I 1 n 1 ~ 1 1 1 1 t' 1 ~ ' 1 '1 1 1 ' ~ r 1 giI10047205~ 845r v ~ ~~ v v' 904 ~ ~ v ~
gi~14749150~ 768~ ' ' ~ '~ ~ v 827 ~ v v ~
S y .y ~
~
y .
~.
y y ~.
~
y r -NOV11 1861 ~~ ~ ~ ~ '~ ~ 1920 ~
gi~183080121 1861 ~~ ~ ~ ~ ~ ~ 1920 ~
gi~9966807~ 1817_________________yL
S_____________G_______________ Q I 1832 S
gi~14749154~ 1817-________________~~L .S_-___________G_______________ Q I 1832 S
gi~10047205~ 905 ~~ ~ ~~ ~ ~ ~ 964 gi~14749150~ 828 ~~ ~ ~~ ~ ~ ~ 887 IS NOV11 1921. ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELICAFGIYLNKIKSCFTK
t gi~18308012~ 1921 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
~
a gi~9966807~ 1833T-__________________________________________________________ gi~14749154~ 1833T-__________________________________________________________ gi~10047205~ 965 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
~
gi~14749150~ 888 ~ y8____________________________________________ v T-~
AL'~'G
QSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
ZS gi~18308012~ 1981MT T ~Q~.~L"~'GQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
gi~9966807~ 1833___________________________________________________________ gi~14749154~ 1833___________________________________________________________ gi~10047205~ 1025MTK T LGKQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
G
gi~14749150~ 903~ ~ ~ 915 L~~"""T 1~C~
I' ________________________________________ gi~18308012~ 2041KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
3S gi~9966807~ 1833-___________________________________________________________ gi~14749154~ 1833____________________________________________________________ gi~10047205~ 1085KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
gi~14749150~ 915___________________________________________________________ gi~183080121 2101LDQLSLSEWDWEWSDDQAVFTFVYDTIQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
gi~9966807~ 1833-___________________________________________________________ 4S gi~14749154~
1833____________________________________________________________ gi~10047205~ 1145LDQLSLSEWDWEWSDDQAVFTFVYDTTQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
gi~14749150~ 915____________________________________________________________ gi.~18308012~ 2161DEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLWHHCRLLGEEIEYL
gi~9966807~ 1833-___________________________________________________________ gi~14749154, 1833____________________________________________________________ SS gi~10047205~
gi~14749150~ 915____________________________________________________________ gi I183080121 giI9966807~ 1833____________________________________________________________ gi~14749154~ 1833____________________________________________________________ gi.'10047205~
6S gi~14749150~
915____________________________________________________________ ....~....~....~....~....~....~....~.
gi~18308012~ 2281DDIATILSKVPLENNYLKNWKQIYQDLFQDCHFYH
giI9966807~ 1833 ____________________________________ 1833 gi~14749154~ 1833 ____________________________________ 1833 gi~100472051 1325 DDIATILSKVPLENNYLKNWICQIYQDLFQDCHFYH 1360 gi~14749150~ 915 ____________________________________ 915 NOV 11 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 11 nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOV 11 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV11 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, Graft vesus host, Cardiovascular diseases, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, as well as other diseases, disorders and conditions.
In addition, various NOV 11 nucleic acids and polypeptides according to the invention are useful, inter olio, as novel members of the protein families according to the presence of sequence relatedness to previously described proteins. The NOV 11 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune, and nerve physiology. As such, the NOV 11 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and nervous system disorders, e.g., Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, Graft vesus host, Cardiovascular diseases, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, as well as other diseases, disorders and conditions.
The NOV11 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 11 nucleic acid is expressed in Adipose, Aorta, Artery, Coronary Artery, Umbilical Vein, Thyroid, Liver, Small Intestine, Duodenum, Colon, Ascending Colon, Bone Marrow, Lymph node, Tonsils, Thymus, Cartilage, Muscle, Brain, Cervix, Uterus, Vulva, Prostate, Testis, Lmig, Bronchus, Urinary Bladder, Kidney, Skin, Epidermis, Dermis.
Additional utilities for NOV 11 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 12 polypeptide has been identified as a Plasma Membrane Protein-like protein (also referred to as CG94282-O1). The disclosed novel NOV12 nucleic acid (SEQ
ID N0:25) of 8811 nucleotides is shown in Table 12A. The novel NOV 12 nucleic acid sequences maps to the chromosome 12.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TAG
codon at nucleotides 4378-4380. A putative untranslated region and/or downstream from the termination codon is underlined in Table 12A, and the start and stop, codons are in bold letters.
Table 12A. NOV12 Nucleotide Sequence (SEQ ID N0:25) ATGCTGTTCAAGCTCCTGCAGAGACAAACCTATACCTGCCTGTCCCACAGGTATGGGCTCTACGTGTGCTTCTT
GGGCGTCGTTGTCACCATCGTCTCCGCCTTCCAGTTCGGAGAGTGGGTAGAAGCCAGGGATCCTGCCAAACATC
CTATAGTGCACAGGACAGCCCCTACAACAAAGAATCATCCAGCCCAAAATGTCGATAGTGCTGAAGTTGAGAAA
TCCGGAATTAGAAGGGGCAAGAATGGCTGCAGGGCAGTTAGTCTACAGGACTGGCCTGGGACTAGAGGATGTGC
CAATTTCACCTTCGCCTTCTGCCATGATTGTAAGTTTTCTGAGGTCTCCCAGAAACGCTTCCTGTACATCCTGC
AGAACTGTCATTGGTTAACTGATTGGGGTTGGACTTGGTTGGCTCTGCTCCACGGGTCTCTCATCCTCCAGGGA
CCAGCCAGCGAACCTGGTTGTGTTCTTCTCAAGGCAAAGGTGGTTCTGGAATGGAGCCGAGATCAATACCATGT
TTTGTTTGATTCCTATAGAGACAATATTGCTGGAAAGTCCTTTCAGAATCGGCTTTGTCTGCCCATGCCGATTG
ACGTTGTTTACACCTGGGTGAATGGCACAGATCTTGAACTACTGAAGGAACTACAGCAGGTCAGAGAACAGATG
GAGGAGGAGCAGAAAGCAATGAGAGAAATCCTTGGGAAAAACACAACGGAACCTACTAAGAAGAGTGAGAAGCA
GTTAGAGTGTTTGCTAACACACTGCATTAAGGTGCCAATGCTTGTCCTGGACCCAGCCCTGCCAGCCAACATCA
CCCTGAAGGACCTGCCATCTCTTTATCCTTCTTTTCATTCTGCCAGTGACATTTTCAATGTTGCAAAACCAAAA
AACCCTTCTACCAATGTCTCAGTTGTTGTTTTTGACAGTACTAAGGATGGGACATTGCTCACTCAGAAGGTGAC
TTTTGAGTGGAAATGTGAAGAAGGTGAGGTAGCCAGCAATGCGAATATCTGGGGAAAGACTGATCTGGGTTCCC
CCAGGAGGCCTTTGCCATGGCCTGTGGCCCTGGAGCCACCTAGGGCTCAGCTCAGCTCTGCCCTACAGATTCTC
ACTAGGCCACGGGTATCTCAGGACAGAGCCAACACAAGTTATGAAATTAAACTAGACACACCCCTTCTTCGAGG
TTACGCCAAGCCAGTGCCTGGGCCTGAAACTGGCCTGCAGCCCCTCAGCTTCGCCCACTGCCTTCCGACCCTGG
ACCTTCGCAAAGTGAACGAGCTTCGGGACTTCGTGAAAATGTATAAGCAGGATCCGAGCATTCTGCATACCAAG
GAAACGTGCTTTCTGAGGGAGCAGGTGGAGAGCATGGGGGAAAGCTATTATAAATCAGAAGAAAATATCAAGGA
ATTAAAAACAGGTAGTAAGAAGGTGGAGGAAAACATAAGCACAGACGAACTATCAAGTGAGGAAAGTGATCTAG
AAATTGATAACGAAGCTGTGATTGAACCAGACACTGATTCCCCTCAAGAAATGGGAGATGGAGAGGCCAGTGTA
GCGCTTCTAAAACTGAATAACCCCAAGGATTTTCAAGAATTGAATAAGCAAACTAAGAAGAACATGACCATTGA
TGGAAAAGAACTGACCATAAGTCCTGCATATTTATTATGGGATCTGAGCGCCATCAGCCAGTCTAAGCAGGATG
AAGACATCTCTGCCAGTCGTTTTGAAGATAACGAAGAACTGAGGTACTCATTGCGATCTATCGAGAGGCATGCA
CCATGGGTTCGGAATATTTTCATTGTCACCAACGGGCAGATTCCATCCTGGCTGAACCTTGACAATCCTCGAGT
GACAATAGTAACACACCAGGATGTTTTTCGAAATTTGAGCCACTTGCCTACCTTTAGTTCACCTGCTATTGAAA
GTCACATTCATCGCATCGAAGGGCTGTCCCAGAAGTTTATTTACCTAAATGATGATGTCATGTTTGGGAAGGAT
GTCTGGCCAGATGATTTTTACAGTCACTCCAAAGGCCAGAAGGTTTATTTGACATGGCCTGTGCCAAACTGTGC
CGAGGGCTGCCCAGGTTCCTGGATTAAGGATGGCTATTGTGACAAGGCTTGTAATAATTCAGCCTGCGATTGGG
ATGGTGGGGATTGCTCTGGAAACAGTGGAGGGAGTCGCTATATTGCAGGAGGTGGAGGTACTGGGAGTATTGGA
GTTGGACAGCCCTGGCAGTTTGGTGGAGGAATAAACAGTGTCTCTTACTGTAATCAGGGATGTGCGAATTCCTG
GCTCGCTGATAAGTTCTGTGACCAAGCATGCAATGTCTTGTCCTGTGGGTTTGATGCTGGCGACTGTGGGCAAG
AAAACTCAGACTCAAAGAATAGGAAAACAGAGGAAAAATGCCCAGTT TCATGTTTCTGTTT
TTTCCTCTAGATCATTTTCATGAATTGTATAAAGTGATCCTTCTCCCAAACCAGACTCACTATATTATTCCAAA
AGGTGAATGCCTGCCTTATTTCAGCTTTGCAGAAGTAGCCAAAAGAGGAGTTGAAGGTGCCTATAGTGACAATC
CAATAATTCGACATGCTTCTATTGCCAACAAGTGGAAAACCATCCACCTCATAATGCACAGTGGAATGAATGCC
ACCACAATACATTTTAATCTCACGTTTCAAAATACAAACGATGAAGAGTTCAAAATGCAGATAACAGTGGAGGT
GGACACAAGGGAGGGACCAAAACTGAATTCTACAGCCCAGAAGGGTTACGAAAATTTAGTTAGTCCCATAACAC
TTCTTCCAGAGGCGGAAATCCTTTTTGAGGATATTCCCAAAGAAAAACGCTTCCCGAAGTTTAAGAGACATGAT
GTTAACTCAACAAGGAGAGCCCAGGAAGAGGTGAAAATTCCCCTGGTAAATATTTCACTCCTTCCAAAAGACGC
CCAGTTGAGTCTCAATACCTTGGATTTGCAACTGGAACATGGAGACATCACTTTGAAAGGATACAATTTGTCCA
AGTCAGCCTTGCTGAGATCATTTCTGATGAACTCACAGCATGCTAAAATAAAAAATCAAGCTATAATAACAGAT
GAAACAAATGACAGTTTGGTGGCTCCACAGGAAAAACAGGTTCATAAAAGCATCTTGCCAAACAGCTTAGGAGT
GTCTGAAAGATTGCAGAGGTTGACTTTTCCTGCAGTGAGTGTAAAAGTGAATGGTCATGACCAGGGTCAGAATC
CACCCCTGGACTTGGAGACCACAGCAAGATTTAGAGTGGAAACTCACACCCAAAAAACCATAGGCGGAAATGTG
ACAAAAGAAAAGCCCCCATCTCTGATTGTTCCACTGGAAAGCCAGATGACAAAAGAAAAGAAAATCACAGGGAA
AGAAAAAGAGAACAGTAGAATGGAGGAAAATGCTGAAAATCACATAGGCGTTACTGAAGTGTTACTTGGAAGAA
AGCTGCAGCATTACACAGATAGTTACTTGGGCTTTTTGCCATGGGAGAAAAAAAAGTATTTCCAAGATCTTCTC
GACGAAGAAGAGTCATTGAAGACACAATTGGCATACTTCACTGATAGCAAAAATACTGGGAGGCAACTAAAAGA
TACATTTGCAGATTCCCTCAGATATGTAAATAAAATTCTAAATAGCAAGTTTGGATTCACATCGCGGAAAGTCC
CTGCTCACATGCCTCACATGATTGACCGGATTGTTATGCAAGAACTGCAAGATATGTTCCCTGAAGAATTTGAC
AAGACGTCATTTCACAAAGTGCGCCATTCTGAGGATATGCAGTTTGCCTTCTCTTATTTTTATTATCTCATGAG
TGCAGTGCAGCCACTGAATATATCTCAAGTCTTTGATGAAGTTGATACAGATCAATCTGGTGTCTTGTCTGACA
GAGAAATCCGAACACTGGCTACCAGAATTCACGAACTGCCGTTAAGTTTGCAGGATTTGACAGGTCTGGAACAC
ATGCTAATAAATTGCTCAAAAATGCTTCCTGCTGATATCACGCAGCTAAATAATATTCCACCAACTCAGGAATC
CTACTATGATCCCAACCTGCCACCGGTCACTAAAAGTCTAGTAACAAACTGTAAACCAGTAACTGACAAAATCC
ACAAAGCATATAAGGACAAAAACAAATATAGGTTTGAAATCATGGGAGAAGAAGAAATCGCTTTTAAAATGATT
CGTACCAACGTTTCTCATGTGGTfiGGCCAGTTGGATGACATAAGAAAAAACCCfiAGGATCTCACTCTGTTGTCC
AAGCTGGAATGCAGTAATGCAAACATGGCTCACTGTAGCCTCGACCTCGTGGGCTCAAGCAATCCTCCCACCTC
AGCCTCCTGACTAGTGGAACCACAGACATGAGCTGCTGCACCCAGCTAAAATGGAGTATTTTTAATTTCTGGGT
CTTTTAAATGCATTTGGAGGTCTfifiAGTTTTACCTCACTGAAATTAGGATTTTAATTATAAATAATCAAAGATG
TGAACCTTACAGACATTTTAAAGCCATTATATTTTTTCTATAAACCCTGTTCTCGTTTGGAGGAGAAAGAAATT
GGAATTTTCP~AAA~1AAATAAAAATACCTTTAACACCTATTTAGTGTCTTTAGTAATCCAGTAAAATACTTGATT
TTTTACTAAATGTTTCCCACAAGCCAAGCAAACCATAAGCTACAATAATAAfiTACCTAGCGTACAGCCCTCTTT
GCATATGCTGTTCCCTCCACTTGAAGTGTACTGTTTAATTTCTTAAAATAACTTTAGCTTTTAAGAACCAATTT
TGATGGGAGTACAGACTTCCCCCATTTTCTTGATGAGTTCTCTCCGTCATGTGTAGTAATAATGTGAGAATTTG
CAGTTTTTAGTTGTAGCCTATACTTTTAGGTCTTTGTGCCAATTTGAAAGTfiATTGGGTTAGAGTATTCATAGA
CATTTTCATGGTACTTAAAGGGACAGGGGTTTAGTAAAAAGACACATGGCAAGCCAGGCTTTTTCCACAGTTTG
CCAGGCCCAGCTGCCTCTTGTGTACCTGAACAGATTTTATCATTAACCCTTGTTTATGTTGTTTTGTTTTATTT
CGACGAAGGCTTATTTTAAGTCAGGCATGGAAAACTAGACTTCAGACTGACTTCAGCTTTAAGGACATGTTTAT
CCCGTTAACAGGGAGTCTGGGATAGACAATCTCCAGGCTTTGTTTTTCTCTGAATTTCTTAGCTCTGCTTGTGA
TGGCTTCATCATCAGGCCACAGACCATTAACACATTCTAGAACTTTAACATTGGTTAAATAATACCATCTAATA
GCCTGTCTTCAGCATTTCCCCAGTTGCCTCCAAATGCCCTTCATAGCTGTTCTCTGCCTCTGTTTGTTTTTAAT
CCAAGATACACTCAAGGCTCATATATTAGGTTGACATAGCTCTTTAGTATCCTTTAATTTAAAGCAGTCTCCAG
GTTTAGAGAAAGATGAATGAGCTTTCACATACCCCTCACTTGTCTTCTTCAGAAGTGTAGGCTACAACTAAAAC
TTCCTTCTTCAGAAGGAAGACAAGTGATTTATATTTATTTACTTCCATTTCTATTTGACCTTGTTTCATCTAAA
CACACCCACTCCACCACTGCTACCTGATTAATATTAAGTGAATCTCAAACATTGTATCATTTTAGCTCCACGTT
TTTTGTATGTATCTCCAAAATATAAAGATTCTTAAAAATATAACCACAATACCATTATCACCCTAAAAAAATCA
ATAATGATTCCTTAATATGACCAACTACTTTGTCAATGTACACCTTTCACTCCTCTTAACTTTCATAAAGACTT
ATGTGTTTTTTTGGTTTTTAAGTTTGTTGGTTTGAAGTTAAATCTATGGGTTTTCCCTCCATCTCTCTTTTTTT
AACCGTATAATTTTTTGCATGTGTATATGAATAAATCTGATTATAGATTCTATAGCTATCTTACACTTTGTCCC
TCTCTGATTGAATCCTAGTTAACAAGTTTCTATGTCTCTTGTATTTCCCATAAATTGGTAGTTGGATCTGAAGG
CTTTATCAGGTTTGTTTGATTTTTTTTTTTTTAATTTTGGCGAATCTACTTCAAAAGTATTGGCCTACCTACAA
GCCACTTTAATGGGCCCTTAGTTTAGTGACCTTTGCCTTGAAAGGAACTTGAAACAAGCAAGGAAGCACCACTG
TAATCTGCTTTTTTGCCAGAACTGTAGCATCTTACAGCTTGGTTAGAGACATAGTAAGCAGAAATTATCAAATT
CATATAATCTGTAGCTATAAGGCACTGTCTCTCTCTCTCAATTATTTACATGATTTTTCTTTGTAATATAACTA
TCATTTCAGAGAACTTGGTTTTGATTTTTTTTTTTTAATCTTTTTGAGACAGAGTCTCGCTTTATCACCCAGGC
TGGAGTGCAGTGGTGCAATCfiAAAGATTGCTGACTGCAACCTCTGCCTCCCGAGTTCAGCAATTCTAGTGCCTC
AGCCTCTCGAGTAGCTGGGATTACAGGCATGCCACCACACCCGGCTAATTTTTTTGTATTTTTAGTAAAGACAG
GGTTTCACCATGTTGGCTAGGCTGGTCTCAAATTTTTGACCTCAAGTAATCAGCCTACCTTGATCTCCCAAAGT
GCTGGGATTACAGGCATGAGCCACCATGCATGGCCTTCAGAGAACTTGGTTTTAGGTACTTACGGATTGTCTTT
CTTTTTTTTCCTCACTGCAGCCTCTCCCTCCCAGGTTCAAGCGATTCTCCTACCTCAGCTTCCTGAAGAGCTGG
GACCACAGGAAGTTTGTTTGCCTGAATGACAACATTGACCACAATCATAAAGATGCTCAGACAGTGAAGGCTGT
TCTCAGGGACTTCTATGAATCCATGTTCCCCATACCTTCCCAATTTGAACTGCCAAGAGAGTATCGAAACCGTT
TCCTTCATATGCATGAGCTGCAGGAATGGAGGGCTTATCGAGACAAATTGAAGTTTTGGACCCATTGTGTACTA
GCAACATTGATTATGTTTACTATATTCTCATTTTTTGCTGAGCAGTTAATTGCACTTAAGCGGAAGATATTTCC
CAGAAGGAGGATACACAAAGAAGCTAGTCCCAATCGAATCAGAGTATAGAAGATCTTCATTTGAAAACCATCTA
CCTCAGCATTTACTGAGCATTTTAAAACTCAGCTTCACAGAGATGTCTTTGTGATGTGATGCTTAGCAGTTTGG
CCCGAAGAAGGAAAATATCCAGTACCATGCTGTTTTGTGGCATGAATATAGCCCACTGACCAGGAATTATTTAA
CCAACCCACTGAAAACTTGTGTGTTG_AGCAGCTCTGAACTGATTTTACTTTTAAAGAATTTGCTCATGGACCTG
TCATCCTTTTTATAAAAAGGCTCACTGACAAGAGACAGCTGTTAATTTCCCACAGCAATCATTGCAGACTAACT
TTATTAGGAGAAGCCTATGCCAGCTGGGAGTGATTGCTAAGAGGCTCCAGTCTTTGCATTCCAAAGCCTTTTGC
TAAAGTTTTGCACTTTTTTTTTTTCATTTCCCATTTTTAAGTAGTTACTAAGTTAACTAGTTATTCTTGCTTCT
GAGTATAACGAATTGGGATGTCTAAACCTATTTTTATAGATGTTATTTAAATAATGCAGCAATATCACCTCTTA
TTGACAATACCTAAATTATGAGTTTTATTAATATTTAAGACTGTAAATGGTCTTAAACCACTAACTACTGAAGA
GCTCAATGATTGACATCTGAAATGCTTTGTAATTATTGACTTCAGCCCCTAAGAATGCTATGATTTCACGTGCA
GGTCTAATTTCAAAGGGCTAGAGTTAGTACTACTTACCAGATGTAATTATGTTTTGGAAATGTACATATTCAAA
CAGAAGTGCCTCATTTTAGAAATGAGTAGTGCTGATGGCACTGGCACATTACAGTGGTGTCTTGTTTAATACTC
ATTGGTATATTCCAGTAGCTATCTCTCTCAGTTGGTTTTTGATAGAACAGAGGCCAGCAAACTTTCTTTGTAAA
AGGCTGGTTAGTAAATTATTGCAGGCCACCTGTGTCTTTGTCATACATTCTTCTTGCTGTTGTTTAGTTTGTTT
TTTTTCAAACAACCCTCTAAAAATGTAAAAACCATGTTTAGCTTGCAGCTGTACAAAAACTGCCCACCAGCCAG
ATGTGACCCTCAGGCCATCATTTGCCAATCACTGAGAATTAGTTTTTGTTGTTGTTGTTGTTGTTGTTTTTGAG
ACAGAGTCTCTCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAACCTCCGCCTCCCGG
GTTCAAGCAGTTCTGTCTCAGCCTTCTGAGTAGCTGGGACTACAGGTGCATGCCACCACACCCTGCTAATTTTT
GTATTTTTAGTAGAGACGGGGGTTCCACCATATTGGTCAGGCTTATCTTGAACTCCTGACCTCAGGTGATCCAC
CTGCCTCTGCCTCCCAAAGTGCTGAGATTACAGGCATAAGCCAGTGCACCCAGCCGAGAATTAGTATTTTTATG
TATGGTTAAACCTTGGCGTCTAGCCATATTTTATGTCATAATACAATGGATTTGTGAAGAGCAGATTCCATGAG
TAACTCTGACAGGTATTTTAGATCATGATCTCAACAATATTCTTCCAAAATGGCATACATCTTTTGTACAAAGA
ACTTGAAATGTAAATACTGTGTTTGTGCTGTAAGAGTTGTGTATTTCAAAAACTGAAATCTCATAAAAAGTTAA
ATTTT
Variant sequences of NOV12 are included in Example 3, Table 24. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 12 protein (SEQ ID N0:26) encoded by SEQ TLS N0:25 is 1459 amino acid residues in length and is presented using the one-letter amino acid code in Table 12B. Psort analysis predicts the NOV 12 protein of the invention to be localized at the plasma membrane with a certainty of 0.6500.
Table 12B. Encoded NOV12 protein sequence (SEQ ID NO:26) MLFKLLQRQTYTCLSHRYGLYVCFLGVWTIVSAFQFGEWVEARDPAKHPIVHRTAPTTKNHPAQ
NVDSAEVEKSGIRRGKNGCRAVSLQDWPGTRGCANFTFAFCHDCKFSEVSQKRFLYILQNCHWLT
DWGWTWLALLHGSLILQGPASEPGCVLLKAKWLEWSRDQYHVLFDSYRDNIAGKSFQNRLCLPM
PIDVVYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKNTTEPTKKSEKQLECLLTHCIKVPM
LVLDPALPANITLKDLPSLYPSFHSASDIFNVAKPKNPSTNVSVWFDSTKDGTLLTQKVTFEWK
CEEGEVASNANIWGKTDLGSPRRPLPWPVALEPPRAQLSSALQILTRPRVSQDRANTSYEIKLDT
PLLRGYAKPVPGPETGLQPLSFAHCLPTLDLRKVNELRDFVKMYKQDPSILHTKETCFLREQVES
MGESYYKSEENIKELKTGSKKVEENISTDELSSEESDLEIDNEAVIEPDTDSPQEMGDGEASVAL
LKLNNPKDFQELNKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDISASRFEDNEELRYSL
RSIERHAPWVRNIFIVTNGQIPSWLNLDNPRVTIVTHQDVFRNLSHLPTFSSPAIESHIHRIEGL
SQKFIYLNDDVMFGKDVWPDDFYSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNSACDW
DGGDCSGNSGGSRYIAGGGGTGSIGVGQPWQFGGGINSVSYCNQGCANSWLADKFCDQACNVLSC
GFDAGDCGQENSDSKNRKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLPY
FSFAEVAKRGVEGAYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNTNDEEFKMQIT
VEVDTREGPKLNSTAQKGYENLVSPITLLPEAEILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKI
PLVNISLLPKDAQLSLNTLDLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITDETND
SLVAPQEKQVHKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDLETTARFRVETHTQKT
IGGNVTKEKPPSLIVPLESQMTKEKKITGKEKENSRMEENAENHIGVTEVLLGRKLQHYTDSYLG
FLPWEKKKYFQDLLDEEESLKTQLAYFTDSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPA
HMPHMIDRIVMQELQDMFPEEFDKTSFHKVRHSEDMQFAFSYFYYLMSAVQPLNTSQVFDEVDTD
QSGVLSDREIRTLATRIHELPLSLQDLTGLEHMLINCSKMLPADTTQLNNIPPTQESYYDPNLPP
VTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIAFKMIRTNVSHWGQLDDIRKNPRISLCC
PSWNAVMQTWLTVASTSWAQAILPPQPPD
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 12C.
Table 12C. results Patp for NOV12 Smallest Sum eading igh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:ABB30279Peptide#2930 encodedby breast cell+1 1900 7.5e-196 >patp:AAM56268Human single +l 1900 7.5e-196 brain exon expressed probe >patp:AAM16457Peptide#2891 encodedby probe +1 1900 7.5e-196 >patp:AAM28952Peptide#2989 encodedby probe +1 1900 7.5e-196 >patp:AAM04186Peptide#2868 encodedby probe +1 1900 7.5e-196 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 6444 of 6447 bases (99%) identical to a gb:GENBANK-m:AB033034~acc:AB033034.1 mRNA from Homo sapiens (mRNA for I~A_A1208 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 663 of 663 amino acid residues (100%) identical to, and 663 of 663 amino acid residues (100%) similar to, the 663 amino acid residue ptnr:SPTREMBL-ACC:Q9ULL2 protein from Homo sapiehs (KIAA1208 PROTEIN).
NOV 12 also has homology to the proteins shown in the BLASTP data in Table 12D.
Table 12D. BLASTresults for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) gi~6382022~dbj~BAA8KIAA1208 protein663 663/663 663/663 0.0 6522.1~(AB033034)[Homo sapiens] (1000 (1000 gi~16551459~dbjIBABunnamed protein847 585/613 585/613 0.0 71102.1~(AK056137)product (95%) (95%) [Homo sapiensl gi~2137411~pir~~I49hypothetical 384 277/400 307/400 e-142 528 protein (69%) (76%) [Mus musculus]
gi~11360271~pir~~T5hypothetical 248 134/137 135/l37 2e-73 0618 protein (97%) (97%) DKFZp762B226.1 [Homo sapiens]
gi~7303923~gb~AAF58CG8027 gene 652 84/l55 114/155 9e-49 967.1~(AE003834)product (54%) (73%) [Drosophila melanogaster]
A multiple sequence alignment is given in Table 12E, with the NOV 12 protein being shown on line 1 in Table 12E in a ClustalW analysis, and comparing the NOV 12 protein with the related protein sequences shown in Table 12D. This BLASTP data is displayed graphically in the ClustalW in Table 12E.
Table 12E. ClustalW Analysis of NOV12 1) > NOV12; SEQ >D N0:26 2) > gi~6382022~/ KIAA1208 protein [Homo sapiens]; SEQ m NO:87 3) > gi~16551459~/ unnamed protein product [Homo sapieras]; SEQ m N0:88 4) > gi~2137411 ~/ hypothetical protein - mouse (fragment); SEQ >D N0:89 5) > gi~11360271~1 hypothetical protein DKFZp762B226.1 - human (fragment); SEQ
II? N0;90 6) > gi~7303923~/ CG8027 gene product [Drosoplaila naelanogaster]; SEQ II7 N0:91 gi~63820221 1 RKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLP------------ 48 gi~165514591 1 ----MLFKLLQRQTYTCLSHRYGLWCFLGVWTIVSAFQF------------------- 37 gi~2137411~ 1 ____________________________________________________-_______ 1 giI113602711 1 ____________________________________________________________ 1 gi~7303923~ 1 ____________________________________________________________ 1 gi~63820221 48 ___________________________________________________________ gi~16551459~ 37 _______________________G____________________________________ 3g gi121374111 1 ____________________________________________________________ 1 gi~11360271~ 1 ____________________________________________________________ 1 gi~7303923~ 1 ___________-________________________________________________ 1 giI63820221 48 ____________________________________________YFSFAEVA--KRGVEG 62 gi~16551459~ 38 --------------------------------------------EWLEWSRDQYHVLFD 54 g I I ______ 1 a. 2137411 1 ______________________________________________________ gi~11360271~ 1 ___________________________________________________________ ' - 1 giI73039231 1 _______________________________________-____________________ 1 gi~6382022~ 63 AYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNT-NDEEFKMQITVEVDTRE
4S gi1165514591 55 SYRDNIAGKSFQNRLCLPMPIDWYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKN
gi~2137411~ 1 ____________________________________________________________ 1 gi~11360271~ 1 __________________________________________________pTRPVFDEVD 10 gi17303923~ 1 ____________________________________________________________ NOV12 237 TTI.~~PTKKSE~QL C LTHCI LVLDPALP~TT;fr~~L'SLYPSFHSASDIFNV~ ~P 296 S giI63820221 122 GP~GLNST~.Q~GY PITL~.,~------- E~~ELFyT~--------------- ,E'' gi ~ 16551459 ~ 115 TT~PTKKSE 'QL C T.~'1~HCI ' VLDPALP~~TI~ ~L
~SLYPSFHSASDIFNV P 174 v L
gi12137411~ 1 - ~-~-ILT~!'~ Y S PVTP~~_- ____QiD~~PFEv ~ _______________ ~E 32 gi ~ 11360271 ~ 1l TD~'~SGVL~D~EIRT V'~!RIHE~ ---- ---~T.iST~QyT---------------gi~73039231 1 ---MEQLRLC~SRR IiALTMTGG---------LC~~WV~;AN------------------ 30 NOV12 297 NPSTNVS .STDGTLLTQKVTFEWKCE~.'GEVASNANIWGKTDLGSPRRPLPWPVAL 356 gi~63820221 158 R--_______ PKF______________H~S-________________________ 169 LS gi~16551459~ 175 NPSTNVS STUD-------------VE~AHS-------G-----------------gi~2137411~ 33 R-________ PKIR______________H~A-________________________ 44 gi~11360271~ 40 _________________G_____________'.____________________________ gi~7303923~ 30 ____________________________________________________________ gi~6382022~ 169 ______________________________-_______________________~R____ gi1165514591 197 ---------LLKGNSRQTVWRGYLTT------D--------KEVPG-LVLMQDLAFLSGF
ZS gi~2137411~ 44 ______________________________________________________TG____ gi~11360271~ 41 ____________________________________________________________ gi~7303923~ 30 ___________________________________________________________ gi~6382022~ 171 ___________________________________________________________172 gi116551459~ 234 P-_____________pTFK--________________ET__N__________QLKT-__246 gi~21374111 46 ___________________________________________________________47 3S gi~11360271~ 41 ____________________________________________________________ 41 giI7303923~ 30 ____________________________________________________________ 30 NOV12 477 VE~ ISTDELSSEESDLE~DNEA'~'IEPDTDSPQEMGDGEASi~F'~- KLNNPKDFQE ..~T
gi I 6382022 ~ 173 AQ E--------------~~CIPI,~----------------I~ P~CDAQLSLNT
~L~L 202 gi~165514591 247 LP ----LSS-----K~t'T~LLQI~Y-----S-------EASE KLNNPKDFQE ~T
gi121374111 48 FQ E______________~IP~-______________~~E P,EAQVRLS vLvL 77 gi~11360271~ 41 __________________~~~L-_______________ __CSNILPADITQ ' IP 64 4S gi~73039231 30 --------------------YLSA--------------------EGQTGGFSSACTAI
~..:
NOV12 537 KN ~. KELTI P~ DL SQ LCQ~DISA'~~FEDN~ L~; . RS'~EiAPWV 595 S0 gi I 6382022 I 203 ~HGDIj --YN~ K~ R-- F I~HAIKNQ~i;II- TD TND -VAPf-----gi l 16551459 l 285 ~KN ~ KELT,I PY DL 5~'Q~DISA~FEDN~ L RSrI~E,~iFiAPWV 343 gi ~ 2137411 I 78 RGD' --YNN~.~ KR-- F.iG'~'~yLDT ~IKPQ --T~ T~fiG E'V'P~-----gi ~ 11360271 ~ 65 PTQESYY~.7PNLPP~TICLV'TN-=-=-CKPVTyIHKAY~----~3 ~ RFEIG~----SS g1 ~ 7303923 I 51 DAVYTW~V~?'1~- --SDPFED- - --IR'T~F~DKYDP~FDDK~~RST.tEEiAAWI 100 ~..v. ~ . . ~,"
NOVl2 596 RNIFIVTNGQIPSWLNL~YNPRVTISTTHQDVFRNLSH PTFSS"'I~ESH.I~R~EGLSQK~I 655 gi ~ 6382022 I 250 -----------------KQVHKS:~~-LPNSLGVSE QRL'I'EF" ' SV
GHI~QGQNPP., 292 E)0 gi ~ 16551459 I 344 RNIFIVTNGQIPSWLNL~NPRVTITHQDVFRNLSH
PT~FSS"IES~R~E~GLS~",'?KQI, 403 gi~21374111 124 ------------- --E"NPSHR---RPHGFAGEHRSER~IT ETVT~KG~ HALNPPP
gi~113602711 110 ------------- --EIAFK-------------IRT,t~VSH~GQiDKNP~. 140 gi I 7303923 I 101 RHVYIVTNGQIPSWLDLSYERVTV~~pHEVLAPDPDQ~PT~SS~31ETFL~t~PKLS~~I~ 160 NOV12 656 Y ~ 'VMFGKDVWPD~FY~HSKGQI~''V~'YLTWP~' CAEGC~ KDGYC~I~ACNNSACDW 715 gi~6382022~ 293 D ETTARFRVETHT''~;,,'3KTIGGNVTKKPPSL~ -----LE QI~TK- ~KKIT-----gi I 16551459 ~ 404 Y ~ VMFGKDVWPDpFYwaHSKG'QKV[l"LTWP~ ' CAEGC
~KDGYC~'JK'ACNNSACDW 463 gi~21374111 165 ET~i'CARL-----APTLGVTVS.'fO~NLSPL~ -----8E H~iPK- ~---------giI11360271~ 141 C ~ IDHN--- ~ ~ Q"TVKAVLRD3k"'YESF ------- -- --------- 169 giI73039231 161 YI~FLGAPLYP~LY~'EAEGV~VQAWGCALDC~I~I'YGDGAC~RHCNIDACQF 220 NOV12 716 DGGC GNSGGRYIAGGGGTGSIGVGQPWQFGGGI S~SYCNQGCANSW ~FCDAC 775 gi~6382022~ 339 KE RMEENE---------------------- H1;G------VTE LG1~KLQHYT 369 gi~165514591 464 DGGT7C GNSGGSRYIAGGGGTGSIGVGQPWQFGGGI S'~t~/,SYCNQGCANSW
~FCD'~~A'C 523 gi~2137411~ 201 - SDRAEG------------------------- VlP------VKEL~PG~'tRCSII 226 l~ gi~11360271~ 169 ___.'________________________________Ip'________SQFEpPREYRNFL 185 giI7303923~ 221 DGG~C~ETGPA--------------------DAHVIPPSKEVLEVQPAA'V'PQSRVH~tFP
IS NOV12 776 NVLSC ~.AGDCGENSDSKNRKTEEKCPVKKKKIMFLFFPLD .YKVILLP~t~THY 835 gi~63820221 370 DSY-L LPWEKK--------------------------- Q~ LDEEESLKT-Q- 397 gi1165514591 524 NVLSC AGDCG----------------------------D~ YKVILLPTHY 555 gi~2137411~ 227 Q--- CPGKKIC-----------------------------~ Q~7 LDAEESLKT-Q-gi~11360271~ 186 H-__________ '._______________________________ QEWRAYR~;7IC___ 199 gi~7303923~ 261 QMGLQKLFRRSSANFKDVMRHRN----------'-------VSTL RRIVERF~T~AKL
NOV12 836 IIPKGEC P FA~VA~ G'~TGA~'S~ :PIIRHASIANKWT~I~L ~ GMN I'IHFNL 895 gi~6382022~ 397 ------- ~'D~~NT ,QL_i~DTF ~ -----------LL KFGF RKVPA 439 gi ~ 16551459 ~ 556 IIPKGEC P FVA G~,GA~~~ PIIRHASIANKWY'~L GMN '.~'IHFNL 615 gi ~ 2137411 ~ 251 ------ D ~CHT Q',~.yDT~.~' y-- -L( L KFGF RKVPA 293 gi I 11360271 I 199 ------- K.~'WT'HCVLATLIFTI~3.~'~ ------------- - ---- 7----gi~7303923~ 303 MSLN--PELETSk~'"PQTTQRHGLRKyDFKSSTD2YSHSLIAT~~RAYGFKARHVLA 360 30 _.~:., ":.~,.~ ...~ ..) ..~....~. ~ .~. ~ ~ .v NOV12 896 TFQT BEEF ~ eIT 'CDT GP ~LTSTAQGY VSPTLLPEAEILFD,IPKEn~~ 955 gi I 6382022 I 440 HMPMI ~R-I ~E~Q~~FP;~''t, FD ~T FH;V~iHS
FAF~aYFYYLMSAVP~sNISE,~ 498 35 gi ~ 16551459 ~ 616 TFQT BEEF ~ITV'C~DT GP ~L~STAQ~~GY
VSPTLLPEAEILF:DI'PKE~ 675 gi ~ 2137411 ~ 294 HMPMI ~R-I ~E'~.cQpt~FP~ FD ~Tc~SFIi~~V~'t'HS
NIFAFi~YFYYLMSAV~P~.rI~TIS'352 gi ~ 11360271 ~ 218 ------- FFAQIP.~jKR'~I_FP~2RRIH~CEASPNR~V------- - --- 248 gi I 7303923 I 361 HVGFLI~K
DI'EAQRRFHt~(;~TLDTAHQF3~2APT~1~,,YAFAYYSFLMSET~SVE'rI~ 419 40 970 980 990 1000 ' 1010 1020 NOV12 956 P'~FKRHD TRR,A,QE.~ICIPL SL.'KDALSLNTLD~:iQLEHG DTLKGYNLSK 1012 gi~6382022~ 499 DVDTD--Q GVLSD IRTLAT HE 'LSLDLTGLEHLINCS ICMLPADITQLN 553 gi~165514591 676 PFKRHD TRR'AQE ~CIPL SL 'KDA~LSLNTLD1.~.~r~,QLEHG DkTLKGYNLSK
gi~2137411~ 353 HVDTD--Q GVLDRF~TLATE2 HD ~LTCI------------ --------- 384 gi~11360271~ 248 -____________.____.__________________________________-__-__ gi~7303923~ 420 DFDTDG--~ATWDRTFLTR~YQP~LDWSAMRYFEEz'~VQNCTRNLGT~HLKVDTVEH 477 NOV12 1013 ALLRSFLMNSQHAKIKNQAITD-ETNDSLVAPQE VHKSILPNSLGVSERLQR'~C.tTFP 1071 gi~6382022~ 554 ~IPPTQESYYDPNLPPVTKS~~V;TN-CKPVTDKIHKAY~KNKYRFEIMGEEEIAFKIRT
gi.~16551459~ 733 kALLRSFLMNSQHAKIKNQA,STD-ETNDSLVAPQEIC~1HKSILPNSLGVSERLQR~TFP 791 gi~2137411~ 384 .______-______,_____________________________________________ gi~11360271~ 248 __________________-_________________________________________ gi~7303923~ 478 TLVYERYEDSNLPTITRDLRCPLLAEALAANFAV1PKYNFHVSPKRTSHSNFM~FLTS 537 CO NOV12 1072 A.'~IS'V G ~QG~7NP'LDLETTARFRVETHTQKTIGGNVTICKPPSLI 'LESQMTKEKK
gi~6382022~ 613 NVS GQL~ I~ 'RISLCCPSWNAVMQTWLTVASTSWA~øAILPPQP'D--------- 663 gi~16551459~ 792 A~~GH~GNP'LDLETTARFRVETHTQKTIGGNVTIC~F.yKPPSLI 'LESQMT----gi~2137411~ 384 _____-______________________________-_______________________ gi~11360271~ 248 ____________________________________________________________ GS gi~7303923~ 538 N~'~',EV~ESLQRLf~RIVQRKFNCINDNLDANRGEDNEMVRHLLDFYLSFFQRRSKFELPPQ 597 giI6382022~ 663 __________-_________________________________________________ gi116551459~ 847 ____________________________________________________________ gi~2137411~ 384 __________________________________________-_____________-___ gi~11360271~ 248 __________________________________,_________________________ gi~7303923~ 598 YR1VRFESWRDFQRWKRRKR.AVLVIGYGVSLLLWCLLRFMCHHKAKLVRRCVQRL-----The NOV 12 Clustal W alignment shown in Table 12E was modified to end at amino residue 1200. The data in Table 1E includes all of the regions overlapping with the NOV12 protein sequences.
NOV12 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV12 nucleic acids and polypeptides can be used to identify proteins that are members of the Plasma Membrane Protein-like Protein Family. The NOV12 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV12 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction. These molecules can be used to treat, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventriculax (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Sclerodenna, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington°s disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
In addition, various NOV 12 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the sequence relatedness to previously described proteins. The NOV 12 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, nerve, and immune physiology. As such, the NOV12 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and nervous system disorders, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
The NOV12 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 12 nucleic acid is expressed in Pancreas, Uterus, Epidermis, Heart, Coronary Artery, Adrenal GlandlSuprarenal gland, Pancreas, Parathyroid Gland, Salivary Glands, Liver, Small Intestine, Bone Marrow, Peripheral Blood, Lymphoid tissue, Lymph node, Cartilage, Brain, Hypothalamus, Spinal Chord, Mammary glandBreast, Uterus, Prostate, Testis, Lung, Kidney, Epidermis, Hair Follicle.
Additional utilities for NOV 12 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV13 polypeptide has been identified as a BHLH Factor MATH6-like protein (also referred to as CG94399-O1). The disclosed novel NOV13 nucleic acid (SEQ
ID N0:27) of 2244 nucleotides is shown in Table 13A. The novel NOV 13 nucleic acid sequences maps to the chromosome 2.
An ORF begins with an ATG initiation codon at nucleotides 105-107 and ends with a TGA codon at nucleotides 1062-1064. A putative untranslated region and/or downstream from the termination codon is underlined in Table 13A, and the start and stop codons are in bold letters.
Table 13A. NOV13 Nucleotide Sequence (SEQ ID N0:27) ACGCGTGAAGGGCGGGCGAAGCGGGAGAGCCAGAGACTCCTCGGCGCTGAGCGCGGCGGCGGCCCGG
GCAGCCCCACGCCCCTGCCTCGCGCGCCGCCCGCGCCATGAAGCACATCCCGGTCCTCGAGGACGGG
.CCGTGGAAGACCGTGTGCGTGAAGGAGCTGAACGGCCTTAAGAAGCTCAAGCGGAAAGGCAAGGAGC
CGGCGCGGCGCGCGAACGGCTATAAAACTTTCCGACTGGACTTGGAAGCGCCCGAGCCCCGCGCCGT
AGCCACCAACGGGCTGCGGGACAGGACCCATCGGCTGCAGCCGGTCCCGGTACCGGTCCGGTGCCAG
TCCCAGTGGCGCCGGCCGTTCCCCCAAGAGGGGGCACGGACACAGCCGGGGAGCGCGGGGGCTCTCG
GGCGCCCGAGGTCTCCGACGCGCGGAAACGTGCTTCGCCCTAGGCGCAGTGGGGCCAGGACTCCCCA
CGCCGCCGCCGCCGCCGCCTCCTGCGCCCCAGAGCCAGGCACCTGGGGGCCCAGAGGCACAGCCTTT
CGGGAGCCGGGTCTGCGTCCTCGCATCTTGCTGTGCGCACCGCCCGCGCCCCGCGCCGTCAGCACCC
CCAGCACCGCCAGCGCCCCCGGAGTCCACTGTGCGCCCTGGCCCCCGACGCGCCCCGGGGAAAGTTC
CTACTCGTCAATTTCACACGTAATTTACAATAACCACCAGGATTCCTCCGCGTCGCCTAGGAAACGA
CCGGGCGAAGCGACTGCCGCCTCCTCCGAGATCAAAGCCCTGCAGCAGACCCGGAGGCTCCTGGCGA
ACGCCAGGGAGCGGACGCGGGTGCACACCATCAGCGCAGCCTTCGAGGCGCTCAGGAAGCAGGTGCC
GTGCTACTCATATGGGCAGAAGCTGTCCAAACTGGCCATCCTGAGGATCGCCTGTAACTACATCCTG
TCCCTGGCGCGGCTGGCTGACCTTGACTACAGTGCCGACCACAGCAACCTCAGCTTCTCCGAGTGTG
TGCAGCGCTGCACCCGCACCCTGCAGGCCGAGGGACGTGCCAAGAAGCGCAAGGAGTGACTGGCTGC
AGGCAAGACCAAGGCCACCACTGTGGGCCCTCCTTCCAGTCAGGCCTGAGGACAAGGTGAGCTCGCT
GAGTCCAGCCTCGTGGTCTTCTCCAAGATGGCGCCCCACTTGGAGCCTACAGCCTCTCAGGGTCGGA
TCGGAGCACGCCTGCCTCCCTCTCCCCTCCGCCCTCACCCAGCCAATCCGAGGCTGCTTCGCACGTT
GCCCTCTGCCTGGTGGGGAGGGGAGAGCTCAGCCCCCGACTCACTCAGACCCCAAGGCCCACTGTCC
AGCTGCAGAAATTCGTTGCCAAAGATTGGACAGAGACACCGAAGGAAATGGGGTGGTGAAACCCCAC
AGCGAAAAGCCACACCGTTGCTCTGTGACTTTTGCTCCTCCTGTTGCCTGAGCCCCATCTCAAGCCA
AAGATGAGTCAGTGGTTCTGCTAGGAACTCATGGAATGGATGGGCATTTGATGACCCCTGGGGGTCA
TCTTGGCCCTCTGACCTGGTGCTCTCTCTCCACTGGGCCTTGTGCTGGTTGAGTGCAAGACAAGCCT
TAGGGGCTGTGAGAGGGAGGCTGGGGTGCCTGGGCGGGGCTGGGAGTGGGACCTGAGATCCCTGCCC
ACTCTCTCCCCTTCATTGGCTTGCCCAGGCCACTGGCCCCAGTTCTCAGTGTCCCTTGGGGTCCAGG
CTCCTTGGGCCCTAAGCATCACCAGAAGGGAGTAAGCAGGGAGAGAAGCAATATTACTCCCTCCCCT
ACACCAGGGACTTGCCCCAGGGCGGCTACCTATGGGTCTTTGCTTCCCCAGCCAGCCTCTCCTCACT
GTGACCCACCCCCATGGGCCCCCGTCCCAGGCAGCCAGCACCATGGGCAGGCCCTGCCATGGACAGA
AAAAGAGTTTTTCTCTTGTTCAGCCTGCACGTGGCCTGAGGAAGGAGTAGAGGCTGGGTTGGCTGGA
GCCGTCCTACTGGGCAAGATGGCGCCCCACTTGGAGGGCGGTGGTCTGTTACAGGGTGTGCAGGGGC
AGAGAAGGAAGGGACCAGGGGACTGGGCCAGTATGTGGAGGATGGGGCCTGCGTGTTCAAAGCCAAG
GCCCGCCCCTTCCTTGTGCTCAAATGGCCAAAGCTGTTCACGTCTGTGCTCAACCATCTGCTTCAAA
TTGAAGTAAAAGCCCCAAAATGTCAAGAAA.AAA
Variant sequences of NOV13 are included in Example 3, Table 25. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 13 protein (SEQ m N0:28) encoded by SEQ m N0:27 is 319 amino acid residues in length and is presented using the one-letter amino acid code in Table 13B. Psort analysis predicts the NOV 13 protein of the invention to be localized at the nucleus with a certainty of 0.7000.
Table 13B. Encoded NOV13 protein sequence (SEQ ID N0:28) MKHIPVLEDGPWKTVCVKELNGLKKLKRKGKEPARRANGYKTFRLDLEAPEPRAVATNGLRDRTH
RLQPVPVPVRCQSQWRRPFPQEGARTQPGSAGALGRPRSPTRGNVLRPRRSGARTPHAAAAAASC
APEPGTWGPRGTAFREPGLRPRILLCAPPAPRAVSTPSTASAPGVHCAPWPPTRPGESSYSSISH
VIYNNHQDSSASPRKRPGEATAASSEIKALQQTRRLLANARERTRVHTISAAFEALRKQVPCYSY
GQKLSKLAILRIACNYILSLARLADLDYSADHSNLSFSECVQRCTRTLQAEGRAKKRKE
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 13C.
Table 13C. Patp results for NOV13 Smallest Sum eadingigh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:AAM93743Humanpolypeptide, SEQ ID NO: +1 1197 2.3e-121 >patp:AAB95274Humanprotein sequence SEQ ID +1 1197 2.3e-121 N0:17476 >patp:AAU16607Humannovel secreted protein, +1 534 4.2e-51 Seq TD 1560 >patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33 #291 >patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33 #291 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 372 of 657 bases (56%) identical to a gb:GENBANK-ID:HSBBICP4A~acc:L14320.1 mRNA from Bovine herpesvirus 1 (Bovine herpesvirus type 1 early-intermediate transcription control protein (BICP4) gene, complete cds). The full amino acid sequence of the protein of the invention was found to have 238 of 322 amino acid residues (73%) identical to, and 244 of 322 amino acid residues (75%) similar to, the 322 amino acid residue ptnr:TREMBLNEW-ACC:BAB39468 protein from Mus musculus (BHLH FACTOR MATH6)..
NOV 13 also has homology to the proteins shown in the BLASTP data in Table 13D.
Table 13D. BLAST
results for Gene Indeac/ Protein/ OrganismLength Identity PositivesEacpect Identifier (aa) (%) (%) giI14249530Iref~NPhypothetical 321 246/321 246/321 2e-93 _ protein FLJ14708 (76%) (76%) 116216.11(NM
[Homo sapiens]
gi~i3383235~dbj~BABbHLH factor 322 233/329 240/329 3e-86 Math6 39468.1~(AB049066)[Mus musculus] (70%) (72%) gi~17864454Iref~NPnet [Drosophila365 55/97 76/97 5e-21 _ melanogaster] (56%) (77%) 524820.1~(NM
gi~7296271~gb~AAF51CG11450 gene 261 55/97 76/97 2e-20 562.11 (AE003590)product (56%) (77%) [Drosophila melanogaster]
gi~18858289Iref~NPatonal homolog 325 36/82 51/82 2e-10 2a _ [Danio rerio] (43%) (61%) 571891.1I(NM
A multiple sequence alignment is given in Table 13E, with the NOV13 protein being shown on line I in Table 13E in a ClustalW analysis, and comparing the NOV13 protein with the related protein sequences shoum in Table 13D. This BLASTP data is displayed graphically in the ClustalW in Table 13E.
Table I3E. CIustalW Analysis of NOVI3 1) > NOV13; SEQ m N0:28 2) > gi~14249530~/ hypothetical protein FLJ14708 [Homo Sapiens]; SEQ m N0:92 3) > gi~13383235~/ bHLH factor Math6 [Mus rrausculus]; SEQ ZD N0:93 4) > gi~ 17864454/ net [Dr~osoplaila rraelanogaster]; SEQ m N0:94 5) > gi~7296271~/ CG11450 gene product [Dr-osophila nrelanogaster~]; SEQ m N0:95 6) > gi~18858289~/ atonal homolog 2a [Danio rerio]; SEQ m N0:96 .1... ~... .I
NOV13 1 __________________________ ' CHI 'LiE~GPKTVCVL'~' ___ ~ 3' ' 28 1~ gi1142495301 1 -------------------------- HI~~T~E~GP~KTVCVLi~==-- ~ ~ ~ 28 gi1133832351 l --------------------------- HI~ ~1E~GP~KTVCVK~#L -- ~ 28 gi~17864454~ 1 MSFAAMANTNTEKLYMQLSASELSAII ~DS~ S ~RDAGFCSASSE"~c~'EGGDDLVVHAv gi172962711 1 ______________________________________________________'=_____ 1 gi1188582891 1 -----------------------MLTLPFED~_~,M~TQ~'GANFPCV~-------D~yG 30 _.
I....I....J. .~.. .L... .~....~....~
NOV13 29 ~F~ANGYKTFRLW,~APE4P-----RAVAT R',t'~RTHRL' ~ PVRCQSQWRRP 83 a V V
~ r V V
gi ~ 14249530 ~ 29 'TtANGYKTFRL~Z~~APP-----RAVA ~ Rx?RTHRLf~ ~ ~ PVPVPVPV ~ 83 2~ gi113383235~ 29 ~ GYKTFRLy.P~LGATVSTTAAT~ R'RT---~'F~IATPVPA'V ~ 85 gi1178644541 61 S SPDI~PKGTDSADSKP~LALVRNKRKSSEPFKV~TTPNSKS ' ~PSSASMN~T ~L
gi172962711 1 __________________.________________________ ~PSSASMNT ~L 16 gi ~ 18858289 1 3l NK~mFEEETLSHVMD~DF~SED-----EDEREQDNGLPRR ~RKKKMTK~RVDR 85 .1...,1... ~ . .1 .I ~.. 1'.
NOVl3 ~ 84 FPQEGARTQPGSAGAG~~R --PT GNLRPRI~SGAR~..,,PHAAAAA ---ASCAPEPGT 136 gi ~ 14249530 ~ 84 VPPRG ~'~;~1GERGGS ~E~"SDAR ~CFALGA'~'GPGLP~~~,~P'P ~PPP----APQ~QAPG- 138 gi1133832351 86 VPPGG L~~~,REFRGI'., ~E~ZDAR ~GFALGT~~GPGLPP~P~P------ASQ LAPG-3~ gi ~ 17864454 ~ 121 KKRIR S~~DSAVV~,'TP ~DSPPPNSC~PSTI~F~LQHEI ~
IYVRHPGV~TLHRS 180 gi ~ 7296271 ~ 17 KKRIRY~S~~~,DSAVVTP'ADSPPPNSC,PSTLr~LQHEI ~ IYVRHPGVfI'TLHRS
gi~18858289~ 86 VKVRRMEAN~RERNRI~HGLNNLDSLE7KV~',PCYSLC~TQKLKIETLR-----..
NOV13 137 WGPRGTAF~EPG~RPRIL PP'i1PRAVTP'u~TASAPG--VHCAPW~PTRP.~ .I 194 gi1142495301 138 -GPEAQPF~EPGPRPRIL~ PPi~1, ~ PyiIPP~,~PPAPP-E TV~P ~PTRP' I
gi1133832351 138 -DPEAHSF~EQ RPRIL PP~i1 ~TQi~PL~~PPAAPQE PV,I2P ~PTRP I 197 gi1178644541 181 LAAHPEQLEPL TTKKQ DQiI ~KIE~F~~iLLIGKQP K~TLKERTQ TS FL 240 4O gi172962711 77 LAAHPEQLEPL VTTKKQ DQ~ KIE~F~LLIGKQP ITLKERTQ T'S FL 136 gi1188582891 141 SEILSTGI~PDL TFVQT KGLQ~TT~1'LV;~,;GCLQLN---ARNFT~DQISF GR~
1.~.~..1.. .I....1 . .1....1 .1. ..~.. .1. .1....1. .1 NOV13 195 HVIY~HQ~S ~PRKRP ET~~SSEI ~Q T ~ L~ ~~~~ ~~ ~Q 254 v i w v gi ~ 14249530 1 197 HVIY~HQrS PRKRP E;T ~SSEI ~Q~T~ ~ 'L~ ~' ~ ~ ~ ~~CQ 256 gi ~ 133832351 198 HVIY~'1HP~5 PRKRP ET ~STEI I QT' ~ ''L~ ~ ~ ~ ~I~Q 257 w a gi1178644541 241 EASL~~3~E-~LNL~.~K~!GLAPISRPHQHQRNY T~E:y.E~ ~~ ~ ~~ T~~A 299 gi172962711 137 EASL~S~E-~LKGLAPISRPHQHQRNY T~E '~E~ ~~ ~ ~ ~'~' T ~A 195 gi (18858289 l 198 PYESVYSTYI3~P~WWTPS~P~VD~1VKPFR~FNYCSSYE-FY SVSPECG'I'PQ
gi1142495301 257 312 gi1133832351 258 313 gi1178644541 300 355 gi172962711 196 251 gi1188582891 257 316 NOV13 311 . . ~'. 1 ~ ~ '~~I 319 gi1142495301 313 ~ ~- 321 gi1133832351 314 ~ ~- 322 gi1178644541 356 ~2 ~E 365 gi172962711 252 ~ ~;E 261 gi~188582891 317 ~DELNTFHN- 325 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 13F lists the domain description from DOMAIN analysis results against NOV 13.
Table 13F
Domain Anal sis of NOV13 Model Region of Score (bits) E value Homology Helix-loop- 234-280 55.8 4.0e-09 helix domain Helix-loop- 229-281 61.4 1.9e-14 helix DNA-binding domain (HLH) . Consistent with other known members of the BHLH Factor MATH6-like family of proteins, NOV 13 has, for example, a Helix-loop-helix domain and a Helix-loop-helix DNA
binding domain (HLH) signature sequence as well as homology to other members of the BHLH Factor MATH6-like Protein Family. NOV 13 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
For example, NOV 13 nucleic acids and polypeptides can be used to identify proteins that are members of the BHLH Factor MATH6-like Protein Family. The NOV 13 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV13 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Inflammatory bowel disease, Diverticular disease, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
In addition, various NOV13 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV13 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the BHLH Factor MATH6-like Protein Family.
A number of eukaryotic proteins, probably sequence specific DNA- binding proteins that act as transcription factors belong to this family. They share a conserved domain that is formed of two amphipathic helices joined by a variable length linker region that could form a loop (Littlewood and Evan, Pf°otein Prof. 2: 621-702 (1995).) This 'helix-loop-helix' (HLH) domain mediates protein dimerization and has been found in a large variety of proteins (Garrell and Campuzano, Bioassays 13: 493-498 (1991); Kato and Dang, FASEB J.
6: 3065-72 (1992).) Most of these proteins have an short basic region adjacent to the HLH domain that specifically binds to DNA. They are referred as basic helix-loop-helix proteins (bHLH), and are classified in two groups: class A (ubiquitous) and class B (tissue-specific). The HLH
proteins lacking the basic domain (Emc, Id) function as negative regulators since they form heterodimers, but fail to bind DNA. The hairy-related proteins (hairy, E(spl), deadpan) also repress transcription although they can bind DNA. The proteins of this subfamily act together with co-repressor proteins, like groucho, through their C-terminal motif WRPW.
MATH6 (moue, et al., Genes to Cells 6: 977-86 (2001)) is a distant homolog of D~osophila proneuronal gene Atonal. Murine expression is higest in developing nervous system (ventricular zone and mantle layer, spinal cord, dorsal root ganglia).
MATH6 is expressed by neuronal precursor cells and designated neurons, e.g., cerebellar Purkinje cells.
The closest mammalian homolog to MATH6 is NeuroD. NeuroD point mutations and NeuroD gene knockout animals have severe diabetes and die perinatally. The NeuroD
knockout anmals lack beta-Islet cells and could not be rescued with insulin administration.
Also, the NeuroD knockout animals are deaf due to a loss of inner ear sensory neurons.
The NOV13 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of metabolism as well as nerve and immune physiology. As such, the nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, hearing, nervous system, immune disorders, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, inflammatory bowel disease, Diverticular disease, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
The NOV13 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 13 nucleic acid is expressed in Pancreas, Umbilical Vein, Small Intestine, Cartilage, Synovium/Synovial membrane, Brain, Placenta, Oviduct/LTterine Tube/Fallopian tube, Lung, Brain, Uterus.
Additional utilities for NOV 13 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 14 polypeptide has been identified as a Putative Protein-Tyrosine Phosphatase-like protein (also referred to as CG94366-O1). The disclosed novel NOV14 nucleic acid (SEQ
ID N0:29) of nucleotides is shown in Table 14A. The novel NOV 14 nucleic acid sequences maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 248-250 and ends with a TAA codon at nucleotides 1679-1681. A putative untranslated region and/or downstream from the termination codon is underlined in Table 14A, and the start and stop codons are in bold letters.
Table 14A. NOV14 Nucleotide Sequence (SEQ ID N0:29) ATTGAGTTTGAAATAACTGCCACCACAAAGTCTGTCACACATTGAGACTGAGGTCATAATAAAGAGG
TTTACTTAAATAGGGAAGCATTACTATTTTCCCCCGCCTAAGATTTTGGTTGTCGCCATATAAATCC
TCATTTCTAATAAAGAGAAAAAGACATTCCAGGTTCCAATAGTGCTATACACATGAATAGTCAGAAA
TTAATTGGTTTCTGTCTAGAATAATGAAAAGTAATTTTTCCAAAATATGAATTCAGAATTAAGTCTC
CTCTCTGACTGTTTTCTCTTATCATCCGCTAGTCCACAGACAAACGAATTTAAAGGAGCAACCGAGG
AGGCACCTGCGAAAGAAAGCCCACACACAGGTGAATTTAAAGGAGCAGCCCTGGTGTCACCTATCAG
TAAAAGAATGTTAGAACGACTTTCCAAGTTTGAAGTTGGAGATGCTGAAAATGTTGCTTCATATGAC
AGCAAGATTAAGAAAATTGTTCATTCAATTGTGTCATCCTTTGCAGTTGGGATATTTGGAGTTTTCC
TGATCTTGTTGGATGTGGCTCTGATCTTTGCTGACCTAATTTTCACTGATAGCAAAGTTTATATTCC
TTTGGAGTATCGTTCTATTTCTCTAGCTATTGCTTTATTTTTTCTCATGGATGTTCTTCTTCGAGTA
TTTGTAGAAAGGAGACAGCATTATTTTTCTGATTTACTTAACATTTTAGATACTGCCGTTACTGTGA
TTATTCTGCTGGTTGATGTCGTTTACATTTTTTTTGACGTTAAGTTTCTTAAGGATATTCCCAGATG
GACACGTTTATTTCGACTTCTACGACTTATCATTCTGATAAGAGTTTTTCGTCTGGCTCATCTAAAA
AGACAACTTGGAAAGCTGATAAGAAGGCTGGTAAGTAGGNGATACGAAAGGGATGGATTTGACCTAG
ACCTCACTTATATTACAGAACGTATTGTCGCTATGTCATTTCCATCTTCGGGAGGCCAGTCTTTCTA
TCGGAATCCAATTAAGGAAGTCGTACAGTTTCTAGACAAGAAACATCCAAACCACTATCGAGTCTAC
AATCTATGCAGTGAAAGAGCTTATGATCCTAAGCACTTCCATAATAGGGTCAGTAGAATCATGATCG
ATGATCATAATGTCCCCACTCTAAGGGAGATGGTAGCATTCTCCAAGGAAGTGTTGGAGTGGATGGC
TCAAGATTCTGAAAACATCGTAGTGATTCACTGTAAAGGAGGCAAAGGTAGAACCGGAACTATGGTT
TGTGCCTGCCTGATTGCCAGTGAAATATTTTTAACTGCAGAGGAAAGATTGTACTATTTTGGAGAAC
GGCGAACAGATAAAACCAATGGCACTAAATATCAGGGAGTAGAAACTCCTTCTCAGAATAGATATGT
TGGATATTTTGCACAAGTGAAACATAGCTACAACTGGAATCTCCCTCCAAGAAAAACACTGTTTATA
AAAAGATTAGTTATTTATTCGATTCATGGTAAGTGTTTAGATCTAAAAGTCCAAATAGTAATGAAGA
AAAAGATTGTCTTTTCCTGCACTTCCTTAAACAGTTGTCGGGTAAGAGAAAACATGGAAACAGACAG
GGTAATAATTGATGTGTTCAACTGTCCACCTCTGTATGATGATGTGAAAGTGCAATTTTTTTTTTCT
TTTTAGGATTTTCCTAAATACTATCACAACTACCCTTTTTTCTTCTGGTTTAACACATCTTTAATAC
AAAATAACAGGCTTTATCTACAAAGAAATGAATTGGATAATCTTCATAAACAAAAAACATGGAAAAT
TTATCAACCAGAATATGCAGTAGAGATATATTTTGATGAGAAATGACTTAAGTTATGTTGTAACTGG
TAGCTGATTAAGTATAGTTCCCTGCACCCCTTCTGGGAAAGAATTATGTTCTTTCTAACCCTGCCAC
ATAGTTATATGTTCTAAATCTTCCTTGCTGGTACATCTATATTGATATATGTATACACATGTTCTTT
ATAAATCTATTAAATATATACAGATAAA
The NOV14 protein (SEQ ID N0:30) encoded by SEQ )D N0:29 is 477 amino acid residues in length and is presented using the one-letter amino acid code in Table 14B. Psort analysis predicts the NOV 14 protein of the invention to be localized at the plasma membrane with a certainty of 0.6000.
Table 145. Encoded NOV14 protein sequence (SEQ ID N0:30) MNSELSLLSDCFLLSSASPQTNEFKGATEEAPAKESPHTGEFKGAALVSPISKRMLERLSKFEVG
DAENVASYDSKIKKIVHSIVSSFAVGIFGVFLILLDVALIFADLIFTDSKVYIPLEYRSISLAIA
LFFLMDVLLRVFVERRQHYFSDLLNILDTAVTVIILLVDVVYIFFDVKFLKDIPRWTRLFRLLRL
IILIRVFRLAHLKRQLGKLIRRLVSRXYERDGFDLDLTYITERIVAMSFPSSGGQSFYRNPIKEV
VQFLDKKHPNHYRVYNLCSERAYDPKHFHNRVSRIMIDDHNVPTLREMVAFSKEVLEWMAQDSEN
IVVIHCKGGKGRTGTMVCACLIASEIFLTAEERLYYFGERRTDKTNGTKYQGVETPSQNRYVGYF
AQVKHSYNWNLPPRKTLFIKRLVIYSIHGKCLDLKVQIVMKKKIVFSCTSLNSCRVRENMETDRV
IIDVFNCPPLYDDVKVQFFFSF
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 14C.
Table 14C. Pat results for NOV14 Smallest Sum eading igh Prob Sequences Score P(N) producing High-scoring Segment Pairs: Frame >patp:AAG67459Amino acid sequence of a human +1 1895 2.5e-195 polypeptide >patp:AAG67638Amino acid sequence of a human +1 1895 2.5e-195 protein >patp:AAB73230Human phosphatase AA493915_h +1 574 1.8e-111 >patp:AAW34402Protein encoded by gene IMAGE +1 473 1.2e-44 clone 264611 >patp:AAY07450Human TS10q23.3 gene bases 453-2243+1 473 1.2e-44 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 1105 of 1427 bases (77%) identical to a gb:GENBANK-m:AF007118~acc:AF007118.1 mRNA from Homo Sapiens (putative tyrosine phosphatase mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 369 of 462 amino acid residues (79%) identical to, and 402 of 462 amino acid residues (87%) similar to, the 551 amino acid residue ptnr:SWISSNEW-ACC:P56180 protein from Ho~rao Sapiens (PUTATIVE PROTEIN-TYROSINE
PHOSPHATASE TPTE (EC 3.1.3.48)).
NOV14 also has homology to the proteins shown in the BLASTP data in Table 14D.
Table 14D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (~) gi~7019559~ref~NPtransmembrane 551 369/462 402/462 0.0 _ phosphatase (79%) (86%) 37447.1I(NM 013315)with tensin homology;
tensin, putative protein-tyrosine phosphatase [Homo Sapiens]
gi~16166555~ref~XPsimilar to 551 367/462 401/462 0.0 _ transmembrane (79%) (86%) 055073.1~(XM_055073 phosphatase with tensin homology [Homo Sapiens]
gi~18640756~refINPsimilar to 445 316/462 343/462 e-156 _ PUTATIVE PROTEIN- (68%) (73%) 570141.1I(NM
TYROSINE
PHOSPHATASE
TPTE
[Homo sapien]
gi~14787415~emb~CACtyrosine 664 275/432 336/432 e-141 44243.11(AJ311311)phosphatase (63%) (77%) isoform A [Mus musculus]
gi~14787417~emb~CACtyrosine 645 275/432 336/432 e-141 44244.1~(AJ311312)phosphatase (63%) (77%) isoform B [Mus musculus]
A multiple sequence alignment is given in Table 14E, with the NOV 14 protein being shown on line 1 in Table 14E in a ClustalW analysis, and comparing the NOV14 protein with the related protein sequences shown in Table 14D. This BLASTP data is displayed graphically in the ClustalW in Table 14E.
Table 14E. ClustalW Analysis of NOV14 1) > NOV14; SEQ ID N0:30 2) > gi~7019559~/ transmembrane phosphatase with tensin homology; tensin, putative protein-tyrosine phosphatase Homo sapieras]; SEQ ID N0:97 3) > gi~16166555~/ similar to transmembrane phosphatase with tensin homology [Homo sapiefts]; SEQ ID N0:98 . 4) > gi~18640756~/ similar to PUTATIVE PROTEIN-TYROSINE PHOSPHATASE TPTE;
similar to transmembrane phosphatase with tensin homology; tensin, putative protein-tyrosine phosphatase [Flomo Sapiens]; SEQ ID N0:99 5) > gi~14787415~/ tyrosine phosphatase isoform A.[Mus musculus]; SEQ ID
NO:100 6) > gi~14787417~/ tyrosine phosphatase isoform B [Mus musculus]; SEQ ID
NO:101 ..
NOV14 1 _________________________________________________ gi~7019559~ 1 _________________________________________________ ~SELSLLS
--~I E~PDPTD
gi~16166555~ 1 ___________________________________________________ i E PDPTD 9 gi~186407561 1 __________________________________________________ __ - i E P-- 5 gi~147874151 1 MYGEKKSHLYLWMEHYGYDMPANIYKMYSQPSRKTDDANKKVSVSASRTIKI~ TGYDT 60 gi1147874171 1 -------------MHFGNINSTNWF------FRDKKHQNKKVSVSASRTI TGYDT 41 w NOV14 9 -DCFL~S- ~SASP~ ' ~. ~~~ G---------------- ~~L 48 giI70195591 10 LAGVI'I!ELGPI7SPv S ~ ... ________________ .. 52 gi~16166555~ 10 LAGIIELGP,SP~ S ~ ~~~ ---------------- ~~, ~ 52 gi~18640756~. 5 ______________~ T ________________ ..L 34 gi1147874151 61 NEQ~ T~.iITNG;SLSYP~ I.~YS,~~,S'YAD~ISTKA~,'' SSVYDPGGASSSTTLY
gi~14787417~ 42 NEQTITNGSLSYPII~S~YAD~ISTKA~S. ,s SSVYDPGGASSSTTLY LNSL~E 101 NOV14 49 ________________________________________________ ~ .'E~'t 58 gi~70195591 53 ________________________________________________ _~g 62 gi~16166555~ 53 ________________________________________________ S n'' 62 gi~18640756~ 35 ________________________________________________ ___'. 39 gi1147874151 121 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ" ~C 180 gi~14787417~ 102 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ;., CI~Q
NOV14 59 ~ ~ ~ FEV ~ A~ . .~K~ . . I=HS3; F~1V I ~ ~ ~ I<F ~ ~ . n 116 gi I 16166555 ~ 63 ~ 'FEVW--AB:NV DK~~.'C, , 'IVHS~ ~ F~G~ ~ nT Il;n ~ ~~.i gi~18640756~ 39 ~-__ _______=__ ____'_'.___R__.______ ~ T I,~~ ~ ~~ 65 i 14787415 181 LYD7WERTt~IQ F GI~Z ' ~ I ~ F ! n ~I 240 gi I 14787417 I 162 ?LYD~7~ERTIQW~~~yF~GII I ~ F ~ ~ ~If I 221 gi~7019559~ 121 180 gi~16166555~ 121 giI186407561 66 94 gi~14787415~ 241 300 gi~14787417~ 222 281 giI7019559~ 181 240 gi~161665551 181 240 giI186407561 94 145 gi~147874151 301 360 gif14787417~ 282 gi~7019559~ 241 55 gi~16166555~ 241 300 gi~18640756~ 146 205 gi~14787415~ 361 gi~14787417~ 342 gi~7019559~ 301 gi~161665551 301 gi~18640756~ 206 360 gi~14787415~ 421 480 gi~14787417~ 402 70 ..
NOVl4 354 413 gi~7019559~ 361 420 gi~16166555~ 361 giI186407561 266 325 gi114787415~ 481 540 gi~14787417~ 462 521 gi~7019559~ 421 gi~161665551 421 476 gi~186407561 326 381 gi~14787415~ 541 600 gi~14787417~ 522 581 NOV14 470 ~~~. F~________F___-____-___-_______________________________ 477 gi~7019559~ 477 ~ Y ~T ~ S L E ~ L Q ~, PSD ~ TL 536 gi~161665551 477 t ~T n S L Ei ~ L Q PSD ~ ~L 536 .k gi I 18640756 ~ 382 ~ S ~ ~ P F F C ' nP 'Q W~ PPE ~ I=L 441 gi114787415~ 601 LSP ~~P F F~R~T ~P~TW~GE ~ D 660 gi ~ 14787417 ~ 582 LSP ' ~ P F''.~!~!~ ~ T P ~TW GE ~ 641 giI7019559~ 537 KMTSSDWAGSD 551 30 gi~16166555~ 537 KMTSSDWAGSD 551 gi~18640756~ 442 i~K----------- 445 gi1147874151 661 ---------- 664 gi~147874171 642 ---------- 645 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uklinterpro~. Table 14F lists the domain description from DOMAIN analysis results against NOV14.
Table 14F
Domain Anal sis of NOV14 Model Region of Score (bits) E value Homology Dual 231-378 -48.8 3.2 specificity phosphatase, catalytic doma Consistent with other known members of the Putative Protein-Tyrosine Phosphatase-like family of proteins, NOV 14 has, for example, a dual specificity protein phosphatase signature sequence and homology to other members of the Putative Protein-Tyrosine Phosphatase-like Protein Family. NOV14 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 14 nucleic acids and polypeptides can be used to identify proteins that are members of the Putative Protein-Tyrosine Phosphatase-like Protein Family. The NOV 14 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV14 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, cystitis, incontinence as well as other diseases, disorders and conditions W addition, various NOV 14 nucleic acids and polypeptides according to the invention are useful, iyater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 14 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Putative Protein-Tyrosine Phosphatase-like Protein Family.
Cellular processes involving growth, differentiation, transformation and metabolism are often regulated in part by protein phosphorylation and dephosphorylation.
The protein tyrosine phosphatases (PTPs), which hydrolyze the phosphate monoesters of tyrosine residues, all share a common active site motif and are classified into 3 groups. These include the receptor-like PTPs, the intracellular PTPs, and the dual-specificity PTPs, which can dephosphorylate at serine and threonine residues as well as at tyrosines.
Diamond et al. (1994) described a PTP from regenerating rat liver that is a member of a fourth class. The gene, which they designated Prll, was one of many immediate-early genes. Overexpression of Prll in stably transfected cells resulted in a transformed phenotype, which suggested that it may play some role in tumorigenesis. By using an ih vitro prenylation screen, Cates et al. (1996) isolated 2 human cDNAs encoding PRL1 homologs, designated PTP(CAAXl) and PTP(CAAX2)(PRL2), that are farnesylated i~a vitro by mammalian farnesyl:protein transferase. Overexpression of these PTPs in epithelial cells caused a transformed phenotype in cultured cells and tumor growth in nude mice. The authors concluded that PTP(CAAX1) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic PTPs. Peng et al.
(1998) reported that the human PTP(CAAX1) gene, or PRL1, is composed of 6 exons and contains 2 promoters. The predicted mouse, rat, and human PRLl proteins are identical. Zeng et al. (1998) determined that the human PRLl and PRL2 proteins share 87% amino acid sequence identity.
The NOV14 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and renal physiology. As such, the NOV 14 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and urogenital system disorders, e.g., Cardiovascular diseases, cystitis, incontinence as well as other diseases, disorders and conditions. The NOV 14 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV14 nucleic acid is expressed in Urinary bladder.
Additional utilities fox NOV 14 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV15 polypeptide has been identified as a Leucine Rich Repeat (LRR)-like protein (also referred to as CG95387-02). The disclosed novel NOV15 nucleic acid (SEQ m N0:31) of 3136 nucleotides is shown in Table 15A. The novel NOV15 nucleic acid sequences maps to the chromosome 19.
An ORF begins with an ATG initiation codon at nucleotides 330-332 and ends with a TAA codon at nucleotides 2331-2333. A putative untranslated region and/or downstream from the termination codon is underlined in Table 15A, and the start and stop codons are in bold letters.
Table 15A. NOV15 Nucleotide Sequence (SEQ ID N0:31) ACTCCTGACCTAAAGTGATCCACTCGCCTTGGCCTCCCAAAGTGCTAGGATTACAGCCCTCATTCTC
TTTTGCTCCTCAGGTGACACAGGACAAGATCATCTGTCTACCCAATCATGAGCTCCAGGAGAACTTA
TCAGAGGCCCCGTGCCAGCAATTGCTGCCTCGGGGGATCCCTGAGCAGATTGGGGCCCTGCAGGAGG
TTAAAGGCCTTAAGAACAATTTGGACCTGCAGCAATACAGCTTTATTAACCAGCTGTGTTATGAGAC
GGCCCTGCACTGGTATGCCAAGTACTTCCCTTACCTCGTGGTCATTCACACACTCATCTTCATGGTC
TGCACCAGTTTCTGGTTCAAGTTCCCTGGCACCAGCTCCAAGATTGAACACTTCATCTCCATCCTGG
GCAAGTGTTTCGACTCTCCATGGACCACCAGGGCCCTATCCGAGGTCTCCGGGGAGAACCAGAAGGG
CCCAGCAGCCACCGAACGGGCTGCGGCATCCATAGTGGCCATGGCAGGGACCGGGCCGGGGAAGGCA
GGGGAGGGTGAGAAGGAGAAAGTGCTGGCGGAACCGGAGAAGGTGGTGACCGAGCCTCCAGTTGTCA
CCCTGTTGGACAAGAAGGAGGGTGAGCAAGCCAAAGCCCTGTTTGAGAAGGTGAAGAAGTTCCGCAT
GCACGTGGAAGAGGGCGACATCCTGTACACCATGTACATCCGACAGACGGTGCTGAAAGTGTGTAAG
TTCCTGGCCATCCTGGTCTACAACCTGGTCTATGTGGAGAAGATCAGTTTCCTGGTGGCCTGTAGGG
TGGAGACGTCAGAGGTCACGGGCTACGCCAGCTTCTGCTGCAACCACACCAAGGCCCACCTCTTCTC
CAAGCTGGCCTTCTGTTACATCTCCTTTGTGTGCATCTACGGACTTACCTGCATCTACACGCTCTAC
TGGCTCTTCCACCGGCCCCTCAAGGAGTACTCCTTCCGTTCCGTGCGGGAGGAGACTGGCATGGGGG
ACATTCCTGACGTCAAGAATGACTTCGCCTTCATGCTGCACCTCATCGATCAGTACGACTCCCTCTA
CTCCAAGCGCTTCGCCGTCTTCCTGTCCGAGGTCAGCGAAAGCCGTCTAAAGCAGCTCAATCTCAAC
CACGAGTGGACCCCCGAGAAGCTTCGACAGAAGCTGCAGCGCAATGCCGCGGGCCGGCTGGAGCTGG
CCCTCTGCATGCTGCCGGGTCTGCCCGACACCGTCTTTGAGCTCAGTGAGGTGGAGTCACTCAGGCT
GGAGGCCATCTGCGATATCACCTTCCCCCCGGGGCTGTCACAGCTGGTGCACTTGCAGGAGCTCAGC
TTGCTCCACTCGCCCGCCAGGCTACCCTTCTCCTTGCAGGTCTTCCTGCGGGACCACCTGAAGGTGA
TGCGCGTCAAATGCGAGGAGCTCCGCGAGGTGCCGCTTTGGGTGTTTGGGCTGCGGGGCTTGGAGGA
GCTGCACCTGGAGGGGCTTTTCCCCCAGGAGCTAGCTCGGGCAGCCACCCTGGAGAGCCTCCGGGAG
CTGAAGCAGCTCAAGGTGTTGTCCCTCCGGAGCAACGCCGGGAAGGTGCCAGCCAGTGTGACCGACG
TTGCTGGCCACCTGCAGAGGCTCAGCCTGCACAACGATGGGGCCCGTCTGGTTGCCCTGAACAGCCT
CAAGAAGCTGGCGGCATTGCGGGAGCTGGAGCTGGTGGCCTGCGGGCTGGAGCGCATCCCCCATGCA
GTGTTCAGCCTGGGTGCGCTGCAGGAACTTGACCTCAAGGACAACCACCTGCGCTCCATCGAGGAAA
TCCTCAGCTTCCAGCACTGCCGGAAGCTGGTCACGCTCAGGCTGTGGCACAACCAGATCGCCTACGT
CCCTGAGCACGTGCGGAAGCTCAGGAGCCTGGAGCAGCTCTACCTCAGCTACAACAAGCTGGAGACC
CTGCCCTCCCAGCTCGGCCTGTGCTCAGGCCTCCGTCTGCTGGATGTGTCCCACAATGGGCTACACT
CCCTGCCACCCGAGGTGGGCCTCCTGCAGAACCTACAGCACCTGGCCCTCTCCTACAATGCCCTGGA
GGCCCTGCCCGAAGAGCTCTTCTTCTGCCGCAAGCTGCGGACGTTGCTTCTGGGCGACAACCAACTG
AGCCAGCTCTCGCCCCACGTGGGTGCCCTCAGAGCCCTCAGCCGCCTGGAGCTCAAAGGCAACCGCT
TAGAGGCGCTGCCAGAAGAACTTGGCAACTGTGGGGGGCTCAAGAAGGCGGGGCTCCTGGTGGAAGA
CACGCTTTACCAGGGTCTGCCGGCAGAAGTGCGGGACAAGATGGAGGAGGAATGAAGCTGGGGTGGG
GCCGTTTTAGGTAGAGCCTTAAAAATGCTTCTGCCCTGGAATCTCAACCATTATCTTCCAAGATAGG
AAGCCAAGTGGGTCTAGGCCAGGAGATGGGGGGGGGCGGGGGCAGCTGTGTCATCTTTCTGGGGCC_C
AGGAGGATCTGGGCTGGTTTGTCTGGGGAGACAGACAGGATGTTGTGGAGGTGGGGTGGAACCTGGT
ATGGAGGGATTAACTCAGTCATGGCATTCTCCGACCAAAACCACACCTGTGTCTCTGGCAGGCTGGC
TGGCCTTGCTCCCATCCCTAGAACTGCTGCCTCTCCCTGGATATTCCAGCTCAATTAGTGCCACATA
TGGGGGAAACGACACATCCCAGTGGGATTTCCAACACTCCCCCTCCCCATGCAACAAAGCAACTTAC
TTCTGGAGTTCTCTCCCAAGGAGAGGACACAGACACAGTTGTTTGCTGTGTTATATGTTAGCTCCGA
ACAATGGTTCTCATTTGGCTAAGCATCAAAATCACCTAGGGAGCCGGTGCAAAACAAAATATCCCAG
TCCCCTCCCCTGAAACACTGACTCAGGAGGTTTGGTTGGGGGCCAGGAGTCTGTTCCTAAATATTCC
AGGTAGTTCTGGTGCAGGTAAGTGGCCCTGAGACAGTATGTTGGGAAATGCTGACGTAAAGGTATCA
GGGCCGGGCGCTGTGGCTCATGACTATAATCCCAGCTGTTTGAGAGGCCAATGCAGGAGGATGGTTG
AGCTCAGGAGTTCGAGATCAGCCTGGGTAACATAGCGAGACCCCACCTCTGCCA
Variant sequences of NOV 15 are included in Example 3, Table 26. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 15 protein (SEQ m NO:32) encoded by SEQ m N0:31 is 667 amino acid residues in length and is presented using the one-letter amino acid code in Table 15B. Psort analysis predicts the NOV15 protein of the invention to be localized at the plasma membrane with a certainty of 0.7900.
Table 15B. Encoded NOV15 protein sequence (SEQ ID N0:32) MVCTSFWFKFPGTSSKIEHFISILGKCFDSPWTTRALSEVSGENQKGPAATERAAASIVAMAGTG
PGKAGEGEKEKVLAEPEKWTEPPVVTLLDKKEGEQAKALFEKVKKFRMHVEEGDILYTMYIRQT
VLKVCKFLAILVYNLVYVEKISFLVACRVETSEVTGYASFCCNHTKAHLFSKLAFCYISFVCIYG
LTCIYTLYWLFHRPLKEYSFRSVREETGMGDIPDVKNDFAFMLHLIDQYDSLYSKRFAVFLSEVS
ESRLKQLNLNHEWTPEKLRQKLQRNAAGRLELALCMLPGLPDTVFELSEVESLRLEAICDITFPP
GLSQLVHLQELSLLHSPARLPFSLQVFLRDHLKVMRVKCEELREVPLWVFGLRGLEELHLEGLFP
QELARAATLESLRELKQLKVLSLRSNAGKVPASVTDVAGHLQRLSLHNDGARLVALNSLKKLAAL
RELELVACGLERIPHAVFSLGALQELDLKDNHLRSIEEILSFQHCRKLVTLRLWHNQIAYVPEHV
RKLRSLEQLYLSYNKLETLPSQLGLCSGLRLLDVSHNGLHSLPPEVGLLQNLQHLALSYNALEAL
PEELFFCRKLRTLLLGDNQLSQLSPHVGALRALSRLELKGNRLEALPEELGNCGGLKKAGLLVED
TLYQGLPAEVRDKMEEE
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 15C.
Table 15C. Patp results for NOV15 Smallest Sum eading igh Prob equences igh-scoring Segment Pairs:Frame Score P(N) producing >patp:AAY70473HumanCNAP-1 +l 2147 5.0e-222 >patp:AAG75413Humancolon cancer antigen protein+1 1882 6.0e-194 >patp:AAM41692Humanpolypeptide SEQ ID NO 6623+1 1878 1.6e-193 >patp:AAU20426Humansecreted protein, Seq ID +1 1835 5.8e-189 No 418 >patp:AAB92855Humanprotein sequence SEQ ID +1 1795 1.0e-184 N0:11424 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2227 of 2228 bases (99%) identical to a gb:GENBANI~-ID:AK.027073~acc:AK027073.1 mRNA from Ho~rao Sapiens (cDNA:
FLJ23420 fis, clone HEP22352). The full amino acid sequence of the protein of the invention was found to have 444 of 444 amino acid residues (100%) identical to, and 444 of 444 amino acid residues (100%) similar to, the 444 amino acid residue ptnr:SPTREMBL-ACC:Q9HSH8 protein from Ho~ao sapiefzs (CDNA: FLJ23420 FIS, CLONE HEP22352).
NOV 15 also has homology to the proteins shown in the BLASTP data in Table 15D.
Table 15D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~13376597~ref~NPhypothetical 444 444/444 444/444 0.0 _ protein FLJ23420 (100%) (100%) 079337.1~(NM_025061 [Homo Sapiens]
gi~14150009~ref~NPhypothetical 708 404/667 520/667 0.0 _ protein (60%) (77%) 115646.1~(NM_032270 DKFZp586J1119 [Homo Sapiens]
gi~19343671~gb~AAH2Similar to 708 404/671 526/671 0.0 5473.1 (BC025473)hypothetical (60%) (78%) protein DKFZp586J1119 [Mus musculus]
gi~7243272~dbj~BAA9KIAA1437 protein811 367/666 84/666 0.0 2675.1 (AB037858)[Homo Sapiens] (55%) (72%) gi~8922442Iref~NPhypothetical 682 345/673 470/673 0.0 _ protein FLJ10470 (51%) (69%) 60573.1I(NM~018103) [Homo Sapiens]
A multiple sequence alignment is given in Table 1 SE, with the NOV 15 protein being shown on line 1 in Table 15E in a ClustalW analysis, and comparing the NOV 15 protein with the related protein sequences shown in Table 15D. This BLASTP data is displayed graphically in the ClustalW in Table 15E.
Table 15E. ClustalW Analysis of NOV15 1) > NOV15; SEQ ID NO:32 2) > gig 13376597/ hypothetical protein FLJ23420 [Homo Sapiens]; SEQ )D N0:102 3) > gig 14150009/ hypothetical protein DKFZp586J1119 [Hofno Sapiens]; SEQ >D
N0:103 4) > gi~19343671~/ Similar to hypothetical protein DKFZp586J1119 [Mus musculus]; SEQ ID N0:104 5) > gi~7243272~/ KIAA1437 protein [Homo sapierts]; SEQ ID NO:105 6) > gi~8922442~/ hypothetical protein FLJ10470 [Homo Sapiens]; SEQ >D N0:106 .1....1....I ..I . .I .1,....1....I. .I. .I. ..1....I
NOV15 l -------------- -'S~GT~~~I ~ ' '~ V~G~ 43 gi1133765971 1 _____________________________.______________________________ 1 v n ~ v V
gi1141500091 27 WYAKYFPYLVLIHTLVF~L GS ~ I ~ ' '~ ~; 86 gi1193436711 27 WYAKYFPYLVLTHTLVF~ G,S t~ ~ ~ ~ " . 86 gi172432721 121 WFAKYFPYLVLLHTLIF' R~' L ~ ~ '~ T~~ 180 gi189224421 1 ______________ y"~,,5 ~ ~K?~C ~ ~ ~ 'tC 43 ..1....1....1....1 [....1....I.. I ...I... 1....1....I
NOV15 44 Q~CGPAATERAAASIVAMAG'GPGA------GEGEKEKVLEP T PPWTL ~ 97 _~
gi1133765971 1 ___________________ ______ __ __________ ___________ 1 gi1141500091 87 EKDNRKNNN1NRSNTIQ-~GP G----- STSQSL~ IP F ~KSTAG ~ 138 gi I 193436711 87 E'EKDNRKNNMNRSGTIQ-~S.'GP G----- N~,r'X~RSQSL ~IP F~tKSAAG ~
gi 17243272 I 181 ~ ~PKPAFSKMNGSMDKKSS'I~,VSE~--VEATVPMIiQRT-- ~S'~RI
G~VC7RSETG ~ 236 gi I 8922442 I 44 y, E<ENKQRITGAQTLPKHVS~"SSD~GSPSASTP~;NKTGF IFS P~~VPSMTI ~
.I. .I. .I. .I....I....I,....1....1....1....1....1....1 NOV15 98 ~ ~ ~ ' ~ L T1~T'~L~CI:i'VONLVYVEICTS~L~ 157 gi1133765971 1 __________________________ __________ ___ _________.______ 1 gi1141500091 139 ~' ' 'L ~ L '~ L I IiI SAL SI~VQ T~ 198 gi1193436711 139 ~~'~ '~ ~ 'L ~ L w .L I LiI VSAL.SK~Q T~,1~~1,~,-~, 198 gi172432721 237 ~~ ' 'T ~ RL '~ TI I I'L I;C TVYY ~~ VD 296 gi I 8922442 I 104 ~ ~ ~ S ~LiI R~i~~~~ ~T I<F IiC~TANF 1~ ~S E 163 I....I....I....I....I ..I. 1....1....1....1....1....1 NOV15 158 R~TSES~C~TES~FC~IiSF~C~~T~~~X~P~T~RS 217 gi1133765971 1 ___________________________________=________________________ 1 gi1141500091 199 N~ltD3 S T 'T S AFC hCF S~ T ~ 'S Y 258 gi I 19343671 I 199 = ~~D , IQ. S T- ' S SFC ~:~1CF ST L Y'S Y 258 gi I 7243272 I 297 TTI~S~, RTE P 'T KI SF 1S1 IF I L ,S K S 356 gi I 8922442 I 164 KPF~VVIiI EVE T '~L LIS ~SICI L F RIP K 223 gi1133765971 1 54 gi1141500091 259 318 gi1193436711 259 318 gi172432721 357 416 gi189224421 224 $5 430 460 gi1133765971 55 gi1141500091 319 378 gi1193436711 319 378 gi17243272) 417 476 gi189224421 284 343 ~I ~~I~
~. .I~
I~ .I~~~~I~
~I ~I
. ~
I
I~~~-I .
~I~ .
.
.
NOV15 338 L SP~RLtPF~LQ _ _ _ _ ' ELFPQ 397 C~E~ L' L ~
F
~
gi 115 BL~SP~R~.iPF,LQV ' , RC,E''1~'' L'~V'L G
E~LFPQ ~L ~ 174 I E' I
gi ~ 14150009 ~ 379 QCS HSL~ ~S n' ~ 'P S SHI3I~438 gi ~ 19343671 ~ 379 QCS ~THS~L KE S n ' L'P ' S SHiIISKNS~ 438 gi ~ 8922442 ~ 344 C~CP~ ~EQT~F~E"H~T T~I~I ~L I L ~T y I~' 403 gi~13376597~ 175 234 gi~14150009~ 439 498 gi~19343671~ 439 498 gi~7243272~ 537 gi~8922442~ 404 463 1$ 610 620 630 640 650 660 gi~13376597~ 235 294 gi~14150009~ 499 558 gi~19343671~ 499 558 gi~7243272~ 597 656 gi~8922442~ 464 523 gi~13376597~ 295 354 gi~14150009~ 559 618 gi~19343671~ 559 618 gi~7243272~ 657 716 gi~89224421 524 583 gi~13376597~ 355 414 gi~141500091 6l9 gi~19343671~ 619 gi~7243272~ 717 gi~8922442~ 584 76 gi113376597~ 415 -------- 444 gi~14150009~ 679 -------- 708 gi~193436711 679 -------- 708 gi~7243272~ 777 KEQA---- 811 gi~8922442~ 644 NIPFANGI 682 . ,I I'.
f f~, G v . ~ ~; ~ T Y~ ~''E-r y~7 ~ ~ ~ , ' SD ' Q I~T
~ 'S~j ' ~~_ PL ~ ~P ~W~
L x... QI
The NOV15 Clustal W alignment shown in Table 15E was modified to begin at amino residue 121. The data in Table 15E includes all of the regions overlapping with the NOV 15 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.ukfinterpron. Table 15F lists the domain description from DOMAIN analysis results against NOV15.
Table 15F
Domain Anal sis of NOV15 Model Region of Score (bits) E value Homology LRR 454-476 5.6 1.5e+02 LRR 477-498 10.8 26 LRR 502-524 11.9 16 LRR 525-547 18.8 0.13 LRR 548-570 15.9 0.99 LRR 571-593 13.9 4 LRR 594-616 12.3 12 LRR 617-639 9.4 42 Consistent with other known members of the LRR-like family of proteins, NOV 15 has, for example, eight Leucine Rich Repeat (LRR) signature sequences and homology to other members of the LRR-like Protein Family. NOV15 nucleic acids, and the encoded polypeptides, according to the invention axe useful in a variety of applications and contexts.
For example, NOV 15 nucleic acids and polypeptides can be used to identify proteins that are members of the LRR-like Protein Family. The NOV 15 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV15 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., cancer, trauma, regeneration (ifz vitro and izz vivo), viral/bacterial/parasitic infections, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, Hirschsprung's disease , Crohn's Disease, appendicitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, autoimmune disease, xerostomia as well as other diseases, disorders and conditions.
In addition, various NOV15 nucleic acids and polypeptides according to the invention are useful, hater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV15 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the LRR-like Protein Family.
LRR Proteins are a family of proteins characterized by a structural motif rich in leucine residues. They are either transmembrane or secreted proteins and are involved in protein-protein interactions. Members of this family have been implicated in extracellular matrix assembly and cellular growth. In addition, several proteins belonging to this family, such as slit, Toll and robo have been shown to mediate key roles in central nervous system development and organogenesis in Drosophila. Vertebrate orthologs of these proteins have also been shown to have similar roles in the CNS as well as other organ systems like kidney..
LRRs are relatively short motifs (22-28 residues in length) found in a variety of cytoplasmic, membrane and extracellular proteins. Although these proteins are associated with widely different functions, a common property involves protein-protein interaction. Little is known about'the 3D structure of LRRs, although it is believed that they can form amphipathic structures with hydrophobic surfaces capable of interacting with membranes.
Irz vitro studies of a synthetic LRR from Drosophila Toll protein have indicated that the peptides form gels by adopting beta-sheet structures that form extended filaments (Packman et al.
FEBS Lett.1991;
291: 87-91). These results are consistent with the idea that LRRs mediate protein-protein interactions and cellular adhesion. Other functions of LRR-containing proteins include, for example, binding to enzymes and vascular repair.
The NOV15 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune, and nerve physiology. As such, the NOV 15 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, nervous, and immune system disorders, e.g., cancer, trauma, regeneration (ih vitro and in vivo), viral/bacterial/parasitic infections, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, Hirschsprung's disease, Crohn's Disease, appendicitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, autoimmune disease, xerostomia as well as other diseases, disorders and conditions.
The NOV15 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 15 nucleic acid is expressed in Coronary Artery, Parotid Salivary glands, Liver, Colon, Bone, Synovium/Synovial membrane, and Brain.
Additional utilities for NOV 15 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV16 polypeptide has been identified as a RhoGEF-like protein (also referred to as CG95419-02). The disclosed novel NOV16 nucleic acid (SEQ B7 N0:33) of 5372 nucleotides is shown in Table 16A. The novel NOV16 nucleic acid sequences maps to the chromosome 5.
An ORF begins with an ATG initiation codon at nucleotides 61-63 and ends with a TAA codon at nucleotides 5179-5181. A putative untranslated region and/or downstream from the termination codon is underlined in Table 16A, and the start and stop codons are in bold letters.
Table 16A. NOV16 Nucleotide Sequence (SEQ ID N0:33) CCATGGGGCCTCCTGCAATAACTTCTCTTGTTTATTATTTTCATTGCAGATGCGAAAGCCATGGAGT
TGAGCTGCAGCGAAGCACCTCTTTACCAGGGGCAGATGATGATCTATGCGAAGTTTGACAAAAATGT
GTATCTTCCTGAAGATGCTGAGTTTTACTTTACTTATGACGGATCTCATCAGCGACATGTCATGATT
GCAGAGCGCATCGAGGATAACGTTCTCCAGTCCAGCGTCCCAGGCCATGGGCTTCAGGAGACGGTGA
CGGTATCTGTGTGCCTCTGCTCGGAAGGTTACTCTCCGGTGACCATGGGCTCTGGCTCAGTGACCTA
CGTGGACAACATGGCTTGCAGGCTGGCTCGTCTGCTGGTGACGCAGGCCAATCGCCTCACAGCCTGC
AGCCACCAGACCCTGCTGACCCCATTTGCCTTGACGGCAGGAGCACTGCCTGCCTTGGATGAGGAGC
TCGTGCTGGCTCTGACCCATCTGGAATTGCCTCTAGAGTGGACTGTGTTGGGAAGTTCTTCACTTGA
AGTATCTTCTCACAGAGAATCTCTTCTACACCTGGCTATGAGATGGGGCCTGGCTAAACTTTCCCAG
TTCTTCTTGTGTCTCCCGGGGGGAGTCCAGGCCTTGGCTTTACCCAACGAAGAGGGTGCCACACCAT
TAGACTTAGCTTTACGTGAAGGACACTCCAAGCTGGTGGAAGACGTCACAAGTTTTCAGGGCAGATG
GTCCCCAAGCTTCTCCCGAGTGCAGCTCAGTGAAGAAGCCTCCTTGCATTACATTCACTCATCGGAA
ACGCTGACCCTGACCCTGAACCACACAGCCGAGCATTTGTTGGAGGCAGATATTAAACTCTTCCGGA
AATACTTTTGGGATAGAGCCTTTCTTGTCAAGGCCTTTGAGCAAGAAGCCAGGCCAGAGGAAAGAAC
AGCTATGCCCTCCAGCGGTGCAGAAACTGAAGAAGAGATTAAGAATTCAGTGTCCAGCAGATCAGCA
GCCGAAAAGGAAGATATAAAGCGTGTCAAAAGCCTGGTGGTTCAACACAATGAACATGAAGACCAGC
ACAGCCTAGATTCTAGATCGCTCCTTCGATATCCTAAAAAATCCAAGCCGCCCTCGACATTGCTTGC
TGCAGGCCGGCTTTCAGACATGCTGAATGGAGGTGATGAAGTCTACGCTAACTGTATGGTGATTGAT
CAGGTTGGTGATTTGGATATCAGCTATATTAATATAGAGGGAATCACTGCCACTACCAGCCCTGAAT
CCAGAGGTTGCACTCTGTGGCCTCAGAGCAGCAAACACACCCTTCCTACAGAAACCAGTCCCAGTGT
GTACCCACTTAGTGAAAATGTCGAAGGGACAGCACACACTGAAGCCCAGCAGTCCTTCATGTCACCA
TCAAGTTCGTGTGCTTCCAACTTGAATCTTTCTTTTGGTTGGCATGGATTTGAAAAGGAACAAAGTC
ATCTAAAGAAAAGAAGTTCTAGCCTTGATGCCTTGGACGCCGACAGTGAAGGGGAAGGGCATTCTGA
GCCATCCCACATCTGTTACACTCCAGGGTCTCAGAGCTCCTCAAGAACTGGGATTCCTAGTGGGGAT
GAATTGGACTCTTTTGAGACTAACACTGAACCGGATTTTAATATCTCCAGGGCTGAATCCCTTCCTC
TATCAAGTAATCTACAGTTGAAGGAATCACTGCTTTCTGGAGTTCGCTCACGTTCTTATTCTTGCTC
GTCACCCAAAATTTCTTTAGGAAAAACTCGTTTGGTGCGTGAATTAACAGTATGCAGTTCAAGTGAA
GAGCAAAAAGCTTACAGCTTATCGGAGCCACCAAGAGAAAACAGGATTCAGGAAGAAGAATGGGATA
AATACATCATACCTGCCAAATCAGAGTCTGAAAAATATAAAGTGAGTCGAACTTTCAGTTTCCTCAT
GAATAGGATGACTAGCCCTCGGAATAAATCAAAGACAAAAAGCAAGGATGCCAAAGATAAAGAGAAG
CTGAATCGACATCAGTTTGCCCCAGGAACATTCTCTGGGGTTCTGCAGTGTTTGGTTTGTGATAAAA
CACTCCTGGGGAAAGAGTCACTGCAGTGTTCTAGTTGTAATGCAAATGTGCACAAAGGTTGTAAAGA
TGCTGCGCCTGCATGCACCAAGAAATTCCAAGAGAAATATAACAAGAACAAACCACAGACCATCCTT
GGAAGTTCTTCATTTAGAGACATCCCACAGCCTGGTCTCTCCTTGCACCCTTCTTCCTCCGTGCCTG
TTGGATTGCCGACTGGAAGGAGGGAGACTGTGGGACAGGTCCATCCATTGTCCAGAAGTGTTCCAGG
TACCACCTTGGAAAGCTTCAGGAGGTCAGCCACATCCTTGGAGTCTGAGAGTGACAATAACAGCTGC
AGAAGCAGGTCTCATTCTGATGAGCTGCTACAGTCCATGGGCTCTTCTCCCTCTACAGAGTCTTTCA
TAATGGAAGATGTTGTGGATTCTTCTCTGTGGAGTGACCTCAGCAGTGATGCCCAGGAGTTTGAAGC
AGAATCTTGGAGTCTTGTGGTGGATCCCTCATTTTGTAATAGGCAGGAGAAGGATGTCATCAAAAGA
CAGGATGTCATTTTTGAGCTAATGCAAACAGAGATGCATCACATCCAGACCCTGTTCATCATGTCTG
AGATCTTCAGGAAAGGCATGAAAGAGGAGCTGCAGCTGGACCACAGCACCGTGGATAAAATTTTCCC
CTGTTTAGATGAGTTGCTTGAAATCCACAGGCATTTCTTCTACAGTATGAAGGAACGAAGGCAGGAA
TCAAGTGCTGGCAGCGACAGGAATTTTGTGATCGACCGAATTGGAGATATTTTGGTACAACAGTTTT
CAGAAGAAAATGCAAGTAAAATGAAGAAAATATATGGAGAATTCTGTTGCCATCATAAAGAAGCTGT
TAACCTCTTTAAAGAACTCCAGCAGAATAAAAAGTTTCAGAATTTTATTAAGCTCCGAAATAGTAAT
CTTTTGGCTCGACGCCGAGGAATTCCAGAATGCATTCTGTTGGTCACTCAGCGTATTACAAAATACC
CTGTCTTGGTGGAAAGGATATTGCAGTACACAAAGGAAAGAACTGAGGAACATAAAGACTTACGCAA
AGCCCTTTGCTTAATTAAAGACATGATTGCAACAGTGGATTTAAAAGTCAATGAATATGAGAAAAAC
CAAAAATGGCTTGAGATCCTAAATAAGATTGAAAACAAAACATACACGAAGCTCAAAAATGGACATG
TGTTTAGGAAGCAGGCACTGATGAGTGAAGAAAGGACTCTGTTATATGATGGCCTTGTTTACTGGAA
AACTGCTACAGGTCGTTTCAAAGATATCCTAGCTCTACTTCTAACTGATGTGCTGCTCTTTTTACAA
TTATTGCTAGAGAAGTTGCTAATGAGGAGAGAGGAATGTTTCTGATCAGTGCTTCATCTGCTGGTCC
TGAGATGTATGAAATTCACACCAATTCCAAGGAGGAACGCAATAACTGGATGAGACGGATCCAGCAG
GCTGTAGAAAGTTGTCCTGAAGAAAAAGGGGGAAGGACAAGTGAATCTGATGAAGACAAGAGGAAAG
CTGAAGCCAGAGTGGCCAAAATTCAGCAATGTCAAGAAATACTCACTAACCAAGACCAACAAATTTG
TGCGTATTTGGAGGAGAAGCTGCATATCTATGCTGAACTTGGAGAACTGAGCGGATTTGAGGACGTC
CATCTAGAGCCCCACCTCCTTATTAAACCTGACCCAGGCGAGCCTCCCCAGGCAGCCTCATTACTGG
CAGCAGCACTGAAAGAAGCTGAGAGCCTACAAGTTGCAGTGAAGGCCTCACAGATGGGCGCCGTGAG
TCAATCATGTGAGGACAGTTGTGGAGACTCTGTCTTGGCGGACACACTCAGTTCTCATGATGTACCA
GGATCACCGACTGCCTCATTAGTCACAGGAGGGAGAGAAGGAAGAGGCTGTTCGGATGTGGATCCCG
GGATCCAGGGTGTGGTAACCGACTTGGCCGTCTCTGATGCAGGGGAGAAGGTGGAATGTAGAAATTT
TCCAGGTTCTTCACAATCAGAGATTATACAAGCCATACAGAATTTAACCCGTCTCTTATACAGCCTT
CAGGCCGCCTTGACCATTCAGGACAGCCACATTGAGATCCACAGGCTGGTTCTCCAGCAGCAGGAGG
GCCTGTCTCTCGGCCACTCTATCCTCCGAGGCGGCCCCTTGCAGGACCAGAAGTCTCGCGACGCGGA
CAGGCAGCATGAGGAGCTGGCCAATGTGCACCAGCTTCAGCACCAGCTCCAGCAGGAGCAGCGGCGC
TGGCTGCGCAGGTGTGAGCAGCAGCAGCGGGCGCAGGCGACCAGGGAGAGCTGGCTGCAGGAGCGGG
AGCGGGAGTGCCAGTCGCAGGAGGAGCTGCTGCTGCGGAGCCGGGGCGAGCTGGACCTCCAGCTCCA
GGAGTACCAGCACAGCCTGGAGCGGCTGAGGGAGGGCCAGCGCCTGGTGGAGAGGGAGCAGGCGAGG
ATGCGGGCCCAGCAGAGCCTGCTGGGCCACTGGAAGCACGGCCGGCAGAGGAGCCTGCCCGCGGTGC
TCCTTCCGGGTGGCCCCGAGGTAATGGAACTTAATCGATCTGAGAGTTTATGTCATGAAAACTCATT
CTTCATCAATGAAGCTTTAGTACAAATGTCATTTAACACTTTCAACAAACTGAATCCGTCAGTTATC
CATCAGGATGCCACTTACCCTACAACTCAATCTCATTCTGACTTGGTGAGGACTAGTGAACATCAAG
TAGACCTCAAGGTGGACCCTTCTCAGCCTTCGAATGTCAGTCACAAACTGTGGACAGCCGCTGGTTC
CGGCCATCAGATACTTCCTTTCCAAGAAAGCAGCAAGGATTCTTGTAAAAATGATTTGGACACCTCC
CACACTGAGTCCCCAACCCCCCATGACTCAAATTCACACCGCCCTCAACTGCAGGCGTTTATAACAG
AAGCAAAGCTAAATCTACCGACAAGGACAATGACCAGACAAGATGGGGAAACTGGAGATGGAGCCAA
AGAAAATATTGTTTACCTCTAATTGTGTTGTCATTTTTCCAAACAAAACAAAACACTGGCACTTTTG
GGAGAAACTTTTTGTCTCCATTCCTTATGTATGTGTGATTGTCTGTGTCCAAATTGCTTTAAGAATA
ATATTTAATATTTCCTGGAAGCTCATTTTTTTGGCATGAGTCTAATTAAATTATTGAAAGCCAAAAA
The NOV16 protein (SEQ 1D N0:34) encoded by SEQ 1D NO:33 is 1706 amino acid residues in length and is presented using the one-letter amino acid code in Table 16B. Psort analysis predicts the NOV16 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 16B. Encoded NOV16 protein sequence (SEQ TD N0:34) MELSCSEAPLYQGQMMIYAKFDKNVYLPEDAEFYFTYDGSHQRHVMIAERIEDNVLQSSVPGHGL
QETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQANRLTACSHQTLLTPFALTAGAL
PALDEELVLALTHLELPLEWTVLGSSSLEVSSHRESLLHLAMRWGLAKLSQFFLCLPGGVQALAL
PNEEGATPLDLALREGHSKLVEDVTSFQGRWSPSFSRVQLSEEASLHYIHSSETLTLTLNHTAEH
LLEADIKLFRKYFWDRAFLVKAFEQEARPEERTAMPSSGAETEEEIKNSVSSRSAAEKEDIKRVK
SLVVQHNEHEDQHSLDSRSLLRYPKKSKPPSTLLAAGRLSDMLNGGDEVYANCMVIDQVGDLDIS
YINIEGITATTSPESRGCTLWPQSSKHTLPTETSPSVYPLSENVEGTAI~TEAQQSFMSPSSSCAS
NLNLSFGWHGFEKEQSHLKKRSSSLDALDADSEGEGHSEPSHICYTPGSQSSSRTGIPSGDELDS
FETNTEPDFNISRAESLPLSSNLQLKESLLSGVRSRSYSCSSPKISLGKTRLVRELTVCSSSEEQ
KAYSLSEPPRENRIQEEEWDKYIIPAKSESEKYKVSRTFSFLMNRMTSPRNKSKTKSKDAKDKEK
LNRHQFAPGTFSGVLQCLVCDKTLLGKESLQCSSCNANVfiKGCKDAAPACTKKFQEKYNKNKPQT
ILGSSSFRDIPQPGLSLHPSSSVPVGLPTGRRETVGQVHPLSRSVPGTTLESFRRSATSLESESD
NNSCRSRSHSDELLQSMGSSPSTESFIMEDVVDSSLWSDLSSDAQEFEAESWSLVVDPSFCNRQE
KDVIKRQDVIFELMQTEMHHIQTLFIMSEIFRKGMKEELQLDHSTVDKIFPCLDELLEIHRHFFY
SMKERRQESSAGSDRNFVIDRIGDILVQQFSEENASKMKKIYGEFCCHHKEAVNLFKELQQNKKF
QNFIKLRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKERTEEHKDLRKALCLIKDMIA
TVDLKVNEYEKNQKWLEILNKIENKTYTKLKNGHVFRKQALMSEERTLLYDGLVYWKTATGRFKD
ILALLLTDVLLFLQEKDQKYIFAAVDQKPSVISLQKLIAREVANEERGMFLISASSAGPEMYEIH
TNSKEERNNWMRRIQQAVESCPEEKGGRTSESDEDKRKAEARVAKIQQCQEILTNQDQQICAYLE
EKLHIYAELGELSGFEDVHLEPHLLIKPDPGEPPQAASLLAAALKEAESLQVAVKASQMGAVSQS
CEDSCGDSVLADTLSSHDVPGSPTASLVTGGREGRGCSDVDPGIQGVVTDLAVSDAGEKVECRNF
PGSSQSEIIQAIQNLTRLLYSLQAALTIQDSHIEIHRLVLQQQEGLSLGHSILRGGPLQDQKSRD
ADRQHEELANVHQLQHQLQQEQRRWLRRCEQQQRAQATRESWLQERERECQSQEELLLRSRGELD
LQLQEYQHSLERLREGQRLVEREQARMRAQQSLLGHWKHGRQRSLPAVLLPGGPEVMELNRSESL
CHENSFFINEALVQMSFNTFNKLNPSVIHQDATYPTTQSHSDLVRTSEHQVDLKVDPSQPSNVSH
KLWTAAGSGHQILPFQESSKDSCKNDLDTSHTESPTPHDSNSHRPQLQAFITEAKLNLPTRTMTR
QDGETGDGAKENIVYL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 16C.
Table 16C. Pat results for NOV16 Smallest Sum eading igh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:ABB44551Humanwound healing related polypeptide+1 1470 2.8e-150 >patp:AAW93941Humanbrx protein +1 1436 1.5e-148 >patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148 #5528 >patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148 #5528 >patp:ABG15870Novelhuman diagnostic protein +1 1447 7.5e-148 #15861 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 4339 of 5274 bases (82%) identical to a gb:GENBANI~-m:MMU73199~acc:U73199.1 mRNA from Mus musculus (Rho-guanine nucleotide exchange factor mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 1350 of 1670 amino acid residues (80%) identical to, and 1460 of 1670 amino acid residues (87%) similar to, the 1693 amino acid residue ptnr:SWISSPROT-ACC:P97433 protein fromMus n2usculus (RHO-GUANINE
NUCLEOTIDE EXCHANGE FACTOR (RHOGEF) (RIP2)).
NOV16 also has homology to the proteins shown in the BLASTP data in Table 16D.
Table 16D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) (~) gi~7106395~ref~NPRho interacting1693 1350/16741460/16740.0 _ protein 2; Rho (80~) (86~) 36156.1~(NM_012026) specific exchange factor [Mus musculus]
gi~18602674~ref~XPhypothetical 669 668/669 668/669 0.0 _ protein FLJ21817 (99~) (99~) 016989.5~(XM
similar to Rhoip2 [Homo Sapiens]
gi~10438441~dbj~BABunnamed protein669 669/669 669/669 0.0 15243.1~(AK025816)product (1000 (1000 [Homo Sapiens]
gi~15341761~gb~AAH1hypothetical 615 609/613 609/613 0.0 2946.1~AAH12946(BCOprotein FLJ21817 (99~) (99~) 12946) similar to Rhoip2 [Homo Sapiens]
gi~17437752~ref~XPsimilar to Rho 590 290/292 291/292 e-170 _ interacting (99$) (99~) 068710.1~(XM
_ protein 2; Rho specific exchange factor [Homo Sapiens]
A multiple sequence alignment is given in Table 16E, with the NOV 16 protein being shown on line 1 in Table 16E in a ClustalW analysis, and comparing the NOV 16 protein with the related protein sequences shown in Table 16D. This BLASTP data is displayed graphically in the ClustalW in Table 16E.
Table 16E. ClustalW Analysis of NOV16 1) > NOV16; SEQ >D NO:34 2) > gi~7106395~/ Rho interacting protein 2; Rho specific exchange factor [Mus musculus]; SEQ >D
N0:107 3) > gig 18602674/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo sapieras]; SEQ >D NO:108 4) > gig 10438441 ~/ unnamed protein product [Horno Sapiens]; SEQ ID N0:109 5) > gi~15341761~/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo Sapiens]; SEQ ID NO:110 6) > gig 17437752/ similar to Rho interacting protein 2; Rho specific exchange factor [Homo Sapiens];
SEQ ID NO:111 ' giI71063951 1 -MELSCSEVPLYGQKTVYAKFGKNVYLPEDAEFYFVYGGSHQRHWIADRVQDNVLQSSI 59 gip8sozs74~ 1 ____________________________________________________________ 1 gi~10438441~ 1 ____________________________________________________________ 1 gi~15341761~ 1 ____________________________________________________________ 1 gi~17437752~ 1 ____________________________________________________________ 1 gi~71063951 60 PGHWLQETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQADRLTACSHQTLL
gi~18602674) 1 ____________________________________________________________ 1 gi~10438441~ 1 ____________________________________________________________ 1 gi~15341761~ 1 ____________________________________________________________ 1 gi~17437752~ 1 ____________________________________________________________ 1 130 140 150 160 170 7.80 NOVl6 121 gi~71063951120 TPFALTVEALPALDEELVLALTQLELPLGWTVLGNSSLEVSLHRESLLHLAVRWALPKLF179 $ giI18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________ gi~15341761~1 ____________________________________________________________1 gi~17437752~1 1 1~ 190 200 210 220 230 240 gi~7106395~ 180 gi~18602674~ 1 ____________________________________________________________1 IS gi~104384411 1 ____________________________________________________________1 gi1153417611 1 ____________________________________________________________1 gi~17437752~ 1 ____________________________________________________________1 giI71063951240 LQFVHSSETLTLTVNHTAEHLLEADIKLFRKYFWDRAFLVKALEQEAKTEKATMPSGAAE299 gi~18602674)1 ____________________________________________________________1 gi~10438441~l ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752)1 ____________________________________________________________1 gi~7106395~300 TEEEVRNLESGRSPSEEEEDAKSIKSQVDGPSEHEDQDRLALDRSFDGLKKSKHVPASLA359 gi~18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~1534176111 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 4O giI71063951360 gi~18602674~1 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi~7106395~420 PDTSPCGRPLIENSEGTLDAAASQSFVTPSSSRTSNLNLSFGLHGFEKEQSHLKKRSSSL479 gi~1860267411 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi ~71063951480 DALVADSEGEGGSEPPICYAVGSQSSPRTG-gi ~1860267411 ____________________________________________________________1 gi ~10438441~1 ____________________________________________________________1 gi ~15341761~1 ____________________________________________________________1 gi ~17437752~1 ____________________________________________________________1 gi I7106395~539 gi ~1860267411 ____________________________________________________________1 gi I10438441~1 ____________________________________________________________1 gi ~15341761~1 ____________________________________________________________1 gi~17437752~ 1 ____________________________________________________________ 1 gi~7106395~
gi~186026741 1 ____________________________________________________________1 gi~10438441~ 1 ____________________________________________________________1 giI15341761~ 1 ____________________________________________________________1 gi117437752~ 1 ____________________________________________________________1 IS
gi~71063951659FSGVLQCSGCDKTLLGKESLQCANCKANTHKGCKDAVPPCTKKFQEKYNKNKPQSILGSS718 gi~1860267411 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 gi~15341761)1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 giI7106395~719SVRDVPAPGLSLHPSSSMPIGLPAGRKEFAAQVHPLSRSVPGTTLESFRR--AVTSLESE776 2S giI1860267411 ____________________________________________________________1 gi ~10438441~1 ____________________________________________________________1 gi~1534176111 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 giI71063951777GDSWRSRSHSDELFQSMGSSPSTESFMMEDWDSSLWIDLSSDAQEFEAESWSLWDPSF836 gi~1860267411 ____________________________________________________________1 3S gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi~71063951837CSRQEKDVIKRQDVIFELMQTEVHHIQTLLIMSEVFRKGMKEELQLDHSTVDKIFPCLDE896 gi~18602674~1 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 4S gi~1534176111 ____________________________________________________________1 gi~1743775211 ____________________________________________________________1 .) giI71063951897LLETHRHFFFSMKERRQESCAGSDRNFVINQIGDILVQQFSEENASKMKRIYGEFCSHHK956 gi~18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi115341761~1 ____________________________________________________________ SS gi~17437752~1 ____________________________________________________________ C70gi~7106395~957EAMSLFKELQQNKKFQNFIKIRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKE10 gi~1860267411 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________ 1030 1040 . 1050 1060 1070 1080 .
' ' a 1077 r rv .n ~ --a ~
~
i a 1073 giI71063951 1017 RTEEHRDLCKALGLIKD ~ ~ --a ~
70 gi~18602674~ 1 ---------------- ~ --a ~ a 40 gi~10438441~ 1 ________________ . , __~ 1 ~ 40 gi~7.5341761~ 1 ________________ . , __~ 1 ~ 40 gi~17437752~ 1 ------------------PDNQNPT CSGET~~ P EGGS---- PAS RCES 38 gi~71063951 1074 1133 gi~186026741 41 100 giI10438441~ 41 100 gi~15341761~ 41 100 gi117437752~ 39 94 gi~7106395~ 1134 1193 gi~18602674~ 101 160 gi~10438441~ 101 160 gi115341761~ 101 160' gi117437752~ 95 148 NOV16 1198 ' 1.1i~- ~ ~. ~ 11 1 ~m 111 s~~ ~~~~ . 1-- 1256 gi~7106395~ 1194 ' 1 ~ ~ ~' ~ 11 1 SPm 111 1T ~ 1 1252 gi~18602674~ 161 1 1~'- ~ ~' ~ 11 1 1111 ~ ~ 1 219 gi~10438441~ 161 1 1 ' ~ ~~ ~ 11 1 1111 ~ 1 219 gi~15341761~ 161 ' 1 1 '- ~ ~~ ~ 11 1 1111 ~ ~ 1 219 gi ~ 17437752 ~ 149 L QSL ~LNIFLPVT~I~QIKFGMFG,- ------FVTIVMSVCKVG~TKE~ 202 ~ y NOV16 1257 ~ 1316 giI7106395~ 1253 1312 gi~18602674~ 220 279 gi~10438441~ 220 279 gi~15341761~ 220 279 gi~17437752~ 203 252 NOV16 1317 . 1375 gi~7106395~ 1313 1371 gi~186026741 280 338 gi~10438441~ 280 338 gi~15341761~ 280 338 gi117437752~ 253 311 gi~7106395~ 1372 1430 gi~186026741 339 397 gi~10438441~ 339 397 gi~15341761~ 339 397 gi~174377521 312 351 gi~7106395~ 1431 1490 giI186026741 398 457 gi~104384411 398 457 gi~15341761~ 398 457 gi~174377521 351 399 ..
NOV16 1495 1~1 1 ~1 1 1~~~ ~11~ ~1 .' 1554 .1. .1..... . 1 . 1 1 1 ~1 . i 1 11 ~
~
.1. .1.. . 1 . 1 E 1 P
. S P T' .1. ..l....~.'~a'a1 ' . 1 1 a a 1 .1. .1..... . 1 . 1 . 1 1 1 . 1 .1. .1..... 1 . 1 . 1 1 1 GQ.".~.:. T- PAEFYFTYD HQRH I-- BRIE..,.QS,'r'"'LPG
~'AKFD _ 1 _ _ _ _ _ _ VY
1 s 1 1 1 r 1 1 1 aee .1 1 1 ' 1 a - 1 1 1 . a G~QEE,,, CLCS- YSPVTGS SVTYVD CRLiRLLVTQA1~ RLTAC QT,'C.e IiT
#PD148197 119-399 1175 e-129 PALADIN GENE
#PD222597 119-231 119 6e-07 DOMAIN
OF UNKNOWN
#PD306865 354-454 132 2e-08 PALADIN GENE
#PD024454 356-445 84 0.007 PLASMID ORFS
#PD325716 400-604 800 6e-86 PALADIN GENE
#PD326847 458-594 97 2e-04 #PD222597 505-602 113 3e-06 bOMAIN
OF UNKNOWN
#PD277963 595-648 97 2e-04 HYDROLASE
CDNA
#PD148197 605-678 340 1e-32 PALADIN GENE
#PD306865 680-856 765 7e-82 PALADIN GENE
#PD325716 751-820 86 0.004 PALADIN GENE
Consistent with other known members of the Paladin-like family of proteins, has, for example, multiple Paladin gene signature sequences and homology to other members of the Paladin-like Protein Family. NOV 1 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 1 nucleic acids and polypeptides can be used to identify proteins that are members of the Paladin like family of proteins. The NOV 1 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 1 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism and signal transduction. These molecules can be used to treat, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atnial septal defect (ASD),atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, hyperthyroidism and hypothyroidism, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmmne disease, allergies, immunodeficiencies, transplantation, graft vesus host, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphedema, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, cancer, trauma, regeneration (in vitro and irz vivo), viral/bacteriallparasitic infections, as well as other diseases, disorders and conditions.
In addition, various NOV 1 nucleic acids and polypeptides according to the invention are useful, izzter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV1 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Paladin-Iike protein family.
Paladin proteins are a family of protein-tyrosine phosphatases. The protein phosphatases can be divided into 2 large families: the serinelthreonine phosphatases, which are metalloproteins, and the protein-tyrosine phosphatases, which proceed via a thiol-phosphate enzyme intermediate. The protein-tyrosine phosphatase family includes the VHl-like dual-specificity phosphatases. These phosphatases dephosphorylate phosphotyrosine-as well as phosphoserine- and phosphothreonine-containing substrates. Members of the dual-specificity phosphatase protein family inactivate mitogen-activated protein (MAP) kinase through dephosphorylation of critical threonine and tyrosine residues. Members of the MAP kinase family play a pivotal role in cellular signal transduction. Using a subtractive screen of mouse gastrulation, Pearce et al. (1996) identified a novel mouse gene, paladin, with similarity to the dual specificity protein phosphatase family.
The NOV1 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and endocrine physiology. As such, the NOV 1 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD),atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, hyperthyroidism and hypothyroidism, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft vesus host, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeflciencies, transplantation, graft versus host disease (GVHD), lymphedema, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, cancer, trauma, regeneration (ira vitro and iya vivo), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
The NOV1 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 1 nucleic acid is expressed in brown adipose, heart, aorta, Ve111, umbilical vein, adrenal gland/suprarenal gland, pancreas, thyroid, salivary glands, parotid salivary glands, stomach, liver, gall bladder, small intestine, colon, bone marrow, lymphoid tissue, spleen, lymph node, tonsils, thymus, cartilage, muscle, brain, thalamus, hypothalamus, pituitary gland, amygdala, substantia nigra, hippocampus, spinal chord, cervix, mammary gland/breast, ovary, placenta, uterus, vulva, prostate, testis, lung, lung pleura, kidney, retina, dermis.
Additional utilities for NOV 1 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV2 polypeptide has been identified as a Plasma Membrane Ring Finger-like protein (also referred to as CG93210-01). The disclosed novel NOV2 nucleic acid (SEQ m N0:3) of 1205 nucleotides is shown in Table 2A. The novel NOV2 nucleic acid sequences maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 17-19 and ends with a ATT codon at nucleotides 1149-1151. A putative untranslated region and/or downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
Table 2A. NOV2 Nucleotide Sequence (SEQ ID N0:3) CTCGCCGGGTCCGGCCATGGGCCCCGCCGCTCGCCCCGCGCTGAGATCGCCGCCGCCGCCTCCGCCG
CCGCCTCCGTCTCCGCTGCTGCTGCTGCTGCCCCTGCTGCCGCTGTGGCTGGGCCTGGCGGGGCCCG
GGGCCGCGGCGGACGGCAGCGAGCCGGCGGCCGGGGCGGGGCGGGGCGGAGCCCGCGCCGTGCGGGT
GGACGTGAGACTGCCGCGCCAGGACGCTCTGGTCCTGGAGGGCGTCAGGATCGGCTCCGAAGCCGAC
CCGGCGCCCCTGCTGGGCGGTCGTCTGCTGCTGATGGACATCGTGGATGCCGAGCAGGAGGCACCAG
TGGAAGGCTGGATTGCAGTGGCATACGTGGGCAAGGAGCAGGCGGCCCAGTTCCACCAGGAGAATAA
GGGCAGTGGCCCGCAGGCCTATCCCAAGGCCCTGGTCCAGCAGATGCGGCGGGCCCTCTTCCTGGGT
GCCTCTGCCCTGCTTCTTCTCATCCTGAACCACAACGTGGTCCGAGAGCTGGACATATCCCAGCTTC
TGCTCAGGCCAGTGATCGTCCTCCATTATTCCTCCAATGTCACCAAGCTGTTGGATGCATTGCTGCA
GAGGACCCAGGCCACGGCTGAGATCACCAGCGGAGAGTCCCTGTCTGCCAATATCGAGTGGAAGTTG
ACCTTGTGGACCACCTGTGGCCTCTCCAAGGATGGCTATGGAGGATGGCAGGACTTGGTCTGCCTTG
GAGGCAGTCGTGCCCAGGAGCAGAAACCCCTGCAGCAGCTGTGGAACGCCATCCTGCTGGTGGCCAT
GCTCCTGTGCACAGGCCTCGTGGTCCAGGCCCAGCGGCAGGCGTCGCGGCAGAGCCAGCGGGAGCTC
GGAGGCCAGGTGGACCTGTTTAAGCGCCGCGTGGTGCGGAGACTGGCATCCCTCAAGACACGGCGCT
GCCGGCTGAGCAGGGCAGCGCAGGGCCTCCCAGATCCGGGTGCTGAGACCTGTGCGGTGTGCCTGGA
CTACTTCTGCAACAAACAGTGGCTCCGGGTGCTGCCCTGTAAGCACGAGTTTCACCGAGACTGTGTG
GACCCCTGGCTGATGCTCCAGCAGACCTGCCCACTGTGCAAATTCAACGTCCTGGGTGAGCACCGCT
ACTCCGATGATTAGCTGCCCAGCTGGACTCTGCACATGGGGATGGACCCCTCCTGCCTGCACCCCG
The NOV2 protein (SEQ ID N0:4) encoded by SEQ ID NO:3 is 378 amino acid residues in length and is presented using the one-letter amino acid code in Table 2B. Psort analysis predicts the NOV2 protein of the invention to be localized at the plasma membrane with a certainty of 0.6400.
Table 2B. Encoded NOV2 protein sequence (SEQ ID N0:4) MGPAARPALRSPPPPPPPPPSPLLLLLPLLPLWLGLAGPGAAADGSEPAAGAGRGGARAVRVDVR
LPRQDALVLEGVRIGSEADPAPLLGGRLLLMDIVDAEQEAPVEGWIAVAYVGKEQAAQFHQENKG
SGPQAYPKALVQQMRRALFLGASALLLLILNHNVVRELDISQLLLRPVIVLHYSSNVTKLLDALL
QRTQATAEITSGESLSANIEWKLTLWTTCGLSKDGYGGWQDLVCLGGSRAQEQKPLQQLWNAILL
VAMLLCTGLVVQAQRQASRQSQRELGGQVDLFKRRWRRLASLKTRRCRLSRAAQGLPDPGAETC
AVCLDYFCNKQWLRVLPCKHEFHRDCVDPWLMLQQTCPLCKFNVLGEHRYSDD
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 2C.
Table 2C. Pat results for Smallest Sum eadingigh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAB42695HumanORFX ORF2459 polypeptide +1 1715 3.0e-276 >patp:AAM79288Humanprotein SEQ TD NO 1950 +1 612 2.3e-59 >patp:AAM80272Humanprotein SEQ TD NO 3918 +1 534 4.2e-51 >patp:AAU28202Novelhuman secretory protein +1 201 5.1e-13 >patp:ABB50251Humantranscription factor TRFX-102+1 148 5.5e-13 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 287 of 489 bases (58%) identical to a gb:GENBANK-1D:SSI132828~acc:AJ132828.1 mRNA from Spermatozopsis similis (mRNA
for p210 protein, partial). The full amino acid sequence of the protein of the invention was found to have 341 of 379 amino acid residues (89%) identical to, and 355 of 379 amino acid residues (93%) similar to, the 379 amino acid residue ptnr:SPTREMBL-ACC:Q9DCW1 protein from Mus musculus (0610009J22RIK PROTEIN).
NOV2 also has homology to the proteins shown in the BLASTP data in Table 2D.
Table 2D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~12832380~dbj~BABdata source:SPTR,379 340/380 354/380 e-173 22082.1 (AK002414)source (89%) (92%) key:Q9Y6U7, evidence:ISS~homo log to WUGSC:H DJ130H16.
PROTEIN(FRAGMENT) putative [Mus musculus]
gi~5441942~gb~AAD43supported by 347 336/336 336/336 e-148 187.1~AC004997 mouse EST (100%) (100%) (AC004997) AA538043 (NID:g2284036) [Homo Sapiens]
gi~17485136~refiXPsimilar to data272 271/283 272/283 e-146 _ source:SPTR, (95%) (95%) 066294.1 (XM 066294) source key:Q9Y6U7 evidence:ISS-homo log to WUGSC:H_DJ130H16.
(FRAGMENT)-putati ve [Homo Sapiens]
giI17861674~gb~AAL3GH20973p 461 26/57 42/57 9e-13 9314.1 (AY069169)[Drosophila (45%) (73%) melanogaster]
gi~18485962~ref~XPsimilar to 461 26/57 42/57 1e-12 - goliath (H. (45%) (73%) 080778.1 (XM 080778) Sapiens) [Drosophila melanogaster]
A multiple sequence alignment is given in Table 2E, with the NOV2 protein being shown on line 1 in Table 2E in a ClustalW analysis, and comparing the NOV2 protein with the related protein sequences shown in Table 2D. This BLASTP data is displayed graphically in the ClustalW in Table 2E.
Table 2E. ClustalW Analysis of NOV2 1) > NOV2; SEQ 1D N0:4 2) >gi~12832380~/ data source:SPTR, source key:Q9Y6U7, evidence:ISS~homolog to WITGSC:H DJ130HI6.6 PROTEIN(FRAGMENT)~putative [Mus musculus]; SEQ ID N0:40 S 3) >gi~5441942[/ supported by mouse EST AA538043 (NID:g2284036) [Homo sapierrs]; SEQ ID
N0:41 4) >gi~17485136~/ similar to data source:SPTR, source key:Q9Y6U7 evidence:ISS~homolog to WUGSC:H DJ130H16.6 PROTEIN (FRAGMENT)~putative [Homo Sapiens]; SEQ ID N0:42 S) >gi~17861674~/ GH20973p [D>"osophila rnelanogaster°]; SEQ ID
N0:43 6) >gi~18485962~/ similar to goliath (H. Sapiens) [Drosophila melarrogaster];
SEQ ID N0:44 1 S NOV2 1 -- GP ~~ P RSP--P ~ 'PPP PPLL ~:f'LPI#LPLWL ~ GPG ~~ . EP ' 52 gi~128323801 1 -- -GS~P RSPSLP " PPS PPLL ~LP~iLPLWL GPG ~EPAT E 54 gi I 5441942 I 1 -- --GP~ARP RSP--P "PPP PPLL T.iLPLPLWLG~AGPGAAAS,EP '.. 52 gi~17485136~ 1 ______~_____________________.____'___________________________ 1 gi ~ 17861674 ~ 1 MYIRKTLL~i1CLVL FGG--L"LTF ~,'TTT AAh?tSIANQD ERYFRPG THSFS
2O gi ~ 18485962 I 1 MYIRKTT~LL~CLVLBFGG--L~LTF~A'~'TTT~AAN~SIANQDBERYFRPG~'~HSF~ 58 p...
NOV2 53 GRGG~i =R----------------VL1't7RLP~.QDj~~I,LVL ------ ~ GS~EAT7PP 87 2S gi ~ 12832380 ~ 55 GRGGf,~~1,PL R=---------------V~T~KLP~.QD~~,LVL ------ ~
gi~5441942~ 53 GRGG~RA~R _______________~RLP~2QD~LVL ________G'EM3PP 87 gi 17485136 Z
gi~178616741 59 EDRI~i1VD YNYAFLNWSYVEHGNMLC~EFA~Q~~,RY KVLNVTGRLH T,ATDFD 118 gi~18485962~ 59 EDRI~~YNYAFLNWSYVEHGNMLC~EFAQFQ~RYG~KVLNVTGRL,~HBT~TDF~.S,D
30 ' gi~12832380~ 90 145 3S gi~5441942~ 88 143 gi~17485136~ 1 48 gi~I7861674~ 119 177 gi~184859621 119 177 gi~128323801 146 203 gi~54419421 144 201 4S gi~174851361 49 106 gi~17861674~ 178 237 gi~18485962~ 178 237 NOV2 202 ~ 1'I' ~ _____________ ______~ "___ 236 gi~12832380~ 204 ~ S ~ _____________ ______~ ~~-__ 238 gi~54419421 202 ~ T _____________ ______~ w-__ 236 gi~17485136~ 107 ~ ~ _______________ _______, w-__ 141 SS gi ~ 17861674 ~ 238 VS FIVL~III~L LFY~'~~~,'IQRFRYMQAKDQQSRNL StrT
~KAIMKIPTKT KE~S~EKD 297 gi ~ 18485962 I 238 VS SFTVL~IISL L~FYIQRFRYMQAKDQQSRNL S~tt'I' ~KAIMKIPTKT
IC~'S~EKD 297 gi~Z2832380~ 238 gi~5441942~ 236 287 gi117485136~ 141 192 gi~178616741 298 357 6S gi~18485962~ 298 357 ~y y ~ .I~ ~~~ ~I. ~~~ .y y y . ~~. y w y v~~w~~ aw ' ~w NOV2 288 sn-- ' '~~Q '~' CDY .KQWLRVLP 342 7~1 ~vV
gi~12832380~ 290 ~E~-- ~ ~~HS~~E''T CDY KQWLRVLP 344 gi~54419421 288 ~ ~-- ~~Q ~~~ C~iDY ------- 334 gi~17485136~ 193 ~ ~-- ' '~~Q ~~~ ~ LRVLP K-------- 239 .YY
gi ~ 17861674 ~ 358 SE~SILE~IYC~PDPP~L,V 'DESAD '~RDF ~FPRVFVLDSGCWGAREMLFPCR
gi~18485962~ 358 ~- --~XQTPSP~HTiP~AIEEVPV~UViVPH~~Q~LQPLQiSN~iSS~APSHYFQSSR
1~ 430 440 450 460 470 NOV2 343 CK---HEFHDC'VD LLsQC~ CF GEHRYS~_?~~D------------- 378 gi~12832380! 345 CK---HEFHR!DC~D L~IL~Q'~C~ CAF GHYSD~-------------- 379 gi~54419421 334 ________ K __~ ~ ApG ___ ____-_________ 347 15 gy 17485136 ~ 239 -----HEFH~DC~DQP1LL~QC~CF G1i.~T:~RYSD~~--------------gi~17861674! 418 IPERSQSSLSLRQARDW,~SLMN E~QQ RMRND MQQVIK-------- 461 gi~18485962~ 413 SP---SSSVQQLTYQPHPQQAASERGRRISAPATMPHAITASHQVTDV 461 20 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Iliterpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 2F lists the domain description from DOMAIN analysis results against NOV2.
Table 2F Domain sis of Anal NOV2 Model Region of Score E value (bits) Homology Ring Finger 325-365 49.3 4.0e-07 zf-C3HC4 325-365 34.7 2.2e-09 PHD 324-368 -10.4 1.1 Consistent with other known members of the Membrane Ring Finger-like family of proteins, NOV2 has, for example, a Ring Finger signature sequence and homology to other members of the Plasma Membrane Ring Finger-Iike Protein Family. NOV 2 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV2 nucleic acids and polypeptides can be used to identify proteins that are members of the Plasma Membrane Ring Finger-like Protein Family. The NOV2 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 1 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism and signal transduction.
These molecules can be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, immunodeficiencies, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cancer, trauma, i regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
In addition, various NOV2 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV2 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Plasma Membrane Ring Finger-like Protein Family .
The NOV2 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of immune and renal physiology. As such, the NOV2 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., anemia, ataxia-telangiectasia, autoimmume disease, immunodeficiencies, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cancer, trauma, regeneration (in vitro and in vdv0), viral/bacterial/parasitic infections, as well as other diseases, disorders and conditions.
The NOV2 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV2 nucleic acid is expressed in peripheral blood, and a pool of various mammalian tissues. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of CuraGen Acc. No. CG93210-Ol.The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-1D: gb:GENBANK-ID:SSI132828~acc:AJ132828.1) a closely related Spe~matozopsis similis mRNA for p210 protein, partial homolog in species Spe~n2atozopsis sirnilis :kidney.
Additional utilities for NOV2 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV3 polypeptide has been identified as a Thrombospondin type 1 (tsp_1) domain containing protein (also referred to as CG93275-OI). The disclosed novel NOV3 nucleic acid (SEQ ID NO:S) of 799 nucleotides is shown in Table 3A. The novel NOV3 nucleic acid sequences maps to the chromosome 16.
An ORF begins with an ATG initiation codon at nucleotides 51-53 and ends with a TGA codon at nucleotides 744-746. A putative untranslated region and/or downstream from the termination codon is underlined in Table 3A, and the start and stop codons are in bold letters.
Table 3A. NOV3 Nucleotide Sequence (SEQ ID NO:S) GAATATATTTAGTGTGTTGTTTTTTTTTTTAATGTGGCTACTGAAACCTAATGGGAATGCAAATAGA
ACTTTTTTGTCTTCTCAAGTGTTCCAAGACCTGTGGACGAGGGGTGAGGAAGCGTGAACTCCTCTGC
AAGGGCTCTGCCGCAGAAACCCTCCCCGAGAGCCAGTGTACCAGTCTCCCCAGACCTGAGCTGCAGG
AGGGCTGTGTGCTTGGACGATGCCCCAAGAACAGCCGGCTACAGTGGGTCGCTTCTTCGTGGAGCGA
GTGTTCTGCAACCTGTGGTTTGGGTGTGAGGAAGAGGGAGATGAAGTGCAGCGAGAAGGGCTTCCAG
GGAAAGCTGATAACTTTCCCAGAGCGAAGATGCCGTAATATTAAGAAACCAAATCTGGACTTGGAAG
AGACCTGCAACCGACGGGCTTGCCCAGCCCATCCAGTGTACAACATGGTAGCTGGATGGTATTCATT
GCCGTGGCAGCAGTGCACAGTCACCTGTGGGGGAGGGGTCCAGACCCGGTCAGTCCACTGTGTTCAG
CAAGGCCGGCCTTCCTCAAGTTGTCTGCTCCATCAGAAACCTCCGGTGCTACGAGCCTGTAATACAA
ACTTCTGTCCAGCTCCTGAAAAGAGAGAGGATCCATCCTGCGTAGATTTCTTCAACTGGTGTCACCT
AGTTCCTCAGCATGGTGTCTGCAACCACAAGTTTTACGGAAAACAATGCTGCAAGTCATGCACAAGG
AAGATCTGATCTTGGTGTCCTCCCCAGCCTTAGGGCCAGGGGCTTACCTTTCAACCTCTAGA
The NOV3 protein (SEQ ID N0:6) encoded by SEQ m N0:5 is 231 amino acid residues in length and is presented using the one-letter amino acid code in Table 3B. Psort analysis predicts the NOV3 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 3B. Encoded NOV3 protein sequence (SEQ ID N0:6) MGMQIELFCLLKCSKTCGRGVRKRELLCKGSAAETLPESQCTSLPRPELQEGCVLGRCPKNSRLQ
WVASSWSECSATCGLGVRKREMKCSEKGFQGKLITFPERRCRNIKKPNLDLEETCNRRACPAHPV
YNMVAGWYSLPWQQCTVTCGGGVQTRSVHCVQQGRPSSSCLLHQKPPVLRACNTNFCPAPEKRED
PSCVDFFNWCHLVPQHGVCNHKFYGKQCCKSCTRKI
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 3C.
Table 3C. Patp results for Smallest Sum eadingigh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAE09696Humangene 7 encoding protein +1 1248 9.2e-127 >patp:AAE09699Humangene 10 encoding protein +1 1245 1.9e-126 >patp:AAU72893Humanmetalloprotease partial +1 1204 4.2e-122 sequence #5 >patp:AAU72891Humanmetalloprotease partial +1 693 3.5e-70 sequence #$3 >patp:AAB21253Humanmetalloproteinase KIAA0605 +1 327 5.5e-28 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 392 of 396 bases (98%) identical to an EST
AA057409 mRNA from human). The full amino acid sequence of the protein of the invention was found to have 74 of 216 amino acid residues (34%) identical to, and 107 of 216 amino acid residues (49%) similar to, the 237 amino acid residue ptnr:SPTREMBL-ACC:Q9HBS6 protein from Homo Sapiens (HYPOTHETICAL 25.7 I~DA PROTEIN.
NOV3 also has homology to the proteins shown in the BLASTP data in Table 3D.
Table 3D. BLAST
results for Gene Index/ Protein/ OrganismLength Tdentity PositivesExpect Tdentifier (aa) (%) (%) gi~18598706~ref~XPhypothetical 1123 181/183 183/183 e-200 _ protein XP_091253 (98%) (99%) 091253.7.~(XM_091253 [Homo Sapiens]
gi~19171150~emb~CACADAMTS18 protein1081 61/62 62/62 4e-27 83612.1~(AJ311903)[Homo Sapiens] (98%) (99%) gi~7662202~ref~NP_0ItIAA0605 gene 951 79/216 99/216 9e-23 55509.11(NM 014694)product (36%) (45%) [Homo Sapiens]
gi~18561227IrefIXPhypothetical 1365 51/112 74/112 4e-21 _ protein XP_094442 (45%) (65%) 094442.1~(XM_094442 [Homo Sapiens]
gi~17432918Isp~Q9H3HUMAN ADAMTS-10223 74/223 104/223 5e-20 24IAT10' precursor (A (33% (46%) disintegrin and metalloproteinase with thrombospondin motifs 10) (ADAM-TS 10) (ADAM-TS10)(Fragment) A multiple sequence alignment is given in Table 3E, with the NOV3 protein being shown on Iine 1 in Table 3E in a ClustalW analysis, and comparing the NOV3 protein with the related protein sequences shown in Table 3D. This BLASTP data is displayed graphically in the ClustalW in Table 3E.
Table 3E. ClustalW Analysis of NOV3 1) > NOV3; SEQ ID N0:6 2) >gi~18598706~/ hypothetical protein XP_091253 [Homo Sapiens]; SEQ >D N0:45 3) >gi~19171150~/ ADAMTS18 protein [Homo Sapiens]; SEQ ID N0:46 4) >gi~7662202~/ I~IAA0605 gene product [Homo Sapiens]; SEQ ID N0:47 5) >gi~ 18561227/ hypothetical protein XP_094442 [Homo Sapiens]; SEQ ID N0:48 6) >gi~17432918~/ AT10_HCTMAN ADAMTS-10 precursor (A disintegrin and metalloproteinase with thrombospondin motifs 10) (ADAM-TS 10) (ADAM-TS10) (Fragment); SEQ IT3 N0:49 NOV3 1 _______________________________________________~__MQIE~iF~LL 11 gi1185987061 769 GGVRSAKVLSLEEWIKSETTL-ARKEQQQPSTGWMPGEQS<TQa~KA~-AeG'IGQQSKIQ~VQ 826 gi1191711501 872 ______________________,__________________________ gi176622021 686 -------------------------------PQWEMSE ~E TA GERSVVT~DSE 715 gi1185612271 986 QGGGCTPSPPADGLVQNQLRLGRHTQAQCLDQSCSVG S ~R.T =~GAQS~t,P~Q TR
S gi 117432918 ~ 807 ______________________________________ Ti ~,AQ
yn~~rGSQV,A'(?'E RN 828 .1....1....1....1....1....1....1....1.. .1. .1. .1.. .1 NOV3 12 ~_________________________________________~ Kip~~ E~',,j,~ G 30 1O gi 118598706 1 827 ~t"jIKPFQKEEAVLHSLCPVST~TQVI~A~NSHA~PPQ~SLGP~SQ Kf 'I~ELL~ G 886 gi1191711501 882 S-_______________________________________E ~ YINVKA~ LR 901 gi176622021 716 DEKLCDP---------NT ' GE~~ TGPP RQ TVSD GP GQ RTI'H'~Y 1T 766 gi1185612271 1045 ~1HYDSE-PVPASLCPQP SSR~ SQS PP SAGP 1'I_' ~ S 1103 gi1174329181 829 LDSSAVAPHYCS-AHSKL KRQTEP PPD G SL R6 'S S ,;, QR 887 .1....1. .1....1.. .1. .1. .1....1. .1. ..1. .1. .1 NOi3 1 31 ----AA'E.''T~ Q L E~.Q~G=~ LG..~KNSRL~ ~-~8S .E L ~ 85 ~ V n VW n V 1.
.1, V V/1 V
gi 18598706 887 ---AAT E Q ~L E~ G LG 'KNSRLiSS E L 941 20 gi1191711501 902 D----QNTQ"NS F SATCT.~ TEPKI AFS ~ ---A 'PGE T _K8 Q~S' v v - ~t c gi176622021 767 ;----DGR .~E Q QMT ~LAIHP- GD ---- ~QD E T ~ 817 gi ~ 17432918 1 8884 RVSAAEE~L ~~A P~~ ~~L~A- LGPT ~KPK P~vLD E 'I'PEP ~~ ' .1. ..1....1....1....1. .1. ..1....1. .1. ..1....1.. .1 NOV3 86 E~I~ S ~GFQ ~LI~FPER RNT' ~.LDL~E~'---- --AHPVYNMVA~ Y 138 gi1185987061 942 E~ S 'GFQ' LI,~~,FPER RNA ~VLDL E~----- --AHPVYNMVA I1Y 994 gi 119171150 1 954 ICS ~ ~KPFQ ~EEAVLHSL P STST~--TQU~?'A---- S -PQ------- S
30 gi176622021 818 L'~L LA- PQ~RSGPE GLA --P EST--- FE'P F---------- Y 858 gi1185612271 1163 FI~~ ~YVS ~YRELASK SHIP ~SLELBR~!~'PHLLLRI~P~GAAGLPHPGLREV P
gi1174329181 942 VjUL KS---ADHRADLPPAH SP°~ -PATMR-____~'"L.~_______p__ 3S ....1....1....1. .1....1. ~1..~...1. ..1....1....1. .1....1 NOV3 139 S--L~ QI,~ ------r;.~ G----- tot's .Q-----------QG.~~ S-- 170 v v gi1185987061 995 S--L~"QQ _____ G_____ ~T.8~ Q___________QG ~~ S-- 1026 gi1191711501 996 L-- ~ S~,'7 ---- V ~R----- ~ 'E~:i~KGSA--------AETL~E Q--gi176622021 859 TS--' S---- ~ ------ ' ~ YQG------------TDIVRG-- 890 gi1185612271 1223 LTRVL~"P PLIHLFRP SGSPCTVP VSYQHNKPIPRRREHPPREHLTQ~'SP 1282 gi1174329181 983 AG--E~G: --- AQ ---- Q~yQ~'7SVR~TS-----------HTGQ~H--E 1015 1.. ~~~~~1~.~ 1 .1. .1 ..1....1....1.. 1 1 ..1 4S NOV3 171 LLHQ ~PVL , ---- TF ~~PEKR--------------DPSC~DFF ~'~'''~~'CH- 206 gi1185987061 1027 LLHQ'~PVL . --------TF ~ PEKR~GEMQAELDSKLSGFQTIS~IWFESEG
gi1191711501 1031 TSLP ELQ~,~,. V --------LG ~KNSRLQ-------------- WVASSG~SE-gi176622021 891 DPLVVGR~,I --------LQP ~TEPPD-----------------SCQDQPGTN- 923 gi1185612271 1283 AKRKYGQKTDLDTLPIPLWAPLS~SPEPRG----PEICEQ----QGLD TECP LLL
SO gi1174329181 1016 TEAL~PTTf,~Q~EAK--- -CD,~PT~GDGPE--------------CKD~NKVACP- 1053 NOV3 206 - 1.~PQHGV~NHKFYGKQCC~I--.TRKI--I_---1----1- 231 SS gi1185987061 1078 NERL~PSFSLHLGGKNGIQYPKRLPBEQKENMALAAIKMLQSTF 1123 gi1191711501 1062 - 'V'WIRSH~RRLRPSWLTQ---------------------- 1081 gi176622021 923 - C ~IKVNL GHWYYSKACC~-- RPPHS------------- 951 gi1185612271 1335 AIG- IiPCQARDTESRPQGPVP~;--P GQDIEK------------ 1365 gi1174329181 1053 --- ,.LKFQF~SRAYFRQMCC'-- QGH--------------- 1077 The NOV3 Clustal W alignment shown in Table 3E was modified to begin at amino residue 1321. The data in Table 3E includes all of the regions overlapping with the NOV3 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (hrip:www.ebi.ac.uk/interpro/). Table 3F lists the domain description from DOMAIN analysis results against NOV3.
Table 3F Domain sis of Anal NOV3 Model Region of Score E value (bits) Homology tsp 1 12-58 -6.8 4.1 tsp 1 66-125 14.6 0.015 tsp 1 141-187 19.3 0.0041 Consistent with other known members of the Thrombospondin type 1 (tsp_1) family of proteins, NOV3 has, for example, three tsp_1 domain signature sequences and homology to other members of the tsp_1 Domain-containing Protein Family. NOV 3 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV3 nucleic acids and polypeptides can be used to identify proteins that are members of the tsp_1 Domain-containing Protein Family. The NOV3 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV3 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, tuberous sclerosis, fertility, hypogonadism, as well as other diseases, disorders and conditions.
In addition, various NOV3 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV3 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the tsp-1 Domain-containing Protein Family.
Thrombospondin type 1 domain (TSP1, IPR000884) is a repeat found in the thrombospondin protein where it is repeated 3 times. Likewise, the tsp_1 domain is repeated three times in the NOV3 polypeptide. Now a number of proteins involved in the complement pathway (properdin, C6, C7, CBA, C8B, C9) (Patthy,L., J. Mol. Biol. 202: 689-696 (1988)) as well as extracellular matrix protein like mindin, F-spondin (Okamoto, et al., Developnaeht 126:
3637-3648 (1999)), SCO-spondin and even the circumsporozoite surface protein 2 and TRAP
proteins of Plasmodium (Wengelnik, et al., EMBO J. 18: 5195-5204 (1999);
Rogers, et al., Mol. Bioclzefn. Parasitol. 53: 45-51 (1992)) contain one or more instance of this repeat. It has been involved in cell-cell interraction, inhibition of angiogenesis (Krutzsch, et al., Circulation 100: 1423-1431 (1999)), apoptosis [Krutzsch, et al., Cancer Res. 57: 1735-1742 (1997)).
The NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of urogenital physiology. As such, the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat reproductive and metabolic disorders, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, tuberous sclerosis, fertility, hypogonadism, as well as other diseases, disorders and conditions.
The NOV3 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV3 nucleic acid is expressed in eye and testis.
Additional utilities for NOV3 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV4 polypeptide has been identified as a Protocadherin Alpha C2 Short Form-like protein (also referred to as CG93187-O1). The disclosed novel NOV4 nucleic acid (SEQ 1D
NO:7) of 600 nucleotides is shown in Table 4A. The novel NOV4 nucleic acid sequences maps to the chromosome 11.
An ORF begins with an ATG initiation codon at nucleotides 41-43 and ends with a TAG codon at nucleotides 2546-2548. A putative untranslated region and/or downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
Table 4A. NOV4 Nucleotide Sequence (SEQ ID N0:7) CACCATAAAAGCTCAGAAAATAGACTTTTCCTCTGCCTCTATGGAGGGGCAGGCCAGATCTGGGGAA
GGGATGGGACAGCCTGGCATGAAGAGCCCCAGGCCCCACCTCCTGCTACCATTGCTGCTGCTGCTGC
TGCTGCTGCTGTCTTCGCCTCGCCGTGCACGCGTGCGCCTCCCAGAGGACCAGCCGCCTGGGCCCGC
GGCTGGCACGCTCCTAGCCCGCGACCCGCATCTGGGCGAGGCTGCACGCGTGTCCTATCGGCTGGCA
TCTGGCGGGGACGGCCACTTCCGGCTGCACTCAAGCACTGGAGCGCTGTCCGTGGTGCGGCCGTTGG
ACCGCGAACAACGAGCTGAGCACGTACTGACAGTGGTGGCCTCAGACCGAGCTCCCCGCCCGCGCTC
GGCCACGCAGGTCCTGACCGTCAGTGTCGCTGACGTCAACGACGAGGCGCCTACTTTCCAGCAGCAG
GAGTACAGCGTCCTCTTGCGTGAGAACAACCCTCCTGGCACATCTCTGCTCACCCTGCGAGCAACCG
ACCCCGACGTGGGGGCCAACGGGCAAGTGACTTATGGAGGCGTCTCTAGCGAAAGCTTTTCTCTGGA
TCCTGACACTGGTGTTCTCACGACTCTTCGGGCCCTGGATCGAGAGGAACAGGAGGAGATCAACCTG
ACAGTGTATGCCCAGGACAGGGGCTCACCTCCTCAGTTAACGCATGTCACTGTTCGAGTGGCTGTGG
AGGATGAGAATGACCATGCACCAACCTTTGGGAGTGCCCATCTCTCTCTGGAGGTGCCTGAGGGCCA
GGACCCCCAGACCCTTACCATGCTTCGGGCCTCTGATCCAGATGTGGGAGCCAATGGGCAGTTGCAG
TACCGCATCCTAGATGGGGACCCATCAGGAGCCTTTGTCCTAGACCTTGCTTCTGGAGAGTTTGGCA
CCATGCGGCCACTAGACAGAGAAGTGGAGCCAGCTTTCCAGCTGAGGATAGAGGCCCGGGATGGAGG
CCAGCCAGCTCTCAGTGCCACGCTGCTTTTGACAGTGACAGTGCTGGATGCCAATGACCATGCTCCA
GCCTTTCCTGTGCCTGCCTACTCGGTGGAGGTGCCGGAGGATGTGCCTGCAGGGACCCTGCTGCTGC
AGCTACAGGCTCATGACCCTGATGCTGGAGCTAATGGCCATGTGACCTACTACCTGGGCGCCGGTAC
AGCAGGAGCCTTCCTGCTGGAGCCCAGCTCTGGAGAACTGGTGTTGCTTGAACCTCTAGACTTTGAA
AGCCTGACACAGTACAATCTAACAGTGGCTGCAGCTGACCGTGGGCAGCCACCCCAAAGCTCAGTCG
TGCCAGTCACTGTCACTGTACTAGATGTCAATGACAACCCACCTGTCTTTACCCGAGCATCCTACCG
TGTGACAGTACCTGAGGACACACCTGTTGGAGCTGAGCTGCTGCATGTAGAGGCCTCTGACGCTGA.C
CCTGCCCTCATGGCCTCCTCAGGCGACCCATCAGGGCTCTTTGAGCTGGATGAGAGCTCAGGCACCT
TGCGACTGGCCCATGCCCTGGACTGTGAGACCCAGGCTCGACATCAGCTTGTAGTACAGGCTGCTGA
CCCTGCTGGTGCACACTTTGCTTTGGCACCAGTGACAATTGAGGTCCAGGATGTGAATGATCATGGC
CCAGCCTTCCCACTGAACTTACTCAGCACCAGCGTGGCCGAGAATCAGCCTCCAGGCACTCTCGTGA
CCACTCTGCATGCAATCGACGGGGATGCTGGGGCTTTTGGGAGGCTCCGTTACAGCCTGTTGGAGGC
TGGGCCAGGACCTGAGGGCCGTGAGGCATTTGCACTGAACAGCTCAACAGGGGAGTTGCGTGCGCGA
GTGCCCTTTGACTATGAGCACACAGAAAGCTTCCGGCTGCTGGTGGGTGCTGCTGATGCTGGGAATC
TCTCAGCCTCTGTCACTGTGTCGGTGCTAGTGACTGGAGAGGATGAGTATGACCCTGTATTTCTGGC
ACCAGCTTTCCACTTCCAAGTGCCCGAAGGTGCCCGGCGTGGCCACAGCTTGGGTCACGTGCAGGCC
ACAGATGAGGATGGGGGTGCCGATGGCCTGGTTCTGTATTCCCTTGCCACCTCTTCCCCCTATTTTG
GTATTAACCAGACTACAGGAGCCCTGTACCTGCGGGTGGACAGTCGGGCACCAGGCAGCGGAACAGC
CACCTCTGGGGGTGGGGGCCGGACCCGGCGGGAAGCACCACGGGAGCTGGGGCTCCACCTGGACTCT
TACCAGAGTCACTCCAAGTCCTGTCTCAGGCAGAATACTCAGATCTATTCCAAGCACCTTCCCTGGG
ATCTCAGGCGCATACTGAGAACCAGTGGGACAGGGTTGAGAGAGAGAGCCAACCGAGAATCTCAAAT
GAACCAAACTGAGAAAGATGCCCCTCAGTGGGGCTACAGACCGACACCCCACCATGGGGCAACAGAA
AAACCAAGACCCCCTCCCCAAAGGAATCAAACCAATCGGGAAAAGGAAGGAGGCGTTGGCCGTGCCT
AGGATAT
The NOV4 protein (SEQ 1D N0:8) encoded by SEQ ID N0:7 is 835 amino acid residues in length and is presented using the one-letter amino acid code in Table 4B. Psort analysis predicts the NOV4 protein of the invention to be localized at the plasma membrane with a certainty of 0.7900.
Table 4B. Encoded NOV4 protein sequence (SEQ ID N0:8) MEGQARSGEGMGQPGMKSPRPHLLLPLLLLLLLLLSSPRRARVRLPEDQPPGPAAGTLLARDPHL
GEAARVSYRLASGGDGHFRLHSSTGALSVVRPLDREQRAEHVLTWASDRAPRPRSATQVLTVSV
ADVNDEAPTFQQQEYSVLLRENNPPGTSLLTLRATDPDVGANGQVTYGGVSSESFSLDPDTGVLT
TLRALDREEQEEINLTWAQDRGSPPQLTHVTVRVAVEDENDHAPTFGSAHLSLEVPEGQDPQTL
TMLRASDPDVGANGQLQYRILDGDPSGAFVLDLASGEFGTMRPLDREVEPAFQLRIEARDGGQPA
LSATLLLTVTVLDANDHAPAFPVPAYSVEVPEDVPAGTLLLQLQAHDPDAGANGHVTYYLGAGTA
GAFLLEPSSGELVLLEPLDFESLTQYNLTVAAADRGQPPQSSWPVTVTVLDVNDNPPVFTRASY
RVTVPEDTPVGAELLHVEASDADPALMASSGDPSGLFELDESSGTLRLAHALDCETQARHQLWQ
AADPAGAHFALAPVTIEVQDVNDHGPAFPLNLLSTSVAENQPPGTLVTTLHAIDGDAGAFGRLRY
SLLEAGPGPEGREAFALNSSTGELRARVPFDYEHTESFRLLVGAADAGNLSASVTVSVLVTGEDE
YDPVFLAPAFHFQVPEGARRGHSLGHVQATDEDGGADGLVLYSLATSSPYFGINQTTGALYLRVD
SRAPGSGTATSGGGGRTRREAPRELGLHLDSYQSHSKSCLRQNTQIYSKHLPWDLRRILRTSGTG
LRERANRESQMNQTEKDAPQWGYRPTPHHGATEKPRPPPQRNQTNREKEGGVGRA
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 4C.
Table 4C. Patp results for Smallest Sum ReadingHigh Prob Sequences producing Frame Score P(N) High-scoring Segment Pairs:
>patp:AAU07054 Flamingo protein +1 968 1.8e-98 Human >patp:AAU07053 Flamingo polypeptide +1 968 2.0e-98 Human >patp:ABG21921 human diagnostic protein +l 642 3.1e-64 Novel #21912 >patp:ABG21921 human diagnostic protein +1 642 3.1e-64 Novel #21912 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 273 of 415 bases (65%) identical to a gb:GENBANI~-ID:AF061573~acc:AF061573.2 mRNA from Homo sapie~rs (protocadherin (PCDHB) mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 273 of 415 amino acid residues (65%) identical to, and 273 of 415 amino acid residues (65%) similar to, the 4076 amino acid residue gb:GENBANK-m:AF061573~acc:AF061573.2 protein from Homo Sapiens (protocadherin (PCDHB) mRNA, complete cds).
NOV4 also has homology to the proteins shown in the BLASTP data in Table 4D.
Table 4D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~17461472~ref~XPsimilar to 1415 459/682 503/682 0.0 _ protocadherin (67%) (73%) 052786.2I(XM 16 [Homo Sapiens]
gi~16933557iref~NPprotocadherin 443/676 490/676 0.0 _ precursor; (65%) (71%) 003728.1~(NM_003737 fibroblast cadherin FIB1;
cadherin 19;
fibroblast cadherin 1;
dachsous homologue [Homo Sapiens]
gi~6753408~ref~NPcadherin EGF 3034 48/693 358/693 1e-98 _ seven-pass G-type (35%) (50%) 34016.1~(NM_009886) receptor [Mus musculus]
gi~13325064~ref~NPcadherin EGF 2923 246/679 345/679 2e-98 LAG
_ seven-pass G-type (36%) (50%) 001399.1~(NM
~
receptor 2;
EGF-like-domain, multiple 2;
epidermal growth factor-like 2;
multiple epidermal growth factor-like domains 3;
cadherin, EGF
LAG
seven-pass G-type receptor 2, flamingo (Drosophila)homol og gi~10727655Igb~AAF5Stan gene product3606 241/700 361/700 3e-98 8763.2 (AE003828)[Drosophila (34% (51%) melanogaster]
A multiple sequence alignment is given in Table 4E, with the NOV4 protein being shown on line 1 in Table 4E in a ClustalW analysis, and comparing the NOV4 protein with the related protein sequences shown in Table 4D. This BLASTP data is displayed graphically in the ClustalW in Table 4E.
Table 4E. ClustalW Analysis of NOV4 1) > NOV4; SEQ >D N0:8 2) >gi~ 17461472/ similar to protocadherin 16 (H, Sapiens) [Homo Sapiens]; SEQ
)D N0:50 3) >gi~16933557~/ protocadherin 16 precursor; fibroblast cadherin FIB1;
cadherin 19; fibroblast cadherin 1; dachsous homologue [Homo sapieras] ; SEQ >D N0:51 4) >gi~6753408~/ cadherin EGF LAG seven-pass G-type receptor [Mus rnusculus] ;
SEQ >D N0:52 5) >gi~13325064~/ cadherin EGF LAG seven-pass G-type receptor 2; EGF-like-domain, multiple 2;
epidermal growth factor-like 2; multiple epidermal growth factor-like domains 3; cadherin, EGF LAG
seven-pass G-type receptor 2, flamingo (Drosoplaila) homolog; ; SEQ )D N0:53 6) >gi~10727655~gb~AAF58763.2~(AE003828) Stan gene product [Dr~osophila rnelanogaster] ; SEQ >D
N0:54 rrov4 1 3 gi~ 17461472~81 ga.~ 16933557~1153 1211 g3.~ 67534081955 1014 gi~
gi~ 10727655~1055 NOV4 4 QARSGE-GMGQ-_______________pGMKS~___________________________ 19 gi1174614721 140 L~ASGAAGGG P ;Q PAR ~P 'I'L~uTTI:iQ~i ~'~ EI GT L T'T~T- PGS
gi I 16933557 I 1212 L~,ASGAAGGG'L~t'Pt2.~Q PIJR ~P TL~TT,LiQ~ ~' ~ E GT L TAT-gi~6753408~ 1015 EKDE--- ~'E~F E~NS~ S IRi ~'~ P Q I Q~~E P Q LL 1068 gi ~ 13325064 ~ 922 Ej~DE--- FDV~F~E~NS~LA~AR~Ti,~~~,T~ ~ ~ T Q Q~VE IP _ Q IF
gi ~ 10727655 ~ 1114 ASDK--- ~CTI~Y PNS SV GE'IHi ~ ~ ~ VUH SI~I DSN S TRP
NOV4 19 __________________ .HL~ _P-________ ___LL~,tL~sL~:,.SS-__ _______ 37 gi~174614721 199 --~ LTAAP IRA-E~~H T~S~HDQGSP~RSSLQ LVQ PSARLAP~~D 253 15 gi~16933557~ 1271 --LTAAP IRA-E~~H TS~,~HDQGSP~RS~SLQ~L. PSARLAFSP~~D 1325 gi I 6753408 ~ 1069 ---~7 RALVE FE- 'RD Qi~i,TS-- ~LVRAT4V~i~~, QN-- ~7 ~ ~E P
gi ~ 13325064 ~ 976 -- TALVD YE-D' ~E Q~TS-- ~LVRAT~'HU7~,~r~DRN-- 't~ ~ G
gi I 10727655 ~ 1168 SERA LTMTE YEST KR~'E u.~R~AS---P ~LRNDAIiE~L'S~TDVN---~3N ~ R 1221 NOV4 37 ____________________________________________________________ 37 gi ~ 17461472 ~ 254 k~RDPAAPVPWLT GL ~GSL S~~P~ GVG' T ~L~~ ;PEGTF ~ 313 gi~169335571 1326 RDPAAPVPWLT EGL ~GSL S, ~P~~ GVG T TL~ PEGTF Afi1385 gi~67534081 1118 FQILFNN----Y KSNSF'SGV,~ ~~P~~,H~ LSD~~a 'PFQ BLS-LLL ~P,A'~
gi I 13325064 ~ 1025 NFEILFNN----YRSSSF'GGA'I R~t P~iH>~~7' ~ ISDS T SFE ~ BLS-LVL G'i' 1079 gi~10727655~ 1222 ~.IFQVIFN--------NFRDHF~SGEiI P~FADVSD RIS ~AN-LL 51272 NOV4 37 ___________________________________________________-_____p__ 38 gi 17461472 314 373 gi~16933557~ 1386 1445 giI6753408~ 1173 1231 giI13325064~ 1080 1138 gi~10727655~ 1273 NOV4 38 ____________________________________________________________ 38 gi117461472~ 374 RTRSPAQRCTLSARRTPTAP TATCATACCARSRPCRASPGRAHRGVSSARPGPRD---gi~16933557~ 1446 PE-NPEPGAALYTF~SDADG'-GPNSDVRY.LLRQEPPVP R'~.iDARTGALSAPRG---gi~6753408~ 1232 EKFLSPLLSLFVEG ~~TVLSTTKDDIFVFNI~NDTDVSS-NI ~ TFSALLPGGTRG--R
giI13325064~ 1139 ERFLSPLLGLFIQAVTLAT~PDHWVFNV~RDTDAPGGHI YSLSVGQPPGPGGGPP
gi~10727655~ 1332 EAFLSPLLNFFLDGL~IIPC KEHIFVFSItDDTDVSS-RI j SFSARRPDVSHE--E
NOV4 38 -_________________ _ __ _ ___ __ R~~a~R~=~~RLP~T'JQP~ 52 gi~17461472~ 431 HSRAAAAGGSHRPA~QRQPPSCFSA;~TSR~QR~TRLS,---P~RLP~QP~ 485 gi ~ 16933557 ~ 1501 LDRETTPALL'~L~1EATDRPANARAARVSARVFVTDEN~3NAPVASPS
~=RLP1QP ~ 1560 gi ~ 6753408 ~ 1289 FFPSEDLQEQ~Y' ~ 'TLLTTI.~~ LP",DD C EPCE'~CV~LRF~SS ~ 1344 gi~133250641 1199 FLPSEDLQERIar9 'SLLTAIAQ LP DN~Cxr EPCERCVS=LRFSS ~ 1254 ' ~ ~ i..
gi~10727655~ 1389 FYTPQYLQER~3Y' ILARLTVEVLP~DN~C,'EPCL~'EECLT~LKFGASE 1444 55 ~ ,.
1630 7.640 1650 1660 1670 1680 . .,I. .~~ ~~~~ .~. ..y y ...I. y . .I.. .y ~V
NOV4 52 GP, P~LGE ~ YRLAS.~ ------~GHF ~S TGAL ' 'P~.iDv -- 101 gi~17461472~ 486 GP~ ~DP LGE YRLAS ------~GHF S TGAL~ ~PhD~ -- 535 gi~16933557~ 1561 GP~~LH ~DPDLGE _YRLAS ------~GHF S TGAL~,~ 'PI~D~ -- 1610 gi~6753408~ 1345 FIST F'PI PITGL~CRCPPGFT ----DYCTEID CY PC ' CRS' GGY 1400 gi~13325064~ 1255 FI~SSF'PI~PVGGL'CRCPPGFT ----DYCTEVD CY RPCGPHGyyCRS~ GGY
gi~10727655~ 1445 FIND F'PI'Y~PVNTFAC~CPEGFT SKEHYLCdTEVD CY DPCQ~1GGTC'tt ' NOV4 101 ____I....~.. .~. .'....~ .._.i....~ I"~ I,, I ~ I,~ ~' --Q ~ L DB=--~pRP ATQ T'.t~~~~SV~TD' FQ~QQY'VL . ~ E 151 gi ~ 17461472 ~ 535 ---_---Q L --~PRP~~ATQ T~S~'~' V~17:: ~ .F~~QQ Y~sVL ~E
gi I 16933557 I 1610 -------Q L DH---GSPP32u~ATQ TVS~ 'TF~,bQf~~Ys.~'r, VL 'E
70 gi I 6753408 ~ 1401 TCECFEDFT CQVNV G~-CAGVC~GGTC4~tN,~L~I,GG~VC
~PG~"YHPYCE~TST 1459 gi ~ 133250641 1311 TCLCRDGYTG~CEVSAR~ -CTPGVCGGTCVNT.~L~GGFKCC~GDFPYCQ~STTT
gi 1107276551 1505 TCVCPSTHTG~IStCE~G~GHrLyPCPETCEGGLSC~fiTYP- ~
QPPPYTA~I'CE~~yA 1558 gi1174614721 586 645 gi1169335571 1661 1720 gi167534081 1460 1518 gi1133250641 1370 1428 gi1107276551 1559 1617 gi1174614721 646 705 gi1169335571 1721 1780 gi16753408~ 1519 1578 gi1133250641 1429 1488 gi1107276551 1618 1677 1.. .~....1....~....1....1....1. .1. ..1. 1....~.. .1 NOV4 272 ~i7GQ ....~;_________-____________-R__-_~.~ ~GAFVLD~' G~ G-Tv P 303 gi1174614721 706 ~iGQ ~1'~' ----------------------R-- D~GAFVLD GG-Tv P 737 gi1169335571 1781 iINGQ ~'----------------------R----~ ~~GAFVLD ' GG-T ~P 1812 gi167534081 1579 JAVA ~._GSYVGNYSCAAQGTQSGSKKSLDLTGPLL ~ LPEDFPH~RQ GC ~ 1638 gi1133250641 1489 TGV 'GSVLGNYSCAAQGTQGGSKKSLDLTGPL DLPESFP'9'RMRQ GC ' 1548 gi1107276551 1678 NRT~LDKRCSLLTETCH-------RFLDLTGPLQT?'?'G~VL~'RIPAHFP~TRGCSD
gi1174614721 738 794 gi1169335571 1813 1869 gi167534081 1639 1698 gi1133250641 1549 1608 giI107276551 1731 1790 .1....1. .1. ..1....1....1 1....1. ..~. ~.~..1....1 NOV4 360 ---------- ~.1'L ~QLQ ~PDAGANG~1V~'GAGT~,~~I,, ~ PSS LVLLEPLD 409 e'E
gi 117461472 1 794 ------- '~L QLQAH~PDAGANGHV'~Y~GAGT~,~, ' ~ PAS ~LR'.AAALD
gi 116933557 1 1869 ------- -~. TL QLQ ~PDAGANG~3"~'Y GAGTi ~ PAS LItAAALD
gi167534081 1699 CEQAMPHPQRFT ES''V~t(LWSDL~ITISVPW'x7~GLMFRTRKEDG ~, gi1133250641 1609 CAQEMANPQHFL ~SAWHGLSLPISQPW~h~L_MFRTRQiDG
S~AI'~RGRS'~.'ITLQL 1668 gi1107276551 1791 CQDNIPAPWRFGS SFNPLLRPIQLPWTT'~5~RTRQKE~ IQI~NSAAVCL 1850 . ..1. ~_ ..1... 1 ..1....1....~ .1. ..1....1.. ~ m..1 NOV4 410 F.SLTQ. L~~ ~--- RG-QP-PQSSWP~Tj~TVLDV~NP------- PV3.:TRA 453 gi1174614721 844 QCPS TFVSA ~--- G,~-~aAGPLSTTVS T TVT~D HA------- PT~PTS 888 gi1169335571 1919 ' QCPS TFSA ~--- G3~-AGPLSTTVS~T T~I2,D~---------PTPTS 1963 gi 1 67534081 1759 LNSYI1;EVYGPS~ ASMQLKRITDGGWHHiL
E~.,~SAKGKDIKYLAVMTLD~'~','GMD 1818 gi1133250641 1669 GHVMLSVEGTGLQASSLRLEPGRANDGDWHHAQ~GASGGPG----HAILSFD~~GQQ
gi110727655~ 1851 '~QGVLY~IFDGEP-----MYLG~SFLSDGEWHRVE~RWS~QGIH------FS-VD'YaGQR 1898 1 ...1.. 1 . ~ ..1.,...1....1....1. 1 ...1....1. ..1 CO NOV4 454 SYR~~J"T~~~~PEDT~- '~GAEL2aHVEASDADP--------ALN~AS~,.'rGD--------'SGL 491 gi 1174614721 889 PLRT~.~RPRP ~SFSTPT~iAI~AT~R.~.A~;bRDAGAN-----ASILY LF~ ------ 'PPG 935 gi1169335571 1964 PLRRI~PRP ~SFSTPT~TRDAGAN-----ASILY'_ --------'PPG 2010 gi167534081 1819 QST~QGNQL~GLKMRTVTGG~VT~KVSVRHG--FRGCMQG ' GE STNIATLNMNDA
gi1133250641 1725 RAEGN~I~GPRLHGLHLSN~T GG~PGPAGGVARG--FRGCLQG ~wD
PEGVNSLD~SHG 1782 65 gi1107276551 1899 SGS~P1~SQKVQGLYVGKT7GSPDGSIGAVPEASPFEGCIQD~ Z~GAG----1.. .1....1....1....1....1. ..1 ...1....1. 1 ...1....1 NOV4 492 FE~'pESS~TLRL~~,HAL------DCETQARHQ. Q ~PAG-AHFALAPVT~!EVQDVN-- 542 70 gi1174614721 936 TT~~SYT~EIRV~RSP------V~LGPRDR IV~T~L~RPARSATG~II'VGLQGEA--32 ~~ _~
gi1169335571 2011 TTU[.1~SYT EIRViRSP------VLGPRDRV~~IVT~L
RPARSATG~iII'S~GLQGEA-- 2062 gi~67534081 1877 LRVKD CDVEDPCASSPCPPHRPCRDTWDS'~SCIC~ YFGKKCVDACL~iNPCKHVAA
gi1133250641 1783 ESIt~VEQ CSLPDPCDSNPCPANYCSNDWDSSCC~P YYGDNCTNVCD~iNPCEHQSV
gi I 107276551 1955 IRE;E~VED CESRi~IQCP-DHCPNHSCQSSWD~STCEC~S
YVGTDCAPCT:StRPCASG-V 2012 .~.
.I.. .1....1....1....1. ..I....I....I....I....~..f..," I....p, NOV4 542 -DH ~ F~LNLLSTSVAE---NQPP ~L~ITTLHAIDGDAG-AF RLRYLF.~AGPGPEG597 gi1174614721 987 -E~ ~RF SEATIRE---NAPP P~~ S~RAVH- G-T ~ITYS~LS --- 1037 1o gi1169335571 2062 -E' ~RF SEATIRE---NAPP P VS~RAVH- 'G-TV ~ITY~TLS V - , 217.2 gi167534081 1937 C ~S~ T GYCECGPG-HYGQYCEN d7L~CPKGWW PVC ~CHC~51'S~ F~PDC
giI107276551 2013 CRANTSL HGYDCECNSSSRHGDYCE~~QQ~CPGGWW ERVC ~CRCD~.tA, YHPDC
.1....I.. .1....I... I ...I I....I....I. ..I.. I . .I
NOV4 598 EAFALNSS GEL,: ~'.AR------VPFD'~'EHTESF LVGAADAGNLSAS~TVSVLVTGEIE651 ga. 11174614721 1038 G"~'FSIQPS GAITVRSA----EGLDF'EVSPRL'~VLQAESGGAFAFT
LiTLTLQDA13N3 1092 giI169335571 2113 G'.~'FSIQPS GAITVRSA----EGLD.~.~EVSPRL'~iVLQAESGGAFAFT~L~TLTLQDA~N 2167 2O gi167534081 1996 TNGQCQCKENYYPPAQDACLPCDCPHGSHS~ CDMDTGQCACKP ~GRQCNRCP2055 gi~133250641 1902 °'GECHCKENHY PPGSPTCLLCDC3~PTGSLS sCDPEDGQCPCKP
~GRQCDRCi~P' 1962 gi110727655~ 2073 TGQCYCKQNHYPPNETACLSCDC~'SIGSFSGACNPLTGQCECREGuGRRCDSCS~P2132 25 ....~....I....I....I....I....I....I ...I. .I. ..1....1....I
NOV4 652 DPVF PAFHFQVPEGARRGH----SLGHaj'QATDEk~GGAD L~7LYATSSP-------- 699 gi1174614721 1093 ~,~~1,PRF RPHY LPES~PL G--- LL~?EADDL~QGS QISY~AASQP ----gi1169335571 2168 ~~,PRF RPHY LPES~PL G----~LLt~EADDL~7QGS 'Q,~SY~cAASQP ----gi~67534081 2056 ~iE=uTSLGCE I'~NGCP~ F GIWW QTFGQPAAVPCP 'S~ GN~,RHCSGE~ -3~ gi1133250641 1962 iiIEuiTTNGCE YDSCP~ I GIWW RTFGLPAAAPCP SFGT,,'RHCDE -gi1107276551 2133 iEuT~SGCE~~DACP~SFAGGWW~RTPiGGVAI!GCPPPARGKGQRSCDVQS SWNTP
.I.. I . .I... 1....I. ..1....I ...1....I. ..~....I....I
3S NOV4 700 YFGINQi~TG - ---- ------ .YLRVDS~ P ---G GT ------------- 725 gi1174614721 1144 LFHVDP~~TGT~TTTAIL ~EIW ~? LMA'D~GP G~ATL ------------ 1190 gi116933557~ 2219 LFHVDP~TGTTTTAILREIW T LMAD~GP GATL ------------ 2265 gi167534081 2115 ELFNCTSGSF~DLKAL LN T GN L L RN=TQGNS------------- 2161 gi~133250641 2021 NLFNCTTTFSELKGFF~~LQ ~ SGF~Q'L~LL RN~~~,TQH~A-------'-----gi110727655~ 2193 DMYNCT~EPFITELRRQLSLEKL LGE~TSFVAIEQ RCEAVDRRGASKDQKISGN
.'..I....I....I....I....I....I....I....I....I....I....I....I
NOV4 725 ______________________________________________gGGG-GRTRREAPR 738 45 gi1174614721 1190 ____________________________________________DTNDNRPTIPQPW
gi1169335571 2265 ____________________________________________~7jOTT7DNRPTIPQPW 2281 gi167534081 2161 ----------------------------------------TLFGNDC~RTAYQLLARILQH
gi1133250641 2067 ----------------------------------------GYFGSDKVAYQLATRLLAH
gi1107276551 2253 GRPNRRYKMESSFLLSNGGNVWSHELEMDYLSDELKFTHDRLYGADLVTEGLLQELINY
.I....I....I....I....I....I....I....I....I....I.'.'I....I
NOV4 739 ~GLHLDSYQS-----------------------HS----------------------KS 753 gi1174614721 1207 RVSED LG~IAQVTG~~DS P YVLSPGP~ DPFSVGRY RVS TGPL~FEQ 1266 gi ~ 16933557 1 2282 RVSED LGI~IAQVTGI~(1'~'DS P YVLSPGP~DPFSVGRY RVS TGPLFEQ
w gi167534081 2182 SRQQGFD REANFHEI~~HT S LAPATE~3SWQIQRSE-- Q LRHFAYF 2239 gi1133250641 2088 STQRGF SA'QDVHFTL'~""iR. S LDTANKRHW_LIQQTE-- TAW LQHYAYA
gi1107276551 2313 QSGL SH~C.~DKYFI~L~AASVLDRKYEEWRRATELIQ- PDD DAFNKYL 2371 .I.. L~. I ...I....I....p....l....l....l....l....~....1 NOV4 754 CLRQNT~IYKHL Ia~_iRRILRTSGTG~iRRA~3RESQ----------------------- 790 gi1174614721 1267 CDRY~LvLLA;E~D ~GRANLT~ Em n APAFSQSI~Y'VML~~~EHTP~GS--------ga.11693355i ~ 2342 CDRYL~LLA'~iD ~GRANLT E~ ~ APAFSQS~.aYVMLEHTP~GS--------gi~6753408 2240 SNVAN~T XL F~TANM~ WF~KL~FTGAQVPFED'~~QEEL~RE-----LES 2294 gi I 133250641 2146 S NMR.I-~TYLS FT. ;
~VTPNI~3~;SWRL~KGFAGAK'~.tPYEA~iRGEQ~PD-----LET 2200 gi ~ 10727655 I 2372 W~S~ID~~'1'TS
~F~ZTQPNMA~GL~~VTTE~LFGYEPE~LSEYHRSKYLICPNAFTTES 2431 ....I....I....I....I....I.'..1....1....1....1....1....1....1 NOV4 790 - -MNQ EIi;D PQW YR-________-_____-______pT~H TE ~RPPPQRN823 gi1174614721 1319 A~LSVS~p G~ ~~~~~,SYH------------------LAS~ FSVD~ GTLF~'~'I
giI16933557~ 2394 A'~Z.tSVS~,D G~~SYH--------------'---LAS~ FSVD~ GTLFTI 2434 gi~6753408~ 2295 StT;SFPAD FPPEKKE~P~VRLTNR--------------RTTPLTAQPEPRAERETSS~~2340 gi~13325064~ 2201 TjVLPE'~UF~;----ETPPRP-----------------------A~ePGEAQEPEELAR~'t 2233 gi~10727655~ 2432 V;VV11PDTGFLQHARQRPISFPKYNNYILDRRKFDQHTKVLV~LEMLGIT~PESDEI~,t~ 2491 l~ NOV4 824 TN~'yIEKEGG Ri-_______________________________________________ 835 gi~17461472~ 1360 VGTVALGHD SGAVD ~~ KHETTG------------------------------------gi~I6933557~ 2435 VGTVALGHD SGAVD LEARDHGAPGRAAR.ATVH-------------------VQLQDQ
giI6753408~ 2341 R ~HPDEPGQFiiIVAL ;;,IYRTLGQLLPEHYDP--------------------------gi~13325064~ 2234 Q HPELSQ~eE~iAS IYRTLAGLLPHNYDP----------------------------1S gi~10727655~ 2492 SG'RGSSHDHRiIVAYAQYKDVGQLLPDLYDETITRRWGVDVELATPILSLQILVPSMER 2551 The NOV4 Clustal W alignment shown in Table 4E was modified to begin at amino residue 1201 and end at amino acid residue 2760. The data in Table 1E includes all of the 20 regions overlapping with the NOV4 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 4F lists the domain description from 25 DOMAIN analysis results against NOV4.
Table 4F Domain Anal sis of NOV4 Model Region of Score (bits) E value Homology cadherin 41-131 97.7 2.4e-25 T25P N 16-223 ~ -I17.2 1.3 cadherin 145-233 104.1 2.7e-27 cadherin 247-337 78.1 1.8e-19 cadherin 351-441 112.9 6e-30 cadherin 455-539 64.7 2e-15 cadherin 553-646 77.5 2.8e-19 cadherin 660-745 15.4 0.036 Consistent with other known members of the Protocadherin Alpha C2 Short Form Protein-like family of proteins, NOV4 has, for example, seven Cadherin domain signature 30 sequences and homology to other members of the Protocadherin Alpha C2 Short Form Protein-Like Protein Family. NOV4 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV4 nucleic acids and polypeptides can be used to identify proteins that are members of the Protocadherin Alpha C2 Short Form Protein-like Protein Family. The NOV4 nucleic acids and polypeptides 35 can also be used to screen for molecules, which inhibit or enhance NOV4 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, fertility, endometriosis, hypogonadism, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimrnune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphaedema, as well as other diseases, disorders and conditions.
In addition, various NOV4 nucleic acids and polypeptides according to the invention are useful, i~atef° alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV4 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Protocadherin Alpha C2 Short Form Protein-like Protein Family.
Cadherins (Takeichi, Arahu. Rev. Biochem. 59: 237-252 (1990); Takeichi Trends Genet. 3: 213-217 (1987)), first discovered in mouse teratocarcinoma cells (Liaw, EMBO.I. 9:
2701-2708 (1990)), are a family of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Cadherins preferentially interact with themselves in a homophilic manner in connecting cells; thus acting as both receptor and ligand. There are a number of different isoforms distributed in a tissue-specific manner in a wide variety of organisms. Cells containing different cadherins tend to segregate ira vitro, while those that contain the same cadherins tend to preferentially aggregate together. This observation is linked to the finding that cadherin expression causes morphological changes involving the positional segregation of cells into layers, suggesting they may play an important role in the sorting of different cell types during morphogenesis, histogenesis and regeneration. They may also be involved in the regulation of tight and gap junctions, and in the control of intercellular spacing. Cadherins are evolutionary related to the desmogleins which are component of intercellular desmosome junctions involved in the interaction of plaque proteins.
Structurally, cadherins comprise a number of domains: these include a signal sequence;
a propeptide of around 130 residues; an extracellular domain of around 600 residues; a single transmembrane domain; and a well-conserved C-terminal cytoplasmic domain of about 150 residues. The extracellular domain can be subdivided into 5 parts, 4 of which are repeats of about 110 residues, and the fifth contains 4 conserved cysteines. The calcium-binding region of cadherins is thought to be located in the extracellular domain. This indicates that the sequence of the invention has properties similar to those of other proteins known to contain this/these domains) and similar to the properties of these domains.
Maniatis et al. has identified 52 novel human cadherin-like genes organized into three closely linked clusters (Wu and Maniatis, Cell 97(6):779-90 (1999).) Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.
The NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of urogenital, nerve, and endocrine physiology. As such, the NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat reproductive and nervous system disorders, e.g., Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, fertility, endometriosis, hypogonadism, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GVHD), lymphaedema, as well as other diseases, disorders and conditions.
The NOV4 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV4 nucleic acid is expressed in Heart, Aorta, Umbilical Vein, Thyroid, Colon, Peripheral Blood, Spleen, Lymph node, Bone, Cartilage, Brain, Left cerebellum, Right Cerebellum, Parietal Lobe, Temporal Lobe, Cerebral Medulla/Cerebral white matter, Hippocampus, Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Testis, Lung, and Retina.
Additional utilities for NOV4 nucleic acids and polypeptides according to the invention are disclosed herein.
NOVS
A NOVS polypeptide has been identified as a Nuclear protein-like protein (also referred to as CG95083-O1). The disclosed novel NOVS nucleic acid (SEQ ID
N0:9) of 2322 nucleotides is shot~cni in Table SA.
An ORF begins with an ATG initiation codon at nucleotides 70-72 and ends with a TAA codon at nucleotides 2320-2322. A putative untranslated region and/or downstream from the termination codon is underlined in Table SA, and the start and stop codons are in bold letters.
Table SA. NOVS Nucleotide Sequence (SEQ ID N0:9) GTGGAAGGACAGTCCAGAGCCCTTGTCATCGCACAGGAACTGCTATCTTCAGAGAAAGCATACGTGG
A_GATGCTCCAGCACTTAAATCTGTTCCTGGCAGAGCAGGCTATCAGCAGGAGAGGCCAGGGCTCCAA
AGCCCCAGGGGAAATCTGCCAAGGAGGACTTGTGCTCAGTCCTATCAACCTGTGGGTAACAGACCTT
TTGGTGTTTCAGGATTTCCATGGAGCTGTCATGAGGGCCTTGGATGACATGGACCATGAAGGCAGAG
ACACATTGGCCCGGGAGGAGCTGAGGCAGGGCCTGAGTGAACTCCCAGCCATCCACGACCTTCATCA
AGGCATCCTGGAGGAGCTGGAGGAAAGGCTGTCAAATTGGGAGAGCCAGCAGAAGGTAGCTGACGTC
TTCCTTGCCCGGGAGCAGGGGTTTGATCACCACGCCACTCACATCCTGCAGTTCGACAGGTACCTAG
GTCTGCTCAGTGAGAATTGCCTCCACTCTCCCCGGCTGGCAGCTGCTGTCCGTGAATTTGAGCAGAG
TGTACAAGGAGGCAGCCAGACTGCGAAGCATCGGCTGCTGCGGGTGGTTCAACGCCTCTTCCAGTAC
CAAGTGCTCCTCACAGACTATTTAAACAACCTTTGTCCGGACTCCGCCGAGTACGACAACACACAGG
GTGCACTGAGCCTCATCTCCAAAGTCACAGACCGTGCCAACGACAGCATGGAGCAAGGGGAAAACCT
GCAGAAGCTGGTCCACATTGAGCACAGCGTCCGGGGCCAAGGGGATCTCCTCCAGCCAGGAAGGGAG
TTTCTGAAGGAAGGGACGCTGATGAAAGTAACAGGGAAAAACAGACGGCCCCGGCACCTATTTCTGA
TGAACGATGTGCTCCTGTACACCTATCCCCAGAAGGATGGGAAGTACCGGCTGAAGAACACATTGGC
TGTGGCCAACATGAAGGCTCTTTACCATGGGGAAGGGGAAGGAGGAAGCACCTTTCTCAGCATGGAG
GTTTGTTCCCTTTTGGAACCAAAGGCTCCACCGAGGAGCCTGTTAGAAAAAGGCATGGGAGACGTGG
TCACTGGCAGGTACTTGTCCAACATGACAGTGCACCTGGGGTTGCCCGGGCTGGGCCCTGAGCATGA
CGCTCTGCAGCCTTCCCAGCGGTGGGTCAGCCGCCCTGTGATGGAGAAAGTGCCCTACGCTCTAAAG
ATTGAGACTTCCGAGTCCTGCCTGATGCTGTCTGCGAGGCTGCAGGTCAGGAAGTCCAAGGTCAAGG
CACTGACTGATTCGGTGTCTGCAGCCCTGGGAGTTAGGGGAATATCATTATTCCAGTGTAAGAAGAA
ACAGACCCAAGGACAGCTAATGGACCAGTGGTCTGCTCGTAAACCTAGTCTGGCAGGTGATCTCTTC
TTTGCTGGTGGTTCTGGGCAGTGTGAGAGGTGCAGGCTCAAGGGGCATCTGAGTGAGAACCTCATCC
ATGCCGAGATGGAGGCCCATGCCCGCAGCTCCTGTGCAGAGAGGGACGAGTGGTATGGCTGTCTGAG
CAGAGCCCTCCCTGAGGACTACAAGGCCCAGGCGCTGGCTGCATTCCACCATAGCGTGGAGATACGA
GAGAGGCTGGGGGTTAGCCTTGGGGAGAGGCCCCCCACCCTGGTGCCTGTCACACACGTCATGATGT
GCATGAACTGCGGCTGCGACTTCTCCCTCACCCTGCGGCGTCATCACTGTCACGCCTGTGGCAAGCA
GATCGTGTGCCGGAACTGTTCGCGGAACAAGTACCCGCTGAAGTACCTGAAGGACAGGATGGCCAAG
GTCTGCGACGGCTGCTTCGGGGAGCTGAAGAAGCGGGGCAGGGCTGTCCCGGGCCTGATGAGAGTTA
CAGAGCGGCCTGTGAGCATGAGCTTCCCGCTGTCTTCACCCCGCTTCTCGGGCAGTGCCTTTTCATC
CGTCTTCCAGAGCATTAACCCCTCGACCTTCAAGAAGCAGAAGAAAGTCCCTTCAGCCCTGACAGAG
GTAGCTGCCTCTGGAGAGGGCTCTGCCATCAGTGGCTATCTCAGCCGGTGTAAGAGGGGCAAGCGGC
ACTGGAAGAAGCTCTGGTTTGTCATCAAAGGCAAAGTTCTCTACACCTACATGGCCAGTGAGGACAA
AGTGGCCTTGGAGAGTATGCCTCTGCTAGGCTTCACCATTGCTCCAGAAAAGGAAGAGGGCAGCAGT
GAAGTAGGACCTATTTTTCACCTTTACCACAAGAAAACCCTATTTTATAGCTTCAAAGCAGAAGATA
CCAATTCATGGATCGAGGCCATGGAAGATGCGAGTGTGTTATAG
Variant sequences of NOVS are included in Example 3, Table 19. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOVS protein (SEQ ID NO:10) encoded by SEQ ID N0:9 is 750 amino acid residues in length and is presented using the one-letter amino acid code in Table SB. Psort analysis predicts the NOVS protein of the invention to be localized in the nucleus with a certainty of 0.3000.
Table 5B. Encoded NOVS protein sequence (SEQ ID N0:10) MLQHLNLFLAEQAISRRGQGSKAPGEICQGGLVLSPINLWVTDLLVFQDFHGAVMRALDDMDHEG
RDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHATHILQFD
RYLGLLSENCLHSPRLAAAVREFEQSVQGGSQTAKHRLLRWQRLFQYQVLLTDYLNNLCPDSAE
YDNTQGALSLISKVTDRANDSMEQGENLQKLVHIEHSVRGQGDLLQPGREFLKEGTLMKVTGKNR
RPRHLFLMNDVLLYTYPQKDGKYRLKNTLAVANMKALYHGEGEGGSTFLSMEVCSLLEPKAPPRS
LLEKGMGDWTGRYLSNMTVHLGLPGLGPEHDALQPSQRWVSRPVMEKVPYALKIETSESCLMLS
ARLQVRKSKVKALTDSVSAALGVRGISLFQCKKKQTQGQLMDQWSARKPSLAGDLFFAGGSGQCE
RCRLKGHLSENLIHAEMEAHARSSCAERDEWYGCLSRALPEDYKAQALAAFHHSVEIRERLGVSL
GERPPTLVPVTHVMMCMNCGCDFSLTLRRHHCHACGKQIVCRNCSRNKYPLKYLKDRMAKVCDGC
FGELKKRGRAVPGLMRVTERPVSMSFPLSSPRFSGSAFSSVFQSINPSTFKKQKKVPSALTEVAA
SGEGSAISGYLSRCKRGKRHWKKLWFVIKGKVLYTYMASEDKVALESMPLLGFTIAPEKEEGSSE
VGPIFHLYHKKTLFYSFKAEDTNSWIEAMEDASVL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table SC.
Table SC. Patp results for NOVS
Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAB93568Human protein sequence SEQ ID +1 577 1.7e-95 N0:12972 >patp:AAY51248Rat actin-binding protein frabin+1 312 1.9e-41 >patp:AAU21630Novel human neoplastic disease +l 256 1.6e-38 polypeptide >patp:AAU27818Human full-length polypeptide +1 300 2.6e-29 #143 >patp:ABG00573Novel human diagnostic protein +1 261 1.8e-26 #564 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 443 of 754 bases (58%) identical to a gb:GENBANI~-ID:AB037783~acc:AB037783.1 mRNA from H~~zo Sapiens (mRNA for KIAA1362 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 114 of 263 amino acid residues (43%) identical to, and 173 of 263 amino acid residues (65%) similar to, the 699 amino acid residue ptnr:SPTREMBL-ACC:Q9P2I5 protein from Ho»ao Sapiens (KIAA1362 PROTElI~.
NOVS also has homology to the proteins shown in the BLASTP data in Table SD.
Table 5D. BLAST
results for NOVS
Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~8922921~ref~NPhypothetical 432 135/284 169/284 5e-57 _ protein FLJ11183 (47%) (58%) 60821.1~(NM_018351) [Homo Sapiens]
gi~16716345~ref~NPethanol decreased431 131/284 171/284 2e-55 _ 4 [Mus musculus] (46%) (60%) 444302.1~(NM
gi~7243105~dbj~BAA9KIAA1362 protein699 111/251 166/251 2e-54 2600.1~(AB037783)[Homo Sapiens] (44%) (65%) gi~13648298~refIXPhypothetical 204 115/222 141/222 1e-49 - protein FLJ11183 (51%) (62%), 012133.21 (XM 012133) [Homo Sapiens]
gi(15426438~gb~AAH1Similar to 376 103/221 129/221 4e-40 3319.1~AAH13319 hypothetical (46%) (57%) (BC013319) protein FLJ11183 [Homo Sapiens]
A multiple sequence alignment is given in Table 5E, with the NOVS protein being shown on line 1 in Table SE in a ClustalW analysis, and comparing the NOVS
protein with the related protein sequences shown in Table SD. This BLASTP data is displayed graphically in the ClustalW in Table SE.
Table 5E. ClustalW Analysis of NOVS
1) > NOVS; SEQ ID NO:10 2) >giJ8922921J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ >D NO:55 3) >giJ16716345J/ ethanol decreased 4 [Mus musculus]; SEQ ID NO:56 4) >giJ7243105J/ KIAA1362 protein [Homo Sapiens]; SEQ ID N0:57 5) >giJ13648298J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ ID N0:58 6) >giJ 15426438J/ Similar to hypothetical protein FLJ11183 [Homo Sapiens];
SEQ m N0:59 ..
NOVS 1 ____________________________________________________________ 1 gi~8922921~ 1 ____________________________________________________________ 1 gi~16716345~ 1 ____________________________________________________________ 1 giI72431051 1 GIESDWQGLLVGEEKRSKPIKAYSTENYSLESQKKRKKSRGQTSAANGLRAESLDDQMLS 60 gi~7.3648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 ..
gi~89229211 1 ____________________________________________________________ 1 gi~16716345~ 1 ________________________________________,______________,____ 1 3O gi~7243105~ 61 RESSSQAPYKSVTSLCAPEYENIRHYEEIPEYENLPFIMATRKTQELEWQNSSSMEDADA
gi~13648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 ....~....J...
gi~8922921J 1 ____________________________________________________________ 1 g1(16716345( l __________________________________________________________ gi~72431051 121 NVYEVEEPYEAPDGQLQLGPRHQHSSSGASQEEQNDLGLGDLPSDEEEIINSSDEDDVSS
gi~13648298~ 1 ____________________________________________________________ 1 gi~15426438~ 1 ____________________________________________________________ 1 J
J
J
J
NOV5 36 .... 64 ....
....J.,..J....J....
....J....J....J....J....J....
-----------------------INLWVTDLLVFQDFHGAVMRAL-----DDMDHE----gi J8922921J1 -___________________________________________________________ gi J16716345J1 -___________________________________________________________ gi J7243105J181ESSKGEPDPLEDKQDEDNGMKSKVHHIAKEIMSSEKVFVDVLKLLHIDFRDAVAHASRQL
giJ13648298J1 ____________________________________________________________ giJ15426438J1 ____________________________________________________________ J
J
J
....
NOV5 65 ....J 124 ....J....J....J....
....J....J....J....J....J....
GRDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHAT
giJ8922921J1 ____________________________________________________________ giJ16716345J1 ____________________________________________________________ IS giJ7243105J241GKPVIEDRILNQILYYLPQLYELNRDLLKELEERMLHWTEQQRIADIFVKKGPYLKMYST
gi~13648298J1 ____________________________________________________________ giJ15426438J1 ____________________________________________________________ 2.0 ....J....J....~....~....~....~.
.J.
.~.
~
.
...J
.
..J
.~
L
~
.
.
NOVS 125HILQFDRYLGLLSENCLHSPRLAAAVREFEQ "~GG,QTA
. r 184 .
.
..
.
._ .
~V
r ~;tFr Q~T
giJ892292111 _____________________________ . . r'.r r 30 .
giJ16716345J1 _____________________________ . v r 30 ~ v giJ7243105J301YIKEFDKNIALLDEQCKKNPGFAAVVREFEi C ~
r 360 r 25 giJ13648298J1 -___________________________________________________________ giJ15426438J1 -_____________________________~
.
.
r.
.r .
r 30 NOVS l85 244 giJ8922921J31 90 giJ16716345J31 90 giJ7243105J361 420 giJ13648298J1 ____________________________________________________________ 35 gi 1542643831 ~I~ilr 90 J J -, r r r r ' r v r v v r r .J. . .J....J. ...J. .~ . .J. . ..J,.. ~J...'....v..W ....J....J
NOV5 245E . T G~LR' E' . r ~~.... I E T YHGEGEG 304 Y ~ A~ i 'HL. QD
40 giJ8922921J91 I ~.. r ~ ~,t,~ 141 ~~_________ giJ16716345J91 T ~ r ~ ~ -------- 141 giJ7243105J421 I t' r ~ -------- 471 giJ13648298J1 ____________________________________________________________ giJ15426438J91 ~I 141 r r r --------J
....J....J....J
NOV5 305....J....J....J....J....J....J....J....J....
GSTFLSMEVCSLLEPKAPPRSLLEKGMGDVVTGRYLSNMTVHLGLPGLGPEHDALQPSQR
giJ8922921Jl41____________________________________________________________ 50 giJ16716345J141___________________________________-____,___________________ giJ7243105J471________________________________________-__________________ giI13648298Jl ____________________________________________________________ giJ15426438J141_____________________________________________ ____________ J
..J.. .J....~....J ....J
NOV5 365W.S~ . ~S SCL~ ....J....J....J....J....J....
RLQVRKSKVKALTDSVSAALGVRGISLFQCKKK
KVPY
giJ8922921J141- ~ ._______________________,_________ ~
giJ16716345J141- ~ ._________________________________ ~
60 giJ7243105J471- r ._________________________________ r giJ13648298J1 ____________________________________________________________ giJ15426438J141- 167 ~
~
._________________________________ 65 ....J....J....J....J....J....J....J....J....J....J..
.~.
.J
..
C r 484 giI8922921J167____________________________________________________ ~T r 174 giJ16716345J167____________________________________________________ r 174 giJ7243105J497____________________________________________________ T r 504 70 giI13648298J1 ____________________________________________________________ gi1154264381 167__,__________________________________________________~T~
.1. ._...~....~'i, ..~.E.P~ ~. 540 S NOV5 485..~....~....~ VF~IR'~RLG- L_ 'G
. 1.
YGCL ..
' -~
T.~P
DY
~ALAAFHH
gi1 89229211175 ~ ~ i ~ .. .~ 234 ..
gi1 167163451175~ S ~ -~ ~ 233 gi1 7243105)505~ ' ~ ~ S ' ~ 564 ' gi1 1364829811 _________________________ _____,______ ____.___________ 6 -154264381175' ~ S 234 gi1 ~ "
~
..
gi1 286 gi1 167163451234 -- ~ 285 gi1 72431051565 --gi1 1364829817 --gi1 154264381235 --..,.~. .~....~. ..~. .~. .~...
NOV5 601 RVTERPSMSFPLS ~RFS ~F FQ PS 660 gi189229211 287 ~ - ~ ~ 341 gi1167163451 286 ~L ~ - ~ 340 gi172431051 617 ~
gi1136482981 59 t - ~ 113 gi1154264381 287 ~ - ~
' ..1....1.,....1.,.. ,1....1 I 'I Y KVY
NOV5 661 ~ S ~ C ERG ~ ~H '1KL ~ ~ ~ :APE E . SEVGP~ 720 gi ~ 89229211 342 ~ ? ~ v . ,.I ~ 3'.. . Q ~ IQ ESK--~. 399 gi1167163451 341 ~ ~ L ~ ~~ ~ ~ Q~~TL ESK--~ 398 gi172431051 672 v v CCK-_-V~_____________________'__ 699 gi 1 13648298 ~ 114 ~ ~ ~ v ~ Q~FIQV~ytl.7~ESK--'~ l71 gi1154264381 342 ~ ~~ R,E~------- -- ------------ 376 NOV5 721 ~ ~ I~KT. ~ 15..:,~:T.. 1 .S~ ~..~. ... 1w~.-; ~A. 750 gi189229211 400 Q L ~ ~E~ .SAQ ~~F~G'.I'~ 432 gi 1167163451 399 Q~L~N~~ r, STQ D~F~G~ 431 gi172431051 699 -_ __________ ___,____ '_ __ 699 gi 1136482981 172 Q~L~mlM~ESAQIt~EiFGT~ 204 gi~15426438~ 376 _________________________________ 376 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table SF lists the domain description from DOMAIN ailalysis results against NOVS.
Table SF Domain Analysis of NOVS
Model Region of Score (bits) E value Homology RhoGEF 33-215 -1.9 1.2e-05 FYVE Ring 525-591 55.6 6.4e-14 Finger Plekstrin 657-748 49.3 8.0e-7 (PH) Consistent with other known members of the Nuclear Protein-like family of proteins, NOVS has, for example, an RhoGEF signature sequence and a FYVE Zinc Finger signature sequence, aw well as homology to other members of the Nuclear Protein-like Protein Family.
NOVS nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOVS nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOVS nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVS activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
In addition, various NOVS nucleic acids and polypeptides according to the invention are useful, ihteY alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOVS
nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Nuclear Protein-like Protein Family.
The NOVS nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and nerve physiology. As such, the NOVS nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
The NOVS nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOVS nucleic acid is expressed in Brown adipose, Vein, Umbilical Vein, Adrenal Gland/Suprarenal gland, Gall Bladder, Small Intestine, Colon, Lymphoid tissue, Spleen, Lymph node, Thymus, Brain, Temporal Lobe, Basal Ganglia/Cerebral nuclei, Substantia Nigra, Spinal Chord, Cervix, Ovary, Uterus, Testis, Lung, Lung Pleura, Larynx, Urinary Bladder, Kidney.
Additional utilities for NOVS nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV6 polypeptide has been identified as a Secretory Protein-like protein (also referred to as CG949~9-O1). The disclosed novel NOV6 nucleic acid (SEQ m NO:l 1) of 2372 nucleotides is shown in Table 6A. The novel NOV6 nucleic acid sequences maps to the chromosome 17.
An ORF begins with an ATG initiation codon at nucleotides 99-101 and ends with a TAA codon at nucleotides 1710-1712. A putative untranslated region and/or downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO:11) CCGGCAAGGATGACGCCTCCGGAGGCCCTGGCCTCACTCCCACCTGGGCGCTAGGAGCCATCCCGGG
GCTCCAGCCAGGAGCCCTGCTGCCCAGGGGCATGGCCAAACCTTTCTTCCGACTCCAGAAGTTTCTC
CGCCGAACACAGTTCCTGCTGTTCTTCCTCACGGCTGCCTACCTGATGACCGGCAGCCTGCTGCTGC
TGCAGCGGGTCCGCGTGGCTCTCCCACAGGGCCCCCGGGCACCCGGCCCCCTGCAGACCTTGCCAGT
GGCCGCCGTGGCGCTGGGCGTGGGCTTGCTGGACAGCAGAGCCCTGCACGACCCTCGAGTCAGCCCA
GAGCTGCTGCTGGGTGTGGACATGCTGCAGAGCCCCCTGACCCGGCCCCGGCCCGGCCCCCGCTGGC
TCCGGAGCCGCAACTCGGAGCTGCGTCAGTTGCGTCGCCGCTGGTTCCACCACTTCATGAGTNGACT
CCCAGGGACCGCCCGCCCTGGGCCCCGAGGCTGCCAGGCCCGCCATCCACAGCCGAGGTCCTATGTC
TACGCCGGCTTGGAGGCCGGGGCGGAGTGTTACTGCGGGAACCGGCTGCCAGCGGTGAGCGTGGGGC
TGGAAGAGTGTAACCATGAGTGCAAAGGCGAGAAGGGCTCTGTGTGCGGGGCTGTGGACCGGCTCTC
CGTGTACCGTGTGGACGAGCTGCAGCCGGGCTCCAGGAAGCGGCGGACCGCCACCTACCGCGGATGC
TTCCGACTGCCAGAGAACATCACACATGCCTTCCCCAGCTCCCTGATACAGGCCAATGTGACCGTGG
GGACTTGCTCGGGCTTTTGTTCCCAGAAAGAGTTCCCCTTGGCCATTCTCAGGGGCTGGGAATGCTA
CTGTGCTTACCCTACCCCCCGGTTCAACCTGCGGGATGCCATGGACAGCTCAGTATGTGGCCAGGAC
CCTGAGGCACAGAGGCTGGCAGAATACTGTGAGGTCTACCAGACACCTGTGCAAGACACTCGTTGTA
CAGACAGGAGGTTCCTGCCTAACAAATCCAAAGTGTTTGTGGCTTTGTCAAGCTTCCCAGGAGCCGG
GAACACGTGGGCACGGCACCTCATTGAGCATGCCACTGGCTTCTATACAGGGAGCTACTACTTTGAT
GGAACCCTCTACAACAAAGGGTTCAAGGGCGAAAAGGACCACTGGCGGAGCCGACGCACCATCTGTG
TCAAAACCCACGAGAGTGGCAGGAGGGAGATTGAGATGTTTGATTCAGCCATCCTGCTAATCCGGAA
CCCATACAGGTCCCTGGTGGCAGAATTCAACAGAAAATGTGCCGGGCACCTGGGATATGCAGCTGAC
CGCAACTGGAAGAGCAAAGAGTGGCCGGACTTTGTCAACAGCTACGCCTCGTGGTGGTCCTCGCACG
TCCTGGACTGGCTCAAGTACGGGAAGCGGCTGCTGGTGGTGCACTACGAGGAGCTGCGGCGCAGCCT
GGTGCCCACGTTACGGGAGATGGTGGCCTTCCTCAACGTGTCTGTGAGCGAGGAGCGGCTGCTCTGC
GTGGAGAACAACAAGGAGGGCAGCTTCCGGCGGCGCGGCCGGCGCTCCCACGACCCTGAGCCCTTCA
CCCCGGAGATGAAAGACTTGATCAATGGCTACATCCGGACGGTGGACCAAGCCCTGCGTGACCACAA
CTGGACGGGGCTGCCCAGGGAGTATGTGCCCAGATGATAGGCCTGGCCCACGCCGCCGCCCCCGCTG
AGTGACGCAATCGCACCACGGGGCTGCGCTCCCCACTCTGATGCTCAGGCCCGTGGCCTCACTGGGA
CGAACGGTGGGTGGGGGGCTCACCCTGGTGCTGCCTCCCGCACAAGGAGACCTGGACACAACAGACA
CACATCACAAGGCGAACACAAATGGACACACATACCTGGCCACGAACCCACACCTCCTCAGACACTC
AGACACCACTCCAGGCTCATAGCCCCGTCTTGATGCAGAGAAGCCACCCACGTGGGGTGTGCCAGGC
ACCCCCAGCTACAAATGCAGCCACGCACAGACGTAACACACAGGTGCCAGGCCGTGTGCTCCTGGAG
GCTGGCTGGCTGTCTCTCTCACACAGATACACGTGCGCTCCCTGGGATCCGGGAGGCCCTGGGCTTC
CTGTGTGTAGCCCTGGCATAGACTTGCTCGTCAGGGTGTTTGACTCTGGGATGCTGGGCCGGGCAGA
CATTTATGCTCTGAGCAGCAAGGACCATTGGGATGGAGGTGGGCACAAAGACTGCTGCTTCCAGGGT
GTGCGGCCCTGGCCGTGTGTCTGACATCCCATAAATGTGTGTGTGGTGTGACTACGGGCACCACAAA
CTCCGC
The NOV6 protein (SEQ m NO: I2) encoded by SEQ m NO:I 1 is 537 amino acid residues in length and is presented using the one-letter amino acid code in Table 6B. Psort analysis predicts the NOV6 protein of the invention to be localized outside the cell with a certainty of 0.6997.
Table 6B. Encoded NOV6 protein sequence (SEQ ID N0:12) MAKPFFRLQKFLRRTQFLLFFLTAAYLMTGSLLLLQRVRVALPQGPRAPGPLQTLPVAAVALGVG
LLDSRALHDPRVSPELLLGVDMLQSPLTRPRPGPRWLRSRNSELRQLRRRWFHHFMSXLPGTARP
GPRGCQARHPQPRSYVYAGLEAGAECYCGNRLPAVSVGLEECNHECKGEKGSVCGAVDRLSVYRV
DELQPGSRKRRTATYRGCFRLPENITHAFPSSLIQANVTVGTCSGFCSQKEFPLAILRGWECYCA
YPTPRFNLRDAMDSSVCGQDPEAQRLAEYCEVYQTPVQDTRCTDRRFLPNKSKVFVALSSFPGAG
NTWARHLIEHATGFYTGSYYFDGTLYNKGFKGEKDHWRSRRTICVKTHESGRREIEMFDSAILLI
RNPYRSLVAEFNRKCAGHLGYAADRNWKSKEWPDFVNSYASWWSSHVLDWLKYGKRLLVVHYEEL
RRSLVPTLREMVAFLNVSVSEERLLCVENNKEGSFRRRGRRSHDPEPFTPEMKDLINGYIRTVDQ
ALRDHNWTGLPREYVPR
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 6C.
Table 6C. Patp results for NOV6 Smallest Sum eading igh Prob Sequences Score P(N) producing High-scoring Segment Pairs: Frame >patp:ABB15485Human nervous system related polypeptide+1 92 0.0036 >patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042 protein >patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042 protein >patp:AAU18674Renal and cardiovascular-associated+1 79 0.085 protein >patp:AAB95341Human protein sequence SEQ ID +1 99 0.17 N0:17621 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2188 of 2189 bases (99%) identical to a gb:GENBANK-ID:AK000243~acc:AK000243.1 mRNA from Horrao sapiehs (cDNA
FLJ20236 fis, clone COLF5810, highly similar to ABOl 1095 Homo Sapiens mRNA
for KIAA.0523 protein). The full amino acid sequence of the protein of the invention was found to have 395 of 395 amino acid residues (100%) identical to, and 395 of 395 amino acid residues (100%) similar to, the 468 amino acid residue ptnr:SPTREMBL-ACC:060276 protein from Homo Sapiens (KIAA0523 PROTEIN)(Fig. 3B).
NOV6 also has homology to the proteins shown in the BLASTP data in Table 6D.
Table 6D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~14602977~gb~AAHOSimilar to 575 523/575 524/575 0.0 9975.1IAAH09975 KIAA0789 gene (90%) (90%) (BC009975) product [Homo Sapiens]
gi~3043570~dbj~BAA2KIAA0523 protein468 417/468 417/468 0.0 5449.1 (AB011095)[Homo Sapiens] (89%) (89%) gi~18489296~ref~XPCG9164 317 76/206 15/206 3e-28 _ [Drosophila (36%) (54%) 082751.1I(XM
melanogaster]
gi~16944644~emb~CADhypothetical 2117 43/131 62/131 5e-08 11404.11(AL513445)protein (32%) (46%) [Neurospora crassa]
gi~11359357~pir~~T4beta-1,3 1032 40/128 55/128 2e-05 3257 exoglucanase (31%) (42%) (EC
3.2.1.-) precursor -fungus (Trichoderma harzianum) A multiple sequence alignment is given in Table 6E, with the NOV6 protein being shown on line 1 in Table 6E in a ClustalW analysis, and comparing the NOV6 protein with the related protein sequences shown in Table 6D. This BLASTP data is displayed graphically in the ClustalW in Table 6E.
Table 6E. ClustalW Analysis of NOV6 1) > NOV6; SEQ ID NO:12 2) >gi~14602977~/ Similar to KIAA0789 gene product [Homo sapiens]; SEQ ID
N0:60 3) >gi~3043570~/ KIAA0523 protein [Homo Sapiens]; SEQ ID N0:61 4) >gi~18489296~/ CG9164 [Drosoplaila melanogaster]; SEQ 117 N0:62 5) >gi~ 16944644/ hypothetical protein [Neurospora cf assa]; SEQ ID N0:63 6) >gi~ 11359357/ beta-1,3 exoglucanase (EC 3.2.1.-) precursor - fungus [Trichodertna harzianum];
SEQ 1D N0:64 p....
NOV6 1 ------------------------MAKPFFR2QKFItRRTQFLLFFLTAAYt'C.yMT~-=--SL 32 gi1146029771 1 ------------------------KPFFRIiQKF~RRTQFLLFFLTAAY~.,MT - --SL
gi130435701 1 ________________________-___________________________________ 1 gi 18489296 1 ____________________________________________________________ 1 gi ~ 16944644 I 841 GTSPGLTTTEVTIRFTNKGGSDLNLI~DKSKPP~'IGSV~iGAQNPSSDLFEGMV2'KP KSESAT 900 gi1113593571 235 TFNGGLIG--------------AA~GNQQYT~RNL~TFNN--CAQPLSAASxaGSB----....~....~....~....~.. .~....~.. .,1....1....1....1..~.1....1 NOV6 33 LLLQRVRV~PQGPRAPGPLQT~.iiPVAAVAL~G~LDSR-ALHDPRVSPE~LLGVDMLQSP 91 gi~14602977~ 33 LLLQRVRV PQGPRAPGPLQTPVAAVAL G~LDSR-ALHDPRVSPE LLGVDMLQSP 91 gi130435701 1 ____________________________________________________________ 1 gi1184892961 1 ____________________________________________________________ 1 IS gi1169446441 901 LFFTPGAA~ADPIVYSGAWTLTFG~Fi7NFIGTLRATKVGPT~PDGSARFKYL 960 gi~11359357~ 275 RAISINNC.','!~lGIDMTAAESITLkr~. SSISGTP G~KTSFRRNQSPATSNS
NOV6 92 LTRPRPGPRWLRSRNS~LR---------------------------------------X7112 giI14602977~ 92 LTRPRPGPRWLRSRNSLR---------------------------------------',#~' gi130435701 1 ________________LR-______________________________________~5 gi1184892961 1 ___________________________________________________________- 1 gi116944644~ 961 GCYRDSSANRLETTQAFPSDNDNGKCQQYAITNKAAFAGTQYTYECWVGRSIPPASLiF
gi1113593571 335 PVAIQSSSGSTILAGGTTT----------------------------------------NOV6 113 RRRWF ~'i~X------------------------------L~GT .~G--PR------- 133 3o gi~146029771 113 RRRWF~~'MT7SQGP--===____ _____= PA'I~ ~E IHRG--=T~I 146 gi 1 30435701 6 RRRWFE., FMSDSQGP-- PAIi ~E ~IHRG-- Trr~~I 39 gi1184892961 1 __ _____________________________________ _________ 1 gi~16944644~ 1021 DDYLCTj~IzCPGDKSQFCGGVGSYMMMWYDTTGYFPENGT'~FRP~ASIC~VVGDW~
gi~11359357~ 355 AAWGQG~QYj~"PNGPTTFQG---------------- SItT SRPSLLGSN T 392 35 _.
.~....~....~....~....~....~....~. _ l~ ~I.
NOV6 134 ~QA--_______R_______________HpQp__ ~~y . ' ~ PA~~ 167 gi1146029771 147 FSDDGHERTLKGAVFYDLRKMTV-SHCQDAC~~'1,E Y~~' ~ PA205 gi~3043570~ 40 FSDDGHERTLKGAVFYDLRKMTV-SHCQDACiIE YV~ ' PA 98 gi~184892961 1 ________________________________~L~G~IR~'g S~TIII I -____'G__ gi1169446441 1081 ~RTDNSASPATRALNDRIVGQSSTNTIESCAQ~iCAG~S'F r .. PG~T 1140 gi~11359357~ 393 RSKPQYETLPVSSFRSVRSAGATGN-AVTDDTA~L~ATAC QI FDAG~ R'~~?S 451 .1. .1. .1. .1. .1.
NOV6 168 .E - E ~ 'E'EKGS~, VF.W . Z7DELQ~ _____________________ 202 gi~14602977~ 206 -- !E KGSi E L VDELQ~ _______________________ 240 gi~3043570! 99 ~ -- _E EKGS~Z~_ I VDELQ~ -____________________ 333 O gy 184892961 21 _ __________ __ __ __ V~~LSMNNI~
gi~16944644~ 1141 VA~KT~YV~ 1~PTEGSG~'yI~TCQKGTVI PSTGVSSSSGTASGTASATAS 1200 gi111359357i 452 T~S------IPP~ -AK~EYP~2IMSSGSFFND----------------------Q 479 . .1...~1. ~~~ .1...y y .~ . ~~. ~ ~~.. y , c - v _ r v-NOV6 203 R j' "I'~~TY~ C~RL~. . ----- !i! ' ~ ~5~~~~ ~ - '~!':2'C~GT S. -Q ~E
gi ~ 146029771 241 R ~TiiITY~ C RL~ _=---- TH~ ~ 5~~~ - --'i~T~TGT ;, -Q ~E
gi ~ 304357Q ~ 134 R WiTY~ C RL E ---- ~'H' ~ S~Ir~~ -- --,~T"~"sIGT ,~ -Q ~E
gi1184892961 34 HP4'PRIE~__ ________ -~ . __-_____ _ ___._ _p_ 53 gi 1 169446441 1201 STSSiIA~TPGN~Q'S'~GQYSSLGCY',~.",Di RSL~GKNTQSNVMSIiDD~T
!GY~yIY 1260 gi~11359357~ 480 SNP~PVVQ TPGQTG-------- QVE~SDM~TVSTQG- -----TQAGAVLIEWNL 520 .1. .1....1....1.. .1.~...~...1.~~ .1. ..1....1....1....1....1 NOV6 247 .PLAIL~~ E YCA ~T'P~tFNL~'~ n SS~ Gy P________________________ 282 gi114602977~ 285 PLAIL E YCAY~'T~P~R,FN~~W ySa GyP3~"'s -______-______________ gi~30435701 178 PLAIL E YCAY~'~P~FN~rw W9~G~',~,?rP~'." ______________________ gi1184892961 53 ____gp________ ~~gTI~ CR~LKYI ~- ________________________ 75 gi116944644~ 1261 ~GTEYSAECF~GNDLLNGAAP1~T~GRC~G QQQICGGSNGLSMYQLNPNGTSSSVT
gi1113593571 521 ATSGTPSe~vIWDVHTRIGGFKGSNLQ'~'~1QCPVTAS~----------------------4~ _:
..J.. .~....~.,...~ ..L ...~....~....~....~....~
NOV6 282 _______ ~QR(~..[~~ E~Q P~'ST;QD RC'DR~~2.F ~ K-___________________ gi~14602977~ 320 ------- Q ~ E EVY'Q PVQD RC DR~F ~ K-------------------- 349 gi~30435701 213 ------- fi~Q E EV~Q P~QD RC~DR~2.~' ~ K--------------------gi 18489296 75 ___________ZPIKSD~'Y ;'PSDVSAAL~S ~ _____________________ 102 gi~16944644) 1321 ASGSATQSATA~GTASGTASSSS ' T TS~AVPT ~ VSVKCPDNNNGTYLSLNGKTF
gi I 11359357 ~ 554 ------- TTt'V.NTA~IGAYMS ,:, I~ASASNL~'~'NIEI~-------------NOV6 311 _____________________________ ~~ . ~ _______________ 325 w gi~14602977~ 349 ______________________________. ~r _______________ 363 w 15 gi~30435701 242 ______________________________r~' S _______________ 256 gi~184892961 102 __-___________________________ pLT. ~ ________________ 114 gi~16944644~ 1381 LLECFTDHEGGDLALAYVDSYALCAEKCSTTDI,rC~ F~ GTGIQAPCYMKKSVGRGF
gi~113593571 582 ______________________________~T~DHDIDDS--__________N___ 597 1450 1460 7.470 1480 1490 1500 ....
NOV6 325 --------------------- F~ K 359 v gi~14602977~ 363 _____________________ y F~ K IC 397 v gi~30435701 256 --------------------- F~ K K 290 gi118489296~ 114 _____________________ I ~Y LKT P 4 148 gi~16944644~ 1441 SAAATMTSAASAMGSNSWGPS TGS TGSATDSTT 1500 gi~11359357~ 598 ------------------FWF ~..'AVEHH QYQ~ANT,638 gi~146029771 398 457 gi~3043570~ 291 350 gi1184892961 148 206 gi~16944644~ 1501 1560 gi~11359357~ 639 697 4o NOV6 420 ----I----I----I 458 gi~14602977~ 458 --------------- 496 gi~3043570~ 351 _,_____________ 389 gi~18489296~ 207 _______________ 249 g3~16944644~ 1561 IAPSENTTPSASVAP 1619 gi~11359357~ 697 --------------- 729 NOV6 458 --------L PT. E1~3't~~ 5~- ERW ~ I$G---------------- 494 giI14602977f 496 -_______L PT ~ES ER,~,~ 'j ' ~G-_______________ 532 gi ~ 3043570 ~ 389 --------L PT ~E~'1'C7~ ~ g~,~,, yG---------------- 425 gi~18489296~ 249 _-______TERE S ~D QFPQLt: IMy" I ~ ~_________________ 284 gi~16944644~ 1620 SNSWPSDS~APSAS~I~iPSA~~ yS~ASV~PSTSIAHS SESVAPAESIAPSASVSS
gi~11359357~ 729 --------N~FD~EGTTN~yNLGTVG,i~VI~IT~ LATS------S--------- 766 NOV6 494 --_________________________-_________-______________________ 494 gi~14602977~ 532 _-_________________________-________________________________ gi~3043570~ 425 ____________________________________________________________ gi118489296~ 284 ____________________________________________________________ gi~16944644~ 1680 GSNTGVAPTNSASVTPTNSASVATTISVSVAPTASDAPTTSITLSVAPGSSSSTTAPAW
gi~11359357~ 766 ____________________________________________________________ NOV6 494 _______________________, ~~~~ PE.: L~~GYIv Q RDHNW G 529 gi~146029771 532 -_____________-_________ ~~E~F PE"IDL~~TGYI~ Q~ RDHNW G 567 gi~3043570~ 425 _________________________ . ~.E. PE ~CDI~Ii~GYI~ ' Q; RDHNW G
gi~18489296~ 284 -------------------------;_~LLSF~1~ ES ~AE~QNR ~I YGL GRQEP--47 _ _ gi~169446441 1740 STTLSAPTVTSVPPAAGTSTTTAAAVT~TTTTTTATSTT TPVLFTTSTT~T 1799 gi~11359357~ 766 -------------------------SNVFADVIALFLASGSGGV~PPP'S~STTKAQT
NOV6 530 LPREY~7'PR-___________,____-__________________________________ 537 giI14602977~ 568 LPRE'IV'PR-___________,____-__________________________________ 575 gi13043570~ 461 LPREYPR-___________,___________________________-___________ gi118489296~ 317 ________________________________________________-___________ 1O gi~16944644~ 1800 gi~11359357~ 802 TFSTI';TSSPPKQTG-------------------------WNFLGCYSDNVNGRTLANQV
The NOV6 Clustal W alignment shown in Table 6E was modified to begin at amino residue 841 and end at amino acid residue 1860. The data in Table 6E includes all of the regions overlapping with the NOV6 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 6F lists the domain description from DOMAIN analysis results against NOV6.
Table 6F Domain Analysis of Model Region of Score (bits) E value Homology Disintegrin 151-159 5.8 0.73 WSC domain 120-186 36.8 5e-07 Peptidase family346-354 -0.2 8.4 Sulfotransferase288-528 -143.3 0.14 proteins Consistent with other known members of the Secretory Protein-like family of proteins, NOV6 has, for example, has homology to other members of the Secretory Protein-like Protein Family. NOV6 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV6 nucleic acids and polypeptides can be used to identify proteins that are members of the Secretory Protein-like Protein Family. The NOV6 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV6 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other diseases, disorders and conditions.
In addition, various NOV6 nucleic acids and polypeptides according to the invention are useful, intes~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV6 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Secretory Protein-like Protein Family.
The NOV6 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac physiology. As such, the NOV6 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiac and vascular system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other diseases, disorders and conditions The NOV6 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV6 nucleic acid is expressed in Aorta.
Additional utilities for NOV6 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV7 polypeptide has been identified as a Transmission Blocking Target Antigen 5230 Precursor-like protein (also referred to as CG9497~-O1). The disclosed novel NOV7 nucleic acid (SEQ )D N0:13) of 1629 nucleotides is shown in Table 7A. The novel NOV7 nucleic acid sequences maps to the chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TGA
codon at nucleotides 1627-1629. A putative untranslated region and/or downstream from the termination codon is underlined in Table 7A, and the start and stop codons are in bold letters.
Table 7A. NOV7 Nucleotide Sequence (SEQ ID N0:13) ATGGCGGTGCCCGGCGAGGCGGAGGAGGAGGCGACAGTTTACCTGGTAGTGAGCGGTATCCCCTCCG
TGTTGCGCTCGGCCCATTTACGGAGCTATTTTAGCCAGTTCCGAGAAGAGCGCGGCGGTGGCTTCCT
CTGTTTCCACTACCGGCATCGGCCTGAGCGGGCCCCTCCGCAGGCCGCTCCTAACTCTGCCCTAATT
CCTACCGACCCAGCCGCTGAGGGCCAGCTTCTCTCTCAGACTTCGGCCACCGATGTCCGGCCTCTCT
CCACTCGAGACTCTACTCCAATCCAGACCCGCACCTGCTGCTGCGTCATCTCGGTAAGGGGGTTGGC
TCAAGCTCAGAGGCTTATTCGCATGTACTCGGGCCGCCGGTGGCTGGATTCTCACGGGACTTGGCTA
CCGGGTCGCTGTCTCATCCGCAGACTTCGGCTACCTACGGAGGCATCAGGTCTGGGCTCCTTTCCCT
TCAAGACCCGGAAGGAACTGCAGAGTTGGAAGGCAGAGAATGAAGCCTTCACCCTGGCTGACCTGAA
GCAACTGCCGGAGCTGAACCCACCAGTGCTGATGCCCAGAGGGAATGTGGGGACTCCCCTGCGGGTC
TTTTTGGAGTTGATCCGGGCCTGCCGCCTACCCCCTCGGATCATCACCCAGCTGCAGCTCCAGTTCC
CCAAGACAGGTTCCTCCCGGCGCTACGGCAATGTGCCTTTTGAGTATGAGGACTCAGAGACTGTGGA
GCAGGAAGAGCTTGTGTATACAGCAGAGGGTGAAGAAATACCCCAAGGAACCTACCTGGCAGATATA
CCAGCCAGCCCCTGTGGAGAGCCTGAGGAAGAAGTGGGGAAGGAAGAGGAAGAAGAGTCTCACTCAG
ATGAGCTGTTCGGGTGTGCTGTGGTCATCCTCCCTGCGCACCTACAGCCGCAGACCGCCGGTGGGGG
GCGGGGGATGCCGGGCTGCCGCATCAGCGCCTGCGGCCCGGGGGCCCAGGAGGGGACGGCAGAGCAG
AGGTCGCCGCCGCCGCCCTGGGATCCCATGCCGTCCTCTCAGCCCCCGCCCCCAACTCCGACCTTGA
CTCCTACCCCGACCCCGGGTCAGTCCCCGCCGCTGCCGGACGCAGCTGGGGCTTCAGCAGGCGCGGC
CGAGGACCAGGAGCTGCAGCGCTGGCGCCAGGGCGCTAGCGGGATCGCGGGGCTCGCCGGCCCCGGA
GGGGGCTCTGGCGCGGCTGCGGGGGCGGGGGGCCGCGCGCTGGAGCTGGCCGAAGCACGGCGGCGGC
TGCTGGAGGTGGAGGGCCGCCGGCGCCTGGTGTCGGAGCTGGAGAGCCGCGTGCTGCAGCTGCACCG
CGTTTTCTTGGCGGCCGAGCTGCGCCTGGCGCACCGCGCGGAGAGCCTGAGCCGCCTGAGCGGCGGC
GTGGCGCAGGCCGAGCTCTACCTGGCGGCTCACGGGTCGCGCCTCAAGAAGGGCCCGCGCCGCGGCC
GCCGCGGCCGACCCCCCGCGCTGCTGGCCTCGGCGCTGGGCCTGGGCGGCTGCGTGCCCTGGGGTGC
CGGGCGACTGCGGCGCGGCCACGGCCCCGAGCCCGACTCGCCCTTCCGCCGCAGCCCGCCCCGCGGC
CCCGCCTCCCCGCAGCGCTGA
The NOV7 protein (SEQ m N0:14) encoded by SEQ m N0:13 is 542 amino acid residues in length and is presented using the one-letter amino acid code in Table 7B. Psort analysis predicts the NOV7 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 7S. Encoded NOV7 protein sequence (SEQ ID N0:14) MAVPGEAEEEATVYLVVSGIPSVLRSAHLRSYFSQFREERGGGFLCFHYRHRPERAPPQAAPNSA
LIPTDPAAEGQLLSQTSATDVRPLSTRDSTPIQTRTCCCVISVRGLAQAQRLIRMYSGRRWLDSH
GTWLPGRCLIRRLRLPTEASGLGSFPFKTRKELQSWKAENEAFTLADLKQLPELNPPVLMPRGNV
GTPLRVFLELIRACRLPPRIITQLQLQFPKTGSSRRYGNVPFEYEDSETVEQEELVYTAEGEEIP
QGTYLADIPASPCGEPEEEVGKEEEEESHSDELFGCAVVILPAHLQPQTAGGGRGMPGCRISACG
PGAQEGTAEQRSPPPPWDPMPSSQPPPPTPTLTPTPTPGQSPPLPDAAGASAGAAEDQELQRWRQ
GASGIAGLAGPGGGSGAAAGAGGRALELAEARRRLLEVEGRRRLVSELESRVLQLHRVFLAAELR
LAHRAESLSRLSGGVAQAELYLAAHGSRLKKGPRRGRRGRPPALLASALGLGGCVPWGAGRLRRG
HGPEPDSPFRRSPPRGPASPQR
A search against the Patp database, a proprietary database that'contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 7C.
Table 7C. Patp results for Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAU33166Novelhuman secreted protein #3657+1 1533 5.8e-157 >patp:AAE04880Humanprotease protein-7 (PRTS-7) +1 1533 5.8e-l57 >patp:AAB94023Humanprotein sequence SEQ ID N0:14157+1 1519 1.8e-155 >patp:AAU33124Novelhuman secreted protein #3615+1 390 7.7e-36 >patp:AAG02700Humansecreted protein, SEQ ID +1 268 3.5e-22 NO: 6781 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 874 of 876 bases (99%) identical to a gb:GENBANK-ID:AK022517~acc:AK022517.1 mRNA from Homo Sapiens (cDNA
FLJ12455 fis, clone NT2RM1000563, weakly similar to TRANSMISSION-BLOCKING
TARGET ANTIGEN 5230 PRECURSOR). The full amino acid sequence of the protein of the invention was found to have 290 of 292 amino acid residues (99%) identical to, and 290 of 292 amino acid residues (99%) similar to, the 525 amino acid residue pW
r:SPTREMBL-ACC:Q9H9Z3 protein from Homo Sapiens (CDNA FLJ12455 FIS, CLONE NT2RM1000563, PRECURSOR).
NOV7 also has homology to the proteins shown in the BLASTP data in Table 7D~
Table 7D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) (%) gi~18545154~ref~XPhypothetical 525 274/274 274/274 e-147 _ protein FLJ12455 (100%) (100%) 084046.1~(XM_084046 [Homo Sapiens]
gi~11545793IrefINPhypothetical 525 272/274 272/274 e-145 _ protein FLJ12455 (99%) (99%) 071361.1I(NM
[Homo Sapiens]
gi~18545156~ref~XPsimilar to 107 83/84 84/84 6e-37 _ hypothetical (98%) (99%) 086159.1~(XM
_ protein FLJ12455 [Homo Sapiens]
giI18545158~ref~XPhypothetical 141 135/137 136/137 2e-33 _ protein XP_097448 (98%) (98%) 097448.1~(XM_097448 [Homo Sapiens]
gi~~7.7562286IrefINPK07B1.7b.p 487 76/237 119/237 3e-30 _ [Caenorhabditis (32%) (50%) 505420.1~(NM_073019 e1 egans]
A multiple sequence alignment is given in Table 7E, with the NOV7 protein being shown on line 1 in Table 7E in a ClustalW analysis, and comparing the NOV7 protein with the select related protein sequences shown in Table 7D. This BLASTP data is displayed graphically in the ClustalW in Table 7E.
Table 7E. ClustalW Analysis of NOV7 1) > NOV7; SEQ m N0:14 2) > gig 18545154/ hypothetical protein FLJ12455 [Horrao Sapiens); SEQ m N0:65 3) > gi~11545793~/ hypothetical protein FLJ12455 [Homo Sapiens]; SEQ ID N0:66 NOV7 l 60 gi1185451541 1 60 gi~11545793~ 1 60 y . ~~~.~.I~ .~~ ~I~ ~I. y NOV7 61 ~~~ ~ ,~~~ ,. ~ ~ . .~ ~ . ~~w ~, 120 TRANSMISSION-FIS
#PD229850 55-90 179 4e-14 BLOCKING
TRANSMISSION-FIS
#PD138963 91-258 872 2e-94 BLOCKING
TRANSMISSION-FIS
Consistent with other known members of the Transmission Blocking Target Antigen 5230 Precursor-like family of proteins, NOV7 has, for example, three Blocking NT2RM1000563 Transmission-FIS Antigen Weakly Precursor Peptidase A2 signature sequences and homology to other members of the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family. NOV7 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV7 nucleic acids and polypeptides can be used to identify proteins that are members of the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family. The I O nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV7 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets fox the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection In addition, various NOV7 nucleic acids and polypeptides according to the invention are useful, irzte~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV7 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Transmission Blocking Target Antigen 5230 Precursor-like Protein Family.
The NOV7 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and nerve physiology. As such, the NOV7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection The NOV7 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV7 nucleic acid is expressed in Adipose, Heart, Aorta, Coronary Artery, Umbilical Vein, Pancreas, Liver, Gall Bladder, Colon, Bone Marrow, Thymus, Bone, Cartilage, Synovium/Synovial membrane, Skeletal Muscle, Brain, Left cerebellum, Right Cerebellum, Thalamus, Hypothalamus, Pituitary Gland, Frontal Lobe, Parietal Lobe, Cerebral Medulla/Cerebral white matter, Basal Ganglia/Cerebral nuclei, Substantia Nigra, Hippocampus, Cervix, Mammary gland/Breast, Uterus, Oviduct/LTterine Tube/Fallopian tube, Prostate, Testis, Lung, Bronchus, Larynx, Kidney, Retina, Skin, Epidermis.
Additional utilities for NOV7 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV8 polypeptide has been identified as a Nuclear Protein-like protein (also referred to as CG94713-O1). The disclosed novel NOV8 nucleic acid (SEQ ZD
NO:15) of 3807 nucleotides is shown in Table 8A. The novel NOV8 nucleic acid sequences maps to the chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 16-18 and ends with a TGA codon at nucleotides 3793-3795. A putative untranslated region and/or downstream from the termination codon is underlined in Table 8A, and the start and stop codons are in bold letters.
Table 8A. NOV8 Nucleotide Sequence (SEQ ID NO:15) ATGATAAAATAGAAGATGAATTGCAAACCTTCTTTACCAGTGATAAAGATGGAAATTACACATGCAT
ACAACCCGAAATCACCACCTACACAAAACTCTTCAGCCAGCAGTGTGAACTGGAATTCTGCCAACCC
AGATGACATGGTGGTTGATTATGAAACTGACCCTGCTGTAGTTACTGGTGAAAATATTTCTTTAAGC
CTTCAGGGTGTTGAAGTATTTGGTCATGAAAAGTCTTCTAGTGATTTCATTAGTAAGCAGGTGTTAG
ATATGCATAAAGATTCTATTTGTCAGTGTCCTGCACTTGTAGGTACTGAGAAGCCCAAATATCTGCA
ACACAGTTGTCATTCCCTAGAAGCAGTTGAGGGCCAGAGTGTTGAGCCATCTTTGCCTTTTGTGTGG
AAGCCTAATGACAATTTGAACTGTGCAGGCTACTGTGATGCCTTGGAGCTGAACCAAACATTTGACA
TGACAGTGGATAAAGTTAACTGCACCTTTATATCACATCATGCCATCGGAAAGAGTCAGTCCTTCCA
TACTGCTGGAAGCCTGCCACCAACTGGTAGGAGAAGTGGAAGTACATCTTCTTTATCCTATTCCACT
TGGACATCTTCCCATTCTGATAAGACGCATGCAAGAGAAACTACTTATGATAGAGAAAGCTTTGAAA
ACCCTCAAGTCACACCATCAGAAGCCCAAGACATGACTTACACAGCATTTTCTGATGTGGTGATGCA
AAGTGAGGTTTTTGTTTCAGATATTGGAAATCAGTGTGCATGTTCTTCAGGAAAGGTCACCAGTGAG
TACACAGATGGATCACAACAAAGACTAGTTGGAGAAAAAGAGACACAAGCACTAACACCAGTTTCTG
ATGGCATGGAAGTCCCCAATGATTCTGCATTACAAGAGTTCTTTTGTTTATCCCATGATGAATCCAA
TAGCGAACCACATTCACAGAGCTCATACAGGCACAAGGAAATGGGCCAAAATCTGAGAGAGACAGTG
TCCTATTGTCTTATTGATGATGAATGCCCTTTAATGGTGCCAGCTTTTGATAAGAGCGAAGCTCAAG
TGCTGAACCCAGAGCATAAAGTCACTGAGACTGAAGACACACAAATGGTCTCCAAAGGAAAGGATTT
GGGAACCCAAAATCATACCTCAGAATTGATTCTAAGTAGCCCGCCAGGACAAAAGGTGGGCTCGTCA
TTTGGACTGACTTGGGATGCAAATGATATGGTCATTAGCACAGACAAAACGATGTGCATGTCAACAC
CAGTCCTAGAACCCACAAAAGTAACCTTTTCTGTTTCACCGATTGAAGCGACGGAGAAATGTAAGAA
AGTGGAGAAGGGTAATCGAGGGCTTAAAAACATACCAGACTCGAAGGAGGCACCTGTGAACCTGTGT
AAACCCAGTTTAGGAAAATCAACAATCAAAACGAATACCCCAATAGGCTGCAAAGTTAGAAAAACTG
AAATTATAAGTTACCCAAGACCAAACTTCAAGAATGTCAAAGCAAAAGTTATGTCTAGAGCAGTGTT
GCAGCCCAAAGATGCTGCTTTATCAAAGGTCACGCCCAGACCTCAGCAGACCAGTGCCTCATCACCC
TCATCAGTGAATTCAAGACAACAAACAGTCTTGAGCAGAACACCGAGATCTGACTTGAATGCAGACA
AAAAAGCAGAAATTCTAATTAACAAGACACATAAGCAGCAGTTTAATAAACTCATTACTAGCCAGGC
TGTGCATGTTACAACTCATTCTAAAAATGCTTCACACAGGGTTCCAAGAACAACATCTGCCGTGAAA
TCGAATCAGGAAGATGTTGACAAAGCCAGTTCTTCTAACTCAGCATGCGAGACCGGGTCCGTTTCTG
CGTTGTTTCAGAAGATCAAAGGCATACTCCCTGTTAAAATGGAAAGTGCAGAATGTTTGGAAATGAC
CTATGTTCCCAACATTGATAGGATTAGCCCTGAAAAGAAGGGTGAAAAAGAAAATGGGACATCTATG
GAAAAACAAGAGCTGAAACAAGAGATTATGAATGAGACTTTTGAATATGGTTCTCTGTTTTTGGGCT
CTGCTTCAAAAACAACGACCACCTCAGGTAGGAATATATCCAAGCCTGACTCCTGCGGTTTGAGGCA
AATAGCTGCTCCAAAAGCCAAAGTGGGGCCCCCTGTTTCCTGTTTGAGGCGGAACAGTGACAATAGA
AATCCCAGTGCTGATCGAGCCGTATCTCCTCAGAGGATCAGGCGTGTGTCCAGTTCTGGAAAGCCTA
CATCCTTGAAAACTGCACAGTCGTCATGGGTGAATTTGCCTAGACCACTTCCTAAATCCAAAGCATC
TTTGAAAAGTCCTGCGCTGCGGAGGACAGGAAGCACCCCCTCAATAGCCAGCACCCACAGTGAGCTG
AGCACTTACAGCAACAATTCTGGTAATGCCGCTGTCATCAAATATGAGGAGAAACCTCCAAAACCAG
CATTTCAGAATGGTTCCTCAGGATCCTTTTATTTGAAGCCTTTGGTATCCAGGGCTCATGTTCACTT
GATGAAAACTCCTCCAAAAGGTCCTTCGAGAAAAAATTTATTTACAGCTCTTAATGCAGTTGAAAAG
AGCAGGCAAAAGAATCCTCGAAGCTTATGTATCCAGCCACAGACAGCTCCCGATGCGCTGCCCCCTG
AGAAAACACTTGAATTGACGCAATATAAAACAAAATGTGAAAACCAAAGTGGATTTATCCTGCAGCT
CAAGCAGCTTCTTGCCTGTGGTAATACCAAGTTTGAGGCATTGACAGTTGTGATTCAGCACCTGCTG
TCTGAGCGGGAGGAAGCACTGAAACAACACAAAACCCTATCTCAAGAACTTGTTAACCTCCGGGGAG
AGCTAGTCACTGCTTCAACCACCTGTGAGAAATTAGAAAAAGCCAGGAATGAGTTACAAACAGTGTA
TGAAGCATTCGTCCAGCAGCACCAGGCTGAAAAAACAGAACGAGAGAATCGGCTTAAAGAGTTTTAC
ACCAGGGAGTATGAAAAGCTTCGGGACACTTACATTGAAGAAGCAGAGAAGTACAAAATGCAATTGC
AAGAGCAGTTTGACAACTTAAATGCTGCGCATGAA.ACCTCTAAGTTGGAAATTGAAGCTAGCCACTC
AGAGAAACTTGAATTGCTAAAGAAGGCCTATGAAGCCTCCCTTTCAGAAATTAAGAAAGGCCATGAA
ATAGAAAAGAAATCGCTTGAAGATTTACTTTCTGAGAAGCAGGAATCGCTAGAGAAGCAAATCAATG
ATCTGAAGAGTGAAAATGATGCTTTAAATGAAAAATTGAAATCAGAAGAACAAAAAAGAAGAGCAAG
AGAAAAAGCAAATTTGAAAAATCCTCAGATCATGTATCTAGAACAGGAGTTAGAAAGCCTGAAAGCT
GTGTTAGAGATCAAGAATGAGAAACTGCATCAACAGGACATCAAGTTAATGAAAATGGAGAAACTGG
TGGACAACAACACAGCATTGGTTGACAAATTGAAGCGTTTCCAGCAGGAGAATGAAGAATTGAAAGC
TCGGATGGACAAGCACATGGCAATCTCAAGGCAGCTTTCCACGGAGCAGGCTGTTCTGCAAGAGTCG
CTGGAGAAGGAGTCGAAAGTCAACAAGCGACTCTCTATGGAAAACGAGGAGCTTCTGTGGAAACTGC
ACAATGGGGACCTGTGTAGCCCCAAGAGATCCCCCACATCCTCCGCCATCCCTTTGCAGTCACCAAG
GAATTCGGGCTCCTTCCCTAGCCCCAGCATTTCACCCAGATGACACCTCCCCAAA
Variant sequences of NOVB are included in Example 3, Table 20. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV8 protein (SEQ m N0:16) encoded by SEQ m NO:15 is 1259 amino acid residues in length and is presented using the one-letter amino acid code in Table 8B. Psort analysis predicts the NOV8 protein of the invention to be localized in the nucleus with a certainty of 0.7600.
Table 8B. Encoded NOV8 protein sequence (SEQ ID N0:16) MNCKPSLPVIKMETTHAYNPKSPPTQNSSASSVNWNSANPDDMVVDYETDPAWTGENISLSLQG
VEVFGHEKSSSDFTSKQVLDMHKDSICQCPALVGTEKPKYLQHSCHSLEAVEGQSVEPSLPFWK
PNDNLNCAGYCDALELNQTFDMTVDKVNCTFISHHAIGKSQSFHTAGSLPPTGRRSGSTSSLSYS
TWTSSHSDKTHARETTYDRESFENPQVTPSEAQDMTYTAFSDWMQSEVFVSDIGNQCACSSGKV
TSEYTDGSQQRLVGEKETQALTPVSDGMEVPNDSALQEFFCLSHDESNSEPHSQSSYRHKEMGQN
LRETVSYCLIDDECPLMVPAFDKSEAQVLNPEHKVTETEDTQMVSKGKDLGTQNHTSELILSSPP
GQKVGSSFGLTWDANDMVISTDKTMCMSTPVLEPTKVTFSVSPIEATEKCKKVEKGNRGLKNIPD
SKEAPVNLCKPSLGKSTIKTNTPIGCKVRKTEIISYPRPNFKNVKAKVMSRAVLQPKDAALSKVT
PRPQQTSASSPSSVNSRQQTVLSRTPRSDLNADKKAEILINKTHKQQFNKLITSQAVHVTTHSKN
ASHRVPRTTSAVKSNQEDVDKASSSNSACETGSVSALFQKIKGILPVKMESAECLEMTYVPNIDR
ISPEKKGEKENGTSMEKQELKQEIMNETFEYGSLFLGSASKTTTTSGRNISKPDSCGLRQIAAPK
AKVGPPVSCLRRNSDNRNPSADRAVSPQRIRRVSSSGKPTSLKTAQSSWVNLPRPLPKSKASLKS
PALRRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPAFQNGSSGSFYLKPLVSRAHVHLM
KTPPKGPSRKNLFTALNAVEKSRQKNPRSLCIQPQTAPDALPPEKTLELTQYKTKCENQSGFILQ
LKQLLACGNTKFEALTWIQHLLSEREEALKQHKTLSQELVNLRGELVTASTTCEKLEKARNELQ
TVYEAFVQQHQAEKTERENRLKEFYTREYEKLRDTYIEEAEKYKMQLQEQFDNLNAAHETSKLEI
EASHSEKLELLKKAYEASLSEIKKGHEIEKKSLEDLLSEKQESLEKQINDLKSENDALNEKLKSE
EQKRRAREKANLKNPQIMYLEQELESLKAVLEIKNEKLHQQDIKLMKMEKLVDNNTALVDKLKRF
QQENEELKARMDKHMAISRQLSTEQAVLQESLEKESKVNKRLSMENEELLWKLHNGDLCSPKRSP
TSSAIPLQSPRNSGSFPSPSISPR
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 8C.
Table 8C. results for Patp NOV8 Smallest Sum eading igh Prob equences Pairs: Frame ScoreP(N) producing High-scoring Segment >patp:AAG63542Aminoacidsequence human ATIP +1 6389 0.0 of a isoform >patp:AAG63529Aminoacidsequence human ATIP +1 6233 0.0 of a isoform >patp:AAG63541Aminoacidsequence human ATIP +1 3928 0.0 of a isoform >patp:AAG63537Aminoacidsequence ATIP isoform +l 3279 0.0 of a >patp:AAG63530Aminoacidsequence human ATIP +1 2954 1.5e-307 of a isoform In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 3751 of 3751 bases (100%) identical to a gb:GENBANI~-m:AB033114~acc:AB033114.1 mRNA from Homo Sapiens (mRNA for KIAA1288 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 1245 of 1245 amino acid residues (100%) identical to, and 1245 of 1245 amino acid residues (100%) similar to, the 1245 amino acid residue ptnr:SPTREMBL-ACC:Q9IJLD2 protein from Horno Sapiens (I~IAA1288 PROTEIN).
NOV8 also has homology to the proteins shown in the BLASTP data in Table 8D.
Table 8D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi163314071dbjIBAABKIAA1288 protein1245 1245/12451245/12450.0 6602.11(AB033114)[Homo Sapiens] (100%) (100%) gi1178656321refINPAT2 receptor- 436 404/436 409/436 0.0 065800.11(NM interacting (92%) (93%) protein 1 [Homo Sapiens]
gi1204367221dbjIBABunnamed protein240 239/240 239/240 e-107 14894.11 (AK024357)product (99%) (99%) [Homo Sapiens]
gi138822691dbjIBAA3KIAA0774 protein1163 135/366 224/366 9e-49 4494.11(AB018317)[Homo Sapiens] (36%) (60%) gi1174756301refIXPKIAA0774 protein901 135/366 224/366 3e-48 ' [Homo Sapiens] (36%) (60%) 029364.31 (XM 029364) A multiple sequence alignment is given in Table 8E, with the NOV8 protein being shown on line 1 in Table 8E in a ClustalW analysis, and comparing the NOVB
protein with the related protein sequences shown in Table 8D. This BLASTP data is displayed graphically in the ClustalW in Table 8E.
Table 8E. ClustalW Analysis of NOVB
1) > NOVB; SEQ m N0:16 2) > gi~6331407~/ KIAA1288 protein [Homo Sapiens]; SEQ ID N0:67 3) > gi~17865632~/ AT2 receptor-interacting protein 1 [Homo Sapiens]; SEQ m NO:68 4) > gi~10436722~/ unnamed protein product [Homo Sapiens]; SEQ m N0:69 5) > gi~3$82269~/ I~IAA0774 protein [Homo Sapiens]; SEQ ID N0:70 6) > gig 17475630/ KIAA0774 protein [Homo Sapiens]; SEQ m N0:71 ....1....1....1....1....1....1....1....1....1....1....1....1 gi163314071 1 ---------------THAYNPKSPPTQNSSASSVNWNSANPDDMWDYETDPAWTG--- 42 gi1178656321 1 ____________________________________________________________ 1 gi1104367221 1 ____________________________________________________________ 1 gi138822691 1 RGQIPGGGEGPQKTLPDHAVPAAFPATDSTSEGKSVRHPKPSTSESKQSTPSETQTVGAH 60 gi1174756301 1 ____________________________________________________________ 1 ....I....I....I....I....I....I....I....I....I....I....I....I
S gi I63314071 42 ---~---ENISLSLQGVEVFGHEKSSSDFISKQVLDMHKDSICQCPALVGTEKPKYLQHSC
gi ~17865632~ 1 _____________________________-______________________________ 1 gi ~10436722~ 1 ____________________________________________________________ gi 13882269~ 61 VLQVCSEHTSHSAHPEPALNLTLASKEIPSKLEAQLGQGKGEAKLDLKYVPPRRVEQEGK
gi ~17475630~ 1 ____________________________________________________________ gi I63314071 97 HSLEAVEGQSVEPSLPFVWKPNDN----LNCAGYCDALELNQTFDMTVDKVNCTFISHHA
15gi 1178656321 1 ____________________________________________________________ gi ~104367221 1 ____________________________________________________________ gi ~3882269~ 121AAQEGYLGCHKEENLSALEGRDPCGEAHPEATDALGHLLNSDLHHLGVGRGNCEEKRGVN
gi I174756301 1 ____________________________________________________________ gi I6331407~ 153IGKSQSFHT---AGSLPPTGRRSGSTSSLSYSTWTSSHSDKTHARETTYDRESFENPQVT
gi ~17865632~ 1 ____________________________________________________________ gi I104367221 1 ____________________________________________________________ gi ~3882269~ 181PGEQDSLHTTPKQGSASLGGADNQPTGKISPCAGEKLGERTSSSFSPGDSHVAFIPNNLT
gi ~174756301 1 ____________________________________________________________ ..
NOV8 224PSEAQDMTYTAFSDVVMQSEVFV'SD'~~ QCACSGKVTSYT~GSQ
~ B ~ LVGEETAL
B
B
giI 6331407~ 210PSEAQDMTYTAFSDVVMQSEVFSD~G QCACSGKVTSYTGSQ
Q
gi~ 17865632~ 1 _________________________.__________________________._______ gi~ 10436722~ 1 ___________________ __ ____ ____________________ ____ 3Sgi1 38822691 241DSKPLDVIEEERRLGSGNKDSVVL~?F PSVGE PL
SEAR~SKVT S 300 KTEVP PQS
gi 174756301 1 ~ ~ ~ ~ S 38 ~ ------------------- ~ PL~pQS ~
VLV'F,,~~,, PSVGE SEARSK~'VT
~KTEVP
g 284~SDGMEU'PN~7~AL Q~FFCLS~
ESNE~HSQSSj~'RHKEGQNL'RE~SYCLID ~ECPL~ 343 i 270SIaGMEU'PN~:?~AL Q ESN HSQSS'Y'RHKE ~ECPL
I SYCLID
i ' __._______.______ gi~ 17865632~ 1 ___________________________________________ i 10436722 1 ____________________________________________________________ B
gi 3882269 301~~.''NRN~~.~iLENADK I~STSAR SVL~TI~--APLPETTiVNMTYP~T
PSSSFQ~S===~ 355 I I ~ ~ s i 1747 I 5 39 NRN I SVLI --APLPETTNMTY PSSSFQ~ 93 g 1 LEN~K STS P T S
~
NOV8 344PAF~KSEAQ~ EHKVTETE QMVSKLGTQNHTSEILS PPGQKVGSSFGLTWD
.. C'~
S0gi~ 63314071 330PAF~KSEAQ~~ EHKVTETE ~QMVSKLGTQNHTSEILS
~PPGQKVGSSFGLTWD 389 gi~ 17865632~ 1 _____________________'_______________________________________ giI 10436722~ 1 ____________________________________________________________ gi1 3882269~ 356FG PTDSARLL SPKVPD KDTPSSQEGMENYQV
~ TCPSGIPKP'T;FTH
GSPL
P
g1~ 17475630~ 94 FGGSPL~P~ PTDSARLL ~SPKVPD~TCPSGIPKP~FTH
~KDTPSSQEGMENYQV 153 SS
NOVS 404ANDMVITDITjNjC 463 I LEPT
i TFSSPIEATEKCKKVEKGN
KNIDKE
L
T
~
A
~
EP~~
~
~
~
~
~
g NDMVITD T S DK 449 I !ICITFSSPIEATEKCKKVEKGNR L
VLEPT
KNI
gi~ 17865632~ 1 ____________________________________________________._______ gi~ 10436722~ 1 __________________________________________________________ gi 3882269 416EKTEER4LI~!:ET ~PI'IP KHVRPIITYRRNPQALGQVDASLVP
~ I ~ ~ PY
B P~''i'CT
~PH
~
~
~
~
gi 17475630 154EKTEERET P~'IPIKHVRP~PH 213 ~ ~ I<ITY~C!RRNPQALGQVDASLVPV
PYA
P~CTl ~
.~....~...
.~..
.~.
_ giI 6331407~ 450C'~P~LKSTIK'hNTPIGCKVRKTIISYPRPNFKNV
PICD Si P 509 SRAVL~
gi~ 178656321 1 _____ 1 _________________~_____________ _____ __'_ _ _ 70gi~ 104367221 1 ____________________________________________________________ gi~174756301 214 EK~GDLKP~ANLYEKFKPDLQKPRVFSSGLMVSGI~PPGHPFS~ S~KF~Q~DH~ 273 .
NOV8 524 Qi~TSAS SSVI~SRQQTVLS AD~CKAEIL'INTCTHKQQFN
gi 510 Q~TSAS ~SSVSRQQTVLS T ~ 569 ~ VHV'~TH,,H., 6331407 'TPR3S~'S'iD
~ ~TPRD~TAD~KAEIL2NT~THKQFNF~T~VHV~~TH
gi~1786563211 ___________'.'________,_________._____________________________ gi ~10436722~1 _ 1 _____ _____________________ _____ __ _________ __ _ IQ gi ~3882269i536 GEEFC PYAYEVPPTF
E --S
LKP~LGLGA~tRLPSA'ECSRT
~
RSAS
MS
~
YB
I
~
~
~
gi~17475630~274 G QLGLGA 329 EFC RLPSATtSRT
A
~
-RSAS
PYA
YEVPPTF
--S
LKP
, I NOV8 584 KIt~ASHRrV~~PRT
S gi~6331407~ 570 . 628 V
QE
~
ShCETGS
.
FQK-IKG~I'LP
E'AEC
EM
ICASHRPRT~AVQEy~
~S~CETGSV~A
FQK-IKGLPV'~EAECEM
gi~1786563211 _=__________________________________________________________ gi~10436722~1 _.___________________________________-______________________ ~ ~ ~
~
~
gi 17475630330 I LYSPS 389 ~ I PPGPTTA y KSNLPI
G
RPPGYSR
P
FGFRS
~S gi~63314071 629 Y~P~T~DRISPEKKG~ITSME~QELKQ
BIMNETFEYGS~ FLGS T~TTSGRNK 688 gi~1786563211 _______________.________.___________________________________ gi~10436722~1 ____________________________________________________________ gi 3882269 652 S~S~Cl'SSTQSGDSAP~ Q~RPATSTFG~EQ--====P~
KASLP~D~PKGAGR'V.A!P 705 I ~ ' ~
' ' gi 17475630390 ,~S Q RPAT~ t~. 443 ~ I SF , KASLP
~SSTQSGDSA~P ,STFG tD
EQ-- PKGAGR
P P
NOV8 703 ~ D~CGLRQI~P~K~GP~CLRRNSDNRNPSADRAV~
~QRI~~SSG ~ 74 6331407 689 D ~
i GLR L
A
I ' ' LRR
DN
S
A
V
#~
g QI KT
( ~ ~
I GP QRI
NS
RNP
ADR
C
P~
C
V
35 gi~17865632~ 1 -------MLLSPFS--------------------LTTHI~LTAKGLLRLR----giI10436722~ 1 ____________._________________________.______________.______ gi 706 S ------------ S 748 ~ -SVTiIPR~;.SLT.iP ~ TSiI ~
3882269 ~ ~ GTR
~ ~ T~PKDD
KPAV
~
~
~~
gi 444 S--SVT 'I-------------- S 486 ~ PR~SLPA TS
17475630 QKD~QD
~ KPAV
GT
Sl0 NOV8 763 SWVNLP ~PI~P~KAS ~
~R~t~,TGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 822 gi~63314071 749 SWVNLP~ P~.iP~KAS ' RtRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 808 gi~178656321 26 ____Lp____________SGFg,~'-_______ST___________________________ gi~10436722~ 1 ______________________=~_____________________________________ gi~3882269~ 748 ___ H GYP_-___________________________________ p ~
TT
gi~174756301 486 ____p~_TTI~ I-I~GYP_-___________________________________ 504 'QKN P'LCTQ'Q TA882 giI6331407~ 809 FQNGSSGSFYLKPLVSRAHVHLMKTPPKGPSRKNLFTALNAVEKS
'QKN P' TA868 LCIQ'~
gi~178656321 35 ____________________________________wFHTVEKS
'QKN P' TA59 LCIQ' giI10436722~ 1 _______________________________________-_______________-____ 1 gi~3882269~ 766 __,________________________________________ T ~PD781 Q -GFP
I
gi~17475630~ 504 ___________________ ~TF~~_GFP~I ~PD519 ______________________Q
gi~6331407~ 869 928 gi~17865632~ 60 gi~104367221 1 _______ 1 ____ _-__ _ _______________________ _____ gi~38822691 782 Q~1RE Q R E QyRQ~GVAGE KRAICT ~FF S
B ~ L ~ ~ ~ ~~ ~~
~ ~ ~
~
gi 520 Q Q V R E Q KRAICGA~AT FF S 578 ( RE L T'RQ7~ {R~
17475630 GVA~GE LV I~
I
70 gi~63314071 929 HST Q~ .' 3.L~VTATT~. E ~~ .'TV . F~~____~H~, KT. .'~ 984 gi I 17865632120 H~yT~Q~~GQI;VTATT~ETV~AFV~----~W
I KT~R~ICE 175 gi~10436722~1 _______ _-__ ___ _______ ________________________ __-__ 1 gi ~ 17475630579 E~E~I~~'~D~VAFHAIQ~EE~~RR~~DE~ L E' QL
~ I~RLGGV~Q~ LQ 638 ~
NOV8 999 F~';T E T~ II~~AEK v ~ ... , 1058 ' v v giI63314071985 F'~T YE 1~TIEAEKY~ ~ ~ '' 1044 ~
gi I 17865632176 F~'~T YE~i~T.I~~I~AEKY~ ~ 235 ~ ~ ~
gi~10436722~1 __________=_'~_______v ~ .. . . 39 v ~
gi ~ 3882269901 Q E QE G t~LLSI~;C~H~~ QDD~DTiK
~ ~ S .7AL TVA'~T~ 960 ~ ~ ,f"D T
gi ~ 17475630639 QLLSIC~ ~ r , QDD~iI7HK
f Qy~ , 698 "',E~Q~E~G vV,Ep T~S AL r a . ~VAT~
gi~6331407~1045 1202 gi~17865632~236 293 gi~10436722~40 97 gi~3882269,961 1020 gi~17475630~699 758 gi~6331407~1103 1162 gi1178656321294 353 giI10436722~98 157 gi~3882269~1021 1080 gi~174756301759 818 gi~633140711163 1222 gi~17865632~354 413 gi~10436722~158 217 gi13882269~1081 1140 gi~17475630~819 878 gi~ 633140711223 1245 gi~ 17865632~414 43b gi~ 104367221218 240 gi~ 3882269~1141 1163 gi~ 17475630~879 901 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the 55 Interpro website (http:www.ebi.ac.uk/interpro~. Table 8F lists the domain description from DOMAIN analysis results against NOVB.
Table 8F Domain Anal sis of NOV8 Model Region of Score (bits) E value Homology RNA polymerase1008-1094 -12.8 5.7 omega subunit Intermediate 967-1193 48.9 2.0e-06 filament Consistent with other known members of the Nuclear Protein-like family of proteins, NOV8 has, for example, an RNA polymerase omega subunit signature sequence and homology to other members of the Nuclear Protein-like Protein Family. NOV8 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV8 nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOVB
nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVB activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Hyperparathyroidism, Hypoparathyroidism as well as other diseases, disorders and conditions.
In addition, various NOV8 nucleic acids and polypeptides according to the invention are useful, ihtef° alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV8 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Nuclear Protein-like Protein Family.
The NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac or endocrine physiology. As such, the NOVB nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat infection, cardiovascular system, immune system, and nervous system disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Hyperparathyroidism, Hypoparathyroidism as well as other diseases, disorders and conditions.
The NOV8 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV8 nucleic acid is expressed in Heart, Aorta, Coronary Artery, Vein, Umbilical Vein, Adrenal GlandlSuprarenal gland, Pancreas, Islets of Langerhans, Parathyroid Gland, Thyroid, Pineal Gland, Tongue, Salivary Glands, Stomach, Liver, Small Intestine, Colon, Ascending Colon, Lymphoid tissue, Spleen, Brain, Thalamus, Hypothalamus, Temporal Lobe, Amygdala, Cerebral Medulla/Cerebral white matter, Basal GangliaJCerebral nuclei, Substantia Nigra, Hippocampus, Spinal Chord, Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Prostate, Testis, Lung, Nasoepithelium, Larynx, Urinary Bladder, Kidney, Kidney Cortex, Retina, Skin, Foreskin, Epidermis, Dermis.
Additional utilities for NOV8 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV9 polypeptide has been identified as a Hemicentin precursor-like protein (also referred to as CG94702-OI). The disclosed novel NOV9 nucleic acid (SEQ ID
N0:17) of 11796 nucleotides is shown in Table 9A. The novel NOV9 nucleic acid sequences maps to the chromosome 9.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TAA
codon at nucleotides 11794-11796. A putative untranslated region and/or downstream from the termination codon is underlined in Table 9A, and the start and stop codons are in bold letters.
Table 9A. NOV9 Nucleotide Sequence (SEQ ID N0:17) ATGTCTGCCTTATTTGCAGCTGTGTACCAGATGCTAAA.ACCACGCCTGGTCCATAACAGCCCACATC
CGGTGACCTATCAAATTGAGGCAAGTTTAAAGCCAGAGCAGCCTGGTGTCACGCTGGTGTCCATCCC
AGTCTTCCTGGCACCTTCCTGGCACAAAGCCTCAGAGCTGATCCCGACCCAGTCCTTCCGAGCACAG
GGGGCAGGGAAGCAGCTCCTCGGCTCTCCTTGCCCCCAAGTGCCCCCCAGCATCCGGGAGGACGGGC
GCAAGGCCAACGTGTCGGGTATGGCCGGGCAGTCCCTGACGCTGGAGTGTGACGCGAACGGCTTTCC
AGTCCCTGAGATCGTGTGGCTGAAGGACGCGCAGCTGATTCCTAAGGTGGGCGGCCACCGCCTCCTG
GACGAGGGCCAGTCCCTCCACTTCCCCAGGATCCAGGAGGGTGATTCTGGGCTCTACTCCTGCCGGG
CAGAGAACCAGGCTGGCACCGCCCAGAGGGACTTCCATCTCCTTGTGCTCACCCCTCCTTCCGTGCT
TGGAGCCGGGGCCGCTCAGGAGGTGCTAGGATTGGCCGGTGCAGACGTGGAGCTGCAGTGTTGGACC
TCAGGGGTCCCCACGCCCCAGGTGGAGTGGACCAAGGACAGGCAGCCTGTCCTTCCGGGAGGCCCTC
ACCTGCAGGTCCAGGAGGATGGCCAGGTTCTCAGGATCACCGGCAGTCACGTGGGGGATGAGGGACG
ATACCAGTGCGTGGCCTTCAGCCCAGCTGGTCAGCAGGCCAGGGACTTCCAGCTCCGAGTTCATGCG
CCCCCCACTATCTGGGGCTCCAACGAGACAGGCGAGGTGGCCGTCATGGAGGACCACCTAGTGCAGC
TCCTGTGTGAGGCTCGAGGAGTGCCCACCCCAAACATCACCTGGTTCAAGGACGGGGCCCTGCTCCC
CACCAGCACCAAGGTGGTCTACACTAGGGGCGGTCGGCAGTTGCAGCTGGGGAGGGCCCAGAGCTCC
GATGCCGGCGTCTACACCTGCAAGGCCAGCAATGCTGTGGGGGCCGCAGAGAAGGCCACCAGGCTGG
ATGTTTATGTCCCACCTACCATCGAGGGCGCCGGTGGAAGACCATACGTGGTGAAGGCTGTGGCTGG
GAGGCCTGTGGCGCTGGAGTGCGTGGCCAGAGGCCACCCGTCCCCCACCCTCTCCTGGCACCACGAG
GGGCTGCCCGTGGCAGAGAGCAACGAGTCGCGGCTGGAGACAGACGGGAGTGTGCTGAGGCTGGAGA
GCCCGGGGGAGGCATCCAGTGGCCTGTACAGCTGTGTGGCCAGCAGTCCTGCCGGGGAAGCCGTCCT
GCAGTACTCCGTGGAGGTTCAGGTGCCCCCACAGCTCCTGGTGGCTGAAGGCTTGGGACAGGTGACC
ACCATCGTGGGACAGCCCCTGGAACTTCCCTGCCAGGCCTCAGGCTCCCCAGTACCCACTATCCAGT
GGCTGCAGAATGGCCGCCCAGCCGAGGAGCTGGCTGGGGTGCAGGTGGCCTCGCAGGGGACCACACT
GCACATTGACCATGTGGAGCTGGACCACTCAGGCCTCTTCGCCTGCCAGGCCACCAATGAGGCGGGC
ACTGCCGGGGCCGAGGTGGAGGTGTCTGTGCATGAGTTCCCATCGGTCAGTATCATTGGGGGTGAGA
ACATCACAGCTCCTTTCCTGCAGCCTGTGACCCTCCAGTGCATAGGGGATGGGGTGCCCACCCCAAG
CCTCCGTTGGTGGAAGGATGGTGTAGCCCTGGCAGCCTTTGGGGGGAACCTACAGATTGAGAAGGTG
GACCTGAGGGACGAGGGCATCTACACTTGTGCTGCTACCAACCTGGCTGGGGAGAGCAAGAGGGAAG
TGGCGCTGAAAGTTTTGGTGCCCCCCAACATCGAGCCAGGCCCAGTCAACAAGGCAGTGCTGGAAAA
TGCCTCAGTGACCTTGGAGTGTCTGGCTTCGGGCGTGCCCCCTCCTGATGTCTCCTGGTTCAAGGGC
CACCAACCTGTCTCTTCATGGATGGGAGTGACAGTATCAGTGGATGGGAGAGTTCTCCGCATTGAGC
AAGCCCAGCTTTCTGATGCTGGGAGCTACCGCTGTGTGGCATCCAATGTGGCAGGTAGCACAGAGCT
GCGGTATGGCCTACGGGTCAATGTGCCCCCTCGAATCACACTGCCACCCAGCCTGCCAGGCCCTGTG
TTGGTCAACACCCCTGTCCGGCTGACCTGCAATGCCACCGGTGCCCCCAGCCCCACACTGATGTGGC
TGAAGGATGGAAACCCTGTGTCCCCTGCAGGGACCCCTGGCCTGCAGGTCTTCCCTGGGGGCCGGGT
CCTCACCTTGGCTAGTGCCCGGGCCTCCGACTCTGGGAGGTACTCCTGCGTGGCTGTGAGCGCGGTG
GGCGAGGACCGCCAGGATGTTGTCCTGCAAGTCCACATGCCCCCGAGTATCCTTGGAGAAGAGCTGA
ATGTGTCCGTTGTGGCCAATGAGTCAGTGGCCCTGGAGTGCCAGAGCCACGCCATGCCCCCTCCTGT
GCTGAGCTGGTGGAAGGACGGGCGGCCCCTGGAACCACGGCCTGGAGTCCACCTCTCCGCAGACAAA
GCCTTGCTGCAGGTGGACAGAGCCGATGTGTGGGATGCGGGCCATTACACCTGTGAGGCACTGAACC
AGGCCGGCCACTCAGAGAAACACTACAATCTGAACGTCTGGGGTCAACCCCTCCCCGGGGAGGGGGC
TGGCCTCCAGCACGTGTCGGCTGTGGGGAGGCTGTTGTACCTGGGACAGGCCCAGCTGGCTCAGGAA
GGAACATACACCTGTGAATGCAGCAACGTGGTGGGGAACAGCAGCCAGGACCTGCAGCTGGAGGTGC
ACGTTCCCCCTCAGATTGCCGGTCCCCGGGAGCCTCCCACACAAGTCTCTGTGGTCCAGGATGGAGT
GGCCACTCTGGAGTGCAACGCCACAGGGAAACCCCCTCCGACAGTGACATGGGAGCGGGACGGCCAG
CCCGTGGGGGCTGAACTGGGCCTGCAGCTGCAGAACCAGGGTCAGAGCCTGCATGTGGAGCGGGCCC
AGGCTGCCCACACTGGACGCTACAGCTGTGTGGCCGAGAACCTGGCTGGGAGGGCAGAGAGGAAGTT
TGAGCTCTCCGTACTGGTGCCCCCAGAGCTCATTGGAGACTTGGACCCGCTGACCAACATCACTGCT
GCCTTGCACAGCCCCTTAACTCTGCTCTGTGAAGCCATGGGGATCCCACCTCCAGCCATCCGCTGGT
TCCGAGGGGAGGAGCCTGTCAGCCCCGGGGAGGACACCTACCTGCTGGCAGGTGGCTGGATGCTGAA
GATGACTCAGACACAGGAGCAAGACAGTGGCCTCTACTCATGCCTGGCAAGCAACGAGGCTGGGGAG
GCACGGAGGAACTTCAGTGTGGAGGTGCTGGTTCCTCCCAGTATTGAGAACGAGGACTTGGAGGAGG
TGATCAAGGTCCTTGATGGACAGACTGCCCATCTTATGTGCAACGTCACAGGCCACCCACAGCCCAA
GCTCACATGGTTCAAAGATGGCCGGCCTCTGGCTAGGGGAGATGCTCACCACATCTCCCCAGACGGA
GTCCTCCTGCAGGTCCTCCAGGCAAACCTGTCCAGTGCTGGCCACTACTCCTGCATTGCAGCCAACG
CTGTTGGGGAGAAGACCAAACACTTCCAGCTCAGTGTCCTGTTGGCTCCCACCATCCTGGGAGGGGC
CGAGGACAGTGCAGATGAGGAGGTGACCGTGACTGTCAACAACCCCATCTCTCTGATCTGCGAGGCC
CTGGCCTTCCCTTCCCCCAACATCACCTGGATGAAGGACGGGGCCCCGTTTGAGGCCTCCAGGAACA
TCCAGCTGCTCCCAGGTACCCACGGGCTGCAGATCCTGAATGCCCAGAAGGAAGATGCTGGCCAGTA
CACCTGCGTGGTCACCAATGAGCTCGGGGAGGCCGTGAAAAACTACCATGTGGAAGTGCTCATCCCC
CCTTCCATCTCCAAAGACGACCCCTTGGCGGAGGTCGGCGTGAAGGAGGTGAAGACCAAGGTCAACA
GCACCTTGACCTTGGAGTGTGAGAGCTGGGCTGTGCCCCCGCCCACCATCCGCTGGTACAAGGATGG
ACAGCCCGTGACCCCCAGCTCGCGGCTGCAGGTCCTGGGTGAAGGGCGACTGCTCCAGATCCAGCCC
ACACAGGTCTCAGACTCGGGGCGGTACCTGTGTGTGGCCACCAATGTGGCTGGCGAGGACGACCAGG
ACTTCAACGTGCTCATCCAGGTGCCCCCCATGTTCCAGAAGGTGGGTGATTTCAGTGCAGCCTTCGA
GATCCTGTCCCGGGAGGAGGAGGCCCGGGGCGGAGTCACGGAATACAGGGAGATCGTGGAGAACAAC
CCAGCCTACCTGTACTGCGACACCAACGCGATCCCACCCCCGGACCTCACCTGGTACAGAGAGGATC
AGCCCCTCTCGGCCGGGGATGAGGTGTCTGTGCTGCAAGGAGGCCGGGTCCTGCAGATCCCCCTGGT
GCGGGCAGAGAACGCCGGGAGGTACTCGTGCAAGGCCTCCAACGAGGTGGGCGAGGACTGGCTGCAC
TACGAGCTGCTGGTGCTGACCCCACCTGTGATCCTGGGTGACACAGAGGAGCTGGTGGAAGAGGTGA
CAGTCAATGCCAGCAGCACCGTCAGCCTGCAGTGCCCGGCCCTGGGAAACCCCGTGCCCACCATCTC
ATGGCTCCAGAATGGGCTGCCTTTCTCCCCGAGCCCACGGCTGCAGGTCCTGGAGGACGGGCAAGTC
TTGCAGGTTTCCACGGCAGAGGTGGCCGACGCCGCCAGCTACATGTGTGTGGCCGAGAACCAGGCGG
GCTCCGCTGAGAAGCTCTTCACCCTCAGGGTTCAAGGCCTGGACTTGGAGCAGGTCACTGCCATCCT
CAACAGCAGCGTCTCCCTCCCTTGCGACGTCCACGCTCACCCAAACCCCGAGGTCACGTGGTACAAG
GACAGCCAGGCCCTCTCCCTGGGTGAAGAGGTCTTCCTCCTGCCTGGCACCCACACGCTGCAGCTGG
GGAGAGCACGGCTGTCGGACTCCGGGATGTACACATGCGAAGCCCTCAATGCTGCCGGCCGAGACCA
GAAGCTGGTGCAGCTCAGTGTTCTGGTTCCCCCGGCCTTCAGGCAGGCTCCCAGAGGTCCCCAGGAT
GCGGTCCTGGTGAGGGTCGGGGACAAAGCTGTCCTGAGCTGCGAGACAGATGCGCTCCCTGAGCCAA
CTGTGACCTGGTACAAGGATGGGCAGCCCCTGGTCCTGGCACAGCGGACCCAGGCTCTGCGGGGTGG
GCAGAGGCTGGAGATCCAGGAAGCCCAGGTATCGGATAAAGGTTTATACAGCTGTAAAGTCAGCAAC
GTGGCTGGGGAGGCCGTGCGGACCTTCACCCTCACCGTCCAGGTGCCCCCAACATTTGAGAACCCCA
AGACAGAGACAGTGAGCCAGGTGGCTGGGAGCCCCCTGGTCCTGACCTGTGATGTGTCCGGGGTCCC
TGCACCCACGGTCACTTGGCTGAAGGACAGGATGCCTGTGGAGAGCAGCGCGGTGCACGGTGTGGTC
TCCCGGGGGGGCCGCCTCCAGCTGAGCCGCCTGCAACCGGCCCAGGCGGGCACCTACACGTGCGTGG
CTGAGAACACCCAGGCTGAGGCCCGCAAGGACTTCGTGGTAGCAGTGCTGGTGGCCCCCCGGATCCG
GAGCTCGGGCGTGGCGCGGGAGCACCATGTCTTGGAAGGGCAGGAGGTGCGGCTGGACTGTGAGGCC
GATGGGCAGCCGCCGCCGGACGTGGCCTGGCTGAAGGACGGCAGCCCGCTGGGCCAGGACATGGGCC
CCCACCTCCGGTTCTACCTGGACGGCGGCTCCCTGGTGCTAAAAGGCCTGAGGGCCTCGGACGCGGG
TGCCTACACCTGCGTGGCCCACAACCCAGCCGGGGAGGACGCCAGGCTGCACACGGTGAATGTGCTG
GTTCCTCCCACCATCAAGCAGGGAGCAGACGGCTCGGGGACCCTGGTGAGCAGGCCTGGGGAGCTGG
TGACCATGGTGTGCCCTGTGCGGGGCTCCCCGCCCATCCACGTGAGCTGGCTCAAGGACGGCCTGCC
CCTCCCGCTCTCCCAGCGCACCCTCCTCCACGGCTCTGGCCACACCCTCAGGATTTCCAAGGTGCAA
TTGGCAGACGCTGGCATCTTCACCTGTGTGGCCGCAAGCCCAGCTGGCGTGGCGGACAGGAACTTCA
CCTTGCAGGTGCAGGTGCCCCCTGTCCTGGAGCCGGTGGAGTTCCAGAATGACGTGGTGGTGGTTCG
TGGCTCCCTGGTGGAACTCCCGTGCGAGGCCCGGGGCGTTCCCCTGCCTCTCGTGTCGTGGATGAAG
GATGGGGAACCCTTGTTGTCCCAGAGCCTCGAGCAGGGGCCCAGCCTGCAGCTGGAGGCAGTGGGAG
CTGGTGACTCGGGGACCTACTCCTGTGTGGCCGTGAGCGAGGCGGGGGAAGCCAGGAGGCATTTCCA
GCTGACCGTCATGGAGCCCCCTCACATTGAGGACTCAGGCCAGCCTACAGAGCTGTCGCTGACCCCC
GGCGCCCCCATGGAGCTCCTCTGTGATGCCCAGGGCACCCCCCAGCCCAACATCACCTGGCATAAGG
ACGGGCAGGCCCTGACCAGGCTGGAGAACAACAGCAGAGCCACACGGGTGCTCCGGGTGGAGAATGT
GCAGGTTAGGGATGCTGGGCTGTACACTTGTCTGGCTGAAAGCCCTGCAGGTGCAATTGAGAAGAGC
TTCCGGGTCAGGGTTCAAGCCCCTCCAAACATTGTTGGGCCCCGAGGCCCCCGCTTTGTGGTCGGCC
TGGCCCCAGGGCAGCTGGTCCTGGAGTGTTCGGTGGAGGCAGAGCCAGCGCCCAAGATCACGTGGCA
CCGAGACGGCATTGTGCTGCAGGAGGACGCCCACACACAATTCCCGGAGCGGGGCAGGTTCCTCCAG
CTGCAGGCCCTGAGCACGGCTGACAGCGGCGACTACAGCTGCACAGCCCGCAACGCCGCAGGCAGCA
CTAGTGTCGCCTTCCGCGTGGAGATCCACACGGTGCCCACCATCCGGTCAGGACCACCTGCAGTGAA
CGTCTCAGTGAACCAGACAGCCCTGCTGCCTTGCCAGGCCGACGGCGTGCCCGCACCCCTCGTGAGC
TGGCGGAAGGACAGGGTCCCCCTGGATCCCAGGAGCCCCAGGGCAACCCCCATCCATTCTAGGTTTG
AAATTCTGCCTGAGGGTTCCCTGAGAATCCAGCCAGTCCTTGCCCAGGACGCCGGCCACTACCTCTG
CCTGGCATCCAACTCTGCTGGCTCCGATCGTCAAGGCCGTGACCTACGGGTCTTGGAGCCTCCAGCC
ATCGCCCCCAGCCCCTCCAACCTGACCCTGACCGCCCACACCCCAGCCTTGCTGCCCTGCGAGGCCA
GCGGCTCCCCTAAGCCCCTGGTGGTCTGGTGGAAGGACGGACAGAAGCTGGACTTCCGCCTGCAGCA
GGGCGCCTACCGGCTCCTGCCCTCCAACGCCCTGCTCCTCACGGCCCCCGGCCCCCAGGACTCAGCC
CAGTTTGAATGCGTGGTGAGCAATGAGGTGGGCGAGGCCCACAGGCTCTACCAGGTGACCGTCCATG
TGCCTCCCACCATTGCCGATGACCAGACAGACTTCACCGTGACCATGATGGCACCTGTGGTCCTCAC
ATGTCACAGCACGGGTATACCAGCTCCGACCGTGTCCTGGAGCAAGGCAGGCGCCCAGCTAGGAGCT
CGGGGGAGTGGCTATCGTGTCTCACCATCGGGCGCCCTGGAGATCGGGCAGGCCCTCCCCATCCACG
CAGGCCGCTACACCTGCTCAGCCCGCAACTCTGCCGGCGTAGCCCACAAGCACGTCTTCCTCACTGT
GCAAGCCTCCCCGGTGGTGAAGCCGCTGCCCAGCGTGGTTCGGGCAGTGGCAGAGGAGGAGGTGCTG
CTGCCCTGCGAGGCCTCAGGCATCCCCCGGCCGACCATCACCTGGCAGAAGGAAGGGCTCAACGTCG
CTACTGGAGTGAGTACCCAGGTCCTACCAGGCGGACAGCTGCGGATTGCCCATGCCAGCCCAGAGGA
TGCTGGAAACTATCTCTGCATCGCTAAGAACAGTGCGGGCAGTGCCATGGGGAAGACGCGGCTGGTG
GTGCAAGTCCCACCAGTGATCGAGAATGGCCTCCCAGACCTGTCCACCACCGAAGGCTCCCACGCCT
TCTTGCCTTGCAAGGCGAGGGGCAGTCCTGAGCCCAACATCACCTGGGACAAAGATGGCCAGCCTGT
GTCGGGCGCCGAGGGGAAGTTCACCATCCAGCCTTCTGGGGAGTTGCTGGTGAAGAACTTGGAGGGC
CAGGACGCAGGCACCTATACCTGTACCGCTGAGAACGCCGTGGGCCGGGCCCGCCGCCGCGTGCACC
TCACCATCCTGGTACTGCCTGTGTTCACCACCCTGCCTGGGGACCGCAGCCTGCGCCTTGGGGACAG
GCTGTGGCTTCGCTGTGCAGCCCGGGGCAGCCCCACCCCTCGCATTGGCTGGACTGTCAACGACCGG
CCAGTCACAGAAGGGGTGTCTGAGCAGGATGGAGGCAGCACGCTGCAGCGGGCCGCTGTCTCCAGAG
AAGACAGCGGGACCTATGTCTGCTGGGCGGAGAACAGAGTGGGCCGCACGCAGGCGGTCAGCTTCGT
CCACGTGAAGGAGGCTCCTGTCCTACAAGGGGAGGCTTTCTCCTACCTGGTGGAACCTGTAGGAGGC
AGCATTCAGCTAGACTGTGTGGTGCGTGGAGACCCAGTGCCGGACATCCACTGGATCAAAGATGGCC
TTCCACTGCGGGGCAGCCACCTCCGGCACCAGCTGCAGAATGGCTCGCTGACCATCCGCAGGACTGA
GGCAAGGCGGGGCCTGGCACCTTGGAGGGACGATGCGGGACGGTACCAGTGCCTGGCAGAGAATGAG
ATGGGCGTGGCGAAGAAAGTGGTGATCCTCGTCCTGCAGACCAGGATGGTGCCAGCAGAGCCCCACT
TGAAGCGCCAACTCCCACCGATCCCCAGCAATAATGAGGCACCCTCCCTGTTCCCGGGTGTCCATGG
AGGCCACGTGGGGAACCCGGACTTCCACTCTCATCTAGCAGAAGTTCTCGCCGTTCAGTTGCTGGCT
GGGTCCCTGCTCTTCTCAGCCAGGGCCATGCCGCAGGCCAGCACAGCAGCCATTTCCCTTTTGGCTC
CTACCAGTTTTGCCCCTTTTCCTGATGATATTTCTCAGGGCATACTTTCATCCTCTACTGCACATCA
AGGCAGCCCCCAGGGGTGGCAAAAGCTGCTGTTTTTCACAGCCATCCCTAATAAAACCACTGTGATG
GTCACGGTGGAGCCCCAGGACATGACAGTGAGATCTGGGGATGACGTGGCCCTGCGGTGCCAGGCCA
CTGGAGAGCCCACACCCACCATTGAATGGCTACAGGCGGGTCAACCCTTGCGGGCCAGCCGGCGGCT
CCGGACCCTGCCCGATGGGAGCCTGTGGCTGGAGAACGTGGAGACTGGGGATGCAGGCACCTACGAC
TGCGTCGCTCACAACCTCCTGGGCTCTGCCACAGCCCGGGCGTTCCTGGTCTGTGCCAGCCACGCCA
TCGTGGGCTCCCGGCATTTCAGAGACCCACAGGTCTTCTGTGAGTTTGTGGTCCCGCCTCCTCATTT
TACAGGGGAGCCCCAGGGGAGCTGGGGCAGCATGACTGGGGTGATAAATGGCCGGAAATTTGGCGTG
GCCACACTCAACACCAGCGTGATGCAGGAGGCACACTCCGGGGTCAGCAGCATCCACAGCAGCATCC
GCCATGTCCCAGCAAACGTGGGGCCTCTGATGCGGGTGCTCGTGGTCACCATCGCCCCCATCTACTG
GGCCCTGGCCAGAGAGAGTGGGGAAGCCCTGAATGGCCACTCTCTGACTGGGGGCAGGTTCCGGCAG
GAGTCACACGTGGAGTTTGCTACAGGGGAGCTGCTCACGATGACCCAGGTGGCCCGGGGTCTGGATC
CCGATGGCCTCCTGCTCCTCGACGTGGTGGTCAATGGCGTTGTCCCCGAGAGCCTGGCTGACGCAGA
TCTTCAAGTGCAGGACTTTGAGGAGCACTACGTGCAAACAGGGCCTGGCCAGCTGTTCGTGGGCTCC
ACACAGCGCTTCTTCCAGGGCGGCCTCCCCTCGTTCCTACGCTGCAACCACAGCATCCAGTACAACG
CGGCCCGGGGCCCCCAGCCCCAGCTGGTGCAGCACCTGCGGGCCTCAGCTATCAGCTCGGCCTTTGA
TCCAGAGGCCGAGGCCCTGCGCTTCCAGCTCGCTACAGCCCTGCAGGCGGAGGAGAACGAGGTCGGC
TGCCCCGAGGGCTTTGAGCTGGACTCCCAGGGAGCGTTTTGTGTGGACAGGGACGAGTGCTCAGGAG
GCCCTAGCCCCTGCTCCCATGCCTGCCTTAATGCACCCGGCCGCTTCTCCTGCACCTGCCCCACTGG
CTTCGCCCTGGCCTGGGATGACAGGAACTGCAGAGATGTGGACGAGTGTGCGTGGGATGCTCACCTC
TGCCGAGAGGGACAGCGCTGTGTGAACCTGCTCGGGTCCTACCGCTGCCTCCCCGACTGTGGGCCTG
GCTTCCGGGTGGCTGATGGGGCCGGCTGTGAAGATGTGGACGAATGCCTGGAGGGGTTGGACGACTG
TCACTACAACCAGCTCTGCGAGAACACCCCAGGCGGTCACCGCTGCAGCTGCCCCAGGGGTTACCGG
ATGCAGGGCCCCAGCCTGCCCTGCCTAGATGTCAATGAGTGCCTGCAGCTGCCCAAGGCCTGCGCCT
ACCAGTGCCACAACCTCCAGGGCAGCTACCGCTGCCTGTGCCCCCCAGGCCAGACCCTCCTTCGCGA
CGGCAAGGCCTGCACCTCACTGGAGCGGAATGGACAAAATGTGACCACCGTCAGCCACCGAGGCCCT
CTATTGCCCTGGCTGCGGCCCTGGGCCTCGATCCCCGGTACCTCCTACCACGCCTGGGTCTCTCTCC
GTCCGGGTCCCATGGCCCTGAGCAGTGTGGGCCGGGCCTGGTGCCCTCCTGGTTTCATCAGGCAGAA
CGGAGTCTGCACAGACCTTGACGAGTGCCGCGTGAGGAACCTGTGTCAGCACGCCTGCCGCAACACT
GAGGGCAGCTACCAGTGCCTGTGCCCCGCCGGCTACCGTCTGCTCCCCAGCGGGAAGAACTGCCAGG
ACATCAACGAGTGCGAGGAGGAGAGCATCGAGTGTGGACCCGGCCAGATGTGCTTCAACACCCGTGG
CAGCTACCAGTGTGTGGACACACCCTGTCCTGCCACCTACCGGCAGGGCCCCAGCCCTGGGACGTGC
TTCCGGCGCTGCTCGCAGGACTGCGGCACGGGCGGCCCCTCTACGCTGCAGTACCGGCTGCTGCCGC
TGCCCCTGGGCGTGCGCGCCCACCACGACGTGGCCCGCCTCACCGCCTTCTCCGAGGTCGGCGTCCC
CGCCAACCGCACCGAGCTCAGCATGCTGGAGCCCGACCCCCGCAGCCCCTTCGCGCTGCGTCCGCTG
CGCGCGGGCCTTGGCGCGGTCTACACCCGTCGCGCGCTCACCCGCGCCGGCCTCTACCGGCTCACCG
TGCGTGCTGCGGCACCGCGCCACCAAAGCGTCTTCGTCTTGCTCATCGCCGTGTCCCCCTACCCCTA
CTAA
Variant sequences of NOV9 are included in Example 3, Table 21. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV9 protein (SEQ m N0:18) encoded by SEQ 1D N0:17 is 3931 amino acid residues in length and is presented using the one-letter amino acid code in Table 9B. Psort analysis predicts the NOV9 protein of the invention to be localized at the plasma membrane IO with a certainty of 0.7300.
Table 9B. Encoded NOV9 protein sequence (SEQ ID N0:18) MSALFAAVYQMLKPRLVHNSPHPVTYQIEASLKPEQPGVTLVSIPVFLAPSWHKASELIPTQSFR
AQGAGKQLLGSPCPQVPPSIREDGRKANVSGMAGQSLTLECDANGFPVPETVWLKDAQLIPKVGG
HRLLDEGQSLHFPRIQEGDSGLYSCRAENQAGTAQRDFHLLVLTPPSVLGAGAAQEVLGLAGADV
ELQCWTSGVPTPQVEWTKDRQPVLPGGPHLQVQEDGQVLRITGSHVGDEGRYQCVAFSPAGQQAR
DFQLRVHAPPTIWGSNETGEVAVMEDHLVQLLCEARGVPTPNITWFKDGALLPTSTKVVYTRGGR
QLQLGRAQSSDAGVYTCKASNAVGAAEKATRLDVYVPPTIEGAGGRPYWKAVAGRPVALECVAR
GHPSPTLSWHHEGLPVAESNESRLETDGSVLRLESPGEASSGLYSCVASSPAGEAVLQYSVEVQV
PPQLLVAEGLGQVTTIVGQPLELPCQASGSPVPTIQWLQNGRPAEELAGVQVASQGTTLHIDHVE
LDHSGLFACQATNEAGTAGAEVEVSVHEFPSVSIIGGENITAPFLQPVTLQCIGDGVPTPSLRWW
KDGVALAAFGGNLQIEKVDLRDEGIYTCAATNLAGESKREVALKVLVPPNIEPGPVNKAVLENAS
VTLECLASGVPPPDVSWFKGHQPVSSWMGVTVSVDGRVLRIEQAQLSDAGSYRCVASNVAGSTEL
RYGLRVNVPPRITLPPSLPGPVLVNTPVRLTCNATGAPSPTLMWLKDGNPVSPAGTPGLQVFPGG
RVLTLASARASDSGRYSCVAVSAVGEDRQDWLQVHMPPSILGEELNVSWANESVALECQSHAM
PPPVLSWWKDGRPLEPRPGVHLSADKALLQVDRADWDAGHYTCEALNQAGHSEKHYNLNVWGQP
LPGEGAGLQHVSAVGRLLYLGQAQLAQEGTYTCECSNWGNSSQDLQLEVHVPPQIAGPREPPTQ
VSWQDGVATLECNATGKPPPTVTWERDGQPVGAELGLQLQNQGQSLHVERAQAAHTGRYSCVAE
NLAGRAERKFELSVLVPPELIGDLDPLTNITAALHSPLTLLCEAMGIPPPAIRWFRGEEPVSPGE
DTYLLAGGWMLKMTQTQEQDSGLYSCLASNEAGEARRNFSVEVLVPPSIENEDLEEVIKVLDGQT
AHLMCNVTGHPQPKLTWFKDGRPLARGDAHHISPDGVLLQVLQANLSSAGHYSCIAANAVGEKTK
HFQLSVLLAPTILGGAEDSADEEVTVTVNNPISLICEALAFPSPNITWMKDGAPFEASRNIQLLP
GTHGLQILNAQKEDAGQYTCWTNELGEAVKNYHVEVLIPPSISKDDPLAEVGVKEVKTKVNSTL
TLECESWAVPPPTIRWYKDGQPVTPSSRLQVLGEGRLLQIQPTQVSDSGRYLCVATNVAGEDDQD
FNVLIQVPPMFQKVGDFSAAFEILSREEEARGGVTEYREIVENNPAYLYCDTNAIPPPDLTWYRE
DQPLSAGDEVSVLQGGRVLQIPLVRAENAGRYSCKASNEVGEDWLHYELLVLTPPVILGDTEELV
EEVTVNASSTVSLQCPALGNPVPTISWLQNGLPFSPSPRLQVLEDGQVLQVSTAEVADAASYMCV
AENQAGSAEKLFTLRVQGLDLEQVTAILNSSVSLPCDVHAHPNPEVTWYKDSQALSLGEEVFLLP
GTHTLQLGRARLSDSGMYTCEALNAAGRDQKLVQLSVLVPPAFRQAPRGPQDAVLVRVGDKAVLS
CETDALPEPTVTWYKDGQPLVLAQRTQALRGGQRLEIQEAQVSDKGLYSCKVSNVAGEAVRTFTL
TVQVPPTFENPKTETVSQVAGSPLVLTCDVSGVPAPTVTWLKDRMPVESSAVHGWSRGGRLQLS
RLQPAQAGTYTCVAENTQAEARKDFWAVLVAPRIRSSGVAREHHVLEGQEVRLDCEADGQPPPD
VAWLKDGSPLGQDMGPHLRFYLDGGSLVLKGLRASDAGAYTCVAHNPAGEDARLHTVNVLVPPTI
KQGADGSGTLVSRPGELVTMVCPVRGSPPIHVSWLKDGLPLPLSQRTLLHGSGHTLRISKVQLAD
AGIFTCVAASPAGVADRNFTLQVQVPPVLEPVEFQNDWWRGSLVELPCEARGVPLPLVSWMKD
GEPLLSQSLEQGPSLQLEAVGAGDSGTYSCVAVSEAGEARRHFQLTVMEPPHIEDSGQPTELSLT
PGAPMELLCDAQGTPQPNITWHKDGQALTRLENNSRATRVLRVENVQVRDAGLYTCLAESPAGAI
EKSFRVRVQAPPNIVGPRGPRFWGLAPGQLVLECSVEAEPAPKITWHRDGIVLQEDAHTQFPER
GRFLQLQALSTADSGDYSCTARNAAGSTSVAFRVEIHTVPTIRSGPPAVNVSVNQTALLPCQADG
VPAPLVSWRKDRVPLDPRSPRATPIHSRFEILPEGSLRIQPVLAQDAGHYLCLASNSAGSDRQGR
DLRVLEPPAIAPSPSNLTLTAHTPALLPCEASGSPKPLWWWKDGQKLDFRLQQGAYRLLPSNAL
LLTAPGPQDSAQFECWSNEVGEAHRLYQVTVHVPPTIADDQTDFTVTMMAPWLTCHSTGIPAP
TVSWSKAGAQLGARGSGYRVSPSGALEIGQALPIHAGRYTCSARNSAGVAHKHVFLTVQASPWK
PLPSWRAVAEEEVLLPCEASGIPRPTITWQKEGLNVATGVSTQVLPGGQLRIAHASPEDAGNYL
CIAKNSAGSAMGKTRLWQVPPVIENGLPDLSTTEGSHAFLPCKARGSPEPNITWDKDGQPVSGA
EGKFTIQPSGELLVKNLEGQDAGTYTCTAENAVGRARRRVHLTILVLPVFTTLPGDRSLRLGDRL
WLRCAARGSPTPRIGWTVNDRPVTEGVSEQDGGSTLQRAAVSREDSGTYVCWAENRVGRTQAVSF
VHVKEAPVLQGEAFSYLVEPVGGSIQLDCWRGDPVPDIHWIKDGLPLRGSHLRHQLQNGSLTIR
RTEARRGLAPWRDDAGRYQCLAENEMGVAKKWILVLQTRMVPAEPHLKRQLPPIPSNNEAPSLF
PGVHGGHVGNPDFHSHLAEVLAVQLLAGSLLFSARAMPQASTAAISLLAPTSFAPFPDDISQGIL
SSSTAHQGSPQGWQKLLFFTAIPNKTTVMVTVEPQDMTVRSGDDVALRCQATGEPTPTIEWLQAG
QPLRASRRLRTLPDGSLWLENVETGDAGTYDCVAHNLLGSATARAFLVCASHAIVGSRHFRDPQV
FCEFWPPPHFTGEPQGSWGSMTGVINGRKFGVATLNTSVMQEAHSGVSSIHSSIRHVPANVGPL
MRVLWTIAPIYWALARESGEALNGHSLTGGRFRQESHVEFATGELLTMTQVARGLDPDGLLLLD
WVNGWPESLADADLQVQDFEEHYVQTGPGQLFVGSTQRFFQGGLPSFLRCNHSIQYNAARGPQ
PQLVQHLRASAISSAFDPEAEALRFQLATALQAEENEVGCPEGFELDSQGAFCVDRDECSGGPSP
CSHACLNAPGRFSCTCPTGFALAWDDRNCRDVDECAWDAHLCREGQRCVNLLGSYRCLPDCGPGF
RVADGAGCEDVDECLEGLDDCHYNQLCENTPGGHRCSCPRGYRMQGPSLPCLDVNECLQLPKACA
YQCHNLQGSYRCLCPPGQTLLRDGKACTSLERNGQNVTTVSHRGPLLPWLRPWASIPGTSYHAWV
SLRPGPMALSSVGRAWCPPGFIRQNGVCTDLDECRVRNLCQHACRNTEGSYQCLCPAGYRLLPSG
KNCQDTNECEEESIECGPGQMCFNTRGSYQCVDTPCPATYRQGPSPGTCFRRCSQDCGTGGPSTL
QYRLLPLPLGVRAHHDVARLTAFSEVGVPANRTELSMLEPDPRSPFALRPLRAGLGAWTRRALT
RAGLYRLTVRAAAPRHQSVFVLLIAVSPYPY
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 9C.
Table 9C. Patp results for NOV9 Smallest Sum eadingigh Prob equences FrameScore P(N) producing High-scoring Segment Pairs:
>patp:AAY53667Sequence gi/3328186 +1 1529 6.5e-244 >patp:AAY87206Human secreted protein sequence +1 2235 6.4e-230 ID N0:245 >patp:AAE06183Human gene 57 encoded secreted +1 2235 6.4e-230 protein >patp:AAY87120Human secreted protein sequence +1 2235 6.4e-230 SEQ ID:159 >patp:AAE06097Human gene 57 secreted protein +1 2235 6.4e-230 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 625 of 1067 bases (58%) identical to a gb:GENBANK-ID:HSLTGFBP4~acc:Y13622.1 mRNA from Homo sapieszs (mRNA for latent transforming growth factor-beta binding protein-4). The full amino acid sequence of the protein of the invention was found to have 502 of 1665 amino acid residues (30%) identical to, and 767 of 1665 amino acid residues (46%) similar to, the 5198 amino acid residue ptnr:SPTREMBL-ACC:076518 protein from Cae~orhabditis elegaf~.s (HEMICENTIN
PRECURSOR).
NOV9 also has homology to the proteins shown in the BLASTP data in Table 9D.
Table 9D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~14575679~gb~AAK6hemicentin 5636 1230/30171785/30170.0 8690.1~AF156100 [Homo Sapiens] (40%) (58%) (AF156100) gi118547943~reflXP_hemicentrin 3645 979/2379 1413/23790.0 053531.3~(XM [Homo Sapiens] (4l%) (59%) gi~17568539~refINP_Ig superfamily 5175 857/3077 1348/30770.0 509636.1~(NM_077235repeats (I-type) (27%) (42%) [Caenorhabditis e1 egans]
gi~17568541IrefINP_IG 5198 857/3077 1348/30770.0 509635.1I(NM (immunoglobulin) (27%) (42%) superfamily (47 domains) [Caenorhabditis e1 egans]
gi~13872813~emb~CACfibulin-6 2673 552/1399 796/1399 0.0 37630.1~(AJ306906)[Homo Sapiens] (39%) (56%) A multiple sequence alignment is given ix~ Table 9E, with the NOV9 protein being shown on line 1 in Table 9E in a ClustalW analysis, and comparing the NOV9 protein with the related protein sequences shown in Table 9D. This BLASTP data is displayed graphically in the ClustalW in Table 9E.
Table 9E. ClustalW Analysis of NOV9 1) > NOV9; SEQ ID N0:18 2) > gig 14575679~J hemicentin [Homo Sapiens]; SEQ 1D N0:72 3) > gi~18547943~/ hemicentrin [Homo Sapiens]; SEQ ID N0:73 4) > gig 17568539/ Ig superfamily repeats (I-type) [Caeraorlaabditfs elegaras]; SEQ ID N0:74 5) > gig 17568541 ~/ IG (inununoglobulin) superfamily (47 domains) [Caenorlaabditis elegans]; SEQ ID
N0:75 6) > gi~13872813~/ fibulin-6 [Homo Sapiens]; SEQ ID N0:76 ..J....J....J....J....J.. ,.J....J. .
NOV9 2602 _________________________ _________ ____ giJ14575679J3929 _________________________ _________ ____ giJ18547943J1938 _________________________ _________ ____ giJ17568539J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
giJ17568541J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
giJ13872813J966 _________________________ _________ ____ ....J....J....J....J....J....J....J....J.. .J., J....J . .J
NOV9 2621---_______________________________________p . GQ~,PI . 2639 ..~
giJ14575679J3948-___,_____________________________________S
.~ TEL 3966 giJ18547943J1957-_________________________________________S
~ TvL ' giJ17568539J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
giJ17568541J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
giJ13872813J985 __________________________________________S
TvL 1003 ~.I
giJ14575679J 3967 4025 giJ18547943J 1976 giJ17568539J 3368 3427 giJ17568541J 3368 3427 giJ13872813J 1004 1062 giJ14575679J 4026 4085 giJ18547943J 2035 2094 giJ17568539J 3428 3486 giJ17568541J 3428 3486 giJ13872813J 1063 1122 ....~....J....J....J,...J....J....J....J...,J....J....J....J
giJ14575679J 4086 4143 ga.J18547943~ 2095 2152 giJ17568539J 3487 3546 giJ17568541J 3487 3546 giJ13872813J 1123 1180 .J....J,...J....J:..,J....J....J....J. .J.. .J....J.. .J
NOV9 2759 __________________ _______ ___ DLTT~~ ~1F ~ ~ S~E~ 2783 H' I v VI r t ~ Vm gi J 14575679 J 4144 T ~ ~ S STST Ti~H ~ RS'I!--E Y , v Q~iI ~ 'D ~T ~ v 4201 gi J 18547943 J 2153 T ' S STST T~~Ii ' RS,~--E ~Y_' QiI ~ iD ~T~ 2210 gi J 17568539 J 3547 I R~iS~E ~ T DIDLI ILL ' DIt'~'NTI PLAIVARTIY E PIS ~Q
~D'S~ 3606 giJ17568541J 3547 ' I RfiS EI~rT DIDLI ~L~~~ DKNTI PLAIVARTIY E PIS ~Q~ 3606 gi J 13872813 ~ 1181 H T ~ ~ S STST TtlIi ~ ~ RS!~--E YiT'VN~Qi~',T ~ iD ~T ~
.J....J....J . . .. .. J....J....J...
NOV9 2784 T~D~GQP~IS,GAE~F'~IQ~S~-QLVK~LEGQ~T~TyE ~ ' ~RARRR~fi~I,L~ 2842 gi~14575679~ 4202 4260 gi1185479431 2211 2269 gi1175685391 3607 3666 gi1175685411 3607 gi1138728131 1239 3666 NOV9 2843 ~...T.:. I..RL1. .1. .~.~S~~____T ~I.~1 'RP~'TEGVSE-I--Q~I 2892 gi1145756791 4261 'T E ' r - ~Q S T I~-----~~KL F I PAHFDS---- 4310 gi1185479431 2270 ~T E ' r - ~Q~ S I'T I~---- 'KL, F~~I~PAHFDS--- 2319 gi ~ 17568539 ~ 3667 ~E DIHGTQP 'KEE ;T~T,T T PIKLAEDIADQ~; . D~t7~
KDRAI7GDLTDNVDIS~J17 3726 gi ~ 27568541 ~ 3667 ~E IHGTQPi~~.KRE TAT T PIKLAEDIADQ'~~'ID;tTS
K'~,~RA~iDGDLTDNVDIS~77D 3726 gi~13872813~ 1298 ~T E ' r 4~ DQfl S It~~lTneI~-____~,. gIpp,HFDS-___~ 1347 a gi1145756791 4311 4369 gi1185479431 2320 2378 gi1175685391 3727 3786 gi1175685411 3727 3786 gi1138728131 1348 1406 gi1145756791 4370 4420 gi1185479431 2379 2429 30 gi~17568539~ 3787 3838 gi1175685411 3787 3838 gi1138728131 1407 1457 gi1145756791 4421 4480 gi1185479431 2430 2489 gi1175685391 3839 3898 gi1175685411 3839 3898 gi1138728131 1458 1517 .'....,....1,...1....1....1....1....1. .1....1,.. .1....1 NOV9 3063 t~GNPr-------------------------------FH- LA'S~QL_' ------ 3083 gi1145756791 4481 SWDrR --S Y A~ ~ErTSF'E ~ L ~P~I Q GF----- 4533 1 7 v r' gi ~ 18547943 ~ 2490 SWDrR ' Y 'yV--S Y ~A ErTS~.~'E V.~',' L V V P ~Q ~l GF---gi1175685391 3899 FDSPDGAR,'~ LKG PHL ~Tr GI?Y'T Q~L 'SEAS S~~L PPEINR17GI
gi 113872813 ~ 1518 SWD~RGLKG-A~ PHL[',J ;E~TS~E!E Q~LI L SEAS ~
P==~~''~~~''Q~~GFNRDGI 1570 .1. ..1. ..1....1....1....1. .1....1. ..1. I . .~.. .1 NOV9 3083 -- LLFj" .PQAST SLL~PTSF F~~IS~GILS~STAHQGSP ------ - 3132 gi I 14575679 ~ 4533 -- WSiiIW~ ~ C~'T GK ~IxCNQ L'L~ GG PCQ DIsEMR'~CQNPCP -D'S 4589 gi ~ 18547943 I 2542 - ~WS~i~ 'CSV'T GK QR~CNQ L~ GC~PCQ D~;EM~CQNPCP -D S
giI175685391 3959 DMSPLP~iQ L'~T.Q LAQG~PVPQ1~RWTLNGTALTHSTP ITASDGTFII SLSD
g7. 175685411 3959 DMSP ~LP~iQ~~v"'L~'IiQ LAQGT~PVPQR.WTLNGTALTHSTP
IT~,ASDixTFI"~3I 'L, SLSD 4018 gi 113872813 ~ 1570 ---~5~'~" C~S"''~T GKGQ~KRR~iCNQ~LGG~T~'PCQ
~iEM'k'i.2~CQNIfPCP -D~S 1626 ~. _..~....~....~....~....~....~
NOV9 3132 - --~QLLCLLFFTAIP~~ --------------- ----- T: TUE'---Q----- 3155 gi 114575679 ~ 4590 WSE~fL E ~CTRSCGRGi Q RTRTCNNPSVQH GPCEGNA'VE~~ ' ~C~ GA---gi118547943~ 2599 WSE~IL CTRSCGRG~QBRTRTCNNPSVQ GPCEGNAiSTE~ ,11 ~C~ GA---gi1175685391 4019 KGV~TC~I,~, AGSDNLMYNVDWQAPVISN T'KQVIEGE E L~EGY' PQVSWL
gi1175685411 4019 KGVY','~CAGSDNLMYNVDWQAPVISN T~QVIEGE~ E L~EGY~ PQVSWL 4078 gi 113872813 1 1627 WSETL~ CTRSCGRG~f~RTRTCNNPSVQH~eG ,PCEGNA3 ,ELI 3 !R~C~ GA-NOV9 3155 ----------------D-------M'VRS DVAL~CATGE~-------------T~TI 3179 gi~14575679~ 4646 ------------ W~'v'AWQPWGTCESC KGTQT' r~LC~ ------------P~ F
gi~18547943~ 2655 ------------ WAW~PWGTCESC KGTQT' _LC ~-------------P~ F 2687 gi~175685391 4079 RNGNRVETGVQGVRYVi!xDG~~MLTIIEARSLDSGIYLCSAT
EAGSAQQAYTLEVLVS~KI 4138 gi~17568541~ 4079 RNGNRVETGVQGVRYV"~'DGMLTIIEARSLDSGIYLCSATGSAQQAYTLEVLVS~KI
gi~13872813~ 1683 ------------ WAWPWGTCESC~KGTQT~LC -----------P~ F 1715 IO
NOV9 3179 -----------------------------E-~LQAGQPLRAS~~L'.LPDGSLWLN--- 3206 gi~14575679~ 4679 G YCDGAETQMQVCNE P~~H KWATW S,,,~ACSVSCG 'Q~ CSDPV~~_~P~YGGR
gi~185479431 2688 G YCDGAETQMQVCNE ' P;IFi KWATW ~~,,,AJCSVSCG _'Q~
CSDP~,PYGGR 2747 gi~17568539~ 4139 IT TPGVLTPSSGSKFSLP '~ YPDPII '15LNGNDIKD ENG I GT~HIEKAE
IS gi~175685411 4139 IT TPGVLTPSSGSKFSLP ~YPDPII 1,,:?~LNGNDIKD ENGH,I
GT~~'.rHIEKAE 4198 gi~13872813~ 1716 G YCDGAETQMQVCNE P~ KWATW ~ACSVSCG 'Q' CSDP~PYGGR 1775 NOV9 3206 - ----- '~GDAGYD . -----------LLGSAT ' L ------ 3236 V ~~;
gi ~ 14575679 I 4739 ---C G~sD~DFC~~P ~~ ~ ~ SGWGTCSRTCNGGQ ~RRT ~P ----- 4788 gi ~ 18547943 ~ 2748 ~----C G~~~~'D1(QDFC~TDP T G ~ ~ SGWGTCSRTCNGGQ ~R'~.'RT
, P------ 2797 gi~17568539~ 4199 e'RHLIY CAKNDAGA~~"'LEF~ QTIU ~KISTSGNRYINGSEGTETVI
°IESESSEF 4258 gi ~ 17568541 ~ 4199 ~RHLI C~'I'AI~NDAGA~.'?~'LEF ~ QTIV KISTSGNRYINGSEGTETVI
gi~13872813~ 1776 ----C G~D~~DFC~IDP ~T~G~W,~~WSGWGTCSRTCNGGQM~'yIR~RT~P------NOV9 3236 -HAI S'HFRD ___________ _________ __________ -VF ~. ~ 3257 r ~ ,~, r r 3~ gi ~ 14575679 ~ 4788 -PPS CG ~SQIQRCNTD1~CP~D~GSWGSWHS~ QCiTS,C GEKT~ IiP ~
gi~18547943~ 2797 -PPS ' CG ~SQIQRCNTD CP~GSWGSWHSQC~~C GEKT ~ 'HP ~ 2856 gi~17568539~ 4259 SWS PLLPSNLIFSEDYKL3~KI~t,(STRLSDQGE~, CTATQT ~GV ~ 4318 gi~17568541~ 4259 SWS PLLPSN~LIFSEDYKL~KISTRLSDQGE'~ CTS ~ ATQ~T GV 4318 gi ~ 13872813 ~ 1825 -PPS~G~ACGG~SQIQRCNTDCP~ST,DGSWGSWHS~J~QC~~~EKT;HP~ 1884 giI14575679~ 4848 gi~7.8547943~ 2857 4900 gi~17568539~ 4319 4378 gi~17568541~ 4319 4378 gi~13872813~ 1885 1937 .,..I
gi~145756791 4901 ----- 4953 gi~18547943~ 2910 ----- 2962 gi~17568539~ 4379 TGIPE 4438 gi~17568541~ 4379 TGIPE 4438 gi~13872813~ 1938 ----- 1990 gi~14575679~ 4953 5008 gi~18547943~ 2962 3017 gi~17568539~ 4439 4498 gi~17568541~ 4439 4498 gi~13872813~ 1990 2045 NOV9 3400 ------- F'~E ' yV ~1 FQ . L(PSFLRCi S~:Q ~ PQ ~Q . Q 3452 a r ...-gi ~ 14575679 ~ 5008 ------- Y'T D Iv ~ ~A ~L I ~ IISIPYT TUF L3Qi ~F E<T 5060 giI185479431 3017 _______ ,~X':T D ~~ ~ Y'AY 'L I~ I',SIPYT T'~F yQil ~,Q~ ~F T~ 3069 giI17568539~ 4499 PSDRPAPI:; CDE~ICG ~KI~TEYMID~GD P~DNPQLLP
KDVEDSSLNGSIAYRC~PGP 4558 gi~17568541~ 4499 PSDRPAPI,, CDE~CG KI~TEYMID'GD P~ NPQLLP KDVEDSSLNGSIAYRC.
gi113872813~ 2045 _______ yT ~~~ ~ v ~;AY 'L I~ I~SIPYT 'T''()F~_Qi~RMOFmE~T~
L, ~ ~ L,. . . I . I . I . I . . . I . . . . I . . . I . . . I . . I . . . I.
NOV9 3453 . ~ TS ~'~PE ~ . Q~.,iAT,~QAENEV . 'E ------ . E n Q n- n S 3505 gi I 14575679 ~ 5061 H~ S'ClIE Y~VQI IHAI~~SKG1~RSNQ 'S ----- ~ P ~-y ' 5113 gi~185479431 3070 H~ SUE D~QI~~T~ ~~HA~"~SKG~1RSNQ 'S ----- ~ P ~-W ~ 3122 gi1175685391 4559 R~ RTVLL~ P~FI~KP TT IGAIVEL S~1AGPPHPTI AKD KLIE~S~F I,~~, gi 1175685411 4559 R~ RTVLL~P~FI'U'KP TT~IGATVEL3 Sn~p,'AGPPHPTI AKD
KLIE~SIfiF Ii1 4618 gi 113872813 I 2098 H~ S~E~DY.~TQI~T~ ~I'HA~~'SKGRSNQ~S~e----- ~ P ~-~ ' 2150 gi1145756791 5114 5172 gi1185479431 3123 3181 gi1175685391 4619 4677 gi1175685411 4619 4677 gi1138728131 2151 2209 .I. .~. ..I....I....I....I....I....I....I....I....I....I
NOV9 3566 '~xiPD .P _______________-________________________________ 3576 gi1145756791 5173 ~ S ~RTS ~i---------------------- Cf~~IECQESPCHQ~CF1~ 5209 gi~18547943~ 3182 ~ S ~RTS~ T~----------------------- C~~IECQESPCHQ'CF3218 gi1175685391 4678 ERNQAYS LTWE ~ ~PMPKNLAGIHFMNNGSLVILDTS L~GLELY(~'CKV 'R 3 gi117568541~ 4678 ERNQAYS LTWE ~ PMPKNLAGIHFMNNGSLVILDTS L~G~LELY'CKV 'RF~
V V ~
gi I 13872813 ~ 2210 ~t~(1R~S 'RTS-----------------------CE~7~IECQESFSPCHQ~'yCF2246 30 ....I....I....I....I....I....1....1....1....1....1....1....1 NOV9 3576 -___________________________________________________________ 3576 gi1145756791 5210 5262 gi~185479431 3219 3271 gi~17568539~ 4738 4797 35 gi1175685411 4738 4797 gi1138728131 2247 2299 .I.. .I... I . .I. .I. .I ...I. ..I. .1....1....I...
NOV9 3577 ADGAG- E~'n L.LDD~. ~.'. PGGv '.S ~'~------------------ 3616 gi114575679i 5263 TKAEG ~W ICC7 T f1 vT RSS,' ~~ __________________ 5304 gii185479431 3272 ~TKAE'GT yI~KT~Q ~~ RGSG,..' ----------------- 3313 gi1175685391 4798 ~LPNNLVL~:~YDANSIGKAFDDTLNVYG------------------- 4838 gi1175685411 4798 ~tLPNNLVL~ICYD~ V~~ ANSIGKAFDDTLNVYE FLPLTGFEGSGINIDDS 4857 gi 113872813 I 2300 TKAE~tGT~T~~ .-, T,~IR~I~RGS----------------- 2341 ....I.
50 gi1145756791 5304 ------ 5353 gi~185479431 3313 ------ 3362 gi1175685391 4838 ----GS 4894 gi1175685411 4858 SNAGGS 4917 gi1138728131 2341 ------ 2390 gi1145756791 5354 5413 gi118547943~ 3363 3422 gi117568539~ 4895 4952 gi117568541~ 4918 4975 gi113872813~ 2391 2450 .. .I.. .1....I., .~. .~. ..~L. .I. .I. .I. .~. .I. .I
NOV9 3718 -- ' W ~P FIR--QG Tn n RVRL.~ E .~~ L.' ~~LP~u.' 3773 gi1145756791 5414 RTI'KT ~ SEA--S~DT ~I~ ENT~3 t F ~ I 'P' Q THE ~ ~T 5471 i 18547943 3423 RTI'KT ' SEA SDT ~I~ ENT v F ~ I P Q TH ~T 3480 gi1175685391 4953 TG-E FAMNPHTRI En_L~AFYQP F I Y G._,~ 'L~E ~Ee~-- 5009 gi~17568541~ 4976TG-E
FAMNPHTRI
E~
~
FYQP
F
I
YD
G
~
L
E
--r gi 7.3872813 2451RTI~'yKT L ~F ~ ~THT~S
~ ~ Z 2508 SEA P~Q
S~IDT~2 ~
ENT~)A
~
gi~14575679~ 5472 5531 gi~18547943~ 3481 3540 gi~17568539~ 5010 5059 gi117568541~ 5033 5082 gi~13872813) 2509 2568 gi~14575679~ 5531 5582 gi~18547943~ 3540 3591 gi~17568539) 5060 5118 gi117568541~ 5083 5141 gi~13872813~ 2568 2619 L, . . ~ ~...v., ~ . . I . . . . I . . . I .
~~ ~ 7 ~ i -NOV9 3883 .~PLRAGL ~T ~GL1~LT " P------- .~~~ .,ltL ~ .P ~ 3931 gi~14575679~ 5583 E-NL T'P RE~ T ' '~~ YS~=-GTIE'~~ T ~~Y ' 5636 V~ i1 gi~185479431 3592 ~ E-NL T~P RE~'T ' ~~S YS,~, -GTIE ~ T IyY ~ 3645 gi1175685391 5119 ' ~AVKRGHAQ G H ~~H DH~,~,TNELHAPK~L GQ ~~ 5175 gi~17568541~ 5142 T~AVKRGHAQ ' GH~K~~H DH~TNELHAPIt~ ~L GQ ~5198 gi ~ 13872813 ~ 2620. ~ E NLI~eV~T P RE~TS~YS ' -GTIEiY~T~~1~Y ' 2673 The NOV9 Clustal W alignment shown in Table 9E was modified to begin at amino residue 4080. The data in Table 9E includes all of the regions overlapping with the NOV9 35 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 9F lists the domain description from 40 DOMAIN analysis results against NOV9.
Table 9F Domain Anal sis of NOV9 Model Region of Score (bits) E value Homology ig 99-157 46.50 6.1e-10 ig 192-251 38.00 2.1e-07 ig 286-344 41.90 1.4e-08 ig 380-438 32,10 1.3e-05 ig 473-531 45.90 8.9e-10 ig 565-615 35.50 1.2e-06 ig 648-706 43.80 4e-09 ig 740-800 39.10 1e-07 ig 833-891 27.10 0.00041 ig 981-1039 42,90 7.1e-09 ig 1075-1133 28.90 0.00012 ig 1168-1226 30.80 3.2e-05 ig 1264-1322 39.60 7.2e-08 ig 1362-1420 43.40 5.2e-09 ig 1473-1531 29.40 8.2e-05 ig 1568-1626 35.70 1e-06 ig 1654-1712 _ 1.5e-09 45.20 ig 1749-1807 43.80 3.8e-09 ig 1841-1899 43.00 6.8e-09 ig 1934-1994 41.40 2e-OS
ig 2030-2088 49.90 5.8e-11 ig 2123-2177 49.60 6.8e-11 ig 2212-2268 37.50 3e-07 ig 2303-2361 28.60 0.00014 ig 2394-2459 28.20 0.00019 ig 2492-2552 32.90 7.2e-06 ig 2585-2643 22.80 0.0081 ig 2676-2733 37.50 3.1e-07 ig 2766-2824 43.10 6.3e-09 ig 2857-2913 44.10 3e-09 ig 2947-3012 18.00 0.22 ig 3162-3219 34.70 2.1e-06 EGF 3504-3539 38.30 1.8e-07 EGF 3589-3626 13.80 1.3 EGF 3632-3667 33.30 5,4e-06 EGF 3739-3773 38.40 1.6e-07 Consistent with other known members of the Hemicentin Precursor-like family of proteins, NOV9 has, for example, thirty-three immunoglobulin (ig) signature sequences and four epidermal growth factor (EGF) signature sequences, as well as homology to other members of the Hemicentin Precursor-like Protein Family. NOV9 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV9 nucleic acids and polypeptides can be used to identify proteins that are members of the Hemicentin Precursor-like Protein Family. The NOV9 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV9 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular differentiation, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Hyperparathyroidism, Hypoparathyroidism, Lymphedema, Allergies as well as other diseases, disorders and conditions..
In addition, various NOV9 nucleic acids and polypeptides according to the invention are useful, ifater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV9 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Hemicentin Precursor-like Protein Family.
Hemicentrin is an extracellular matrix protein with a modular sturcture. Like NOV9, the hemicentrin structure includes many immunoglobulin domains flanked by EGF
domains.
The protein is likely involved in cellular differentiation of epithelial tissue.
The NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune and endocrine physiology. As such, the NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and endocrine disorders, e.g., Cardiovascular diseases, Hyperparathyroidism, Hypoparathyroidism, Lymphedema, Allergies as well as other diseases, disorders and conditions.
The NOV9 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV9 nucleic acid is expressed in Adipose, Thyroid, Colon, Lymph node, Bone, Myometrium, Prostate, Testis, Aorta, Vein.
Additional utilities for NOV9 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV10 polypeptide has been identified as a Selectin-like protein. The novel nucleic acid sequences maps to the chromosome 9. Two alternative novel NOV10, NOVlOa and NOVlOb, nucleic acids and encoded polypeptides are provided.
NOVlOa A NOV10 variant is NOVlOa (alternatively referred to herein as CG94661-Ol), which includes the 1268 nucleotide sequence (SEQ m N0:19) shown in Table 10A. A NOV
1 Oa ORF begins with a ATG initiation codon at nucleotides 145-147 and ends with a TGA codon at nucleotides 871-873. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 10A, and the start and stop codons are in bold letters.
Table 10A. NOVlOa Nucleotide Sequence (SEQ ID N0:19) GCGGCCGCCACCCTCCGTGGCAAGGCGAGGCCCCGGGGGCGGGCCGGGGTCACCACGCCTGTCCCAG
GGAACCGCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAG
ATGAAGTGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCACGTGC
GCTAAGCTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGA
CCGTGCTCATGTTCCGCTGCCCCTCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTG
GAAGGGGAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACC
TTTGGCTTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGG
CCTTCCTCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGC
CCAGCTGTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAG
CACTTCAACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATG
GTGAGAGCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGC
TCTAAGCCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGA
CAGCCCCTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGT
GACCACGCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCA
GCTACAACTCCACATCAACTCCACATGCGCCCAGCTCGAGACTGATGAGTGGAATCAGCTTCCAGGT
GTAGGGACCCCTTGAGGGGCCGAGCTGACATCCAAGGCTGAGGACCCCAGTGGGGAGTGTTCTGTTC
CGGCATATCCTGGCCGTAACGATTTTTATAGTTATGGACTACTTGAAACCACTACTGAGGGTAATTT
ACTAGCTGTGGCCTCCCACTAACTAGCATTCCTTTAAAGAGACTGGGAAATGTTTTAAGCAAATCTA
GTTTTGTATAATAAAATAAGAAAATAGCAATAAACTTCTTTTCAGCAACTACF,~~e~AAAAAAA
The NOV 10a polypeptide (SEQ ID N0:20) encoded by SEQ ID N0:19 is 242 amino acid residues in length and is presented using the one-letter amino acid code in Table l OB. The Psort profile for the NOVlOa predicts that this peptide is likely to be localized at the plasma membrane with a certainty of 0.7000.
Table 10B. NOVlOa protein sequence (SEQ ID N0:20) MKDTIGLVMEWEIPEIICTCAKLRLPPQATFQVLRGNGASVGTVLMFRCPSNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOVlOb Alternatively, a NOV 10 variant is the novel NOV l Ob (alternatively referred to herein as CG94661-02), which includes the 887 nucleotide sequence (SEQ TD N0:21) shown in Table l OC. NOV l Ob was created by polymerase chain reaction (PCR) using the primers detailed in Example 1, Table 17. Primers were designed based on ih silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clone 143260::COR100348691 extn.698976.C20.
The NOVlOb ORF begins with a Kozak consensus ATG initiation colon at nucleotides 72-74 and ends with a TGA colon at nucleotides 1958-1960. Putative untranslated regions upstream from the initiation colon and downstream from the termination colon are underlined in Table l OC, and the start and stop colons are in bold letters.
Table IOC. NOVlOb Nucleotide Sequence (SEQ ID N0:21) GCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAGATGAAG
TGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCATGTGCGCTAAG
CTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGACCGTGC
TCATGTTCCGCTGCCCCCCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTGGAAGGG
GAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACCTTTGGC
TTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGGCCTTCC
TCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGCCCAGCT
GTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAGCACTTC
AACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATGGTGAGA
GCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGCTCTAAG
CCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGACAGCCC
CTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGTGACCAC
GCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCAGCTACA
ACTCCACATCAACTCC
Variant sequences of NOVlOb are included in Example 3, Table 22. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP
originates as a cDNA.
The NOV l Ob protein (SEQ ID N0:22) encoded by SEQ ID NO:21 is 242 amino acid residues in length and is presented using the one-letter code in Table l OD.
The Psort profile for NOV l Ob predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.7000.
Table 10D. NOVlOb protein sequence (SEQ ID N0:22) MKNIGLVMEWEIPEIICMCAKLRLPPQATFQVLRGNGASVGTVLMFRCPPNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOV10 Clones Unless specifically addressed as NOVlOa or NOVlOb, any reference to NOV10 is assumed to encompass all variants. NOVlOa differs from NOVlOb at amino acid position 18 (T>M) and amino acid position 50 (S>P) as shown in Tables 10B and l OD.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 10E.
Table 10E. Pat results for NOV10 Smallest Sum eading igh Prob equences Frame ScoreP(N) producing High-scoring Segment Pairs:
>patp:AAM93054Human digestive system antigen +1 210 7.2e-17 >patp:AAR05494Endothelial leukocyte adhesion +1 113 0.0016 molecule-1 >patp:AAR08116Endothelial leucocyte adhesion +1 l13 0.0016 molecule-1 >patp:AAW18839E-selectin +1 113 0.0016 >patp:AAW46733Endothelial leukocyte adhesion +1 113 0.0016 molecule-1 hi a BLAST search of public sequence databases, it was found, for example, that the NOVlOa nucleic acid sequence of this invention has 438 of 447 bases (97%) identical to a gb:GENBANI~-m:HSM802384~acc:AL137623.1 mRNA from Homo sapieras (cDNA
DKFZp434J1812 (from clone DKFZp434J1812)). The full amino acid sequence of the protein of the invention was found to have 110 of 139 amino acid residues (79%) identical to, and 123 of 139 amino acid residues (88%) similar to, the 269 amino acid residue ptnr:SPTREMBL-ACC:Q9D176 protein from Mus musculus (1700017I11R1K PROTEIN).
Similarly, it was found, for example, the NOV l Ob nucleic acid sequence of this invention has 438 of 447 bases (97%) identical to a gb:GENBANK-ID:HSM802384~acc:AL137623.1 mRNA from Homo Sapiens (cDNA DKFZp434J1812 (from clone DKFZp434J1812)). The full amino acid sequence of the protein of the invention was found to have 108 of 138 amino acid residues (78%) identical to, and 121 of 138 amino acid residues (87%) similar to, the 269 amino acid residue ptnr:SPTREMBL-ACC:Q9D176 protein from Mus musculus (1700017I11RIK PROTEIN.
Additional BLAST results are shown in Table 10F.
Table 10F. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier as >gi~15779059~gb~ASimilar to RIKEN255 192/225 192/225 e-101 AH14601.1~AAH1460cDNA 1700017I11 (85%) (85%) 1(BC014601) gene [Homo Sapiens]
>gi~12834785~dbj~Sushi domain 269 130/246 142/246 8e-56 BAB23043.1~(AK003(SCR repeat) (52%) (56%) 860) containing protein~data source:Pfam, source key:PF00084, evidence:ISS-put ative [Mus musculus]
>gi~128505441dbjlSushi domain 170 71/102 77/102 6e-35 BAB28764.1I(AK013(SCR repeat) (69%) (74%) 276) containing protein-.data source:Pfam, source key:PF00084, evidence:ISS-put ative [Mus musculus]
>gi~128389761dbjlSushi domain 149 55/73 61/73 2e-26 ' BAB24394.1~(AK006(SCR repeat) (75%) (83%) 068) containing protein-data source:Pfam, source key:PF00084, evidence:ISS-.put ative [Mus musculus]
>gi~7494498~pir~~scavenger 2043 30/87 42/87 2e-04 T18524 receptor (34%) (47%) cysteine-rich protein homolog srcrm2 - Geodia cydonium A multiple sequence alignment is given in Table 1 OG, with the NOV 10 protein of the invention being shown on line 1, in a ClustalW analysis comparing NOV10 with related protein sequences disclosed in Table IOF.
Table 10G. Information for the ClustalW proteins:
1) > NOVlOa; SEQ ID N0:20 2) > NOV l Ob; SEQ ID N0:22 3) > gi~1577905/ similar to RTKFN cDNA 1700017I11 gene [Homo Sapiens]; SEQ ID
N0:77 4) > gig 1283478/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:78 5) > gig 1285054/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:79 6) > gi~1283897/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084: ISS-putative [Mus nausculus]; SEQ ID N0:80 7) > gi~7494498/ scavenger receptor cysteine-rich protein homolog srcrm2-Geodia cydohiu~ra; SEQ ll~
NO:81 NOVlOa ___ IGLVMEWEIP--_____________________EIIC
RL
~ v NOVlOb -- NIGLVMEWEIP-----------------------EIIC
RL
K
.
gi~ 1577905--- WAAATLRGKARPRGRA--------- GNRT
RL
R TT
P
gi~ 1283478--- RTSATLRGRARPRWRA--------- VNQT Q
R TT P
P
V
V
~S gi~ 1285054--- RTSATLRGRARPRWRA--------- VNQT
' Q
R TT P
P
gi~ 1283897--- RTSATLRGRAR.PRWRA--------- VNQT
Q
IR TT P
P
gi1 7494498GRYTQDTGWIACEDGYQPTEGAADVLCTED PACSVS
P
TWSRT QV
NOVlOa ~ F~ -__________________________________________ NOVlOb v F~ -__________________________________________ gi~1577905 'v Fv -_____________________________-____________ gi~1283478 ~~v.~L~ ___________________________________________ giI1285054 ~ 7 -__________________________________________ ~~ L~
gi~1283897 ~~,.'~~1IL~ -__________________________________________ gi~7494498 SLSSPDTIPGLWSLVCDKGYTYDASSDGDFSWCGLDGEWNSTLG
NOVlOa _________,___________ L. .
NOVlOb _____________________ L. .p gi~ 1577905--___________________ L.
gi1 1283478--__________________ T L I
v n g7.I 1285054--__________________ T L I 1 gi1 1283897--___________________ T L I ' gi1 7494498TCKLVLCPAYSFNVTTNLRVSLTQ S TT T S 'FH SVI
S
.
.
NOVlOa T IAE ~L
' NOVlOb T IAE ~L ' . ' ' gi~ 1577905T IAE ~L
gi~ 1283478 TVD ' ' gi~1285054 TVD ' gi~1283897 TVD ~ --VPSSC~CPW
gi17494498 S S ---T GTV'H SRE " TKCPTLTISDHVTAS SETTINTVVS~
.) NOVlOa ------ S ' 'S ~SAQ .SQLKDED -ET ~~~Y G
NOVlOb ------ S ' 'S 'SAQ SQLKDED -ETi~~ y G
giI1577905 ------ S ' 'S 'SAQ SQLKDED -ET ~' Y G
1O gi~1283478 ------ Q~ E " TAQ YQLRGED -ET s~ Y G
giI1285054 ------ Q~ E " ~-------YSG-- -MSF~KLC
gi~1283897 PSS-__________p~SSSVC'KMSG--_______G_________ T
gi~7494498 TCDNGYFLKGDKI~E~LSTGVWNGTAPTCS~PNSCPSLI~SDH~TiSSTD
NOVlOa LKHFN-------------------KPVSGP ~ NHSF TDHGESTSKL
NOVlOb LKHFN-------------------KPVSGP ~ DNHSF TDHGESTSKL
g1~1577905 LKHFN-------------------KPVSGP t DNHSF TDHGESTSKL
2O gi~1283478 LKGHI~NSSSVGGGNGGPSGGGGKPGIQH ~ DNHSF TDPGD-IREQ
gi~1285054 VR-__-__-________-_______LLITSC~ PWG--____________ gi~1283897 GR-________________________ GTS-___-__-________ gi~7494498 TRINAVVTFTCD--DDRYTLNGNKIIACQSTGVWNGTAP~CKEIPTCPEL
NOVlOa ASVT KD~GIPR----------------------------ALSLSGS
NOVlOb ASVT DKD~GIPR----------------------------ALSLSGS
gi~1577905 ASVT TLD'GIPR----------------------------ALSLSGS
3O gi11283478 AGVTH DKD' TFR--------------------------MGTPGPGGC
giI1285054 ____- ___________________________________________ gi~1283897 -______________________-________________,_________ gi~7494498 TPSSHVIPST~DNSVGAEVSFQCEDGYTLQGEKKITCLPTQKWSANPPSC
NOVlOa S S~QAQ PRQPLP---AS T PQQPAAYALG---------NOVlOb S S'QAQ PRQPLP---AS T PQQPAAYALG---------gi'1577905 S S'QAQ PRQPLP---AS T PQQPAAYALG-________ 40 gi11283478 S S'GTYVMVHAL ---------S P PGRPKVYLPG--------gi~1285054 _____-___-_______-________________________________ gi~1283897 ___________,______________________________________ g1~7494498 G~TSQPLSNND~GSGTKVGPIVG~IG~ILVIVLIIVATAILFWKLSS
The NOV 10 Clustal W alignment shown in Table l OF was modified to begin at amino residue 1600 and end at amino acid residue 2000. The data in Table 1 OF
includes all of the regions overlapping with the NOV 10 protein sequences.
The NOV 10 Clustal W alignment shown in Table l OG was modified to begin at amino residue 1601. The data in Table l OG includes all of the regions overlapping with the NOV 10 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uklinterpro~. Table lOH lists the domain description from DOMAIN analysis results against NOV10.
Table lOH
Domain Anal sis of NOV10 Model Region of Score (bits) E value Homology Sushi domain 19-78 15.8 0.0075 (SCR repeat) Consistent with other known members of the Selectin-like family of proteins, has, for example, a Sushi domain (SCR repeat) signature sequences and homology to other members of the Selectin-like Protein Family. NOV 10 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
For example, NOV10 nucleic acids and polypeptides can be used to identify proteins that are members of the Selectin-like Protein Family. The NOV 10 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV 10 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular adhesion and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous I5 sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Diabetes, Autoimmune disease, Renal artery stenosis, lilterstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Systemic lupus erythematosus, Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
In addition, various NOV 10 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 10 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Selectin-like Protein Family.
The NOV 10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and immune physiology. As such, the NOV10 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and immune disorders, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic ~0 stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Diabetes, Autoixnmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
The NOV 10 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 10 nucleic acid is expressed in Heart, Thyroid, Parotid Salivaxy glands, Liver, Colon, Ascending Colon, Bone Marrow, Peripheral Blood, Lymphoid tissue, Spleen, Lymph node, Tonsils, Thymus, Cerebellum, Spinal Chord, Cervix, Mammary glandBreast, Ovary, Placenta, Uterus, Oviduct/LTterine Tube/Fallopian tube, Vulva, Prostate, Testis, Lung, Kidney, Kidney Cortex, Retina, Skin.
Additional utilities for NOV 10 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 11 polypeptide has been identified as a Nucleax Protein-like protein (also referred to as CG94325-O1). The disclosed novel NOV11 nucleic acid (SEQ ID
NO:23) of 8670 nucleotides is shown in Table 11A. The novel NOV 11 nucleic acid sequences maps to the chromosome 15.
An ORF begins with an ATG initiation codon at nucleotides 204-206 and ends with a TAA codon at nucleotides 7152-7154. A putative untranslated region and/or downstream from the termination codon is underlined in Table 11A, and the start and stop codons are in bold letters.
Table 11A. NOVll Nucleotide Sequence (SEQ ID N0:23) ACGCGTAGAGCCGCTTTGCGCGTGCGCATCACCTAGGCGGTTAGATTTGAATACTTCACTGAGGCGA
GCCGGGCGTTGTGAGCGGACTGCTAGAGGCGGCTGTCTGTTTCCGCTCTAAGGAAACTCAGAGCGTG
TGGACCCCAAACAAGTCTGCGCAAAATTTGTCGAGGAGGTTTGCCGCGGCAGAAAAGTTTTCTTCAA
AAATGGATGGGGTGTCTTCAGAGGCTAATGAAGAAAATGACAATATAGAGAGACCTGTTAGAAGACG
GCATTCTTCAATATTGAAACCCCCAAGGAGTCCTCTTCAGGACCTCAGAGGTGGGAATGAAAGAGTT
CAGGAATCCAATGCTTTGAGAAATAAGAAAAACTCTCGTCGAGTCAGCTTTGCAGATACTATAAAGG
TATTCCAGACGGAGTCTCATATGAAAATAGTGAGAAAGTCAGAAATGGAAGAAACAGAAACAGGAGA
AAATCTTCTTTTGATACAGAATAAGAAATTAGAAGATAATTACTGTGAAATTACTGGGATGAACACA
TTGCTTTCTGCTCCCATTCATACCCAGATGCAACAGAAGGAGTTTTCAATTATAGAACATACCCGTG
AAAGGAAACATGCAAATGACCAGACAGTCATTTTTTCAGATGAAAACCAGATGGACCTGACATCAAG
$1 TCACACTGTAATGATTACCAAAGGCCTTTTAGATAATCCCATAAGTGAAAAGTCCACCAAGATAGAT
ACCACATCATTTCTAGCTAATTTAAAGCTTCACACCGAGGACTCAAGAATGAAAAAAGAAGTAAATT
TTTCCGTGGATCAAAACACTTCTTCAGAAAATAAAATAGATTTCAATGACTTCATAAAAAGATTGAA
AACAGGAAAATGTAGTGCTTTTCCTGATGTGCCTGATAAAGAAAATTTTGAGATACCTATTTATTCC
AAGGAACCGAACAGTGCCTCTTCTACACATCAAATGCATGTATCTCTTAAGGAAGATGAAAATAACA
GTAATATTACTAGGCTCTTTAGAGAAAAAGATGATGGGATGAATTTCACCCAGTGTCATACAGCCAA
TATTCAGACATTGATTCCCACATCCAGTGAGACCAACTCACGGGAATCTAAAGGTAATGATATTACA
ATTTATGGCAATGACTTTATGGACTTGACATTTAACCACACTTTGCAGATCTTACCTGCAACAGGTA
ATTTTTCTGAAATAGAAAATCAAACTCAGAATGCCATGGATGTAACAACAGGTTATGGAACTAAAGC
TTCAGGAAATAAAACAGTTTTTAAGAGTAAACAAAATACTGCTTTTCAAGACCTTTCCATAAACTCT
GCAGACAAAATACATATTACCAGAAGTCATATTATGGGGGCAGAAACTCACATAGTCTCACAGACTT
GTAATCAGGATGCCAGAATATTAGCCATGACCCCAGAATCTATATATTCTAATCCATCTATTCAAGG
TTGTAAGACTGTTTTCTATTCTAGTTGTAATGATGCCATGGAAATGACCAAATGTCTCTCAAATATG
AGAGAGGAGAAAAATTTGCTAAAGCATGACAGTAATTATTCTAAAATGTATTGCAATCCAGATGCTA
TGTCTTCTCTCACAGAGAAAACTATTTATTCCGGAGAGGAGAACATGGACATTACCAAGAGTCATAC
AGTTGCAATAGATAATCAAATTTTTAAACAAGATCAATCAAATGTGCAAATAGCAGCTGCACCAACA
CCCGAAAAAGAAATGATGCTCCAAAATCTTATGACCACATCAGAAGATGGGAAAATGAATGTAAATT
GTAACTCAGTTCCTCATGTATCTAAGGAAAGAATACAGCAGAGCCTGTCAAATCCTTTGTCTATTTC
ATTGACTGATAGAAAGACTGAACTCTTATCAGGTGAAAATACGGATTTGACTGAAAGTCACACAAGT
AACTTAGGAAGTCAGGTTCCTCTTGCAGCTTATAATCTAGCACCGGAGAGTACCAGTGAATCTCACT
CTCAGAGCAAAAGCTCTTCAGATGAATGTGAAGAAATTACCAAAAGTCGTAATGAACCATTTCAGCG
ATCAGACATAATAGCCAAAAACAGCTTAACCGACACCTGGAACAAAGACAAAGATTGGGTTTTGAAG
ATTTTGCCCTACCTTGATAAAGATTCTCCTCAGTCAGCTGATTGTAATCAGGAGATAGCAACAAGCC
ATAATATAGTCTACTGTGGTGGAGTTCTTGATAAACAAATAACTAATAGAAATACAGTATCATGGGA
ACAATCTTTGTTTTCTACCACAAAGCCATTATTTTCATCAGGACAGTTCTCTATGAAAAATCATGAT
ACTGCTATAAGTAGTCATACAGTGAAATCTGTACTAGGCCAGAATTCTAAACTGGCTGAGCCACTGA
GGAAAAGTTTAAGCAATCCCACACCTGACTATTGCCATGACAAGATGATTATATGTTCAGAGGAAGA
GCAAAATATGGATCTAACAAAGAGCCACACTGTCGTCATTGGATTTGGTCCTTCTGAACTACAAGAA
CTTGGTAAAACTAATTTAGAACACACTACTGGCCAGCTAACAACAATGAACAGACAGATAGCTGTAA
AAGTTGAAAAATGTGGTAAAAGTCCCATAGAAAAAAGTGGAGTGCTTAAATCTAACTGTATTATGGA
TGTGTTAGAGGACGAAAGTGTACAGAAACCTAAATTTCCAAAGGAAAAGCAAAATGTCAAAATTTGG
GGAAGGAAAAGTGTTGGTGGACCAAAAATTGATAAGACTATTGTATTTTCAGAAGACGATAAGAATG
ATATGGATATCACTAAGAGTTATACAATAGAAATAAACCATAGACCTTTATTAGAGAAACGTGATTG
TCATTTGGTGCCATTGGCAGGAACTTCTGAAACTATTTTATATACATGTGGGCAGGATGACATGGAG
ATCACTAGAAGTCACACAACTGCCTTAGAATGTAAAACTGTCTCACCAGATGAAATAACTACTAGGC
CTATGGACAAAACTGTAGTGTTTGTAGATAATCATGTTGAACTAGAAATGACAGAGTCCCATACTGT
TTTCATTGACTACCAAGAAAAGGAAAGAACAGACAGACCTAACTTTGAACTATCCCAAAGGAAAAGC
CTAGGAACACCAACAGTGATATGTACTCCTACTGAGGAGAGTGTTTTCTTTCCAGGAAATGGTGAAA
GTGACCGTCTAGTAGCAAATGACAGCCAGCTAACCCCTCTGGAGGAATGGTCTAATAATAGGGGCCC
TGTAGAGGTAGCTGATAACATGGAATTGTCTAAATCAGCCACTTGCAAAAACATCAAAGATGTACAA
AGTCCTGGATTTCTGAATGAACCTCTATCAAGCAAAAGTCAGAGAAGAAAAAGCCTTAAGCTAAAAA
ATGACAAGACCATTGTATTTTCAGAGAATCATAAAAATGATATGGATATTACCCAGAGTTGTATGGT
GGAAATAGATAACGAAAGTGCCCTGGAGGATAAAGAGGACTTCCATTTGGCAGGGGCTTCTAAAACT
ATTTTGTATTCATGTGGGCAGGATGACATGGAGATCACTAGGAGTCACACAACTGCCTTAGAATGTA
AAACTCTCCTGCCAAACGAAATAGCTATTAGGCCCATGGACAAAACCGTATTGTTCACAGATAATTA
CAGTGATCTGGAAGTCACCGATTCCCATACTGTTTTCATTGACTGTCAAGCCACAGAGAAAATACTT
GAAGAAAACCCTAAATTTGGAATAGGAAAAGGAAAAAACTTGGGTGTTTCCTTTCCTAAGGATAATA
GCTGTGTTCAAGAAATCGCTGAAAAACAAGCACTGGCTGTAGGAAACAAAATAGTTCTTCACACCGA
GCAAAAGCAACAACTCTTTGCTGCTACTAATAGAACTACTAATGAAATCATCAAATTTCATAGTGCT
GCTATGGATGAAAAGGTCATAGGGAAAGTTGTAGACCAGGCCTGTACATTGGAAAAAGCGCAAGTTG
AAAGCTGTCAGTTAAATAATAGAGATAGAAGAAATGTGGACTTTACAAGTAGTCATGCAACTGCTGT
TTGTGGATCCAGTGATAATTATTCCTGTTTACCAAATGTTATTTCCTGTACTGATAATTTGGAGGGT
AGTGCCATGCTCTTATGTGATAAAGATGAGGAAAAAGCCAATTATTGCCCAGTGCAAAATGATCTTG
CTTATGCAAATGATTTTGCCAGTGAATATTACTTGGAATCTGAGGGACAGCCTCTCTCTGCTCCTTG
TCCTTTGTTAGAGAAGGAAGAAGTTATTCAAACCAGTACCAAAGGACAGTTAGACTGTGTTATAACA
CTGCACAAAGATCAAGATCTGATTAAGGATCCACGAAATCTATTGGCTAATCAAACTTTAGTATATA
GTCAAGATCTGGGGGAGATGACTAAACTTAATTCAAAGCGAGTATCTTTTAAGCTTCCAAAGGATCA
AATGAAAGTCTATGTTGATGACATTTATGTTATTCCTCAGCCTCATTTCTCAACCGACCAACCTCCA
TTACCTAAAAAAGGACAGAGTAGTATCAATAAAGAAGAAGTAATACTGTCTAAAGCTGGAAATAAGA
GTTTAAATATTATAGAAAATTCCTCTGCACCCATATGTGAAAACAAGCCCAAAATACTCAATAGTGA
GGAATGGTT'T'GCTGCAGCCTGTAAAAAAGAACTGAAGGAAAATATTCAAACAACTAACTATAATACA
GCTCTAGATTTCCACAGTAACTCAGACGTAACTAAGCAAGTCATTCAAACTCATGTCAATGCTGGAG
AAGCACCAGATCCTGTAATTACATCTAATGTTCCATGTTTTCATAGTATCAAACCAAATCTGAATAA
TTTGAATGGAAAAACTGGAGAGTTTTTAGCCTTTCAAACTGTTCATCTACCACCCCTTCCAGAGCAA
TTACTTGAATTAGGAAATAAGGCACACAATGATATGCATATAGTGCAAGCTACAGAAATACATAATA
TTAACATAATCTCCAGCAATGCTAAAGATAGTAGAGATGAGGAAAATAAAAAGTCTCATAATGGAGC
TGAAACCACCTCTCTACCGCCAAAGACAGTTTTTAAAGATAAAGTAAGGAGATGTTCTTTGGGAATC
TTTTTGCCTAGATTGCCCAACAAGAGAAATTGTAGTGTCACTGGTATTGATGACCTGGAACAGATTC
CAGCAGACACAACTGATATAAATCACTTAGAAACTCAGCCGGTCTCTAGCAAAGATTCAGGCATTGG
ATCTGTTGCAGGTAAACTGAACCTAAGTCCTTCTCAATATATAAATGAGGAAAATCTTCCTGTATAT
CCTGATGAGATCAATTCTTCAGACTCTATTAACATAGAAACTGAGGAAAAGGCCTTGATTGAGACAT
ACCAAAAAGAGATTTCACCATATGAAAATAAAATGGGAAAAACTTGCAATAGCCAAAAAAGAACGTG
GGTACAAGAAGAAGAAGATATTCATAAGGAGAAAAAAATCAGAAAAAATGAGATTAAGTTTAGTGAT
ACGACACAAGATCGGGAGATTTTTGATCACCATACTGAAGAGGATATAGATAAAAGTGCTAACAGTG
TATTGATAAAAAACCTGAGCAGGACCCCATCTAGTTGCAGCAGCTCTCTGGATTCAATCAAGGCTGA
TGGGACCTCTCTGGACTTCAGCACTTACCGCAGTAGTCAAATGGAATCACAGTTTCTCAGAGATACT
ATTTGTGAAGAGAGCTTGAGGGAGAAACTCCAAGATGGGAGAATAACAATAAGGGAGTTCTTTATAC
TTCTCCAGGTCCACATCTTGATACAGAAACCCCGACAGAGCAATCTCCCAGGCAATTTTACTGTAAA
CACACCACCTACTCCAGAAGACCTGATGTTAAGTCAATATGTTTACCGACCCAAGATACAGATTTAT
AGAGAAGATTGTGAGGCTCGTCGCCAAAAGATTGAAGAATTAAAGCTTTCTGCATCGAACCAAGATA
AGCTGTTGGTTGATATAAATAAGAACCTGTGGGAAAAAATGAGACACTGCTCTGACAAAGAGCTGAA
GGCCTTTGGAATTTATCTTAACAAAATAAAGTCATGTTTTACCAAGATGACTAAAGTCTTCACTCAC
CAAGGAAAAGTGGCTCTGTATGGCAAGCTGGTGCAGTCAGCTCAGAATGAGAGGGAGAAACTTCAAA
TAAAGATAGATGAGATGGATAAAATACTTAAGAAGATCGATAACTGCCTCACTGAGATGGAAACAGA
AACTAAGAATTTGGAGGATGAAGAGAAAAACAATCCTGTGGAAGAATGGGATTCTGAAATGAGAGCT
GCAGAAAAAGAATTGGAACAGCTGAAAACTGAAGAAGAGGAGCTTCAAAGAAATCTCTTAGAACTGG
AGGTACAAAAAGAGCAGACCCTTGCTCAAATAGACTTTATGCAAAAACAAAGAAATAGAACTGAAGA
GCTACTGGATCAGTTGAGCTTGTCTGAGTGGGATGTCGTTGAGTGGAGTGATGATCAAGCTGTATTC
ACCTTTGTTTATGACACGATACAACTCACCATCACCTTTGAAGAGTCAGTTGTTGGTTTCCCTTTCC
TGGACAAGCGTTATAGGAAGATTGTTGATGTCAATTTTCAATCTCTGTTAGATGAGGATCAAGCTCC
TCCTTCCTCCCTTTTAGTTCATAAGCTTATTTTCCAGTACGTTGAAGAAAAGGAATCCTGGAAGAAG
ACATGTACAACCCAGCATCAGTTACCCAAGATGCTTGAAGAATTCTCACTGGTAGTGCACCATTGCA
GACTCCTTGGAGAGGAGATTGAGTATTTAAAGAGATGGGGACCAAATTATAACCTAATGAACATAGA
TATTAATAAT.AATGAATTGAGACTTTTATTCTCTAGCTCCGCAGCATTTGCAAAGTTTGAAATAACT
TTGTTTCTCTCAGCCTATTATCCATCTGTACCATTACCTTCCACCATTCAGAATCACGTTGGGAACA
CTAGCCAAGATGATATTGCTACCATTCTATCTAAAGTGCCACTGGAGAACAACTACCTGAAGAATGT
AGTCAAGCAAATTTACCAAGATCTGTTTCAGGACTGCCATTTCTACCACTAGACCCTTGGACCACCA
TTGGAACAACCAAGCAGAATGTACTTGATATTATTTCAGGGTCCCATTGCTGTTCAGCCTTTGTTTT
TACGTCATTACAAGCTGAGTAAAATTCCTTCTGATGATGTTATAGTTAATCTGTATGTTTTTTATAT
CTCTGCAGAATGATGGTGATGAAGTCTGGATGGTAGGCCTCATAGCCTACTATCAACTTACTCATCT
TTGTACCAAAGGTTTAAGTAATAGGACACTTAGGAAAAATGTCTCCTAACTAAACTAGTGCTTTCTG
CTTTAGTACAAGCCCTAAGGATTAACTTAAGTATAAGAAGTGTTATCACTGACAAGAACATTAGCCA
TTTTCCCATAACTAGATAGAGCTATGATTTTTTAGGTTGCCTGGCTTCTGCCTAGCAGATATTTCTG
GAGTAGAAATGTATCTGTCTACAAACTATTATCCTTTTTCTCCGTTACTAAAATGCTATTAAGAGAA
AGTAGGGCTGGGTGTGAGCCACCACACCCAGCAATGTTTTCTTAATAAGTATAGTTTTTCTAGGGAA
AGTTAATTCATTTTTGTCTAGTACATATATGTAAATATATTAATGTTGTTTTTGTGTTTGTGATGTA
GTAAGGAGATGTACATAGAAATTCATTGAGGTATATAGATACTCATCTGTCTAGGCAGTTCCCAATT
TTCTGAAGAATGTTTTACAGCAAAATTTTCTATTTTCTTTTATTAAATAGTGACACGTCAAACAATG
TCACATCCAAAACACTAGTTTCATCAATTTCTAGCAGTAATAATAGACTTGCTGTAAGTATTGTTTT
CTGATGCCATACCCTTGTCATACATATTATTAAATGACCAATATTATGTATGAAGTAGACAAAAAAA
TTTACTCAAACTTCATTCAAATCCTAATTGTGATAATTTTTGTTTTATATTTAATTATAAACCAAAA
TACATTTGCATTTTTAAGCTAATTTGTCTCAAAATTTTGCTTTATATTTTTGGATCAGGTTAAAGTC
CTGTGGATCCCCTGAATGTTATTGTCCCTCTTGATTGGTTTTTACTTCTGAGCTATACGTCAAAAGA
CACATAAGCTTCAAAAGTCAAGACAAACCTCATTTGCCATAAAAATCAAGATATAGATGTTCTGTTC
CGTAAACTCCTTGAAAAACATTTTAAAGTCATCAATATGATCTGTTTCCCATGAAACTTAAGTTAGC
TTTCTTATTGGAGTTATTTCTTTTCTGTAAGTCTGAAAAGTAGAGATTTTGTTTTACGCATTTTAGT
AACCTGCAACAACCAACTCTAAAAA.AGATTTGGCTTGTAATGACGGTCTCTGCTTTTTTGGGTTTGG
AGTACACAATTGTAATATTTACTTAGTTATTTGTGTTTTTCTTTGTTCAAGGTATTGACTAGTTTCA
TAAATTTTTTGCAAGTTTTTCTTTCATTGGTTGGAAAGCAGATTACATTTTGCACTATTAAAATAAG
TTTATTACTTTP,AAAP~AAGTCGACG
Variant sequences of NOV11 are included in Example 3, Table 23. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 11 protein (SEQ m N0:24) encoded by SEQ m N0:23 is 2316 amino acid residues in length and is presented using the one-letter amino acid code in Table 11B. Psort analysis predicts the NOV 11 protein of the invention to be localized at the nucleus with a certainty of 0.8800.
Table 11B. Encoded NOVll protein sequence (SEQ ID N0:24) MDGVSSEANEENDNIERPVRRRHSSILKPPRSPLQDLRGGNERVQESNALRNKKNSRRVSFADTI
KVFQTESHMKIVRKSEMEETETGENLLLIQNKKLEDNYCEITGMNTLLSAPIHTQMQQKEFSIIE
HTRERKHANDQTVIFSDENQMDLTSSHTVMITKGLLDNPISEKSTKIDTTSFLANLKLHTEDSRM
KKEVNFSVDQNTSSENKIDFNDFIKRLKTGKCSAFPDVPDKENFEIPIYSKEPNSASSTHQMHVS
LKEDENNSNITRLFREKDDGMNFTQCHTANIQTLIPTSSETNSRESKGNDITIYGNDFMDLTFNH
TLQILPATGNFSEIENQTQNAMDVTTGYGTKASGNKTVFKSKQNTAFQDLSINSADKIHITRSHI
MGAETHIVSQTCNQDARILAMTPESIYSNPSIQGCKTVFYSSCNDAMEMTKCLSNMREEKNLLKH
DSNYSKMYCNPDAMSSLTEKTIYSGEENMDITKSHTVAIDNQIFKQDQSNVQIAAAPTPEKEMML
QNLMTTSEDGKMNVNCNSVPHVSKERIQQSLSNPLSISLTDRKTELLSGENTDLTESHTSNLGSQ
VPLAAYNLAPESTSESHSQSKSSSDECEEITKSRNEPFQRSDIIAKNSLTDTWNKDKDWVLKILP
YLDKDSPQSADCNQEIATSHNIVYCGGVLDKQITNRNTVSWEQSLFSTTKPLFSSGQFSMKNHDT
AISSHTVKSVLGQNSKLAEPLRKSLSNPTPDYCHDKMIICSEEEQNMDLTKSHTWIGFGPSELQ
ELGKTNLEHTTGQLTTMNRQIAVKVEKCGKSPIEKSGVLKSNCIMDVLEDESVQKPKFPKEKQNV
KIWGRKSVGGPKIDKTIVFSEDDKNDMDITKSYTIEINHRPLLEKRDCHLVPLAGTSETILYTCG
QDDMEITRSHTTALECKTVSPDEITTRPMDKTVVFVDNHVELEMTESHTVFIDYQEKERTDRPNF
ELSQRKSLGTPTVICTPTEESVFFPGNGESDRLVANDSQLTPLEEWSNNRGPVEVADNMELSKSA
TCKNIKDVQSPGFLNEPLSSKSQRRKSLKLKNDKTIVFSENHKNDMDITQSCMVEIDNESALEDK
EDFHLAGASKTILYSCGQDDMEITRSHTTALECKTLLPNEIAIRPMDKTVLFTDNYSDLEVTDSH
TVFIDCQATEKILEENPKFGIGKGKNLGVSFPKDNSCVQEIAEKQALAVGNKIVLHTEQKQQLFA' ATNRTTNEIIKFHSAAMDEKVIGKVVDQACTLEKAQVESCQLNNRDRRNVDFTSSHATAVCGSSD
NYSCLPNVISCTDNLEGSAMLLCDKDEEKANYCPVQNDLAYANDFASEYYLESEGQPLSAPCPLL
EKEEVIQTSTKGQLDCVITLHKDQDLIKDPRNLLANQTLVYSQDLGEMTKLNSKRVSFKLPKDQM
KVYVDDIYVIPQPHFSTDQPPLPKKGQSSINKEEVILSKAGNKSLNIIENSSAPICENKPKILNS
EEWFAAACKKELKENIQTTNYNTALDFHSNSDVTKQVIQTHVNAGEAPDPVITSNVPCFHSIKPN
LNNLNGKTGEFLAFQTVHLPPLPEQLLELGNKAHNDMHIVQATEIHNINIISSNAKDSRDEENKK
SHNGAETTSLPPKTVFKDKVRRCSLGIFLPRLPNKRNCSVTGIDDLEQIPADTTDINHLETQPVS
SKDSGIGSVAGKLNLSPSQYINEENLPVYPDEINSSDSINIETEEKALIETYQKEISPYENKMGK
TCNSQKRTWVQEEEDIHKEKKIRKNEIKFSDTTQDREIFDHHTEEDIDKSANSVLIKNLSRTPSS
CSSSLDSIKADGTSLDFSTYRSSQMESQFLRDTICEESLREKLQDGRITIREFFILLQVHILIQK
PRQSNLPGNFTVNTPPTPEDLMLSQYVYRPKIQIYREDCEARRQKIEELKLSASNQDKLLVDINK
NLWEKMRHCSDKELKAFGIYLNKIKSCFTKMTKVFTHQGKVALYGKLVQSAQNEREKLQIKIDEM
DKILKKIDNCLTEMETETKNLEDEEKNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQK
EQTLAQIDFMQKQRNR'T'EELLDQLSLSEWDVVEWSDDQAVFTFVYDTIQLTITFEESVVGFPFLD
KRYRKIVDVNFQSLLDEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLVVHHC
RLLGEEIEYLKRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHV
GNTSQDDIATILSKVPLENNYLKN~TVKQIYQDLFQDCHFYH
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 11 C.
Table 11C. results Pat fox NOVll Smallest Sum eading igh Prob equences igh-scoringegment Frame ScoreP(N) producing Pairs:
>patp:AAW8839.8Humantestis +1 2444 1.7e-253 secreted protein dol5 4 >patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253 >patp:AAU71961Humanbone marrowtissuepolypeptide#39+1 2444 1.7e-253 >patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253 >patp:AAU71961Humanbone marrowtissuepolypeptide#39+l 2444 1.7e-253 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 5584 of 5584 bases (100%) identical to a gb:GENBANK-m:AB046790~acc:AB046790.1 mRNA from Homo Sapiens (mRNA for KTA_A_1570 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 1790 of 1793 amino acid residues (99%) identical to, and 1792 of 1793 amino acid residues (99%) similar to, the 1833 amino acid residue ptnr:SPTREMBL-ACC:Q9NR92 protein from Horno sapieras (AF15Q14 PROTEIN).
NOV11 also has homology to the proteins shown in the BLASTP data in Table 11D.
Table 11D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) giI18308012~gb~AAL6AF15q14 isoform2316 2316/23162316/23160.0 7803.1~AF461041 [Homo Sapiens] (100%) (100%) 1(A
F461041) gi~9966807~refINPAF15q14 protein1833 1790/17931792/17930.0 _ [Homo Sapiens] (99%) (99%) 65113.1~(NM_020380) gi~14749154Iref~XP_AF15q14 protein1833 1789/17931791/17930.0 031524.1~(XM [Homo Sapiens] (99%) (99%) giI10047205~dbj~BABKIAA1570 protein1360 1360/13601360/13600.0 13396.1~(AB046790)[Homo Sapiens] (100%) (100%) gi~14749150~ref~XPsimilar to 915 900/900 900/900 0.0 _ KIAA1570 protein (100%) (100%) 012461.3 (XM 012461) [Homo Sapiens]
A multiple sequence alignment is given in Table 11E, with the NOV11 protein being shown on line 1 in Table 11E in a ClustalW analysis, and comparing the NOV11 protein with the related protein sequences shown in Table 11D. This BLASTP data is displayed graphically in the ClustalW in Table 11E.
Table 11E. ClustalW Analysis of NOVll 1) > NOVl 1; SEQ JD N0:24 2) > gi~18308012~/ AF15q14 isoform 2 [Homo Sapiens]; SEQ ID N0:82 3) > gi~9966807~/ AF15q14 protein [Homo Sapiens]; SEQ ID N0:83 4) > gi~14749154~/ AF15q14 protein [Homo Sapiens]; SEQ >D N0:84 5) > gi~10047205~/ KIAA1570 protein [Homo Sapiens]; SEQ >D N0:85 6) > gi~14749150~/ similar to KIAA1570 protein [Hoyno Sapiens]; SEQ >D N0:~6 .(. .( . .(..(. .~. .(..( .(.
NOV11 1 1 r 1 ~ 60 gi(18308012(1 1 1 1 ~ 60 gi(9966807(1 1 1 1 ~ 60 gi(14749154(1 1 1 1 ' ~ 60 gi(10047205(1 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ gi(14749150(1 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ ( ( ( ( ( ( ( ( ( ' ( ( gi(18308012(61 120 gi(9966807(6l gi(14749154(61 gi(1004720511 _____ ______ _______ ____ ___ ________ _________ ______ _____ _______ gi(14749150~1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ ( ( ( . (. .I. .( I. y .( ( ( .~ .
gi~183080121121 180 gi~9966807~121 gi(14749154(121 180 gi~10047205(1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ gi(14749150~1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( ( ( ( ( ( ( ( ( ( ( ( gi(18308012(181 240 gi~9966807(181 240 gi(14749154(181 240 gi(10047205(1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ gi(14749150(1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( . ( .( y .(.I ~ .( y y .
gi(183080121 241 300 gi(9966807( 241 45 gi(14749154( 241 300 giI10047205( 1 _____ ______ _______ ____ ___ ________ __________ _____ _____ _______ gi(147491501 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ 50 ( gi(18308012( 301 360 gi(9966807( 301 gi(14749154( 301 360 gi(10047205~ 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ gi(14749150( 1 _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ ( .( .( ( .(.. ( gi(18308012(361 gi(9966807(361 gi(14749154(361 g1(10047205(1 1 6$ _____ ______ _______ ____ ____ _______ __________ _____ _____ _______ giI14749150(1 1 ( ( ( ( ( NOV11 421 .( . . ~.. . . .(. .(..(. . . .(..
.(. . 1 . . r 1 . . .. 480 8~ i1' ~6 1 v v 1 I v . y~ 11 1 1I'Il1 / '1 1 1 1 1 1 1 ! 1 1 1 r o v I Ir r 1 a 1 ~ -11 1 1 1 r 1 ,~ I 11 1 1 1 9~ I 11 1 1 11 ~ I ~ 1W 1 1 ~1 a 1 r 1 11 y 1 v 1 11 I v v .(. .(.. .(. (. . .(.
.(. . .( .(. .(..
.~.
.( r 1 1 v 1. v IvI 1 1 1 1 1 . 1 , 1 W 11 1 y1.
~
1 11 1 v ~~i1 gi(18308012( 421 m 1 1 SI '1' 480 gi(9966807( 42l v . 1 1 1 1 SI 1 480 gi (14749154( 421 . 1 1 ~ 1 1 1 1 480 gi(10047205( 1 ______ _____ ___________ ____ ________ ____ _____ _____ ____ ____ 1 _____ gi(14749150( 1 ______ _____ ___________ ____ ________ ________ _____ _____ ____ ____ 1 gi(18308012( 481 540 gi (9966807( 481 540 gi(14749154( 481 540 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ( (. .( .( (. .(. .(. . ( .( NOV11 541 Iv . 600 T
gi(18308012( 541 I TI i . 600 gi(9966807( 541 1 TI
gi(14749154( 541 1 ' ' 600 gi(10047205( 1 -_____ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 (..( ~ ~. .( . ~ ~..( . ( gi(18308012~ 601 660 gi(9966807( 601 660 30 gi(14749154( 601 660 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ _ gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(18308012( 661 720 gi(9966807( 661 720 gi(14749154( 661 720 40 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ___ gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 (. .( .(. ( .( ( (. ( .(. .
.(. . .( giI18308012( 721 780 gi~9966807( 721 780 gi(14749154( 721 780 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ( ( .~ .(. ( (. ( ( ~. .( .~.
5$ gi(18308012( 781 840 gi(9966807( 781 840 gi(14749154( 781 840 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 ( ~ ( ( ( ( NOV11 841 .. .(. .( . .~. .(. . . . . . . . .(.. 900 .( . . . .
gi(18308012( 841 900 65 gi(9966807( 841 900 gi(14749154( 841 900 gi(10047205( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 gi(14749150( 1 ______ ______ __________ ____ ________ ________ _____ _____ ____ ____ 1 g7 r ~ 1 1 1 1 1 ~I' v v ~ 0 r .(. .(. . . (.. . .(..
.~ .(. . .(. .(. .( .(.
.( 1 v v 1 ~ I 1 n 1 ~ 1 1 1 1 t' 1 ~ ' 1 '1 1 1 ' ~ r 1 giI10047205~ 845r v ~ ~~ v v' 904 ~ ~ v ~
gi~14749150~ 768~ ' ' ~ '~ ~ v 827 ~ v v ~
S y .y ~
~
y .
~.
y y ~.
~
y r -NOV11 1861 ~~ ~ ~ ~ '~ ~ 1920 ~
gi~183080121 1861 ~~ ~ ~ ~ ~ ~ 1920 ~
gi~9966807~ 1817_________________yL
S_____________G_______________ Q I 1832 S
gi~14749154~ 1817-________________~~L .S_-___________G_______________ Q I 1832 S
gi~10047205~ 905 ~~ ~ ~~ ~ ~ ~ 964 gi~14749150~ 828 ~~ ~ ~~ ~ ~ ~ 887 IS NOV11 1921. ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELICAFGIYLNKIKSCFTK
t gi~18308012~ 1921 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
~
a gi~9966807~ 1833T-__________________________________________________________ gi~14749154~ 1833T-__________________________________________________________ gi~10047205~ 965 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
~
gi~14749150~ 888 ~ y8____________________________________________ v T-~
AL'~'G
QSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
ZS gi~18308012~ 1981MT T ~Q~.~L"~'GQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
gi~9966807~ 1833___________________________________________________________ gi~14749154~ 1833___________________________________________________________ gi~10047205~ 1025MTK T LGKQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
G
gi~14749150~ 903~ ~ ~ 915 L~~"""T 1~C~
I' ________________________________________ gi~18308012~ 2041KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
3S gi~9966807~ 1833-___________________________________________________________ gi~14749154~ 1833____________________________________________________________ gi~10047205~ 1085KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
gi~14749150~ 915___________________________________________________________ gi~183080121 2101LDQLSLSEWDWEWSDDQAVFTFVYDTIQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
gi~9966807~ 1833-___________________________________________________________ 4S gi~14749154~
1833____________________________________________________________ gi~10047205~ 1145LDQLSLSEWDWEWSDDQAVFTFVYDTTQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
gi~14749150~ 915____________________________________________________________ gi.~18308012~ 2161DEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLWHHCRLLGEEIEYL
gi~9966807~ 1833-___________________________________________________________ gi~14749154, 1833____________________________________________________________ SS gi~10047205~
gi~14749150~ 915____________________________________________________________ gi I183080121 giI9966807~ 1833____________________________________________________________ gi~14749154~ 1833____________________________________________________________ gi.'10047205~
6S gi~14749150~
915____________________________________________________________ ....~....~....~....~....~....~....~.
gi~18308012~ 2281DDIATILSKVPLENNYLKNWKQIYQDLFQDCHFYH
giI9966807~ 1833 ____________________________________ 1833 gi~14749154~ 1833 ____________________________________ 1833 gi~100472051 1325 DDIATILSKVPLENNYLKNWICQIYQDLFQDCHFYH 1360 gi~14749150~ 915 ____________________________________ 915 NOV 11 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 11 nucleic acids and polypeptides can be used to identify proteins that are members of the Nuclear Protein-like Protein Family. The NOV 11 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV11 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, Graft vesus host, Cardiovascular diseases, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, as well as other diseases, disorders and conditions.
In addition, various NOV 11 nucleic acids and polypeptides according to the invention are useful, inter olio, as novel members of the protein families according to the presence of sequence relatedness to previously described proteins. The NOV 11 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune, and nerve physiology. As such, the NOV 11 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and nervous system disorders, e.g., Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, Graft vesus host, Cardiovascular diseases, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, as well as other diseases, disorders and conditions.
The NOV11 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 11 nucleic acid is expressed in Adipose, Aorta, Artery, Coronary Artery, Umbilical Vein, Thyroid, Liver, Small Intestine, Duodenum, Colon, Ascending Colon, Bone Marrow, Lymph node, Tonsils, Thymus, Cartilage, Muscle, Brain, Cervix, Uterus, Vulva, Prostate, Testis, Lmig, Bronchus, Urinary Bladder, Kidney, Skin, Epidermis, Dermis.
Additional utilities for NOV 11 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 12 polypeptide has been identified as a Plasma Membrane Protein-like protein (also referred to as CG94282-O1). The disclosed novel NOV12 nucleic acid (SEQ
ID N0:25) of 8811 nucleotides is shown in Table 12A. The novel NOV 12 nucleic acid sequences maps to the chromosome 12.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a TAG
codon at nucleotides 4378-4380. A putative untranslated region and/or downstream from the termination codon is underlined in Table 12A, and the start and stop, codons are in bold letters.
Table 12A. NOV12 Nucleotide Sequence (SEQ ID N0:25) ATGCTGTTCAAGCTCCTGCAGAGACAAACCTATACCTGCCTGTCCCACAGGTATGGGCTCTACGTGTGCTTCTT
GGGCGTCGTTGTCACCATCGTCTCCGCCTTCCAGTTCGGAGAGTGGGTAGAAGCCAGGGATCCTGCCAAACATC
CTATAGTGCACAGGACAGCCCCTACAACAAAGAATCATCCAGCCCAAAATGTCGATAGTGCTGAAGTTGAGAAA
TCCGGAATTAGAAGGGGCAAGAATGGCTGCAGGGCAGTTAGTCTACAGGACTGGCCTGGGACTAGAGGATGTGC
CAATTTCACCTTCGCCTTCTGCCATGATTGTAAGTTTTCTGAGGTCTCCCAGAAACGCTTCCTGTACATCCTGC
AGAACTGTCATTGGTTAACTGATTGGGGTTGGACTTGGTTGGCTCTGCTCCACGGGTCTCTCATCCTCCAGGGA
CCAGCCAGCGAACCTGGTTGTGTTCTTCTCAAGGCAAAGGTGGTTCTGGAATGGAGCCGAGATCAATACCATGT
TTTGTTTGATTCCTATAGAGACAATATTGCTGGAAAGTCCTTTCAGAATCGGCTTTGTCTGCCCATGCCGATTG
ACGTTGTTTACACCTGGGTGAATGGCACAGATCTTGAACTACTGAAGGAACTACAGCAGGTCAGAGAACAGATG
GAGGAGGAGCAGAAAGCAATGAGAGAAATCCTTGGGAAAAACACAACGGAACCTACTAAGAAGAGTGAGAAGCA
GTTAGAGTGTTTGCTAACACACTGCATTAAGGTGCCAATGCTTGTCCTGGACCCAGCCCTGCCAGCCAACATCA
CCCTGAAGGACCTGCCATCTCTTTATCCTTCTTTTCATTCTGCCAGTGACATTTTCAATGTTGCAAAACCAAAA
AACCCTTCTACCAATGTCTCAGTTGTTGTTTTTGACAGTACTAAGGATGGGACATTGCTCACTCAGAAGGTGAC
TTTTGAGTGGAAATGTGAAGAAGGTGAGGTAGCCAGCAATGCGAATATCTGGGGAAAGACTGATCTGGGTTCCC
CCAGGAGGCCTTTGCCATGGCCTGTGGCCCTGGAGCCACCTAGGGCTCAGCTCAGCTCTGCCCTACAGATTCTC
ACTAGGCCACGGGTATCTCAGGACAGAGCCAACACAAGTTATGAAATTAAACTAGACACACCCCTTCTTCGAGG
TTACGCCAAGCCAGTGCCTGGGCCTGAAACTGGCCTGCAGCCCCTCAGCTTCGCCCACTGCCTTCCGACCCTGG
ACCTTCGCAAAGTGAACGAGCTTCGGGACTTCGTGAAAATGTATAAGCAGGATCCGAGCATTCTGCATACCAAG
GAAACGTGCTTTCTGAGGGAGCAGGTGGAGAGCATGGGGGAAAGCTATTATAAATCAGAAGAAAATATCAAGGA
ATTAAAAACAGGTAGTAAGAAGGTGGAGGAAAACATAAGCACAGACGAACTATCAAGTGAGGAAAGTGATCTAG
AAATTGATAACGAAGCTGTGATTGAACCAGACACTGATTCCCCTCAAGAAATGGGAGATGGAGAGGCCAGTGTA
GCGCTTCTAAAACTGAATAACCCCAAGGATTTTCAAGAATTGAATAAGCAAACTAAGAAGAACATGACCATTGA
TGGAAAAGAACTGACCATAAGTCCTGCATATTTATTATGGGATCTGAGCGCCATCAGCCAGTCTAAGCAGGATG
AAGACATCTCTGCCAGTCGTTTTGAAGATAACGAAGAACTGAGGTACTCATTGCGATCTATCGAGAGGCATGCA
CCATGGGTTCGGAATATTTTCATTGTCACCAACGGGCAGATTCCATCCTGGCTGAACCTTGACAATCCTCGAGT
GACAATAGTAACACACCAGGATGTTTTTCGAAATTTGAGCCACTTGCCTACCTTTAGTTCACCTGCTATTGAAA
GTCACATTCATCGCATCGAAGGGCTGTCCCAGAAGTTTATTTACCTAAATGATGATGTCATGTTTGGGAAGGAT
GTCTGGCCAGATGATTTTTACAGTCACTCCAAAGGCCAGAAGGTTTATTTGACATGGCCTGTGCCAAACTGTGC
CGAGGGCTGCCCAGGTTCCTGGATTAAGGATGGCTATTGTGACAAGGCTTGTAATAATTCAGCCTGCGATTGGG
ATGGTGGGGATTGCTCTGGAAACAGTGGAGGGAGTCGCTATATTGCAGGAGGTGGAGGTACTGGGAGTATTGGA
GTTGGACAGCCCTGGCAGTTTGGTGGAGGAATAAACAGTGTCTCTTACTGTAATCAGGGATGTGCGAATTCCTG
GCTCGCTGATAAGTTCTGTGACCAAGCATGCAATGTCTTGTCCTGTGGGTTTGATGCTGGCGACTGTGGGCAAG
AAAACTCAGACTCAAAGAATAGGAAAACAGAGGAAAAATGCCCAGTT TCATGTTTCTGTTT
TTTCCTCTAGATCATTTTCATGAATTGTATAAAGTGATCCTTCTCCCAAACCAGACTCACTATATTATTCCAAA
AGGTGAATGCCTGCCTTATTTCAGCTTTGCAGAAGTAGCCAAAAGAGGAGTTGAAGGTGCCTATAGTGACAATC
CAATAATTCGACATGCTTCTATTGCCAACAAGTGGAAAACCATCCACCTCATAATGCACAGTGGAATGAATGCC
ACCACAATACATTTTAATCTCACGTTTCAAAATACAAACGATGAAGAGTTCAAAATGCAGATAACAGTGGAGGT
GGACACAAGGGAGGGACCAAAACTGAATTCTACAGCCCAGAAGGGTTACGAAAATTTAGTTAGTCCCATAACAC
TTCTTCCAGAGGCGGAAATCCTTTTTGAGGATATTCCCAAAGAAAAACGCTTCCCGAAGTTTAAGAGACATGAT
GTTAACTCAACAAGGAGAGCCCAGGAAGAGGTGAAAATTCCCCTGGTAAATATTTCACTCCTTCCAAAAGACGC
CCAGTTGAGTCTCAATACCTTGGATTTGCAACTGGAACATGGAGACATCACTTTGAAAGGATACAATTTGTCCA
AGTCAGCCTTGCTGAGATCATTTCTGATGAACTCACAGCATGCTAAAATAAAAAATCAAGCTATAATAACAGAT
GAAACAAATGACAGTTTGGTGGCTCCACAGGAAAAACAGGTTCATAAAAGCATCTTGCCAAACAGCTTAGGAGT
GTCTGAAAGATTGCAGAGGTTGACTTTTCCTGCAGTGAGTGTAAAAGTGAATGGTCATGACCAGGGTCAGAATC
CACCCCTGGACTTGGAGACCACAGCAAGATTTAGAGTGGAAACTCACACCCAAAAAACCATAGGCGGAAATGTG
ACAAAAGAAAAGCCCCCATCTCTGATTGTTCCACTGGAAAGCCAGATGACAAAAGAAAAGAAAATCACAGGGAA
AGAAAAAGAGAACAGTAGAATGGAGGAAAATGCTGAAAATCACATAGGCGTTACTGAAGTGTTACTTGGAAGAA
AGCTGCAGCATTACACAGATAGTTACTTGGGCTTTTTGCCATGGGAGAAAAAAAAGTATTTCCAAGATCTTCTC
GACGAAGAAGAGTCATTGAAGACACAATTGGCATACTTCACTGATAGCAAAAATACTGGGAGGCAACTAAAAGA
TACATTTGCAGATTCCCTCAGATATGTAAATAAAATTCTAAATAGCAAGTTTGGATTCACATCGCGGAAAGTCC
CTGCTCACATGCCTCACATGATTGACCGGATTGTTATGCAAGAACTGCAAGATATGTTCCCTGAAGAATTTGAC
AAGACGTCATTTCACAAAGTGCGCCATTCTGAGGATATGCAGTTTGCCTTCTCTTATTTTTATTATCTCATGAG
TGCAGTGCAGCCACTGAATATATCTCAAGTCTTTGATGAAGTTGATACAGATCAATCTGGTGTCTTGTCTGACA
GAGAAATCCGAACACTGGCTACCAGAATTCACGAACTGCCGTTAAGTTTGCAGGATTTGACAGGTCTGGAACAC
ATGCTAATAAATTGCTCAAAAATGCTTCCTGCTGATATCACGCAGCTAAATAATATTCCACCAACTCAGGAATC
CTACTATGATCCCAACCTGCCACCGGTCACTAAAAGTCTAGTAACAAACTGTAAACCAGTAACTGACAAAATCC
ACAAAGCATATAAGGACAAAAACAAATATAGGTTTGAAATCATGGGAGAAGAAGAAATCGCTTTTAAAATGATT
CGTACCAACGTTTCTCATGTGGTfiGGCCAGTTGGATGACATAAGAAAAAACCCfiAGGATCTCACTCTGTTGTCC
AAGCTGGAATGCAGTAATGCAAACATGGCTCACTGTAGCCTCGACCTCGTGGGCTCAAGCAATCCTCCCACCTC
AGCCTCCTGACTAGTGGAACCACAGACATGAGCTGCTGCACCCAGCTAAAATGGAGTATTTTTAATTTCTGGGT
CTTTTAAATGCATTTGGAGGTCTfifiAGTTTTACCTCACTGAAATTAGGATTTTAATTATAAATAATCAAAGATG
TGAACCTTACAGACATTTTAAAGCCATTATATTTTTTCTATAAACCCTGTTCTCGTTTGGAGGAGAAAGAAATT
GGAATTTTCP~AAA~1AAATAAAAATACCTTTAACACCTATTTAGTGTCTTTAGTAATCCAGTAAAATACTTGATT
TTTTACTAAATGTTTCCCACAAGCCAAGCAAACCATAAGCTACAATAATAAfiTACCTAGCGTACAGCCCTCTTT
GCATATGCTGTTCCCTCCACTTGAAGTGTACTGTTTAATTTCTTAAAATAACTTTAGCTTTTAAGAACCAATTT
TGATGGGAGTACAGACTTCCCCCATTTTCTTGATGAGTTCTCTCCGTCATGTGTAGTAATAATGTGAGAATTTG
CAGTTTTTAGTTGTAGCCTATACTTTTAGGTCTTTGTGCCAATTTGAAAGTfiATTGGGTTAGAGTATTCATAGA
CATTTTCATGGTACTTAAAGGGACAGGGGTTTAGTAAAAAGACACATGGCAAGCCAGGCTTTTTCCACAGTTTG
CCAGGCCCAGCTGCCTCTTGTGTACCTGAACAGATTTTATCATTAACCCTTGTTTATGTTGTTTTGTTTTATTT
CGACGAAGGCTTATTTTAAGTCAGGCATGGAAAACTAGACTTCAGACTGACTTCAGCTTTAAGGACATGTTTAT
CCCGTTAACAGGGAGTCTGGGATAGACAATCTCCAGGCTTTGTTTTTCTCTGAATTTCTTAGCTCTGCTTGTGA
TGGCTTCATCATCAGGCCACAGACCATTAACACATTCTAGAACTTTAACATTGGTTAAATAATACCATCTAATA
GCCTGTCTTCAGCATTTCCCCAGTTGCCTCCAAATGCCCTTCATAGCTGTTCTCTGCCTCTGTTTGTTTTTAAT
CCAAGATACACTCAAGGCTCATATATTAGGTTGACATAGCTCTTTAGTATCCTTTAATTTAAAGCAGTCTCCAG
GTTTAGAGAAAGATGAATGAGCTTTCACATACCCCTCACTTGTCTTCTTCAGAAGTGTAGGCTACAACTAAAAC
TTCCTTCTTCAGAAGGAAGACAAGTGATTTATATTTATTTACTTCCATTTCTATTTGACCTTGTTTCATCTAAA
CACACCCACTCCACCACTGCTACCTGATTAATATTAAGTGAATCTCAAACATTGTATCATTTTAGCTCCACGTT
TTTTGTATGTATCTCCAAAATATAAAGATTCTTAAAAATATAACCACAATACCATTATCACCCTAAAAAAATCA
ATAATGATTCCTTAATATGACCAACTACTTTGTCAATGTACACCTTTCACTCCTCTTAACTTTCATAAAGACTT
ATGTGTTTTTTTGGTTTTTAAGTTTGTTGGTTTGAAGTTAAATCTATGGGTTTTCCCTCCATCTCTCTTTTTTT
AACCGTATAATTTTTTGCATGTGTATATGAATAAATCTGATTATAGATTCTATAGCTATCTTACACTTTGTCCC
TCTCTGATTGAATCCTAGTTAACAAGTTTCTATGTCTCTTGTATTTCCCATAAATTGGTAGTTGGATCTGAAGG
CTTTATCAGGTTTGTTTGATTTTTTTTTTTTTAATTTTGGCGAATCTACTTCAAAAGTATTGGCCTACCTACAA
GCCACTTTAATGGGCCCTTAGTTTAGTGACCTTTGCCTTGAAAGGAACTTGAAACAAGCAAGGAAGCACCACTG
TAATCTGCTTTTTTGCCAGAACTGTAGCATCTTACAGCTTGGTTAGAGACATAGTAAGCAGAAATTATCAAATT
CATATAATCTGTAGCTATAAGGCACTGTCTCTCTCTCTCAATTATTTACATGATTTTTCTTTGTAATATAACTA
TCATTTCAGAGAACTTGGTTTTGATTTTTTTTTTTTAATCTTTTTGAGACAGAGTCTCGCTTTATCACCCAGGC
TGGAGTGCAGTGGTGCAATCfiAAAGATTGCTGACTGCAACCTCTGCCTCCCGAGTTCAGCAATTCTAGTGCCTC
AGCCTCTCGAGTAGCTGGGATTACAGGCATGCCACCACACCCGGCTAATTTTTTTGTATTTTTAGTAAAGACAG
GGTTTCACCATGTTGGCTAGGCTGGTCTCAAATTTTTGACCTCAAGTAATCAGCCTACCTTGATCTCCCAAAGT
GCTGGGATTACAGGCATGAGCCACCATGCATGGCCTTCAGAGAACTTGGTTTTAGGTACTTACGGATTGTCTTT
CTTTTTTTTCCTCACTGCAGCCTCTCCCTCCCAGGTTCAAGCGATTCTCCTACCTCAGCTTCCTGAAGAGCTGG
GACCACAGGAAGTTTGTTTGCCTGAATGACAACATTGACCACAATCATAAAGATGCTCAGACAGTGAAGGCTGT
TCTCAGGGACTTCTATGAATCCATGTTCCCCATACCTTCCCAATTTGAACTGCCAAGAGAGTATCGAAACCGTT
TCCTTCATATGCATGAGCTGCAGGAATGGAGGGCTTATCGAGACAAATTGAAGTTTTGGACCCATTGTGTACTA
GCAACATTGATTATGTTTACTATATTCTCATTTTTTGCTGAGCAGTTAATTGCACTTAAGCGGAAGATATTTCC
CAGAAGGAGGATACACAAAGAAGCTAGTCCCAATCGAATCAGAGTATAGAAGATCTTCATTTGAAAACCATCTA
CCTCAGCATTTACTGAGCATTTTAAAACTCAGCTTCACAGAGATGTCTTTGTGATGTGATGCTTAGCAGTTTGG
CCCGAAGAAGGAAAATATCCAGTACCATGCTGTTTTGTGGCATGAATATAGCCCACTGACCAGGAATTATTTAA
CCAACCCACTGAAAACTTGTGTGTTG_AGCAGCTCTGAACTGATTTTACTTTTAAAGAATTTGCTCATGGACCTG
TCATCCTTTTTATAAAAAGGCTCACTGACAAGAGACAGCTGTTAATTTCCCACAGCAATCATTGCAGACTAACT
TTATTAGGAGAAGCCTATGCCAGCTGGGAGTGATTGCTAAGAGGCTCCAGTCTTTGCATTCCAAAGCCTTTTGC
TAAAGTTTTGCACTTTTTTTTTTTCATTTCCCATTTTTAAGTAGTTACTAAGTTAACTAGTTATTCTTGCTTCT
GAGTATAACGAATTGGGATGTCTAAACCTATTTTTATAGATGTTATTTAAATAATGCAGCAATATCACCTCTTA
TTGACAATACCTAAATTATGAGTTTTATTAATATTTAAGACTGTAAATGGTCTTAAACCACTAACTACTGAAGA
GCTCAATGATTGACATCTGAAATGCTTTGTAATTATTGACTTCAGCCCCTAAGAATGCTATGATTTCACGTGCA
GGTCTAATTTCAAAGGGCTAGAGTTAGTACTACTTACCAGATGTAATTATGTTTTGGAAATGTACATATTCAAA
CAGAAGTGCCTCATTTTAGAAATGAGTAGTGCTGATGGCACTGGCACATTACAGTGGTGTCTTGTTTAATACTC
ATTGGTATATTCCAGTAGCTATCTCTCTCAGTTGGTTTTTGATAGAACAGAGGCCAGCAAACTTTCTTTGTAAA
AGGCTGGTTAGTAAATTATTGCAGGCCACCTGTGTCTTTGTCATACATTCTTCTTGCTGTTGTTTAGTTTGTTT
TTTTTCAAACAACCCTCTAAAAATGTAAAAACCATGTTTAGCTTGCAGCTGTACAAAAACTGCCCACCAGCCAG
ATGTGACCCTCAGGCCATCATTTGCCAATCACTGAGAATTAGTTTTTGTTGTTGTTGTTGTTGTTGTTTTTGAG
ACAGAGTCTCTCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAACCTCCGCCTCCCGG
GTTCAAGCAGTTCTGTCTCAGCCTTCTGAGTAGCTGGGACTACAGGTGCATGCCACCACACCCTGCTAATTTTT
GTATTTTTAGTAGAGACGGGGGTTCCACCATATTGGTCAGGCTTATCTTGAACTCCTGACCTCAGGTGATCCAC
CTGCCTCTGCCTCCCAAAGTGCTGAGATTACAGGCATAAGCCAGTGCACCCAGCCGAGAATTAGTATTTTTATG
TATGGTTAAACCTTGGCGTCTAGCCATATTTTATGTCATAATACAATGGATTTGTGAAGAGCAGATTCCATGAG
TAACTCTGACAGGTATTTTAGATCATGATCTCAACAATATTCTTCCAAAATGGCATACATCTTTTGTACAAAGA
ACTTGAAATGTAAATACTGTGTTTGTGCTGTAAGAGTTGTGTATTTCAAAAACTGAAATCTCATAAAAAGTTAA
ATTTT
Variant sequences of NOV12 are included in Example 3, Table 24. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 12 protein (SEQ ID N0:26) encoded by SEQ TLS N0:25 is 1459 amino acid residues in length and is presented using the one-letter amino acid code in Table 12B. Psort analysis predicts the NOV 12 protein of the invention to be localized at the plasma membrane with a certainty of 0.6500.
Table 12B. Encoded NOV12 protein sequence (SEQ ID NO:26) MLFKLLQRQTYTCLSHRYGLYVCFLGVWTIVSAFQFGEWVEARDPAKHPIVHRTAPTTKNHPAQ
NVDSAEVEKSGIRRGKNGCRAVSLQDWPGTRGCANFTFAFCHDCKFSEVSQKRFLYILQNCHWLT
DWGWTWLALLHGSLILQGPASEPGCVLLKAKWLEWSRDQYHVLFDSYRDNIAGKSFQNRLCLPM
PIDVVYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKNTTEPTKKSEKQLECLLTHCIKVPM
LVLDPALPANITLKDLPSLYPSFHSASDIFNVAKPKNPSTNVSVWFDSTKDGTLLTQKVTFEWK
CEEGEVASNANIWGKTDLGSPRRPLPWPVALEPPRAQLSSALQILTRPRVSQDRANTSYEIKLDT
PLLRGYAKPVPGPETGLQPLSFAHCLPTLDLRKVNELRDFVKMYKQDPSILHTKETCFLREQVES
MGESYYKSEENIKELKTGSKKVEENISTDELSSEESDLEIDNEAVIEPDTDSPQEMGDGEASVAL
LKLNNPKDFQELNKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDISASRFEDNEELRYSL
RSIERHAPWVRNIFIVTNGQIPSWLNLDNPRVTIVTHQDVFRNLSHLPTFSSPAIESHIHRIEGL
SQKFIYLNDDVMFGKDVWPDDFYSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNSACDW
DGGDCSGNSGGSRYIAGGGGTGSIGVGQPWQFGGGINSVSYCNQGCANSWLADKFCDQACNVLSC
GFDAGDCGQENSDSKNRKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLPY
FSFAEVAKRGVEGAYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNTNDEEFKMQIT
VEVDTREGPKLNSTAQKGYENLVSPITLLPEAEILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKI
PLVNISLLPKDAQLSLNTLDLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITDETND
SLVAPQEKQVHKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDLETTARFRVETHTQKT
IGGNVTKEKPPSLIVPLESQMTKEKKITGKEKENSRMEENAENHIGVTEVLLGRKLQHYTDSYLG
FLPWEKKKYFQDLLDEEESLKTQLAYFTDSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPA
HMPHMIDRIVMQELQDMFPEEFDKTSFHKVRHSEDMQFAFSYFYYLMSAVQPLNTSQVFDEVDTD
QSGVLSDREIRTLATRIHELPLSLQDLTGLEHMLINCSKMLPADTTQLNNIPPTQESYYDPNLPP
VTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIAFKMIRTNVSHWGQLDDIRKNPRISLCC
PSWNAVMQTWLTVASTSWAQAILPPQPPD
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 12C.
Table 12C. results Patp for NOV12 Smallest Sum eading igh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:ABB30279Peptide#2930 encodedby breast cell+1 1900 7.5e-196 >patp:AAM56268Human single +l 1900 7.5e-196 brain exon expressed probe >patp:AAM16457Peptide#2891 encodedby probe +1 1900 7.5e-196 >patp:AAM28952Peptide#2989 encodedby probe +1 1900 7.5e-196 >patp:AAM04186Peptide#2868 encodedby probe +1 1900 7.5e-196 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 6444 of 6447 bases (99%) identical to a gb:GENBANK-m:AB033034~acc:AB033034.1 mRNA from Homo sapiens (mRNA for I~A_A1208 protein, partial cds). The full amino acid sequence of the protein of the invention was found to have 663 of 663 amino acid residues (100%) identical to, and 663 of 663 amino acid residues (100%) similar to, the 663 amino acid residue ptnr:SPTREMBL-ACC:Q9ULL2 protein from Homo sapiehs (KIAA1208 PROTEIN).
NOV 12 also has homology to the proteins shown in the BLASTP data in Table 12D.
Table 12D. BLASTresults for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) gi~6382022~dbj~BAA8KIAA1208 protein663 663/663 663/663 0.0 6522.1~(AB033034)[Homo sapiens] (1000 (1000 gi~16551459~dbjIBABunnamed protein847 585/613 585/613 0.0 71102.1~(AK056137)product (95%) (95%) [Homo sapiensl gi~2137411~pir~~I49hypothetical 384 277/400 307/400 e-142 528 protein (69%) (76%) [Mus musculus]
gi~11360271~pir~~T5hypothetical 248 134/137 135/l37 2e-73 0618 protein (97%) (97%) DKFZp762B226.1 [Homo sapiens]
gi~7303923~gb~AAF58CG8027 gene 652 84/l55 114/155 9e-49 967.1~(AE003834)product (54%) (73%) [Drosophila melanogaster]
A multiple sequence alignment is given in Table 12E, with the NOV 12 protein being shown on line 1 in Table 12E in a ClustalW analysis, and comparing the NOV 12 protein with the related protein sequences shown in Table 12D. This BLASTP data is displayed graphically in the ClustalW in Table 12E.
Table 12E. ClustalW Analysis of NOV12 1) > NOV12; SEQ >D N0:26 2) > gi~6382022~/ KIAA1208 protein [Homo sapiens]; SEQ m NO:87 3) > gi~16551459~/ unnamed protein product [Homo sapieras]; SEQ m N0:88 4) > gi~2137411 ~/ hypothetical protein - mouse (fragment); SEQ >D N0:89 5) > gi~11360271~1 hypothetical protein DKFZp762B226.1 - human (fragment); SEQ
II? N0;90 6) > gi~7303923~/ CG8027 gene product [Drosoplaila naelanogaster]; SEQ II7 N0:91 gi~63820221 1 RKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLP------------ 48 gi~165514591 1 ----MLFKLLQRQTYTCLSHRYGLWCFLGVWTIVSAFQF------------------- 37 gi~2137411~ 1 ____________________________________________________-_______ 1 giI113602711 1 ____________________________________________________________ 1 gi~7303923~ 1 ____________________________________________________________ 1 gi~63820221 48 ___________________________________________________________ gi~16551459~ 37 _______________________G____________________________________ 3g gi121374111 1 ____________________________________________________________ 1 gi~11360271~ 1 ____________________________________________________________ 1 gi~7303923~ 1 ___________-________________________________________________ 1 giI63820221 48 ____________________________________________YFSFAEVA--KRGVEG 62 gi~16551459~ 38 --------------------------------------------EWLEWSRDQYHVLFD 54 g I I ______ 1 a. 2137411 1 ______________________________________________________ gi~11360271~ 1 ___________________________________________________________ ' - 1 giI73039231 1 _______________________________________-____________________ 1 gi~6382022~ 63 AYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNT-NDEEFKMQITVEVDTRE
4S gi1165514591 55 SYRDNIAGKSFQNRLCLPMPIDWYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKN
gi~2137411~ 1 ____________________________________________________________ 1 gi~11360271~ 1 __________________________________________________pTRPVFDEVD 10 gi17303923~ 1 ____________________________________________________________ NOV12 237 TTI.~~PTKKSE~QL C LTHCI LVLDPALP~TT;fr~~L'SLYPSFHSASDIFNV~ ~P 296 S giI63820221 122 GP~GLNST~.Q~GY PITL~.,~------- E~~ELFyT~--------------- ,E'' gi ~ 16551459 ~ 115 TT~PTKKSE 'QL C T.~'1~HCI ' VLDPALP~~TI~ ~L
~SLYPSFHSASDIFNV P 174 v L
gi12137411~ 1 - ~-~-ILT~!'~ Y S PVTP~~_- ____QiD~~PFEv ~ _______________ ~E 32 gi ~ 11360271 ~ 1l TD~'~SGVL~D~EIRT V'~!RIHE~ ---- ---~T.iST~QyT---------------gi~73039231 1 ---MEQLRLC~SRR IiALTMTGG---------LC~~WV~;AN------------------ 30 NOV12 297 NPSTNVS .STDGTLLTQKVTFEWKCE~.'GEVASNANIWGKTDLGSPRRPLPWPVAL 356 gi~63820221 158 R--_______ PKF______________H~S-________________________ 169 LS gi~16551459~ 175 NPSTNVS STUD-------------VE~AHS-------G-----------------gi~2137411~ 33 R-________ PKIR______________H~A-________________________ 44 gi~11360271~ 40 _________________G_____________'.____________________________ gi~7303923~ 30 ____________________________________________________________ gi~6382022~ 169 ______________________________-_______________________~R____ gi1165514591 197 ---------LLKGNSRQTVWRGYLTT------D--------KEVPG-LVLMQDLAFLSGF
ZS gi~2137411~ 44 ______________________________________________________TG____ gi~11360271~ 41 ____________________________________________________________ gi~7303923~ 30 ___________________________________________________________ gi~6382022~ 171 ___________________________________________________________172 gi116551459~ 234 P-_____________pTFK--________________ET__N__________QLKT-__246 gi~21374111 46 ___________________________________________________________47 3S gi~11360271~ 41 ____________________________________________________________ 41 giI7303923~ 30 ____________________________________________________________ 30 NOV12 477 VE~ ISTDELSSEESDLE~DNEA'~'IEPDTDSPQEMGDGEASi~F'~- KLNNPKDFQE ..~T
gi I 6382022 ~ 173 AQ E--------------~~CIPI,~----------------I~ P~CDAQLSLNT
~L~L 202 gi~165514591 247 LP ----LSS-----K~t'T~LLQI~Y-----S-------EASE KLNNPKDFQE ~T
gi121374111 48 FQ E______________~IP~-______________~~E P,EAQVRLS vLvL 77 gi~11360271~ 41 __________________~~~L-_______________ __CSNILPADITQ ' IP 64 4S gi~73039231 30 --------------------YLSA--------------------EGQTGGFSSACTAI
~..:
NOV12 537 KN ~. KELTI P~ DL SQ LCQ~DISA'~~FEDN~ L~; . RS'~EiAPWV 595 S0 gi I 6382022 I 203 ~HGDIj --YN~ K~ R-- F I~HAIKNQ~i;II- TD TND -VAPf-----gi l 16551459 l 285 ~KN ~ KELT,I PY DL 5~'Q~DISA~FEDN~ L RSrI~E,~iFiAPWV 343 gi ~ 2137411 I 78 RGD' --YNN~.~ KR-- F.iG'~'~yLDT ~IKPQ --T~ T~fiG E'V'P~-----gi ~ 11360271 ~ 65 PTQESYY~.7PNLPP~TICLV'TN-=-=-CKPVTyIHKAY~----~3 ~ RFEIG~----SS g1 ~ 7303923 I 51 DAVYTW~V~?'1~- --SDPFED- - --IR'T~F~DKYDP~FDDK~~RST.tEEiAAWI 100 ~..v. ~ . . ~,"
NOVl2 596 RNIFIVTNGQIPSWLNL~YNPRVTISTTHQDVFRNLSH PTFSS"'I~ESH.I~R~EGLSQK~I 655 gi ~ 6382022 I 250 -----------------KQVHKS:~~-LPNSLGVSE QRL'I'EF" ' SV
GHI~QGQNPP., 292 E)0 gi ~ 16551459 I 344 RNIFIVTNGQIPSWLNL~NPRVTITHQDVFRNLSH
PT~FSS"IES~R~E~GLS~",'?KQI, 403 gi~21374111 124 ------------- --E"NPSHR---RPHGFAGEHRSER~IT ETVT~KG~ HALNPPP
gi~113602711 110 ------------- --EIAFK-------------IRT,t~VSH~GQiDKNP~. 140 gi I 7303923 I 101 RHVYIVTNGQIPSWLDLSYERVTV~~pHEVLAPDPDQ~PT~SS~31ETFL~t~PKLS~~I~ 160 NOV12 656 Y ~ 'VMFGKDVWPD~FY~HSKGQI~''V~'YLTWP~' CAEGC~ KDGYC~I~ACNNSACDW 715 gi~6382022~ 293 D ETTARFRVETHT''~;,,'3KTIGGNVTKKPPSL~ -----LE QI~TK- ~KKIT-----gi I 16551459 ~ 404 Y ~ VMFGKDVWPDpFYwaHSKG'QKV[l"LTWP~ ' CAEGC
~KDGYC~'JK'ACNNSACDW 463 gi~21374111 165 ET~i'CARL-----APTLGVTVS.'fO~NLSPL~ -----8E H~iPK- ~---------giI11360271~ 141 C ~ IDHN--- ~ ~ Q"TVKAVLRD3k"'YESF ------- -- --------- 169 giI73039231 161 YI~FLGAPLYP~LY~'EAEGV~VQAWGCALDC~I~I'YGDGAC~RHCNIDACQF 220 NOV12 716 DGGC GNSGGRYIAGGGGTGSIGVGQPWQFGGGI S~SYCNQGCANSW ~FCDAC 775 gi~6382022~ 339 KE RMEENE---------------------- H1;G------VTE LG1~KLQHYT 369 gi~165514591 464 DGGT7C GNSGGSRYIAGGGGTGSIGVGQPWQFGGGI S'~t~/,SYCNQGCANSW
~FCD'~~A'C 523 gi~2137411~ 201 - SDRAEG------------------------- VlP------VKEL~PG~'tRCSII 226 l~ gi~11360271~ 169 ___.'________________________________Ip'________SQFEpPREYRNFL 185 giI7303923~ 221 DGG~C~ETGPA--------------------DAHVIPPSKEVLEVQPAA'V'PQSRVH~tFP
IS NOV12 776 NVLSC ~.AGDCGENSDSKNRKTEEKCPVKKKKIMFLFFPLD .YKVILLP~t~THY 835 gi~63820221 370 DSY-L LPWEKK--------------------------- Q~ LDEEESLKT-Q- 397 gi1165514591 524 NVLSC AGDCG----------------------------D~ YKVILLPTHY 555 gi~2137411~ 227 Q--- CPGKKIC-----------------------------~ Q~7 LDAEESLKT-Q-gi~11360271~ 186 H-__________ '._______________________________ QEWRAYR~;7IC___ 199 gi~7303923~ 261 QMGLQKLFRRSSANFKDVMRHRN----------'-------VSTL RRIVERF~T~AKL
NOV12 836 IIPKGEC P FA~VA~ G'~TGA~'S~ :PIIRHASIANKWT~I~L ~ GMN I'IHFNL 895 gi~6382022~ 397 ------- ~'D~~NT ,QL_i~DTF ~ -----------LL KFGF RKVPA 439 gi ~ 16551459 ~ 556 IIPKGEC P FVA G~,GA~~~ PIIRHASIANKWY'~L GMN '.~'IHFNL 615 gi ~ 2137411 ~ 251 ------ D ~CHT Q',~.yDT~.~' y-- -L( L KFGF RKVPA 293 gi I 11360271 I 199 ------- K.~'WT'HCVLATLIFTI~3.~'~ ------------- - ---- 7----gi~7303923~ 303 MSLN--PELETSk~'"PQTTQRHGLRKyDFKSSTD2YSHSLIAT~~RAYGFKARHVLA 360 30 _.~:., ":.~,.~ ...~ ..) ..~....~. ~ .~. ~ ~ .v NOV12 896 TFQT BEEF ~ eIT 'CDT GP ~LTSTAQGY VSPTLLPEAEILFD,IPKEn~~ 955 gi I 6382022 I 440 HMPMI ~R-I ~E~Q~~FP;~''t, FD ~T FH;V~iHS
FAF~aYFYYLMSAVP~sNISE,~ 498 35 gi ~ 16551459 ~ 616 TFQT BEEF ~ITV'C~DT GP ~L~STAQ~~GY
VSPTLLPEAEILF:DI'PKE~ 675 gi ~ 2137411 ~ 294 HMPMI ~R-I ~E'~.cQpt~FP~ FD ~Tc~SFIi~~V~'t'HS
NIFAFi~YFYYLMSAV~P~.rI~TIS'352 gi ~ 11360271 ~ 218 ------- FFAQIP.~jKR'~I_FP~2RRIH~CEASPNR~V------- - --- 248 gi I 7303923 I 361 HVGFLI~K
DI'EAQRRFHt~(;~TLDTAHQF3~2APT~1~,,YAFAYYSFLMSET~SVE'rI~ 419 40 970 980 990 1000 ' 1010 1020 NOV12 956 P'~FKRHD TRR,A,QE.~ICIPL SL.'KDALSLNTLD~:iQLEHG DTLKGYNLSK 1012 gi~6382022~ 499 DVDTD--Q GVLSD IRTLAT HE 'LSLDLTGLEHLINCS ICMLPADITQLN 553 gi~165514591 676 PFKRHD TRR'AQE ~CIPL SL 'KDA~LSLNTLD1.~.~r~,QLEHG DkTLKGYNLSK
gi~2137411~ 353 HVDTD--Q GVLDRF~TLATE2 HD ~LTCI------------ --------- 384 gi~11360271~ 248 -____________.____.__________________________________-__-__ gi~7303923~ 420 DFDTDG--~ATWDRTFLTR~YQP~LDWSAMRYFEEz'~VQNCTRNLGT~HLKVDTVEH 477 NOV12 1013 ALLRSFLMNSQHAKIKNQAITD-ETNDSLVAPQE VHKSILPNSLGVSERLQR'~C.tTFP 1071 gi~6382022~ 554 ~IPPTQESYYDPNLPPVTKS~~V;TN-CKPVTDKIHKAY~KNKYRFEIMGEEEIAFKIRT
gi.~16551459~ 733 kALLRSFLMNSQHAKIKNQA,STD-ETNDSLVAPQEIC~1HKSILPNSLGVSERLQR~TFP 791 gi~2137411~ 384 .______-______,_____________________________________________ gi~11360271~ 248 __________________-_________________________________________ gi~7303923~ 478 TLVYERYEDSNLPTITRDLRCPLLAEALAANFAV1PKYNFHVSPKRTSHSNFM~FLTS 537 CO NOV12 1072 A.'~IS'V G ~QG~7NP'LDLETTARFRVETHTQKTIGGNVTICKPPSLI 'LESQMTKEKK
gi~6382022~ 613 NVS GQL~ I~ 'RISLCCPSWNAVMQTWLTVASTSWA~øAILPPQP'D--------- 663 gi~16551459~ 792 A~~GH~GNP'LDLETTARFRVETHTQKTIGGNVTIC~F.yKPPSLI 'LESQMT----gi~2137411~ 384 _____-______________________________-_______________________ gi~11360271~ 248 ____________________________________________________________ GS gi~7303923~ 538 N~'~',EV~ESLQRLf~RIVQRKFNCINDNLDANRGEDNEMVRHLLDFYLSFFQRRSKFELPPQ 597 giI6382022~ 663 __________-_________________________________________________ gi116551459~ 847 ____________________________________________________________ gi~2137411~ 384 __________________________________________-_____________-___ gi~11360271~ 248 __________________________________,_________________________ gi~7303923~ 598 YR1VRFESWRDFQRWKRRKR.AVLVIGYGVSLLLWCLLRFMCHHKAKLVRRCVQRL-----The NOV 12 Clustal W alignment shown in Table 12E was modified to end at amino residue 1200. The data in Table 1E includes all of the regions overlapping with the NOV12 protein sequences.
NOV12 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV12 nucleic acids and polypeptides can be used to identify proteins that are members of the Plasma Membrane Protein-like Protein Family. The NOV12 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV12 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and signal transduction. These molecules can be used to treat, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventriculax (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Sclerodenna, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington°s disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
In addition, various NOV 12 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the sequence relatedness to previously described proteins. The NOV 12 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, nerve, and immune physiology. As such, the NOV12 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, immune, and nervous system disorders, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other diseases, disorders and conditions.
The NOV12 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 12 nucleic acid is expressed in Pancreas, Uterus, Epidermis, Heart, Coronary Artery, Adrenal GlandlSuprarenal gland, Pancreas, Parathyroid Gland, Salivary Glands, Liver, Small Intestine, Bone Marrow, Peripheral Blood, Lymphoid tissue, Lymph node, Cartilage, Brain, Hypothalamus, Spinal Chord, Mammary glandBreast, Uterus, Prostate, Testis, Lung, Kidney, Epidermis, Hair Follicle.
Additional utilities for NOV 12 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV13 polypeptide has been identified as a BHLH Factor MATH6-like protein (also referred to as CG94399-O1). The disclosed novel NOV13 nucleic acid (SEQ
ID N0:27) of 2244 nucleotides is shown in Table 13A. The novel NOV 13 nucleic acid sequences maps to the chromosome 2.
An ORF begins with an ATG initiation codon at nucleotides 105-107 and ends with a TGA codon at nucleotides 1062-1064. A putative untranslated region and/or downstream from the termination codon is underlined in Table 13A, and the start and stop codons are in bold letters.
Table 13A. NOV13 Nucleotide Sequence (SEQ ID N0:27) ACGCGTGAAGGGCGGGCGAAGCGGGAGAGCCAGAGACTCCTCGGCGCTGAGCGCGGCGGCGGCCCGG
GCAGCCCCACGCCCCTGCCTCGCGCGCCGCCCGCGCCATGAAGCACATCCCGGTCCTCGAGGACGGG
.CCGTGGAAGACCGTGTGCGTGAAGGAGCTGAACGGCCTTAAGAAGCTCAAGCGGAAAGGCAAGGAGC
CGGCGCGGCGCGCGAACGGCTATAAAACTTTCCGACTGGACTTGGAAGCGCCCGAGCCCCGCGCCGT
AGCCACCAACGGGCTGCGGGACAGGACCCATCGGCTGCAGCCGGTCCCGGTACCGGTCCGGTGCCAG
TCCCAGTGGCGCCGGCCGTTCCCCCAAGAGGGGGCACGGACACAGCCGGGGAGCGCGGGGGCTCTCG
GGCGCCCGAGGTCTCCGACGCGCGGAAACGTGCTTCGCCCTAGGCGCAGTGGGGCCAGGACTCCCCA
CGCCGCCGCCGCCGCCGCCTCCTGCGCCCCAGAGCCAGGCACCTGGGGGCCCAGAGGCACAGCCTTT
CGGGAGCCGGGTCTGCGTCCTCGCATCTTGCTGTGCGCACCGCCCGCGCCCCGCGCCGTCAGCACCC
CCAGCACCGCCAGCGCCCCCGGAGTCCACTGTGCGCCCTGGCCCCCGACGCGCCCCGGGGAAAGTTC
CTACTCGTCAATTTCACACGTAATTTACAATAACCACCAGGATTCCTCCGCGTCGCCTAGGAAACGA
CCGGGCGAAGCGACTGCCGCCTCCTCCGAGATCAAAGCCCTGCAGCAGACCCGGAGGCTCCTGGCGA
ACGCCAGGGAGCGGACGCGGGTGCACACCATCAGCGCAGCCTTCGAGGCGCTCAGGAAGCAGGTGCC
GTGCTACTCATATGGGCAGAAGCTGTCCAAACTGGCCATCCTGAGGATCGCCTGTAACTACATCCTG
TCCCTGGCGCGGCTGGCTGACCTTGACTACAGTGCCGACCACAGCAACCTCAGCTTCTCCGAGTGTG
TGCAGCGCTGCACCCGCACCCTGCAGGCCGAGGGACGTGCCAAGAAGCGCAAGGAGTGACTGGCTGC
AGGCAAGACCAAGGCCACCACTGTGGGCCCTCCTTCCAGTCAGGCCTGAGGACAAGGTGAGCTCGCT
GAGTCCAGCCTCGTGGTCTTCTCCAAGATGGCGCCCCACTTGGAGCCTACAGCCTCTCAGGGTCGGA
TCGGAGCACGCCTGCCTCCCTCTCCCCTCCGCCCTCACCCAGCCAATCCGAGGCTGCTTCGCACGTT
GCCCTCTGCCTGGTGGGGAGGGGAGAGCTCAGCCCCCGACTCACTCAGACCCCAAGGCCCACTGTCC
AGCTGCAGAAATTCGTTGCCAAAGATTGGACAGAGACACCGAAGGAAATGGGGTGGTGAAACCCCAC
AGCGAAAAGCCACACCGTTGCTCTGTGACTTTTGCTCCTCCTGTTGCCTGAGCCCCATCTCAAGCCA
AAGATGAGTCAGTGGTTCTGCTAGGAACTCATGGAATGGATGGGCATTTGATGACCCCTGGGGGTCA
TCTTGGCCCTCTGACCTGGTGCTCTCTCTCCACTGGGCCTTGTGCTGGTTGAGTGCAAGACAAGCCT
TAGGGGCTGTGAGAGGGAGGCTGGGGTGCCTGGGCGGGGCTGGGAGTGGGACCTGAGATCCCTGCCC
ACTCTCTCCCCTTCATTGGCTTGCCCAGGCCACTGGCCCCAGTTCTCAGTGTCCCTTGGGGTCCAGG
CTCCTTGGGCCCTAAGCATCACCAGAAGGGAGTAAGCAGGGAGAGAAGCAATATTACTCCCTCCCCT
ACACCAGGGACTTGCCCCAGGGCGGCTACCTATGGGTCTTTGCTTCCCCAGCCAGCCTCTCCTCACT
GTGACCCACCCCCATGGGCCCCCGTCCCAGGCAGCCAGCACCATGGGCAGGCCCTGCCATGGACAGA
AAAAGAGTTTTTCTCTTGTTCAGCCTGCACGTGGCCTGAGGAAGGAGTAGAGGCTGGGTTGGCTGGA
GCCGTCCTACTGGGCAAGATGGCGCCCCACTTGGAGGGCGGTGGTCTGTTACAGGGTGTGCAGGGGC
AGAGAAGGAAGGGACCAGGGGACTGGGCCAGTATGTGGAGGATGGGGCCTGCGTGTTCAAAGCCAAG
GCCCGCCCCTTCCTTGTGCTCAAATGGCCAAAGCTGTTCACGTCTGTGCTCAACCATCTGCTTCAAA
TTGAAGTAAAAGCCCCAAAATGTCAAGAAA.AAA
Variant sequences of NOV13 are included in Example 3, Table 25. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 13 protein (SEQ m N0:28) encoded by SEQ m N0:27 is 319 amino acid residues in length and is presented using the one-letter amino acid code in Table 13B. Psort analysis predicts the NOV 13 protein of the invention to be localized at the nucleus with a certainty of 0.7000.
Table 13B. Encoded NOV13 protein sequence (SEQ ID N0:28) MKHIPVLEDGPWKTVCVKELNGLKKLKRKGKEPARRANGYKTFRLDLEAPEPRAVATNGLRDRTH
RLQPVPVPVRCQSQWRRPFPQEGARTQPGSAGALGRPRSPTRGNVLRPRRSGARTPHAAAAAASC
APEPGTWGPRGTAFREPGLRPRILLCAPPAPRAVSTPSTASAPGVHCAPWPPTRPGESSYSSISH
VIYNNHQDSSASPRKRPGEATAASSEIKALQQTRRLLANARERTRVHTISAAFEALRKQVPCYSY
GQKLSKLAILRIACNYILSLARLADLDYSADHSNLSFSECVQRCTRTLQAEGRAKKRKE
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 13C.
Table 13C. Patp results for NOV13 Smallest Sum eadingigh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:AAM93743Humanpolypeptide, SEQ ID NO: +1 1197 2.3e-121 >patp:AAB95274Humanprotein sequence SEQ ID +1 1197 2.3e-121 N0:17476 >patp:AAU16607Humannovel secreted protein, +1 534 4.2e-51 Seq TD 1560 >patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33 #291 >patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33 #291 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 372 of 657 bases (56%) identical to a gb:GENBANK-ID:HSBBICP4A~acc:L14320.1 mRNA from Bovine herpesvirus 1 (Bovine herpesvirus type 1 early-intermediate transcription control protein (BICP4) gene, complete cds). The full amino acid sequence of the protein of the invention was found to have 238 of 322 amino acid residues (73%) identical to, and 244 of 322 amino acid residues (75%) similar to, the 322 amino acid residue ptnr:TREMBLNEW-ACC:BAB39468 protein from Mus musculus (BHLH FACTOR MATH6)..
NOV 13 also has homology to the proteins shown in the BLASTP data in Table 13D.
Table 13D. BLAST
results for Gene Indeac/ Protein/ OrganismLength Identity PositivesEacpect Identifier (aa) (%) (%) giI14249530Iref~NPhypothetical 321 246/321 246/321 2e-93 _ protein FLJ14708 (76%) (76%) 116216.11(NM
[Homo sapiens]
gi~i3383235~dbj~BABbHLH factor 322 233/329 240/329 3e-86 Math6 39468.1~(AB049066)[Mus musculus] (70%) (72%) gi~17864454Iref~NPnet [Drosophila365 55/97 76/97 5e-21 _ melanogaster] (56%) (77%) 524820.1~(NM
gi~7296271~gb~AAF51CG11450 gene 261 55/97 76/97 2e-20 562.11 (AE003590)product (56%) (77%) [Drosophila melanogaster]
gi~18858289Iref~NPatonal homolog 325 36/82 51/82 2e-10 2a _ [Danio rerio] (43%) (61%) 571891.1I(NM
A multiple sequence alignment is given in Table 13E, with the NOV13 protein being shown on line I in Table 13E in a ClustalW analysis, and comparing the NOV13 protein with the related protein sequences shoum in Table 13D. This BLASTP data is displayed graphically in the ClustalW in Table 13E.
Table I3E. CIustalW Analysis of NOVI3 1) > NOV13; SEQ m N0:28 2) > gi~14249530~/ hypothetical protein FLJ14708 [Homo Sapiens]; SEQ m N0:92 3) > gi~13383235~/ bHLH factor Math6 [Mus rrausculus]; SEQ ZD N0:93 4) > gi~ 17864454/ net [Dr~osoplaila rraelanogaster]; SEQ m N0:94 5) > gi~7296271~/ CG11450 gene product [Dr-osophila nrelanogaster~]; SEQ m N0:95 6) > gi~18858289~/ atonal homolog 2a [Danio rerio]; SEQ m N0:96 .1... ~... .I
NOV13 1 __________________________ ' CHI 'LiE~GPKTVCVL'~' ___ ~ 3' ' 28 1~ gi1142495301 1 -------------------------- HI~~T~E~GP~KTVCVLi~==-- ~ ~ ~ 28 gi1133832351 l --------------------------- HI~ ~1E~GP~KTVCVK~#L -- ~ 28 gi~17864454~ 1 MSFAAMANTNTEKLYMQLSASELSAII ~DS~ S ~RDAGFCSASSE"~c~'EGGDDLVVHAv gi172962711 1 ______________________________________________________'=_____ 1 gi1188582891 1 -----------------------MLTLPFED~_~,M~TQ~'GANFPCV~-------D~yG 30 _.
I....I....J. .~.. .L... .~....~....~
NOV13 29 ~F~ANGYKTFRLW,~APE4P-----RAVAT R',t'~RTHRL' ~ PVRCQSQWRRP 83 a V V
~ r V V
gi ~ 14249530 ~ 29 'TtANGYKTFRL~Z~~APP-----RAVA ~ Rx?RTHRLf~ ~ ~ PVPVPVPV ~ 83 2~ gi113383235~ 29 ~ GYKTFRLy.P~LGATVSTTAAT~ R'RT---~'F~IATPVPA'V ~ 85 gi1178644541 61 S SPDI~PKGTDSADSKP~LALVRNKRKSSEPFKV~TTPNSKS ' ~PSSASMN~T ~L
gi172962711 1 __________________.________________________ ~PSSASMNT ~L 16 gi ~ 18858289 1 3l NK~mFEEETLSHVMD~DF~SED-----EDEREQDNGLPRR ~RKKKMTK~RVDR 85 .1...,1... ~ . .1 .I ~.. 1'.
NOVl3 ~ 84 FPQEGARTQPGSAGAG~~R --PT GNLRPRI~SGAR~..,,PHAAAAA ---ASCAPEPGT 136 gi ~ 14249530 ~ 84 VPPRG ~'~;~1GERGGS ~E~"SDAR ~CFALGA'~'GPGLP~~~,~P'P ~PPP----APQ~QAPG- 138 gi1133832351 86 VPPGG L~~~,REFRGI'., ~E~ZDAR ~GFALGT~~GPGLPP~P~P------ASQ LAPG-3~ gi ~ 17864454 ~ 121 KKRIR S~~DSAVV~,'TP ~DSPPPNSC~PSTI~F~LQHEI ~
IYVRHPGV~TLHRS 180 gi ~ 7296271 ~ 17 KKRIRY~S~~~,DSAVVTP'ADSPPPNSC,PSTLr~LQHEI ~ IYVRHPGVfI'TLHRS
gi~18858289~ 86 VKVRRMEAN~RERNRI~HGLNNLDSLE7KV~',PCYSLC~TQKLKIETLR-----..
NOV13 137 WGPRGTAF~EPG~RPRIL PP'i1PRAVTP'u~TASAPG--VHCAPW~PTRP.~ .I 194 gi1142495301 138 -GPEAQPF~EPGPRPRIL~ PPi~1, ~ PyiIPP~,~PPAPP-E TV~P ~PTRP' I
gi1133832351 138 -DPEAHSF~EQ RPRIL PP~i1 ~TQi~PL~~PPAAPQE PV,I2P ~PTRP I 197 gi1178644541 181 LAAHPEQLEPL TTKKQ DQiI ~KIE~F~~iLLIGKQP K~TLKERTQ TS FL 240 4O gi172962711 77 LAAHPEQLEPL VTTKKQ DQ~ KIE~F~LLIGKQP ITLKERTQ T'S FL 136 gi1188582891 141 SEILSTGI~PDL TFVQT KGLQ~TT~1'LV;~,;GCLQLN---ARNFT~DQISF GR~
1.~.~..1.. .I....1 . .1....1 .1. ..~.. .1. .1....1. .1 NOV13 195 HVIY~HQ~S ~PRKRP ET~~SSEI ~Q T ~ L~ ~~~~ ~~ ~Q 254 v i w v gi ~ 14249530 1 197 HVIY~HQrS PRKRP E;T ~SSEI ~Q~T~ ~ 'L~ ~' ~ ~ ~ ~~CQ 256 gi ~ 133832351 198 HVIY~'1HP~5 PRKRP ET ~STEI I QT' ~ ''L~ ~ ~ ~ ~I~Q 257 w a gi1178644541 241 EASL~~3~E-~LNL~.~K~!GLAPISRPHQHQRNY T~E:y.E~ ~~ ~ ~~ T~~A 299 gi172962711 137 EASL~S~E-~LKGLAPISRPHQHQRNY T~E '~E~ ~~ ~ ~ ~'~' T ~A 195 gi (18858289 l 198 PYESVYSTYI3~P~WWTPS~P~VD~1VKPFR~FNYCSSYE-FY SVSPECG'I'PQ
gi1142495301 257 312 gi1133832351 258 313 gi1178644541 300 355 gi172962711 196 251 gi1188582891 257 316 NOV13 311 . . ~'. 1 ~ ~ '~~I 319 gi1142495301 313 ~ ~- 321 gi1133832351 314 ~ ~- 322 gi1178644541 356 ~2 ~E 365 gi172962711 252 ~ ~;E 261 gi~188582891 317 ~DELNTFHN- 325 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 13F lists the domain description from DOMAIN analysis results against NOV 13.
Table 13F
Domain Anal sis of NOV13 Model Region of Score (bits) E value Homology Helix-loop- 234-280 55.8 4.0e-09 helix domain Helix-loop- 229-281 61.4 1.9e-14 helix DNA-binding domain (HLH) . Consistent with other known members of the BHLH Factor MATH6-like family of proteins, NOV 13 has, for example, a Helix-loop-helix domain and a Helix-loop-helix DNA
binding domain (HLH) signature sequence as well as homology to other members of the BHLH Factor MATH6-like Protein Family. NOV 13 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
For example, NOV 13 nucleic acids and polypeptides can be used to identify proteins that are members of the BHLH Factor MATH6-like Protein Family. The NOV 13 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV13 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Inflammatory bowel disease, Diverticular disease, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
In addition, various NOV13 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV13 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the BHLH Factor MATH6-like Protein Family.
A number of eukaryotic proteins, probably sequence specific DNA- binding proteins that act as transcription factors belong to this family. They share a conserved domain that is formed of two amphipathic helices joined by a variable length linker region that could form a loop (Littlewood and Evan, Pf°otein Prof. 2: 621-702 (1995).) This 'helix-loop-helix' (HLH) domain mediates protein dimerization and has been found in a large variety of proteins (Garrell and Campuzano, Bioassays 13: 493-498 (1991); Kato and Dang, FASEB J.
6: 3065-72 (1992).) Most of these proteins have an short basic region adjacent to the HLH domain that specifically binds to DNA. They are referred as basic helix-loop-helix proteins (bHLH), and are classified in two groups: class A (ubiquitous) and class B (tissue-specific). The HLH
proteins lacking the basic domain (Emc, Id) function as negative regulators since they form heterodimers, but fail to bind DNA. The hairy-related proteins (hairy, E(spl), deadpan) also repress transcription although they can bind DNA. The proteins of this subfamily act together with co-repressor proteins, like groucho, through their C-terminal motif WRPW.
MATH6 (moue, et al., Genes to Cells 6: 977-86 (2001)) is a distant homolog of D~osophila proneuronal gene Atonal. Murine expression is higest in developing nervous system (ventricular zone and mantle layer, spinal cord, dorsal root ganglia).
MATH6 is expressed by neuronal precursor cells and designated neurons, e.g., cerebellar Purkinje cells.
The closest mammalian homolog to MATH6 is NeuroD. NeuroD point mutations and NeuroD gene knockout animals have severe diabetes and die perinatally. The NeuroD
knockout anmals lack beta-Islet cells and could not be rescued with insulin administration.
Also, the NeuroD knockout animals are deaf due to a loss of inner ear sensory neurons.
The NOV13 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of metabolism as well as nerve and immune physiology. As such, the nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, hearing, nervous system, immune disorders, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, inflammatory bowel disease, Diverticular disease, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as well as other diseases, disorders and conditions.
The NOV13 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 13 nucleic acid is expressed in Pancreas, Umbilical Vein, Small Intestine, Cartilage, Synovium/Synovial membrane, Brain, Placenta, Oviduct/LTterine Tube/Fallopian tube, Lung, Brain, Uterus.
Additional utilities for NOV 13 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV 14 polypeptide has been identified as a Putative Protein-Tyrosine Phosphatase-like protein (also referred to as CG94366-O1). The disclosed novel NOV14 nucleic acid (SEQ
ID N0:29) of nucleotides is shown in Table 14A. The novel NOV 14 nucleic acid sequences maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 248-250 and ends with a TAA codon at nucleotides 1679-1681. A putative untranslated region and/or downstream from the termination codon is underlined in Table 14A, and the start and stop codons are in bold letters.
Table 14A. NOV14 Nucleotide Sequence (SEQ ID N0:29) ATTGAGTTTGAAATAACTGCCACCACAAAGTCTGTCACACATTGAGACTGAGGTCATAATAAAGAGG
TTTACTTAAATAGGGAAGCATTACTATTTTCCCCCGCCTAAGATTTTGGTTGTCGCCATATAAATCC
TCATTTCTAATAAAGAGAAAAAGACATTCCAGGTTCCAATAGTGCTATACACATGAATAGTCAGAAA
TTAATTGGTTTCTGTCTAGAATAATGAAAAGTAATTTTTCCAAAATATGAATTCAGAATTAAGTCTC
CTCTCTGACTGTTTTCTCTTATCATCCGCTAGTCCACAGACAAACGAATTTAAAGGAGCAACCGAGG
AGGCACCTGCGAAAGAAAGCCCACACACAGGTGAATTTAAAGGAGCAGCCCTGGTGTCACCTATCAG
TAAAAGAATGTTAGAACGACTTTCCAAGTTTGAAGTTGGAGATGCTGAAAATGTTGCTTCATATGAC
AGCAAGATTAAGAAAATTGTTCATTCAATTGTGTCATCCTTTGCAGTTGGGATATTTGGAGTTTTCC
TGATCTTGTTGGATGTGGCTCTGATCTTTGCTGACCTAATTTTCACTGATAGCAAAGTTTATATTCC
TTTGGAGTATCGTTCTATTTCTCTAGCTATTGCTTTATTTTTTCTCATGGATGTTCTTCTTCGAGTA
TTTGTAGAAAGGAGACAGCATTATTTTTCTGATTTACTTAACATTTTAGATACTGCCGTTACTGTGA
TTATTCTGCTGGTTGATGTCGTTTACATTTTTTTTGACGTTAAGTTTCTTAAGGATATTCCCAGATG
GACACGTTTATTTCGACTTCTACGACTTATCATTCTGATAAGAGTTTTTCGTCTGGCTCATCTAAAA
AGACAACTTGGAAAGCTGATAAGAAGGCTGGTAAGTAGGNGATACGAAAGGGATGGATTTGACCTAG
ACCTCACTTATATTACAGAACGTATTGTCGCTATGTCATTTCCATCTTCGGGAGGCCAGTCTTTCTA
TCGGAATCCAATTAAGGAAGTCGTACAGTTTCTAGACAAGAAACATCCAAACCACTATCGAGTCTAC
AATCTATGCAGTGAAAGAGCTTATGATCCTAAGCACTTCCATAATAGGGTCAGTAGAATCATGATCG
ATGATCATAATGTCCCCACTCTAAGGGAGATGGTAGCATTCTCCAAGGAAGTGTTGGAGTGGATGGC
TCAAGATTCTGAAAACATCGTAGTGATTCACTGTAAAGGAGGCAAAGGTAGAACCGGAACTATGGTT
TGTGCCTGCCTGATTGCCAGTGAAATATTTTTAACTGCAGAGGAAAGATTGTACTATTTTGGAGAAC
GGCGAACAGATAAAACCAATGGCACTAAATATCAGGGAGTAGAAACTCCTTCTCAGAATAGATATGT
TGGATATTTTGCACAAGTGAAACATAGCTACAACTGGAATCTCCCTCCAAGAAAAACACTGTTTATA
AAAAGATTAGTTATTTATTCGATTCATGGTAAGTGTTTAGATCTAAAAGTCCAAATAGTAATGAAGA
AAAAGATTGTCTTTTCCTGCACTTCCTTAAACAGTTGTCGGGTAAGAGAAAACATGGAAACAGACAG
GGTAATAATTGATGTGTTCAACTGTCCACCTCTGTATGATGATGTGAAAGTGCAATTTTTTTTTTCT
TTTTAGGATTTTCCTAAATACTATCACAACTACCCTTTTTTCTTCTGGTTTAACACATCTTTAATAC
AAAATAACAGGCTTTATCTACAAAGAAATGAATTGGATAATCTTCATAAACAAAAAACATGGAAAAT
TTATCAACCAGAATATGCAGTAGAGATATATTTTGATGAGAAATGACTTAAGTTATGTTGTAACTGG
TAGCTGATTAAGTATAGTTCCCTGCACCCCTTCTGGGAAAGAATTATGTTCTTTCTAACCCTGCCAC
ATAGTTATATGTTCTAAATCTTCCTTGCTGGTACATCTATATTGATATATGTATACACATGTTCTTT
ATAAATCTATTAAATATATACAGATAAA
The NOV14 protein (SEQ ID N0:30) encoded by SEQ )D N0:29 is 477 amino acid residues in length and is presented using the one-letter amino acid code in Table 14B. Psort analysis predicts the NOV 14 protein of the invention to be localized at the plasma membrane with a certainty of 0.6000.
Table 145. Encoded NOV14 protein sequence (SEQ ID N0:30) MNSELSLLSDCFLLSSASPQTNEFKGATEEAPAKESPHTGEFKGAALVSPISKRMLERLSKFEVG
DAENVASYDSKIKKIVHSIVSSFAVGIFGVFLILLDVALIFADLIFTDSKVYIPLEYRSISLAIA
LFFLMDVLLRVFVERRQHYFSDLLNILDTAVTVIILLVDVVYIFFDVKFLKDIPRWTRLFRLLRL
IILIRVFRLAHLKRQLGKLIRRLVSRXYERDGFDLDLTYITERIVAMSFPSSGGQSFYRNPIKEV
VQFLDKKHPNHYRVYNLCSERAYDPKHFHNRVSRIMIDDHNVPTLREMVAFSKEVLEWMAQDSEN
IVVIHCKGGKGRTGTMVCACLIASEIFLTAEERLYYFGERRTDKTNGTKYQGVETPSQNRYVGYF
AQVKHSYNWNLPPRKTLFIKRLVIYSIHGKCLDLKVQIVMKKKIVFSCTSLNSCRVRENMETDRV
IIDVFNCPPLYDDVKVQFFFSF
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 14C.
Table 14C. Pat results for NOV14 Smallest Sum eading igh Prob Sequences Score P(N) producing High-scoring Segment Pairs: Frame >patp:AAG67459Amino acid sequence of a human +1 1895 2.5e-195 polypeptide >patp:AAG67638Amino acid sequence of a human +1 1895 2.5e-195 protein >patp:AAB73230Human phosphatase AA493915_h +1 574 1.8e-111 >patp:AAW34402Protein encoded by gene IMAGE +1 473 1.2e-44 clone 264611 >patp:AAY07450Human TS10q23.3 gene bases 453-2243+1 473 1.2e-44 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 1105 of 1427 bases (77%) identical to a gb:GENBANK-m:AF007118~acc:AF007118.1 mRNA from Homo Sapiens (putative tyrosine phosphatase mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 369 of 462 amino acid residues (79%) identical to, and 402 of 462 amino acid residues (87%) similar to, the 551 amino acid residue ptnr:SWISSNEW-ACC:P56180 protein from Ho~rao Sapiens (PUTATIVE PROTEIN-TYROSINE
PHOSPHATASE TPTE (EC 3.1.3.48)).
NOV14 also has homology to the proteins shown in the BLASTP data in Table 14D.
Table 14D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (~) gi~7019559~ref~NPtransmembrane 551 369/462 402/462 0.0 _ phosphatase (79%) (86%) 37447.1I(NM 013315)with tensin homology;
tensin, putative protein-tyrosine phosphatase [Homo Sapiens]
gi~16166555~ref~XPsimilar to 551 367/462 401/462 0.0 _ transmembrane (79%) (86%) 055073.1~(XM_055073 phosphatase with tensin homology [Homo Sapiens]
gi~18640756~refINPsimilar to 445 316/462 343/462 e-156 _ PUTATIVE PROTEIN- (68%) (73%) 570141.1I(NM
TYROSINE
PHOSPHATASE
TPTE
[Homo sapien]
gi~14787415~emb~CACtyrosine 664 275/432 336/432 e-141 44243.11(AJ311311)phosphatase (63%) (77%) isoform A [Mus musculus]
gi~14787417~emb~CACtyrosine 645 275/432 336/432 e-141 44244.1~(AJ311312)phosphatase (63%) (77%) isoform B [Mus musculus]
A multiple sequence alignment is given in Table 14E, with the NOV 14 protein being shown on line 1 in Table 14E in a ClustalW analysis, and comparing the NOV14 protein with the related protein sequences shown in Table 14D. This BLASTP data is displayed graphically in the ClustalW in Table 14E.
Table 14E. ClustalW Analysis of NOV14 1) > NOV14; SEQ ID N0:30 2) > gi~7019559~/ transmembrane phosphatase with tensin homology; tensin, putative protein-tyrosine phosphatase Homo sapieras]; SEQ ID N0:97 3) > gi~16166555~/ similar to transmembrane phosphatase with tensin homology [Homo sapiefts]; SEQ ID N0:98 . 4) > gi~18640756~/ similar to PUTATIVE PROTEIN-TYROSINE PHOSPHATASE TPTE;
similar to transmembrane phosphatase with tensin homology; tensin, putative protein-tyrosine phosphatase [Flomo Sapiens]; SEQ ID N0:99 5) > gi~14787415~/ tyrosine phosphatase isoform A.[Mus musculus]; SEQ ID
NO:100 6) > gi~14787417~/ tyrosine phosphatase isoform B [Mus musculus]; SEQ ID
NO:101 ..
NOV14 1 _________________________________________________ gi~7019559~ 1 _________________________________________________ ~SELSLLS
--~I E~PDPTD
gi~16166555~ 1 ___________________________________________________ i E PDPTD 9 gi~186407561 1 __________________________________________________ __ - i E P-- 5 gi~147874151 1 MYGEKKSHLYLWMEHYGYDMPANIYKMYSQPSRKTDDANKKVSVSASRTIKI~ TGYDT 60 gi1147874171 1 -------------MHFGNINSTNWF------FRDKKHQNKKVSVSASRTI TGYDT 41 w NOV14 9 -DCFL~S- ~SASP~ ' ~. ~~~ G---------------- ~~L 48 giI70195591 10 LAGVI'I!ELGPI7SPv S ~ ... ________________ .. 52 gi~16166555~ 10 LAGIIELGP,SP~ S ~ ~~~ ---------------- ~~, ~ 52 gi~18640756~. 5 ______________~ T ________________ ..L 34 gi1147874151 61 NEQ~ T~.iITNG;SLSYP~ I.~YS,~~,S'YAD~ISTKA~,'' SSVYDPGGASSSTTLY
gi~14787417~ 42 NEQTITNGSLSYPII~S~YAD~ISTKA~S. ,s SSVYDPGGASSSTTLY LNSL~E 101 NOV14 49 ________________________________________________ ~ .'E~'t 58 gi~70195591 53 ________________________________________________ _~g 62 gi~16166555~ 53 ________________________________________________ S n'' 62 gi~18640756~ 35 ________________________________________________ ___'. 39 gi1147874151 121 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ" ~C 180 gi~14787417~ 102 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ;., CI~Q
NOV14 59 ~ ~ ~ FEV ~ A~ . .~K~ . . I=HS3; F~1V I ~ ~ ~ I<F ~ ~ . n 116 gi I 16166555 ~ 63 ~ 'FEVW--AB:NV DK~~.'C, , 'IVHS~ ~ F~G~ ~ nT Il;n ~ ~~.i gi~18640756~ 39 ~-__ _______=__ ____'_'.___R__.______ ~ T I,~~ ~ ~~ 65 i 14787415 181 LYD7WERTt~IQ F GI~Z ' ~ I ~ F ! n ~I 240 gi I 14787417 I 162 ?LYD~7~ERTIQW~~~yF~GII I ~ F ~ ~ ~If I 221 gi~7019559~ 121 180 gi~16166555~ 121 giI186407561 66 94 gi~14787415~ 241 300 gi~14787417~ 222 281 giI7019559~ 181 240 gi~161665551 181 240 giI186407561 94 145 gi~147874151 301 360 gif14787417~ 282 gi~7019559~ 241 55 gi~16166555~ 241 300 gi~18640756~ 146 205 gi~14787415~ 361 gi~14787417~ 342 gi~7019559~ 301 gi~161665551 301 gi~18640756~ 206 360 gi~14787415~ 421 480 gi~14787417~ 402 70 ..
NOVl4 354 413 gi~7019559~ 361 420 gi~16166555~ 361 giI186407561 266 325 gi114787415~ 481 540 gi~14787417~ 462 521 gi~7019559~ 421 gi~161665551 421 476 gi~186407561 326 381 gi~14787415~ 541 600 gi~14787417~ 522 581 NOV14 470 ~~~. F~________F___-____-___-_______________________________ 477 gi~7019559~ 477 ~ Y ~T ~ S L E ~ L Q ~, PSD ~ TL 536 gi~161665551 477 t ~T n S L Ei ~ L Q PSD ~ ~L 536 .k gi I 18640756 ~ 382 ~ S ~ ~ P F F C ' nP 'Q W~ PPE ~ I=L 441 gi114787415~ 601 LSP ~~P F F~R~T ~P~TW~GE ~ D 660 gi ~ 14787417 ~ 582 LSP ' ~ P F''.~!~!~ ~ T P ~TW GE ~ 641 giI7019559~ 537 KMTSSDWAGSD 551 30 gi~16166555~ 537 KMTSSDWAGSD 551 gi~18640756~ 442 i~K----------- 445 gi1147874151 661 ---------- 664 gi~147874171 642 ---------- 645 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uklinterpro~. Table 14F lists the domain description from DOMAIN analysis results against NOV14.
Table 14F
Domain Anal sis of NOV14 Model Region of Score (bits) E value Homology Dual 231-378 -48.8 3.2 specificity phosphatase, catalytic doma Consistent with other known members of the Putative Protein-Tyrosine Phosphatase-like family of proteins, NOV 14 has, for example, a dual specificity protein phosphatase signature sequence and homology to other members of the Putative Protein-Tyrosine Phosphatase-like Protein Family. NOV14 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 14 nucleic acids and polypeptides can be used to identify proteins that are members of the Putative Protein-Tyrosine Phosphatase-like Protein Family. The NOV 14 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV14 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., Cardiovascular diseases, cystitis, incontinence as well as other diseases, disorders and conditions W addition, various NOV 14 nucleic acids and polypeptides according to the invention are useful, iyater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 14 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the Putative Protein-Tyrosine Phosphatase-like Protein Family.
Cellular processes involving growth, differentiation, transformation and metabolism are often regulated in part by protein phosphorylation and dephosphorylation.
The protein tyrosine phosphatases (PTPs), which hydrolyze the phosphate monoesters of tyrosine residues, all share a common active site motif and are classified into 3 groups. These include the receptor-like PTPs, the intracellular PTPs, and the dual-specificity PTPs, which can dephosphorylate at serine and threonine residues as well as at tyrosines.
Diamond et al. (1994) described a PTP from regenerating rat liver that is a member of a fourth class. The gene, which they designated Prll, was one of many immediate-early genes. Overexpression of Prll in stably transfected cells resulted in a transformed phenotype, which suggested that it may play some role in tumorigenesis. By using an ih vitro prenylation screen, Cates et al. (1996) isolated 2 human cDNAs encoding PRL1 homologs, designated PTP(CAAXl) and PTP(CAAX2)(PRL2), that are farnesylated i~a vitro by mammalian farnesyl:protein transferase. Overexpression of these PTPs in epithelial cells caused a transformed phenotype in cultured cells and tumor growth in nude mice. The authors concluded that PTP(CAAX1) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic PTPs. Peng et al.
(1998) reported that the human PTP(CAAX1) gene, or PRL1, is composed of 6 exons and contains 2 promoters. The predicted mouse, rat, and human PRLl proteins are identical. Zeng et al. (1998) determined that the human PRLl and PRL2 proteins share 87% amino acid sequence identity.
The NOV14 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac and renal physiology. As such, the NOV 14 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular and urogenital system disorders, e.g., Cardiovascular diseases, cystitis, incontinence as well as other diseases, disorders and conditions. The NOV 14 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV14 nucleic acid is expressed in Urinary bladder.
Additional utilities fox NOV 14 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV15 polypeptide has been identified as a Leucine Rich Repeat (LRR)-like protein (also referred to as CG95387-02). The disclosed novel NOV15 nucleic acid (SEQ m N0:31) of 3136 nucleotides is shown in Table 15A. The novel NOV15 nucleic acid sequences maps to the chromosome 19.
An ORF begins with an ATG initiation codon at nucleotides 330-332 and ends with a TAA codon at nucleotides 2331-2333. A putative untranslated region and/or downstream from the termination codon is underlined in Table 15A, and the start and stop codons are in bold letters.
Table 15A. NOV15 Nucleotide Sequence (SEQ ID N0:31) ACTCCTGACCTAAAGTGATCCACTCGCCTTGGCCTCCCAAAGTGCTAGGATTACAGCCCTCATTCTC
TTTTGCTCCTCAGGTGACACAGGACAAGATCATCTGTCTACCCAATCATGAGCTCCAGGAGAACTTA
TCAGAGGCCCCGTGCCAGCAATTGCTGCCTCGGGGGATCCCTGAGCAGATTGGGGCCCTGCAGGAGG
TTAAAGGCCTTAAGAACAATTTGGACCTGCAGCAATACAGCTTTATTAACCAGCTGTGTTATGAGAC
GGCCCTGCACTGGTATGCCAAGTACTTCCCTTACCTCGTGGTCATTCACACACTCATCTTCATGGTC
TGCACCAGTTTCTGGTTCAAGTTCCCTGGCACCAGCTCCAAGATTGAACACTTCATCTCCATCCTGG
GCAAGTGTTTCGACTCTCCATGGACCACCAGGGCCCTATCCGAGGTCTCCGGGGAGAACCAGAAGGG
CCCAGCAGCCACCGAACGGGCTGCGGCATCCATAGTGGCCATGGCAGGGACCGGGCCGGGGAAGGCA
GGGGAGGGTGAGAAGGAGAAAGTGCTGGCGGAACCGGAGAAGGTGGTGACCGAGCCTCCAGTTGTCA
CCCTGTTGGACAAGAAGGAGGGTGAGCAAGCCAAAGCCCTGTTTGAGAAGGTGAAGAAGTTCCGCAT
GCACGTGGAAGAGGGCGACATCCTGTACACCATGTACATCCGACAGACGGTGCTGAAAGTGTGTAAG
TTCCTGGCCATCCTGGTCTACAACCTGGTCTATGTGGAGAAGATCAGTTTCCTGGTGGCCTGTAGGG
TGGAGACGTCAGAGGTCACGGGCTACGCCAGCTTCTGCTGCAACCACACCAAGGCCCACCTCTTCTC
CAAGCTGGCCTTCTGTTACATCTCCTTTGTGTGCATCTACGGACTTACCTGCATCTACACGCTCTAC
TGGCTCTTCCACCGGCCCCTCAAGGAGTACTCCTTCCGTTCCGTGCGGGAGGAGACTGGCATGGGGG
ACATTCCTGACGTCAAGAATGACTTCGCCTTCATGCTGCACCTCATCGATCAGTACGACTCCCTCTA
CTCCAAGCGCTTCGCCGTCTTCCTGTCCGAGGTCAGCGAAAGCCGTCTAAAGCAGCTCAATCTCAAC
CACGAGTGGACCCCCGAGAAGCTTCGACAGAAGCTGCAGCGCAATGCCGCGGGCCGGCTGGAGCTGG
CCCTCTGCATGCTGCCGGGTCTGCCCGACACCGTCTTTGAGCTCAGTGAGGTGGAGTCACTCAGGCT
GGAGGCCATCTGCGATATCACCTTCCCCCCGGGGCTGTCACAGCTGGTGCACTTGCAGGAGCTCAGC
TTGCTCCACTCGCCCGCCAGGCTACCCTTCTCCTTGCAGGTCTTCCTGCGGGACCACCTGAAGGTGA
TGCGCGTCAAATGCGAGGAGCTCCGCGAGGTGCCGCTTTGGGTGTTTGGGCTGCGGGGCTTGGAGGA
GCTGCACCTGGAGGGGCTTTTCCCCCAGGAGCTAGCTCGGGCAGCCACCCTGGAGAGCCTCCGGGAG
CTGAAGCAGCTCAAGGTGTTGTCCCTCCGGAGCAACGCCGGGAAGGTGCCAGCCAGTGTGACCGACG
TTGCTGGCCACCTGCAGAGGCTCAGCCTGCACAACGATGGGGCCCGTCTGGTTGCCCTGAACAGCCT
CAAGAAGCTGGCGGCATTGCGGGAGCTGGAGCTGGTGGCCTGCGGGCTGGAGCGCATCCCCCATGCA
GTGTTCAGCCTGGGTGCGCTGCAGGAACTTGACCTCAAGGACAACCACCTGCGCTCCATCGAGGAAA
TCCTCAGCTTCCAGCACTGCCGGAAGCTGGTCACGCTCAGGCTGTGGCACAACCAGATCGCCTACGT
CCCTGAGCACGTGCGGAAGCTCAGGAGCCTGGAGCAGCTCTACCTCAGCTACAACAAGCTGGAGACC
CTGCCCTCCCAGCTCGGCCTGTGCTCAGGCCTCCGTCTGCTGGATGTGTCCCACAATGGGCTACACT
CCCTGCCACCCGAGGTGGGCCTCCTGCAGAACCTACAGCACCTGGCCCTCTCCTACAATGCCCTGGA
GGCCCTGCCCGAAGAGCTCTTCTTCTGCCGCAAGCTGCGGACGTTGCTTCTGGGCGACAACCAACTG
AGCCAGCTCTCGCCCCACGTGGGTGCCCTCAGAGCCCTCAGCCGCCTGGAGCTCAAAGGCAACCGCT
TAGAGGCGCTGCCAGAAGAACTTGGCAACTGTGGGGGGCTCAAGAAGGCGGGGCTCCTGGTGGAAGA
CACGCTTTACCAGGGTCTGCCGGCAGAAGTGCGGGACAAGATGGAGGAGGAATGAAGCTGGGGTGGG
GCCGTTTTAGGTAGAGCCTTAAAAATGCTTCTGCCCTGGAATCTCAACCATTATCTTCCAAGATAGG
AAGCCAAGTGGGTCTAGGCCAGGAGATGGGGGGGGGCGGGGGCAGCTGTGTCATCTTTCTGGGGCC_C
AGGAGGATCTGGGCTGGTTTGTCTGGGGAGACAGACAGGATGTTGTGGAGGTGGGGTGGAACCTGGT
ATGGAGGGATTAACTCAGTCATGGCATTCTCCGACCAAAACCACACCTGTGTCTCTGGCAGGCTGGC
TGGCCTTGCTCCCATCCCTAGAACTGCTGCCTCTCCCTGGATATTCCAGCTCAATTAGTGCCACATA
TGGGGGAAACGACACATCCCAGTGGGATTTCCAACACTCCCCCTCCCCATGCAACAAAGCAACTTAC
TTCTGGAGTTCTCTCCCAAGGAGAGGACACAGACACAGTTGTTTGCTGTGTTATATGTTAGCTCCGA
ACAATGGTTCTCATTTGGCTAAGCATCAAAATCACCTAGGGAGCCGGTGCAAAACAAAATATCCCAG
TCCCCTCCCCTGAAACACTGACTCAGGAGGTTTGGTTGGGGGCCAGGAGTCTGTTCCTAAATATTCC
AGGTAGTTCTGGTGCAGGTAAGTGGCCCTGAGACAGTATGTTGGGAAATGCTGACGTAAAGGTATCA
GGGCCGGGCGCTGTGGCTCATGACTATAATCCCAGCTGTTTGAGAGGCCAATGCAGGAGGATGGTTG
AGCTCAGGAGTTCGAGATCAGCCTGGGTAACATAGCGAGACCCCACCTCTGCCA
Variant sequences of NOV 15 are included in Example 3, Table 26. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV 15 protein (SEQ m NO:32) encoded by SEQ m N0:31 is 667 amino acid residues in length and is presented using the one-letter amino acid code in Table 15B. Psort analysis predicts the NOV15 protein of the invention to be localized at the plasma membrane with a certainty of 0.7900.
Table 15B. Encoded NOV15 protein sequence (SEQ ID N0:32) MVCTSFWFKFPGTSSKIEHFISILGKCFDSPWTTRALSEVSGENQKGPAATERAAASIVAMAGTG
PGKAGEGEKEKVLAEPEKWTEPPVVTLLDKKEGEQAKALFEKVKKFRMHVEEGDILYTMYIRQT
VLKVCKFLAILVYNLVYVEKISFLVACRVETSEVTGYASFCCNHTKAHLFSKLAFCYISFVCIYG
LTCIYTLYWLFHRPLKEYSFRSVREETGMGDIPDVKNDFAFMLHLIDQYDSLYSKRFAVFLSEVS
ESRLKQLNLNHEWTPEKLRQKLQRNAAGRLELALCMLPGLPDTVFELSEVESLRLEAICDITFPP
GLSQLVHLQELSLLHSPARLPFSLQVFLRDHLKVMRVKCEELREVPLWVFGLRGLEELHLEGLFP
QELARAATLESLRELKQLKVLSLRSNAGKVPASVTDVAGHLQRLSLHNDGARLVALNSLKKLAAL
RELELVACGLERIPHAVFSLGALQELDLKDNHLRSIEEILSFQHCRKLVTLRLWHNQIAYVPEHV
RKLRSLEQLYLSYNKLETLPSQLGLCSGLRLLDVSHNGLHSLPPEVGLLQNLQHLALSYNALEAL
PEELFFCRKLRTLLLGDNQLSQLSPHVGALRALSRLELKGNRLEALPEELGNCGGLKKAGLLVED
TLYQGLPAEVRDKMEEE
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 15C.
Table 15C. Patp results for NOV15 Smallest Sum eading igh Prob equences igh-scoring Segment Pairs:Frame Score P(N) producing >patp:AAY70473HumanCNAP-1 +l 2147 5.0e-222 >patp:AAG75413Humancolon cancer antigen protein+1 1882 6.0e-194 >patp:AAM41692Humanpolypeptide SEQ ID NO 6623+1 1878 1.6e-193 >patp:AAU20426Humansecreted protein, Seq ID +1 1835 5.8e-189 No 418 >patp:AAB92855Humanprotein sequence SEQ ID +1 1795 1.0e-184 N0:11424 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 2227 of 2228 bases (99%) identical to a gb:GENBANI~-ID:AK.027073~acc:AK027073.1 mRNA from Ho~rao Sapiens (cDNA:
FLJ23420 fis, clone HEP22352). The full amino acid sequence of the protein of the invention was found to have 444 of 444 amino acid residues (100%) identical to, and 444 of 444 amino acid residues (100%) similar to, the 444 amino acid residue ptnr:SPTREMBL-ACC:Q9HSH8 protein from Ho~ao sapiefzs (CDNA: FLJ23420 FIS, CLONE HEP22352).
NOV 15 also has homology to the proteins shown in the BLASTP data in Table 15D.
Table 15D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (%) (%) gi~13376597~ref~NPhypothetical 444 444/444 444/444 0.0 _ protein FLJ23420 (100%) (100%) 079337.1~(NM_025061 [Homo Sapiens]
gi~14150009~ref~NPhypothetical 708 404/667 520/667 0.0 _ protein (60%) (77%) 115646.1~(NM_032270 DKFZp586J1119 [Homo Sapiens]
gi~19343671~gb~AAH2Similar to 708 404/671 526/671 0.0 5473.1 (BC025473)hypothetical (60%) (78%) protein DKFZp586J1119 [Mus musculus]
gi~7243272~dbj~BAA9KIAA1437 protein811 367/666 84/666 0.0 2675.1 (AB037858)[Homo Sapiens] (55%) (72%) gi~8922442Iref~NPhypothetical 682 345/673 470/673 0.0 _ protein FLJ10470 (51%) (69%) 60573.1I(NM~018103) [Homo Sapiens]
A multiple sequence alignment is given in Table 1 SE, with the NOV 15 protein being shown on line 1 in Table 15E in a ClustalW analysis, and comparing the NOV 15 protein with the related protein sequences shown in Table 15D. This BLASTP data is displayed graphically in the ClustalW in Table 15E.
Table 15E. ClustalW Analysis of NOV15 1) > NOV15; SEQ ID NO:32 2) > gig 13376597/ hypothetical protein FLJ23420 [Homo Sapiens]; SEQ )D N0:102 3) > gig 14150009/ hypothetical protein DKFZp586J1119 [Hofno Sapiens]; SEQ >D
N0:103 4) > gi~19343671~/ Similar to hypothetical protein DKFZp586J1119 [Mus musculus]; SEQ ID N0:104 5) > gi~7243272~/ KIAA1437 protein [Homo sapierts]; SEQ ID NO:105 6) > gi~8922442~/ hypothetical protein FLJ10470 [Homo Sapiens]; SEQ >D N0:106 .1....1....I ..I . .I .1,....1....I. .I. .I. ..1....I
NOV15 l -------------- -'S~GT~~~I ~ ' '~ V~G~ 43 gi1133765971 1 _____________________________.______________________________ 1 v n ~ v V
gi1141500091 27 WYAKYFPYLVLIHTLVF~L GS ~ I ~ ' '~ ~; 86 gi1193436711 27 WYAKYFPYLVLTHTLVF~ G,S t~ ~ ~ ~ " . 86 gi172432721 121 WFAKYFPYLVLLHTLIF' R~' L ~ ~ '~ T~~ 180 gi189224421 1 ______________ y"~,,5 ~ ~K?~C ~ ~ ~ 'tC 43 ..1....1....1....1 [....1....I.. I ...I... 1....1....I
NOV15 44 Q~CGPAATERAAASIVAMAG'GPGA------GEGEKEKVLEP T PPWTL ~ 97 _~
gi1133765971 1 ___________________ ______ __ __________ ___________ 1 gi1141500091 87 EKDNRKNNN1NRSNTIQ-~GP G----- STSQSL~ IP F ~KSTAG ~ 138 gi I 193436711 87 E'EKDNRKNNMNRSGTIQ-~S.'GP G----- N~,r'X~RSQSL ~IP F~tKSAAG ~
gi 17243272 I 181 ~ ~PKPAFSKMNGSMDKKSS'I~,VSE~--VEATVPMIiQRT-- ~S'~RI
G~VC7RSETG ~ 236 gi I 8922442 I 44 y, E<ENKQRITGAQTLPKHVS~"SSD~GSPSASTP~;NKTGF IFS P~~VPSMTI ~
.I. .I. .I. .I....I....I,....1....1....1....1....1....1 NOV15 98 ~ ~ ~ ' ~ L T1~T'~L~CI:i'VONLVYVEICTS~L~ 157 gi1133765971 1 __________________________ __________ ___ _________.______ 1 gi1141500091 139 ~' ' 'L ~ L '~ L I IiI SAL SI~VQ T~ 198 gi1193436711 139 ~~'~ '~ ~ 'L ~ L w .L I LiI VSAL.SK~Q T~,1~~1,~,-~, 198 gi172432721 237 ~~ ' 'T ~ RL '~ TI I I'L I;C TVYY ~~ VD 296 gi I 8922442 I 104 ~ ~ ~ S ~LiI R~i~~~~ ~T I<F IiC~TANF 1~ ~S E 163 I....I....I....I....I ..I. 1....1....1....1....1....1 NOV15 158 R~TSES~C~TES~FC~IiSF~C~~T~~~X~P~T~RS 217 gi1133765971 1 ___________________________________=________________________ 1 gi1141500091 199 N~ltD3 S T 'T S AFC hCF S~ T ~ 'S Y 258 gi I 19343671 I 199 = ~~D , IQ. S T- ' S SFC ~:~1CF ST L Y'S Y 258 gi I 7243272 I 297 TTI~S~, RTE P 'T KI SF 1S1 IF I L ,S K S 356 gi I 8922442 I 164 KPF~VVIiI EVE T '~L LIS ~SICI L F RIP K 223 gi1133765971 1 54 gi1141500091 259 318 gi1193436711 259 318 gi172432721 357 416 gi189224421 224 $5 430 460 gi1133765971 55 gi1141500091 319 378 gi1193436711 319 378 gi17243272) 417 476 gi189224421 284 343 ~I ~~I~
~. .I~
I~ .I~~~~I~
~I ~I
. ~
I
I~~~-I .
~I~ .
.
.
NOV15 338 L SP~RLtPF~LQ _ _ _ _ ' ELFPQ 397 C~E~ L' L ~
F
~
gi 115 BL~SP~R~.iPF,LQV ' , RC,E''1~'' L'~V'L G
E~LFPQ ~L ~ 174 I E' I
gi ~ 14150009 ~ 379 QCS HSL~ ~S n' ~ 'P S SHI3I~438 gi ~ 19343671 ~ 379 QCS ~THS~L KE S n ' L'P ' S SHiIISKNS~ 438 gi ~ 8922442 ~ 344 C~CP~ ~EQT~F~E"H~T T~I~I ~L I L ~T y I~' 403 gi~13376597~ 175 234 gi~14150009~ 439 498 gi~19343671~ 439 498 gi~7243272~ 537 gi~8922442~ 404 463 1$ 610 620 630 640 650 660 gi~13376597~ 235 294 gi~14150009~ 499 558 gi~19343671~ 499 558 gi~7243272~ 597 656 gi~8922442~ 464 523 gi~13376597~ 295 354 gi~14150009~ 559 618 gi~19343671~ 559 618 gi~7243272~ 657 716 gi~89224421 524 583 gi~13376597~ 355 414 gi~141500091 6l9 gi~19343671~ 619 gi~7243272~ 717 gi~8922442~ 584 76 gi113376597~ 415 -------- 444 gi~14150009~ 679 -------- 708 gi~193436711 679 -------- 708 gi~7243272~ 777 KEQA---- 811 gi~8922442~ 644 NIPFANGI 682 . ,I I'.
f f~, G v . ~ ~; ~ T Y~ ~''E-r y~7 ~ ~ ~ , ' SD ' Q I~T
~ 'S~j ' ~~_ PL ~ ~P ~W~
L x... QI
The NOV15 Clustal W alignment shown in Table 15E was modified to begin at amino residue 121. The data in Table 15E includes all of the regions overlapping with the NOV 15 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.ukfinterpron. Table 15F lists the domain description from DOMAIN analysis results against NOV15.
Table 15F
Domain Anal sis of NOV15 Model Region of Score (bits) E value Homology LRR 454-476 5.6 1.5e+02 LRR 477-498 10.8 26 LRR 502-524 11.9 16 LRR 525-547 18.8 0.13 LRR 548-570 15.9 0.99 LRR 571-593 13.9 4 LRR 594-616 12.3 12 LRR 617-639 9.4 42 Consistent with other known members of the LRR-like family of proteins, NOV 15 has, for example, eight Leucine Rich Repeat (LRR) signature sequences and homology to other members of the LRR-like Protein Family. NOV15 nucleic acids, and the encoded polypeptides, according to the invention axe useful in a variety of applications and contexts.
For example, NOV 15 nucleic acids and polypeptides can be used to identify proteins that are members of the LRR-like Protein Family. The NOV 15 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV15 activity or function.
Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., cancer, trauma, regeneration (ifz vitro and izz vivo), viral/bacterial/parasitic infections, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, Hirschsprung's disease , Crohn's Disease, appendicitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, autoimmune disease, xerostomia as well as other diseases, disorders and conditions.
In addition, various NOV15 nucleic acids and polypeptides according to the invention are useful, hater alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV15 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the LRR-like Protein Family.
LRR Proteins are a family of proteins characterized by a structural motif rich in leucine residues. They are either transmembrane or secreted proteins and are involved in protein-protein interactions. Members of this family have been implicated in extracellular matrix assembly and cellular growth. In addition, several proteins belonging to this family, such as slit, Toll and robo have been shown to mediate key roles in central nervous system development and organogenesis in Drosophila. Vertebrate orthologs of these proteins have also been shown to have similar roles in the CNS as well as other organ systems like kidney..
LRRs are relatively short motifs (22-28 residues in length) found in a variety of cytoplasmic, membrane and extracellular proteins. Although these proteins are associated with widely different functions, a common property involves protein-protein interaction. Little is known about'the 3D structure of LRRs, although it is believed that they can form amphipathic structures with hydrophobic surfaces capable of interacting with membranes.
Irz vitro studies of a synthetic LRR from Drosophila Toll protein have indicated that the peptides form gels by adopting beta-sheet structures that form extended filaments (Packman et al.
FEBS Lett.1991;
291: 87-91). These results are consistent with the idea that LRRs mediate protein-protein interactions and cellular adhesion. Other functions of LRR-containing proteins include, for example, binding to enzymes and vascular repair.
The NOV15 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cardiac, immune, and nerve physiology. As such, the NOV 15 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cardiovascular, nervous, and immune system disorders, e.g., cancer, trauma, regeneration (ih vitro and in vivo), viral/bacterial/parasitic infections, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, Hirschsprung's disease, Crohn's Disease, appendicitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, autoimmune disease, xerostomia as well as other diseases, disorders and conditions.
The NOV15 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 15 nucleic acid is expressed in Coronary Artery, Parotid Salivary glands, Liver, Colon, Bone, Synovium/Synovial membrane, and Brain.
Additional utilities for NOV 15 nucleic acids and polypeptides according to the invention are disclosed herein.
A NOV16 polypeptide has been identified as a RhoGEF-like protein (also referred to as CG95419-02). The disclosed novel NOV16 nucleic acid (SEQ B7 N0:33) of 5372 nucleotides is shown in Table 16A. The novel NOV16 nucleic acid sequences maps to the chromosome 5.
An ORF begins with an ATG initiation codon at nucleotides 61-63 and ends with a TAA codon at nucleotides 5179-5181. A putative untranslated region and/or downstream from the termination codon is underlined in Table 16A, and the start and stop codons are in bold letters.
Table 16A. NOV16 Nucleotide Sequence (SEQ ID N0:33) CCATGGGGCCTCCTGCAATAACTTCTCTTGTTTATTATTTTCATTGCAGATGCGAAAGCCATGGAGT
TGAGCTGCAGCGAAGCACCTCTTTACCAGGGGCAGATGATGATCTATGCGAAGTTTGACAAAAATGT
GTATCTTCCTGAAGATGCTGAGTTTTACTTTACTTATGACGGATCTCATCAGCGACATGTCATGATT
GCAGAGCGCATCGAGGATAACGTTCTCCAGTCCAGCGTCCCAGGCCATGGGCTTCAGGAGACGGTGA
CGGTATCTGTGTGCCTCTGCTCGGAAGGTTACTCTCCGGTGACCATGGGCTCTGGCTCAGTGACCTA
CGTGGACAACATGGCTTGCAGGCTGGCTCGTCTGCTGGTGACGCAGGCCAATCGCCTCACAGCCTGC
AGCCACCAGACCCTGCTGACCCCATTTGCCTTGACGGCAGGAGCACTGCCTGCCTTGGATGAGGAGC
TCGTGCTGGCTCTGACCCATCTGGAATTGCCTCTAGAGTGGACTGTGTTGGGAAGTTCTTCACTTGA
AGTATCTTCTCACAGAGAATCTCTTCTACACCTGGCTATGAGATGGGGCCTGGCTAAACTTTCCCAG
TTCTTCTTGTGTCTCCCGGGGGGAGTCCAGGCCTTGGCTTTACCCAACGAAGAGGGTGCCACACCAT
TAGACTTAGCTTTACGTGAAGGACACTCCAAGCTGGTGGAAGACGTCACAAGTTTTCAGGGCAGATG
GTCCCCAAGCTTCTCCCGAGTGCAGCTCAGTGAAGAAGCCTCCTTGCATTACATTCACTCATCGGAA
ACGCTGACCCTGACCCTGAACCACACAGCCGAGCATTTGTTGGAGGCAGATATTAAACTCTTCCGGA
AATACTTTTGGGATAGAGCCTTTCTTGTCAAGGCCTTTGAGCAAGAAGCCAGGCCAGAGGAAAGAAC
AGCTATGCCCTCCAGCGGTGCAGAAACTGAAGAAGAGATTAAGAATTCAGTGTCCAGCAGATCAGCA
GCCGAAAAGGAAGATATAAAGCGTGTCAAAAGCCTGGTGGTTCAACACAATGAACATGAAGACCAGC
ACAGCCTAGATTCTAGATCGCTCCTTCGATATCCTAAAAAATCCAAGCCGCCCTCGACATTGCTTGC
TGCAGGCCGGCTTTCAGACATGCTGAATGGAGGTGATGAAGTCTACGCTAACTGTATGGTGATTGAT
CAGGTTGGTGATTTGGATATCAGCTATATTAATATAGAGGGAATCACTGCCACTACCAGCCCTGAAT
CCAGAGGTTGCACTCTGTGGCCTCAGAGCAGCAAACACACCCTTCCTACAGAAACCAGTCCCAGTGT
GTACCCACTTAGTGAAAATGTCGAAGGGACAGCACACACTGAAGCCCAGCAGTCCTTCATGTCACCA
TCAAGTTCGTGTGCTTCCAACTTGAATCTTTCTTTTGGTTGGCATGGATTTGAAAAGGAACAAAGTC
ATCTAAAGAAAAGAAGTTCTAGCCTTGATGCCTTGGACGCCGACAGTGAAGGGGAAGGGCATTCTGA
GCCATCCCACATCTGTTACACTCCAGGGTCTCAGAGCTCCTCAAGAACTGGGATTCCTAGTGGGGAT
GAATTGGACTCTTTTGAGACTAACACTGAACCGGATTTTAATATCTCCAGGGCTGAATCCCTTCCTC
TATCAAGTAATCTACAGTTGAAGGAATCACTGCTTTCTGGAGTTCGCTCACGTTCTTATTCTTGCTC
GTCACCCAAAATTTCTTTAGGAAAAACTCGTTTGGTGCGTGAATTAACAGTATGCAGTTCAAGTGAA
GAGCAAAAAGCTTACAGCTTATCGGAGCCACCAAGAGAAAACAGGATTCAGGAAGAAGAATGGGATA
AATACATCATACCTGCCAAATCAGAGTCTGAAAAATATAAAGTGAGTCGAACTTTCAGTTTCCTCAT
GAATAGGATGACTAGCCCTCGGAATAAATCAAAGACAAAAAGCAAGGATGCCAAAGATAAAGAGAAG
CTGAATCGACATCAGTTTGCCCCAGGAACATTCTCTGGGGTTCTGCAGTGTTTGGTTTGTGATAAAA
CACTCCTGGGGAAAGAGTCACTGCAGTGTTCTAGTTGTAATGCAAATGTGCACAAAGGTTGTAAAGA
TGCTGCGCCTGCATGCACCAAGAAATTCCAAGAGAAATATAACAAGAACAAACCACAGACCATCCTT
GGAAGTTCTTCATTTAGAGACATCCCACAGCCTGGTCTCTCCTTGCACCCTTCTTCCTCCGTGCCTG
TTGGATTGCCGACTGGAAGGAGGGAGACTGTGGGACAGGTCCATCCATTGTCCAGAAGTGTTCCAGG
TACCACCTTGGAAAGCTTCAGGAGGTCAGCCACATCCTTGGAGTCTGAGAGTGACAATAACAGCTGC
AGAAGCAGGTCTCATTCTGATGAGCTGCTACAGTCCATGGGCTCTTCTCCCTCTACAGAGTCTTTCA
TAATGGAAGATGTTGTGGATTCTTCTCTGTGGAGTGACCTCAGCAGTGATGCCCAGGAGTTTGAAGC
AGAATCTTGGAGTCTTGTGGTGGATCCCTCATTTTGTAATAGGCAGGAGAAGGATGTCATCAAAAGA
CAGGATGTCATTTTTGAGCTAATGCAAACAGAGATGCATCACATCCAGACCCTGTTCATCATGTCTG
AGATCTTCAGGAAAGGCATGAAAGAGGAGCTGCAGCTGGACCACAGCACCGTGGATAAAATTTTCCC
CTGTTTAGATGAGTTGCTTGAAATCCACAGGCATTTCTTCTACAGTATGAAGGAACGAAGGCAGGAA
TCAAGTGCTGGCAGCGACAGGAATTTTGTGATCGACCGAATTGGAGATATTTTGGTACAACAGTTTT
CAGAAGAAAATGCAAGTAAAATGAAGAAAATATATGGAGAATTCTGTTGCCATCATAAAGAAGCTGT
TAACCTCTTTAAAGAACTCCAGCAGAATAAAAAGTTTCAGAATTTTATTAAGCTCCGAAATAGTAAT
CTTTTGGCTCGACGCCGAGGAATTCCAGAATGCATTCTGTTGGTCACTCAGCGTATTACAAAATACC
CTGTCTTGGTGGAAAGGATATTGCAGTACACAAAGGAAAGAACTGAGGAACATAAAGACTTACGCAA
AGCCCTTTGCTTAATTAAAGACATGATTGCAACAGTGGATTTAAAAGTCAATGAATATGAGAAAAAC
CAAAAATGGCTTGAGATCCTAAATAAGATTGAAAACAAAACATACACGAAGCTCAAAAATGGACATG
TGTTTAGGAAGCAGGCACTGATGAGTGAAGAAAGGACTCTGTTATATGATGGCCTTGTTTACTGGAA
AACTGCTACAGGTCGTTTCAAAGATATCCTAGCTCTACTTCTAACTGATGTGCTGCTCTTTTTACAA
TTATTGCTAGAGAAGTTGCTAATGAGGAGAGAGGAATGTTTCTGATCAGTGCTTCATCTGCTGGTCC
TGAGATGTATGAAATTCACACCAATTCCAAGGAGGAACGCAATAACTGGATGAGACGGATCCAGCAG
GCTGTAGAAAGTTGTCCTGAAGAAAAAGGGGGAAGGACAAGTGAATCTGATGAAGACAAGAGGAAAG
CTGAAGCCAGAGTGGCCAAAATTCAGCAATGTCAAGAAATACTCACTAACCAAGACCAACAAATTTG
TGCGTATTTGGAGGAGAAGCTGCATATCTATGCTGAACTTGGAGAACTGAGCGGATTTGAGGACGTC
CATCTAGAGCCCCACCTCCTTATTAAACCTGACCCAGGCGAGCCTCCCCAGGCAGCCTCATTACTGG
CAGCAGCACTGAAAGAAGCTGAGAGCCTACAAGTTGCAGTGAAGGCCTCACAGATGGGCGCCGTGAG
TCAATCATGTGAGGACAGTTGTGGAGACTCTGTCTTGGCGGACACACTCAGTTCTCATGATGTACCA
GGATCACCGACTGCCTCATTAGTCACAGGAGGGAGAGAAGGAAGAGGCTGTTCGGATGTGGATCCCG
GGATCCAGGGTGTGGTAACCGACTTGGCCGTCTCTGATGCAGGGGAGAAGGTGGAATGTAGAAATTT
TCCAGGTTCTTCACAATCAGAGATTATACAAGCCATACAGAATTTAACCCGTCTCTTATACAGCCTT
CAGGCCGCCTTGACCATTCAGGACAGCCACATTGAGATCCACAGGCTGGTTCTCCAGCAGCAGGAGG
GCCTGTCTCTCGGCCACTCTATCCTCCGAGGCGGCCCCTTGCAGGACCAGAAGTCTCGCGACGCGGA
CAGGCAGCATGAGGAGCTGGCCAATGTGCACCAGCTTCAGCACCAGCTCCAGCAGGAGCAGCGGCGC
TGGCTGCGCAGGTGTGAGCAGCAGCAGCGGGCGCAGGCGACCAGGGAGAGCTGGCTGCAGGAGCGGG
AGCGGGAGTGCCAGTCGCAGGAGGAGCTGCTGCTGCGGAGCCGGGGCGAGCTGGACCTCCAGCTCCA
GGAGTACCAGCACAGCCTGGAGCGGCTGAGGGAGGGCCAGCGCCTGGTGGAGAGGGAGCAGGCGAGG
ATGCGGGCCCAGCAGAGCCTGCTGGGCCACTGGAAGCACGGCCGGCAGAGGAGCCTGCCCGCGGTGC
TCCTTCCGGGTGGCCCCGAGGTAATGGAACTTAATCGATCTGAGAGTTTATGTCATGAAAACTCATT
CTTCATCAATGAAGCTTTAGTACAAATGTCATTTAACACTTTCAACAAACTGAATCCGTCAGTTATC
CATCAGGATGCCACTTACCCTACAACTCAATCTCATTCTGACTTGGTGAGGACTAGTGAACATCAAG
TAGACCTCAAGGTGGACCCTTCTCAGCCTTCGAATGTCAGTCACAAACTGTGGACAGCCGCTGGTTC
CGGCCATCAGATACTTCCTTTCCAAGAAAGCAGCAAGGATTCTTGTAAAAATGATTTGGACACCTCC
CACACTGAGTCCCCAACCCCCCATGACTCAAATTCACACCGCCCTCAACTGCAGGCGTTTATAACAG
AAGCAAAGCTAAATCTACCGACAAGGACAATGACCAGACAAGATGGGGAAACTGGAGATGGAGCCAA
AGAAAATATTGTTTACCTCTAATTGTGTTGTCATTTTTCCAAACAAAACAAAACACTGGCACTTTTG
GGAGAAACTTTTTGTCTCCATTCCTTATGTATGTGTGATTGTCTGTGTCCAAATTGCTTTAAGAATA
ATATTTAATATTTCCTGGAAGCTCATTTTTTTGGCATGAGTCTAATTAAATTATTGAAAGCCAAAAA
The NOV16 protein (SEQ 1D N0:34) encoded by SEQ 1D NO:33 is 1706 amino acid residues in length and is presented using the one-letter amino acid code in Table 16B. Psort analysis predicts the NOV16 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500.
Table 16B. Encoded NOV16 protein sequence (SEQ TD N0:34) MELSCSEAPLYQGQMMIYAKFDKNVYLPEDAEFYFTYDGSHQRHVMIAERIEDNVLQSSVPGHGL
QETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQANRLTACSHQTLLTPFALTAGAL
PALDEELVLALTHLELPLEWTVLGSSSLEVSSHRESLLHLAMRWGLAKLSQFFLCLPGGVQALAL
PNEEGATPLDLALREGHSKLVEDVTSFQGRWSPSFSRVQLSEEASLHYIHSSETLTLTLNHTAEH
LLEADIKLFRKYFWDRAFLVKAFEQEARPEERTAMPSSGAETEEEIKNSVSSRSAAEKEDIKRVK
SLVVQHNEHEDQHSLDSRSLLRYPKKSKPPSTLLAAGRLSDMLNGGDEVYANCMVIDQVGDLDIS
YINIEGITATTSPESRGCTLWPQSSKHTLPTETSPSVYPLSENVEGTAI~TEAQQSFMSPSSSCAS
NLNLSFGWHGFEKEQSHLKKRSSSLDALDADSEGEGHSEPSHICYTPGSQSSSRTGIPSGDELDS
FETNTEPDFNISRAESLPLSSNLQLKESLLSGVRSRSYSCSSPKISLGKTRLVRELTVCSSSEEQ
KAYSLSEPPRENRIQEEEWDKYIIPAKSESEKYKVSRTFSFLMNRMTSPRNKSKTKSKDAKDKEK
LNRHQFAPGTFSGVLQCLVCDKTLLGKESLQCSSCNANVfiKGCKDAAPACTKKFQEKYNKNKPQT
ILGSSSFRDIPQPGLSLHPSSSVPVGLPTGRRETVGQVHPLSRSVPGTTLESFRRSATSLESESD
NNSCRSRSHSDELLQSMGSSPSTESFIMEDVVDSSLWSDLSSDAQEFEAESWSLVVDPSFCNRQE
KDVIKRQDVIFELMQTEMHHIQTLFIMSEIFRKGMKEELQLDHSTVDKIFPCLDELLEIHRHFFY
SMKERRQESSAGSDRNFVIDRIGDILVQQFSEENASKMKKIYGEFCCHHKEAVNLFKELQQNKKF
QNFIKLRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKERTEEHKDLRKALCLIKDMIA
TVDLKVNEYEKNQKWLEILNKIENKTYTKLKNGHVFRKQALMSEERTLLYDGLVYWKTATGRFKD
ILALLLTDVLLFLQEKDQKYIFAAVDQKPSVISLQKLIAREVANEERGMFLISASSAGPEMYEIH
TNSKEERNNWMRRIQQAVESCPEEKGGRTSESDEDKRKAEARVAKIQQCQEILTNQDQQICAYLE
EKLHIYAELGELSGFEDVHLEPHLLIKPDPGEPPQAASLLAAALKEAESLQVAVKASQMGAVSQS
CEDSCGDSVLADTLSSHDVPGSPTASLVTGGREGRGCSDVDPGIQGVVTDLAVSDAGEKVECRNF
PGSSQSEIIQAIQNLTRLLYSLQAALTIQDSHIEIHRLVLQQQEGLSLGHSILRGGPLQDQKSRD
ADRQHEELANVHQLQHQLQQEQRRWLRRCEQQQRAQATRESWLQERERECQSQEELLLRSRGELD
LQLQEYQHSLERLREGQRLVEREQARMRAQQSLLGHWKHGRQRSLPAVLLPGGPEVMELNRSESL
CHENSFFINEALVQMSFNTFNKLNPSVIHQDATYPTTQSHSDLVRTSEHQVDLKVDPSQPSNVSH
KLWTAAGSGHQILPFQESSKDSCKNDLDTSHTESPTPHDSNSHRPQLQAFITEAKLNLPTRTMTR
QDGETGDGAKENIVYL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 16C.
Table 16C. Pat results for NOV16 Smallest Sum eading igh Prob equences Frame Score P(N) producing High-scoring Segment Pairs:
>patp:ABB44551Humanwound healing related polypeptide+1 1470 2.8e-150 >patp:AAW93941Humanbrx protein +1 1436 1.5e-148 >patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148 #5528 >patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148 #5528 >patp:ABG15870Novelhuman diagnostic protein +1 1447 7.5e-148 #15861 In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 4339 of 5274 bases (82%) identical to a gb:GENBANI~-m:MMU73199~acc:U73199.1 mRNA from Mus musculus (Rho-guanine nucleotide exchange factor mRNA, complete cds). The full amino acid sequence of the protein of the invention was found to have 1350 of 1670 amino acid residues (80%) identical to, and 1460 of 1670 amino acid residues (87%) similar to, the 1693 amino acid residue ptnr:SWISSPROT-ACC:P97433 protein fromMus n2usculus (RHO-GUANINE
NUCLEOTIDE EXCHANGE FACTOR (RHOGEF) (RIP2)).
NOV16 also has homology to the proteins shown in the BLASTP data in Table 16D.
Table 16D. BLAST
results for Gene Index/ Protein/ OrganismLength Identity PositivesExpect Identifier (aa) (~) (~) gi~7106395~ref~NPRho interacting1693 1350/16741460/16740.0 _ protein 2; Rho (80~) (86~) 36156.1~(NM_012026) specific exchange factor [Mus musculus]
gi~18602674~ref~XPhypothetical 669 668/669 668/669 0.0 _ protein FLJ21817 (99~) (99~) 016989.5~(XM
similar to Rhoip2 [Homo Sapiens]
gi~10438441~dbj~BABunnamed protein669 669/669 669/669 0.0 15243.1~(AK025816)product (1000 (1000 [Homo Sapiens]
gi~15341761~gb~AAH1hypothetical 615 609/613 609/613 0.0 2946.1~AAH12946(BCOprotein FLJ21817 (99~) (99~) 12946) similar to Rhoip2 [Homo Sapiens]
gi~17437752~ref~XPsimilar to Rho 590 290/292 291/292 e-170 _ interacting (99$) (99~) 068710.1~(XM
_ protein 2; Rho specific exchange factor [Homo Sapiens]
A multiple sequence alignment is given in Table 16E, with the NOV 16 protein being shown on line 1 in Table 16E in a ClustalW analysis, and comparing the NOV 16 protein with the related protein sequences shown in Table 16D. This BLASTP data is displayed graphically in the ClustalW in Table 16E.
Table 16E. ClustalW Analysis of NOV16 1) > NOV16; SEQ >D NO:34 2) > gi~7106395~/ Rho interacting protein 2; Rho specific exchange factor [Mus musculus]; SEQ >D
N0:107 3) > gig 18602674/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo sapieras]; SEQ >D NO:108 4) > gig 10438441 ~/ unnamed protein product [Horno Sapiens]; SEQ ID N0:109 5) > gi~15341761~/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo Sapiens]; SEQ ID NO:110 6) > gig 17437752/ similar to Rho interacting protein 2; Rho specific exchange factor [Homo Sapiens];
SEQ ID NO:111 ' giI71063951 1 -MELSCSEVPLYGQKTVYAKFGKNVYLPEDAEFYFVYGGSHQRHWIADRVQDNVLQSSI 59 gip8sozs74~ 1 ____________________________________________________________ 1 gi~10438441~ 1 ____________________________________________________________ 1 gi~15341761~ 1 ____________________________________________________________ 1 gi~17437752~ 1 ____________________________________________________________ 1 gi~71063951 60 PGHWLQETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQADRLTACSHQTLL
gi~18602674) 1 ____________________________________________________________ 1 gi~10438441~ 1 ____________________________________________________________ 1 gi~15341761~ 1 ____________________________________________________________ 1 gi~17437752~ 1 ____________________________________________________________ 1 130 140 150 160 170 7.80 NOVl6 121 gi~71063951120 TPFALTVEALPALDEELVLALTQLELPLGWTVLGNSSLEVSLHRESLLHLAVRWALPKLF179 $ giI18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________ gi~15341761~1 ____________________________________________________________1 gi~17437752~1 1 1~ 190 200 210 220 230 240 gi~7106395~ 180 gi~18602674~ 1 ____________________________________________________________1 IS gi~104384411 1 ____________________________________________________________1 gi1153417611 1 ____________________________________________________________1 gi~17437752~ 1 ____________________________________________________________1 giI71063951240 LQFVHSSETLTLTVNHTAEHLLEADIKLFRKYFWDRAFLVKALEQEAKTEKATMPSGAAE299 gi~18602674)1 ____________________________________________________________1 gi~10438441~l ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752)1 ____________________________________________________________1 gi~7106395~300 TEEEVRNLESGRSPSEEEEDAKSIKSQVDGPSEHEDQDRLALDRSFDGLKKSKHVPASLA359 gi~18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~1534176111 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 4O giI71063951360 gi~18602674~1 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi~7106395~420 PDTSPCGRPLIENSEGTLDAAASQSFVTPSSSRTSNLNLSFGLHGFEKEQSHLKKRSSSL479 gi~1860267411 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi ~71063951480 DALVADSEGEGGSEPPICYAVGSQSSPRTG-gi ~1860267411 ____________________________________________________________1 gi ~10438441~1 ____________________________________________________________1 gi ~15341761~1 ____________________________________________________________1 gi ~17437752~1 ____________________________________________________________1 gi I7106395~539 gi ~1860267411 ____________________________________________________________1 gi I10438441~1 ____________________________________________________________1 gi ~15341761~1 ____________________________________________________________1 gi~17437752~ 1 ____________________________________________________________ 1 gi~7106395~
gi~186026741 1 ____________________________________________________________1 gi~10438441~ 1 ____________________________________________________________1 giI15341761~ 1 ____________________________________________________________1 gi117437752~ 1 ____________________________________________________________1 IS
gi~71063951659FSGVLQCSGCDKTLLGKESLQCANCKANTHKGCKDAVPPCTKKFQEKYNKNKPQSILGSS718 gi~1860267411 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 gi~15341761)1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 giI7106395~719SVRDVPAPGLSLHPSSSMPIGLPAGRKEFAAQVHPLSRSVPGTTLESFRR--AVTSLESE776 2S giI1860267411 ____________________________________________________________1 gi ~10438441~1 ____________________________________________________________1 gi~1534176111 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 giI71063951777GDSWRSRSHSDELFQSMGSSPSTESFMMEDWDSSLWIDLSSDAQEFEAESWSLWDPSF836 gi~1860267411 ____________________________________________________________1 3S gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________1 gi~71063951837CSRQEKDVIKRQDVIFELMQTEVHHIQTLLIMSEVFRKGMKEELQLDHSTVDKIFPCLDE896 gi~18602674~1 ____________________________________________________________1 gi~1043844111 ____________________________________________________________1 4S gi~1534176111 ____________________________________________________________1 gi~1743775211 ____________________________________________________________1 .) giI71063951897LLETHRHFFFSMKERRQESCAGSDRNFVINQIGDILVQQFSEENASKMKRIYGEFCSHHK956 gi~18602674~1 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi115341761~1 ____________________________________________________________ SS gi~17437752~1 ____________________________________________________________ C70gi~7106395~957EAMSLFKELQQNKKFQNFIKIRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKE10 gi~1860267411 ____________________________________________________________1 gi~10438441~1 ____________________________________________________________1 gi~15341761~1 ____________________________________________________________1 gi~17437752~1 ____________________________________________________________ 1030 1040 . 1050 1060 1070 1080 .
' ' a 1077 r rv .n ~ --a ~
~
i a 1073 giI71063951 1017 RTEEHRDLCKALGLIKD ~ ~ --a ~
70 gi~18602674~ 1 ---------------- ~ --a ~ a 40 gi~10438441~ 1 ________________ . , __~ 1 ~ 40 gi~7.5341761~ 1 ________________ . , __~ 1 ~ 40 gi~17437752~ 1 ------------------PDNQNPT CSGET~~ P EGGS---- PAS RCES 38 gi~71063951 1074 1133 gi~186026741 41 100 giI10438441~ 41 100 gi~15341761~ 41 100 gi117437752~ 39 94 gi~7106395~ 1134 1193 gi~18602674~ 101 160 gi~10438441~ 101 160 gi115341761~ 101 160' gi117437752~ 95 148 NOV16 1198 ' 1.1i~- ~ ~. ~ 11 1 ~m 111 s~~ ~~~~ . 1-- 1256 gi~7106395~ 1194 ' 1 ~ ~ ~' ~ 11 1 SPm 111 1T ~ 1 1252 gi~18602674~ 161 1 1~'- ~ ~' ~ 11 1 1111 ~ ~ 1 219 gi~10438441~ 161 1 1 ' ~ ~~ ~ 11 1 1111 ~ 1 219 gi~15341761~ 161 ' 1 1 '- ~ ~~ ~ 11 1 1111 ~ ~ 1 219 gi ~ 17437752 ~ 149 L QSL ~LNIFLPVT~I~QIKFGMFG,- ------FVTIVMSVCKVG~TKE~ 202 ~ y NOV16 1257 ~ 1316 giI7106395~ 1253 1312 gi~18602674~ 220 279 gi~10438441~ 220 279 gi~15341761~ 220 279 gi~17437752~ 203 252 NOV16 1317 . 1375 gi~7106395~ 1313 1371 gi~186026741 280 338 gi~10438441~ 280 338 gi~15341761~ 280 338 gi117437752~ 253 311 gi~7106395~ 1372 1430 gi~186026741 339 397 gi~10438441~ 339 397 gi~15341761~ 339 397 gi~174377521 312 351 gi~7106395~ 1431 1490 giI186026741 398 457 gi~104384411 398 457 gi~15341761~ 398 457 gi~174377521 351 399 ..
NOV16 1495 1~1 1 ~1 1 1~~~ ~11~ ~1 .' 1554 .1. .1..... . 1 . 1 1 1 ~1 . i 1 11 ~
~
.1. .1.. . 1 . 1 E 1 P
. S P T' .1. ..l....~.'~a'a1 ' . 1 1 a a 1 .1. .1..... . 1 . 1 . 1 1 1 . 1 .1. .1..... 1 . 1 . 1 1 1 GQ.".~.:. T- PAEFYFTYD HQRH I-- BRIE..,.QS,'r'"'LPG
~'AKFD _ 1 _ _ _ _ _ _ VY
1 s 1 1 1 r 1 1 1 aee .1 1 1 ' 1 a - 1 1 1 . a G~QEE,,, CLCS- YSPVTGS SVTYVD CRLiRLLVTQA1~ RLTAC QT,'C.e IiT
$ ~ I 56 NOV16 1555 ~ ' ~ ~~~ -- ~ ~ -m 1611 giI71063951 1551 ~FGH L S ~P ~ C,'~PW~~--- ~LE F~ S-PT 1606 gi~18602674~ 518 ~ ~ ~~~ - ~ ~ -~ 574 giI10438441~ 518 ~ t ~s~ - ~ ~ -~ 574 gi~15341761~ 518 ~ ~ ~ ~ ~~~ -- ~ ~F ~ -~ 574 gi ~ 17437752 ~ 457 KLFRKYFW~3 ~, LV'KAFEPARPEERTAts'~PS
GAET~:EARLK2VLT~KQFL~I'~'LG PDA 516 . ..
y ~~~ .y ~~~ ~~. ~~.~ ~~. y ~r vr --w n ~~~ ~ ~ ~v v NOV16 1612 ~~~ ~~1~~S ~ ~ o~~~ --~ vTSHT P'~ ~~~ H 1668 giI7106395~ 1607 rI~I ~~P -STG-P ~RPAL~ ~ Y~ r yFQS S~Q~ QR 1664 gi ~ 18602674 ~ 575 ~ ~ ~ ~ ~S ~'~ . ~ w. r ~ - r ~TPH~' P'~ ~ ~ 631 gi I 10438441 ~ 575 ~ ~ ~ v ~S ~ ~ ~ ~ o ~ --~ ~'SFi':G' P'T~ ~ H 631 gi~15341761~ 575 ~ L ~~ ~~L ~~ ~ ~ ~ --GN---------------- 615 gi~17437752~ 517 L ~LDLP~QTAL ~PREFAFT ~LWVLAT y,;GAT~YPAAQ~IYILTT'~'V~R 576 NOV16 1669 (~ ~Q~FI~rE~,~ rLNLPTRT ~QDGETGDGAKENIVYL 1706 gi~71063951 1665 W~PATDTHNR~ TKSSDGGW ~ RCWRRG--------- 1693 giI186026741 632 Q~FI~E' LNLPTRT ~QDGETGDGAKENIVYL 669 gi~10438441~ 632 Q ~ FI E LNLPTRT ~QDGETGDGAKENIVYL 669 gi~15341761~ 615 ______________________________________ 615 gi~17437752~ 577 F~~KED~RCARG-_________________-_____ 590 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 16F lists the domain description from DOMAIN analysis results against NOV 16.
Tab le 16F Domain Anal sis of NQV16 Model Region of Score (bits) E value Homology Phorbol 654-700 40.1 4.9e-08 esters/diacylglycerol binding dom (DAG
PE-bind) PHD-finger (PHD) 666-703 1.4 0.06 Phorbol 654-698 46.2 2.0e-05 esters/diacylglycerol binding domain (DAG
PE-bind) Cl, Protein kinase654-698 45.8 2.0e-05 C
conserved region RhoGEF domain 854-1044 78.4 1.5e-19 (RhoGEF) bZIP transcription1022-1048 5.0 7.1 factor (bZIP) PH domain (PH) 1088-1189 40.9 4.5e-10 gi~7106395~ 1491 1 gi~18602674~ 458 gi~10438441~ 458 5 gi~153417611 458 5 gi 17437752 399 4 Consistent with other known members of the subunit family of proteins, NOV 16 has, for example, a RhoGEF signature sequence and homology to other members of the RhoGEF-like Protein Family. NOV 16 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 16 nucleic acids and polypeptides can be used to identify proteins that are members of the RhoGEF-like Protein Family. The NOV16 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV16 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., cancer, trauma, regeneration (ifa vitro and in vivo), viral/bacterial/parasitic infections, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, anemia , bleeding disorders, scleroderma, transplantation, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cirrhosis, transplantation, adrenoleukodystrophy , congenital adrenal hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmme disease, allergies, immunodeficiencies, transplantation, graft versus host disease, lyrnphedema , allergies, immunodeficiencies, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, ARDS, endometriosis, fertility, hyperthyroidism, hypothyroidism, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, alopecia, pigmentation disorders, endocrine disorders as well as other diseases, disorders and conditions.
In addition, various NOV 16 nucleic acids and polypeptides according to the invention are useful, ifate~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 16 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the RhoGEF-like Protein Family.
GEF (Guanine nucleotide exchange factor) for Rho/Rac/Cdc42-like GTPases is also called Dbl-homologous (DH) domain. It appears that PH (pleckstrin homology) domains invariably occur C-terminal to RhoGEF/DH domains. Although the exact function of PH
domains is unclear, several choices include binding to the beta/gamma subunit of heterotrimeric G proteins, binding to lipids, e.g. phosphatidylinositol-4,5-bisphosphate, binding to phosphorylated Ser/Thr residues, attachment to membranes by an unknown mechanism. The DAG PE-binding domain binds two zinc ions; the ligands of these metal ions are probably the six cysteines and two histidines that are conserved in this domain and can regulate signal transduction by the PKC family of kinases.
NOV 16 belongs to the guanine nucleotide exchange factor family of proteins which play a signficant role in signal transduction. The guanine nucleotide exchange factor (GEF) domain that regulates GTP binding protein signaling. The GEF domain regulates positively the signaling cascades that utilize GTP-binding proteins (such as those of the ras superfamily) that function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. An example of a protein containing GEF
and PH
domains is FGD1 (faciogenital dyplasia protein) Experiments have shown that the GEF and (PH) domains of FGDl can bind specifically to the Rho family GTPase Cdc42Hs and stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. The GEF
domain of FGD1 has also been shown to activate 2 kinases involved in cell proliferation;
the Jun NH2-terminal kinase and the p70 S6 kinase (~heng et al.; J. Biol. Chem 1996 Dec 27;271(52):33169-72). Thus, NOV16 polypeptide may play an important role in normal development as well as disease. This class of molecules (GEFs) is also being considered as a good drug target as the guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters and is bound by bryostatin 1, a compound currently in cliW cal trials (Lorenzo et al.; Mol. Pharmacol 2000 May; 57(5):840-6). The homolog of RhoGEF, DRhoGEF2 fail to gastrulate due to a defect in cell shape changes required for tissue invagination and the mRNA is found throughout oogenesis and embryogenesis (Barrett et al.;
Cell 1997; 91(7):905-15; Werner et al.; Gene 1997; 187(1):107-14). RhoGEF also interacts with c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1). JIP-1 might function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3 (Meyer et al.; J
Biol Chem 1999; 274(49):35113-8).
The NOV 16 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of blood and nerve physiology. As such, the NOV 16 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat blood and nervous system disorders, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, anemia , bleeding disorders, scleroderma, transplantation, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cirrhosis, transplantation, adrenoleukodystrophy , congenital adrenal hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease, lymphedema allergies, immunodeficiencies, osteoporosis, hypercalceirriia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, ARDS, endometriosis, fertility, hyperthyroidism, hypothyroidism, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubulax acidosis, IgA
nephropathy, hypercalceimia, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, alopecia, pigmentation disorders, endocrine disorders as well as other diseases, disorders and conditions.
The NOV16 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 16 nucleic acid is expressed in Adipose, Umbilical Vein, Pancreas, Thymus, Brain, Lung, Kidney, Adrenal Gland/Suprarenal gland, Peripheral Blood, Lymph node, Cartilage, Mammary gland/Breast, Uterus, Prostate, Trachea, Cochlea, I~ermis, Heart, Aorta, Coronary Artery, Thyroid, Liver, Bone, Bone Marrow, Spinal Cord, Cervix, and Retina.
Additional utilities for NOV16 nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-s stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation colon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes axe generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, wluch is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i. e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 199; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement thereof.
Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ll~
NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ m NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isofonns of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX
polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG
"start" codon and terminates with one of the three "stop" codons, naively, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fzde cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, S0, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; or an anti-sense strand nucleotide sequence of SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; or of a naturally occurring mutant of SEQ m NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an NOVX polypeptide"
refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, that encodes a polypeptide having an NOVX
biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 due to degeneracy of the genetic code and thus encode the same NOVX
proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
In addition to the human NOVX nucleotide sequences shown in SEQ 1T7 NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX
polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well knovcni in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, SO% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formainide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, rohrl Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, SX Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990;
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ )D NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, SX SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vo1) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA
78:
6789-6792.
Conservative Mutations In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins.
For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX
proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; still more preferably at least about 80% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QIiRK, MILV, MTLF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof. An "antisense"nucleic acid comprises a nucleotide sequence that is complementary to a ".sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, or antisense nucleic acids complementary to aii NOVX nucleic acid sequence of SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an NOVX
protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA.
For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylinethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladeiune, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated ih situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an oc-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. I5:
6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215: 327-330.
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX
mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33). For example, a derivative of a Tetrahynaena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA
can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX
promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6:
569-84; Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polyrnerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996.
supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment.
See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., I~rol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988.
Phar~n. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ 1D NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX
antibodies. Tn one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellulax material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX
proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein.
Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
Tn an embodiment, the NOVX protein has an amino acid sequence shown SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. In other embodiments, the NOVX protein is substantially homologous to SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and retains the functional activity of the protein of SEQ ~
NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and retains the functional activity of the NOVX proteins of SEQ m NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
Determining Homology Between Two or More Seguences I O To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal aligmnent with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMoI Bi~l 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of S.0 and GAP
extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS: I, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or T, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i. e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins The invention also provides NOVX chimeric or fusion proteins. As used herein, an NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34), whereas a "non-NOVX
polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein. In another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein. In yet another embodiment, an NOVX
fusion protein comprises at least three biologically-active portions of an NOVX protein.
Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX
polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX
polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX
polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence. , In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction i:2 vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as imrnunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are Iigated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX A~onists and Antagonists The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein).
An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific 14g biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subj ect with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subj ect relative to treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Ayauu. Rev. Bioehem. 53: 323; Itakura, et al., 1984.
Scieyace 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeutide Libraries In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX
3 S proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants.
See, e.g., Arkin and Yourvan, 1992. Pnoc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et al., 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and imrnunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab~ and F~ab~>a fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGI, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated NOVX-related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Py~oc. Nat. Acad.
Sci. USA 78:
3824-3828; I~yte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immmospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immmlostimulatory agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affiuty for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Inanaunol., 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLTCATIONS, MarCel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an ira vitf°o binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. BioclZefn., 107:220 (I980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of marine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous marine sequences ((J.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combiung site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechrnann et al., 1988; and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Itnmunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barn Virus ih vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J: Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (BiolTechn.ology 10, 779-783 (1992));
Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-I3 (1994));
Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Natune Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. IynmurZOl. 13 6S-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light imrnunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO
96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins.
The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chairr locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F~~b~~2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F,, fragments.
Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')z bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies .
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal ditluols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')a molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. US'A 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Imrnunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. ImnZUnol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7); or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTLTBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
For example, irmnunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No.
4,676,980.
Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.
Examples S include magi, 131h t3yl~ 90~,~ and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Caxbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is adminstered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein possessing such a domain. Thus, antibodies that axe specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NOVX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an NOVX
polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-NOVX antibody can facilitate the purification of natural NOVX
polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells.
Moreover, an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NOVX
protein. Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detectiomcan be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzynes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/fiotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include lash i3ih ass or 3H.
NOVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) axe integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX
proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Eschey~ichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated iya vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Eschericlaia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Geue 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Geyze 69:301-315) and pET 11 d (Studier et al., GENE EXPRESSION
TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-211$). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Sacclaaro~cyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:
933-943), pJRY8f (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMB (Seed, 1987. Nature 329: 840) and pMT2PC (I~aufinan, et al., 1987. EMBO
J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Imyrl.unol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983.
Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Scieyace 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.
264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (I~essel and Gruss, 1990. Science 249: 374-379) and the ~-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either.
mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the teens "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G4I8, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinj ection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 can be introduced as a transgene into the genome of a non-human animal.
Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX
gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequences) can be operably-linked to the NOVX
transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866;
4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING TIC MousE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant anmal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7, 9, 11; 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33), but more preferably, is a non-human homologue of a human NOVX gene.
For example, a mouse homologue of human NOVX gene of SEQ m NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein;
also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the NOVX
gene to allow for homologous recombination to occur between the exogenous NOVX
gene earned by the vector and an endogenous NOVX gene in an embryonic stem cell.
The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX
gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described fixrther in Bradley, 1991. Curs. Opiya. Biotechhol. 2: 823-829; PCT
International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the. cre/loxP recombinase system of bacteriophage P 1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Pf~oc. Natl.
Aead. Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP
recombinase system is used to. regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wihnut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable Garner" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable Garners are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5%
human serum albumin. Liposornes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid Garner is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the Iike can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
m For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal admiiustration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods fox preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including Iiposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated;
each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such am active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,32,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. P~oc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity..In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supna.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or iuubitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX
protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. S'ee, e.g., Lam, 1997. AnticanceY Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. P~oc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. TLS'.A. 91: 11422; Zuckermaim, et al., 1994. J. Med.
Chezn. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.
Izzt. Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chezn. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Clzem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Bioteclzhiques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. P~oc. Natl. Acad. Sci. USA. 87: 6378-6382;
Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lash 3sS, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX
protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a "target molecule" is a molecule with which an NOVX
protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide of the invention. In one embodiment, an NOVX
target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that 'has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an NOVX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalyticJenzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determW ed either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein determining the ability of the test compound to interact with an NOVX
protein comprises determining the ability of the test compound to preferentially bind to NOVX
or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX
protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to an NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supYa.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of an NOVX
target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton° X-114, Thesit~, Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein ox its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the.test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.
Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its taxget molecule, can be derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX
protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of N~VX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its 17~
absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mltNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate.
compound is identified as an inhibitor of NOVX mIZNA or protein expression.
The level of NOVX mIZNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. G'hem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8:
1693-1696; and Brent WO 94110300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains of the transcription.factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention fiu they pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences, SEQ ID
NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome.
The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers 1 S (preferably 15-25 by in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
1 S Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR
primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 are used, a more appropriate number of primers for positive individual identification would be S00-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX
protein andlor nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assavs An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA
or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')a) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i. e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA
include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX
protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subj ect or genomic DNA molecules from the test subj ect. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
Tn another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent 1~4 capable of detecting NOVX protein, mItNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard., The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity.
Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subj ect that can be administered the agent to treat a disorder associated with aberrant NOVX
expression or activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR maybe desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Q(3 Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or Ioss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.
Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, 1 S RNA/DNA duplexes can.be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzyrnatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroXide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g:, Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids.
See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766;
Cotton,1993. Mutat. Res.
285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA
fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g:, Keen, et al., 1991. Trends Genet. 7:
S.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the laiown mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163;
Saiki, et a1.,1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerise extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acid. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match.at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX
protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linden, 1997. Clin.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT~2) and cytochrome P450 enzymes CI~P2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CI'I'2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified.
Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i. e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the DEMANDE OU BREVET VOLUMINEUX
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PLUS D'UN TOME.
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VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
GAET~:EARLK2VLT~KQFL~I'~'LG PDA 516 . ..
y ~~~ .y ~~~ ~~. ~~.~ ~~. y ~r vr --w n ~~~ ~ ~ ~v v NOV16 1612 ~~~ ~~1~~S ~ ~ o~~~ --~ vTSHT P'~ ~~~ H 1668 giI7106395~ 1607 rI~I ~~P -STG-P ~RPAL~ ~ Y~ r yFQS S~Q~ QR 1664 gi ~ 18602674 ~ 575 ~ ~ ~ ~ ~S ~'~ . ~ w. r ~ - r ~TPH~' P'~ ~ ~ 631 gi I 10438441 ~ 575 ~ ~ ~ v ~S ~ ~ ~ ~ o ~ --~ ~'SFi':G' P'T~ ~ H 631 gi~15341761~ 575 ~ L ~~ ~~L ~~ ~ ~ ~ --GN---------------- 615 gi~17437752~ 517 L ~LDLP~QTAL ~PREFAFT ~LWVLAT y,;GAT~YPAAQ~IYILTT'~'V~R 576 NOV16 1669 (~ ~Q~FI~rE~,~ rLNLPTRT ~QDGETGDGAKENIVYL 1706 gi~71063951 1665 W~PATDTHNR~ TKSSDGGW ~ RCWRRG--------- 1693 giI186026741 632 Q~FI~E' LNLPTRT ~QDGETGDGAKENIVYL 669 gi~10438441~ 632 Q ~ FI E LNLPTRT ~QDGETGDGAKENIVYL 669 gi~15341761~ 615 ______________________________________ 615 gi~17437752~ 577 F~~KED~RCARG-_________________-_____ 590 The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro~. Table 16F lists the domain description from DOMAIN analysis results against NOV 16.
Tab le 16F Domain Anal sis of NQV16 Model Region of Score (bits) E value Homology Phorbol 654-700 40.1 4.9e-08 esters/diacylglycerol binding dom (DAG
PE-bind) PHD-finger (PHD) 666-703 1.4 0.06 Phorbol 654-698 46.2 2.0e-05 esters/diacylglycerol binding domain (DAG
PE-bind) Cl, Protein kinase654-698 45.8 2.0e-05 C
conserved region RhoGEF domain 854-1044 78.4 1.5e-19 (RhoGEF) bZIP transcription1022-1048 5.0 7.1 factor (bZIP) PH domain (PH) 1088-1189 40.9 4.5e-10 gi~7106395~ 1491 1 gi~18602674~ 458 gi~10438441~ 458 5 gi~153417611 458 5 gi 17437752 399 4 Consistent with other known members of the subunit family of proteins, NOV 16 has, for example, a RhoGEF signature sequence and homology to other members of the RhoGEF-like Protein Family. NOV 16 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV 16 nucleic acids and polypeptides can be used to identify proteins that are members of the RhoGEF-like Protein Family. The NOV16 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV16 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular replication, and signal transduction. These molecules can be used to treat, e.g., cancer, trauma, regeneration (ifa vitro and in vivo), viral/bacterial/parasitic infections, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, anemia , bleeding disorders, scleroderma, transplantation, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cirrhosis, transplantation, adrenoleukodystrophy , congenital adrenal hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmme disease, allergies, immunodeficiencies, transplantation, graft versus host disease, lyrnphedema , allergies, immunodeficiencies, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, ARDS, endometriosis, fertility, hyperthyroidism, hypothyroidism, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, alopecia, pigmentation disorders, endocrine disorders as well as other diseases, disorders and conditions.
In addition, various NOV 16 nucleic acids and polypeptides according to the invention are useful, ifate~ alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV 16 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the RhoGEF-like Protein Family.
GEF (Guanine nucleotide exchange factor) for Rho/Rac/Cdc42-like GTPases is also called Dbl-homologous (DH) domain. It appears that PH (pleckstrin homology) domains invariably occur C-terminal to RhoGEF/DH domains. Although the exact function of PH
domains is unclear, several choices include binding to the beta/gamma subunit of heterotrimeric G proteins, binding to lipids, e.g. phosphatidylinositol-4,5-bisphosphate, binding to phosphorylated Ser/Thr residues, attachment to membranes by an unknown mechanism. The DAG PE-binding domain binds two zinc ions; the ligands of these metal ions are probably the six cysteines and two histidines that are conserved in this domain and can regulate signal transduction by the PKC family of kinases.
NOV 16 belongs to the guanine nucleotide exchange factor family of proteins which play a signficant role in signal transduction. The guanine nucleotide exchange factor (GEF) domain that regulates GTP binding protein signaling. The GEF domain regulates positively the signaling cascades that utilize GTP-binding proteins (such as those of the ras superfamily) that function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. An example of a protein containing GEF
and PH
domains is FGD1 (faciogenital dyplasia protein) Experiments have shown that the GEF and (PH) domains of FGDl can bind specifically to the Rho family GTPase Cdc42Hs and stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. The GEF
domain of FGD1 has also been shown to activate 2 kinases involved in cell proliferation;
the Jun NH2-terminal kinase and the p70 S6 kinase (~heng et al.; J. Biol. Chem 1996 Dec 27;271(52):33169-72). Thus, NOV16 polypeptide may play an important role in normal development as well as disease. This class of molecules (GEFs) is also being considered as a good drug target as the guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters and is bound by bryostatin 1, a compound currently in cliW cal trials (Lorenzo et al.; Mol. Pharmacol 2000 May; 57(5):840-6). The homolog of RhoGEF, DRhoGEF2 fail to gastrulate due to a defect in cell shape changes required for tissue invagination and the mRNA is found throughout oogenesis and embryogenesis (Barrett et al.;
Cell 1997; 91(7):905-15; Werner et al.; Gene 1997; 187(1):107-14). RhoGEF also interacts with c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1). JIP-1 might function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3 (Meyer et al.; J
Biol Chem 1999; 274(49):35113-8).
The NOV 16 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of blood and nerve physiology. As such, the NOV 16 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat blood and nervous system disorders, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, anemia , bleeding disorders, scleroderma, transplantation, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cirrhosis, transplantation, adrenoleukodystrophy , congenital adrenal hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease, lymphedema allergies, immunodeficiencies, osteoporosis, hypercalceirriia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, ARDS, endometriosis, fertility, hyperthyroidism, hypothyroidism, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubulax acidosis, IgA
nephropathy, hypercalceimia, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, alopecia, pigmentation disorders, endocrine disorders as well as other diseases, disorders and conditions.
The NOV16 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a NOV 16 nucleic acid is expressed in Adipose, Umbilical Vein, Pancreas, Thymus, Brain, Lung, Kidney, Adrenal Gland/Suprarenal gland, Peripheral Blood, Lymph node, Cartilage, Mammary gland/Breast, Uterus, Prostate, Trachea, Cochlea, I~ermis, Heart, Aorta, Coronary Artery, Thyroid, Liver, Bone, Bone Marrow, Spinal Cord, Cervix, and Retina.
Additional utilities for NOV16 nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-s stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation colon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes axe generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, wluch is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i. e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 199; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement thereof.
Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ll~
NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ m NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isofonns of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX
polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG
"start" codon and terminates with one of the three "stop" codons, naively, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fzde cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, S0, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; or an anti-sense strand nucleotide sequence of SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; or of a naturally occurring mutant of SEQ m NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an NOVX polypeptide"
refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, that encodes a polypeptide having an NOVX
biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 due to degeneracy of the genetic code and thus encode the same NOVX
proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
In addition to the human NOVX nucleotide sequences shown in SEQ 1T7 NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX
polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well knovcni in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, SO% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formainide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, rohrl Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, SX Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990;
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ )D NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, SX SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vo1) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA
78:
6789-6792.
Conservative Mutations In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins.
For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX
proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; still more preferably at least about 80% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QIiRK, MILV, MTLF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives thereof. An "antisense"nucleic acid comprises a nucleotide sequence that is complementary to a ".sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, or antisense nucleic acids complementary to aii NOVX nucleic acid sequence of SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an NOVX
protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA.
For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylinethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladeiune, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated ih situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an oc-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. I5:
6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215: 327-330.
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX
mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33). For example, a derivative of a Tetrahynaena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA
can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX
promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6:
569-84; Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polyrnerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996.
supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment.
See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., I~rol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988.
Phar~n. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ 1D NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX
antibodies. Tn one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellulax material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX
proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein.
Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
Tn an embodiment, the NOVX protein has an amino acid sequence shown SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. In other embodiments, the NOVX protein is substantially homologous to SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and retains the functional activity of the protein of SEQ ~
NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and retains the functional activity of the NOVX proteins of SEQ m NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
Determining Homology Between Two or More Seguences I O To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal aligmnent with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMoI Bi~l 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of S.0 and GAP
extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS: I, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or T, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i. e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins The invention also provides NOVX chimeric or fusion proteins. As used herein, an NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34), whereas a "non-NOVX
polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein. In another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein. In yet another embodiment, an NOVX
fusion protein comprises at least three biologically-active portions of an NOVX protein.
Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX
polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX
polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX
polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence. , In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction i:2 vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as imrnunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are Iigated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX A~onists and Antagonists The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein).
An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific 14g biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subj ect with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subj ect relative to treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Ayauu. Rev. Bioehem. 53: 323; Itakura, et al., 1984.
Scieyace 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeutide Libraries In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX
3 S proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants.
See, e.g., Arkin and Yourvan, 1992. Pnoc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et al., 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and imrnunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab~ and F~ab~>a fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGI, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated NOVX-related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Py~oc. Nat. Acad.
Sci. USA 78:
3824-3828; I~yte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immmospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immmlostimulatory agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affiuty for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Inanaunol., 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLTCATIONS, MarCel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an ira vitf°o binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. BioclZefn., 107:220 (I980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of marine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous marine sequences ((J.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combiung site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechrnann et al., 1988; and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Itnmunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barn Virus ih vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J: Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (BiolTechn.ology 10, 779-783 (1992));
Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-I3 (1994));
Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Natune Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. IynmurZOl. 13 6S-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light imrnunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO
96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins.
The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chairr locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F~~b~~2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F,, fragments.
Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')z bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies .
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal ditluols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')a molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. US'A 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Imrnunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. ImnZUnol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7); or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTLTBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
For example, irmnunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No.
4,676,980.
Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.
Examples S include magi, 131h t3yl~ 90~,~ and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Caxbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is adminstered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein possessing such a domain. Thus, antibodies that axe specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NOVX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an NOVX
polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-NOVX antibody can facilitate the purification of natural NOVX
polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells.
Moreover, an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NOVX
protein. Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detectiomcan be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzynes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/fiotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include lash i3ih ass or 3H.
NOVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) axe integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX
proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Eschey~ichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated iya vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Eschericlaia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Geue 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Geyze 69:301-315) and pET 11 d (Studier et al., GENE EXPRESSION
TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-211$). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Sacclaaro~cyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:
933-943), pJRY8f (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMB (Seed, 1987. Nature 329: 840) and pMT2PC (I~aufinan, et al., 1987. EMBO
J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Imyrl.unol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983.
Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Scieyace 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.
264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (I~essel and Gruss, 1990. Science 249: 374-379) and the ~-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either.
mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the teens "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G4I8, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinj ection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 can be introduced as a transgene into the genome of a non-human animal.
Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX
gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequences) can be operably-linked to the NOVX
transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866;
4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING TIC MousE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant anmal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7, 9, 11; 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33), but more preferably, is a non-human homologue of a human NOVX gene.
For example, a mouse homologue of human NOVX gene of SEQ m NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein;
also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the NOVX
gene to allow for homologous recombination to occur between the exogenous NOVX
gene earned by the vector and an endogenous NOVX gene in an embryonic stem cell.
The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX
gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described fixrther in Bradley, 1991. Curs. Opiya. Biotechhol. 2: 823-829; PCT
International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the. cre/loxP recombinase system of bacteriophage P 1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Pf~oc. Natl.
Aead. Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP
recombinase system is used to. regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wihnut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable Garner" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable Garners are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5%
human serum albumin. Liposornes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid Garner is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the Iike can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
m For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal admiiustration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods fox preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including Iiposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated;
each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such am active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,32,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. P~oc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity..In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supna.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or iuubitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX
protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. S'ee, e.g., Lam, 1997. AnticanceY Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. P~oc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. TLS'.A. 91: 11422; Zuckermaim, et al., 1994. J. Med.
Chezn. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.
Izzt. Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chezn. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Clzem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Bioteclzhiques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. P~oc. Natl. Acad. Sci. USA. 87: 6378-6382;
Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lash 3sS, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX
protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a "target molecule" is a molecule with which an NOVX
protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide of the invention. In one embodiment, an NOVX
target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that 'has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an NOVX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalyticJenzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determW ed either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX
protein, wherein determining the ability of the test compound to interact with an NOVX
protein comprises determining the ability of the test compound to preferentially bind to NOVX
or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX
protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to an NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supYa.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of an NOVX
target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton° X-114, Thesit~, Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein ox its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the.test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.
Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its taxget molecule, can be derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX
protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of N~VX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its 17~
absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mltNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate.
compound is identified as an inhibitor of NOVX mIZNA or protein expression.
The level of NOVX mIZNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. G'hem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8:
1693-1696; and Brent WO 94110300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains of the transcription.factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention fiu they pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences, SEQ ID
NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome.
The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers 1 S (preferably 15-25 by in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
1 S Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR
primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 are used, a more appropriate number of primers for positive individual identification would be S00-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX
protein andlor nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assavs An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA
or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')a) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i. e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA
include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX
protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subj ect or genomic DNA molecules from the test subj ect. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
Tn another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent 1~4 capable of detecting NOVX protein, mItNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard., The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity.
Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX
expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subj ect that can be administered the agent to treat a disorder associated with aberrant NOVX
expression or activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR maybe desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Q(3 Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or Ioss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.
Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, 1 S RNA/DNA duplexes can.be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzyrnatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroXide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g:, Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids.
See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766;
Cotton,1993. Mutat. Res.
285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA
fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g:, Keen, et al., 1991. Trends Genet. 7:
S.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the laiown mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163;
Saiki, et a1.,1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerise extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acid. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match.at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX
protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linden, 1997. Clin.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT~2) and cytochrome P450 enzymes CI~P2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CI'I'2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified.
Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i. e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the DEMANDE OU BREVET VOLUMINEUX
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Claims (41)
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34; and (d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4,-6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid. residues from said amino acid sequence.
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34; and (d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4,-6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid. residues from said amino acid sequence.
2. The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
4. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and (f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34;
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and (f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ
ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
9. The nucleic acid molecule of claim S, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence selected from the group consisting of SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33;
(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, provided that no more than 20% of the nucleotides differ from said nucleotide sequence;
(c) a nucleic acid fragment of (a); and (d) a nucleic acid fragment of (b).
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33;
(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, provided that no more than 20% of the nucleotides differ from said nucleotide sequence;
(c) a nucleic acid fragment of (a); and (d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting of SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of (a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;
(b) an isolated second polynucleotide that is a complement of the first polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
(b) an isolated second polynucleotide that is a complement of the first polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that immunospecifically-binds to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
(a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid molecule;
and (c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid molecule;
and (c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
20. A method of identifying an agent that binds to a polypeptide of claim 1, the method comprising:
(a) contacting said polypeptide with said agent; and (b) determining whether said agent binds to said polypeptide.
(a) contacting said polypeptide with said agent; and (b) determining whether said agent binds to said polypeptide.
21. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1, the method comprising:
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent; and (c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent; and (c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
22. A method for modulating the activity of the polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.
23. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
24. The method of claim 23, wherein said subject is a human.
25. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
26. The method of claim 25, wherein said subject is a human.
27. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
28. The method of claim 27, wherein the subject is a human.
29. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
30. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
31. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
32. A kit comprising in one or more containers, the pharmaceutical composition of claim 29.
33. A kit comprising in one or more containers, the pharmaceutical composition of claim 30.
34. A kit comprising in one or more containers, the pharmaceutical composition of claim 31.
35. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a NOVX-associated disorder, wherein said therapeutic is selected from the group consisting of a NOVX polypeptide, a NOVX nucleic acid, and a NOVX antibody.
36. A method for screening for a modulator of activity or of latency or predisposition to a NOVX-associated disorder, said method comprising:
(a) administering a test compound to a test animal at increased risk for a NOVX-associated disorder, wherein said test animal recombinantly expresses the polypeptide of claim 1;
(b) measuring the activity of said polypeptide in said test animal after administering the compound of step (a);
(c) comparing the activity of said protein in said test animal with the activity of said polypeptide in a control animal not administered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator of latency of or predisposition to a NOVX-associated disorder.
(a) administering a test compound to a test animal at increased risk for a NOVX-associated disorder, wherein said test animal recombinantly expresses the polypeptide of claim 1;
(b) measuring the activity of said polypeptide in said test animal after administering the compound of step (a);
(c) comparing the activity of said protein in said test animal with the activity of said polypeptide in a control animal not administered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator of latency of or predisposition to a NOVX-associated disorder.
37. The method of claim 36, wherein said test animal is a recombinant test animal that expresses a test protein transgene or expresses said transgene under the control of a promoter at an increased level relative to a wild-type test animal, and wherein said promoter is not the native gene promoter of said transgene.
38. A method for determining the presence of or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject, the method comprising:
(a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and (b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
(a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and (b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
39. A method for determining the presence of or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:
(a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and (b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease;
wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
(a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and (b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease;
wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
40. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a biologically active fragment thereof.
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a biologically active fragment thereof.
41. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
Applications Claiming Priority (13)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US28574801P | 2001-04-23 | 2001-04-23 | |
| US60/285,748 | 2001-04-23 | ||
| US28606801P | 2001-04-24 | 2001-04-24 | |
| US60/286,068 | 2001-04-24 | ||
| US28629201P | 2001-04-25 | 2001-04-25 | |
| US60/286,292 | 2001-04-25 | ||
| US28833401P | 2001-05-03 | 2001-05-03 | |
| US60/288,334 | 2001-05-03 | ||
| US29124101P | 2001-05-16 | 2001-05-16 | |
| US60/291,241 | 2001-05-16 | ||
| US32228401P | 2001-09-14 | 2001-09-14 | |
| US60/322,284 | 2001-09-14 | ||
| PCT/US2002/011634 WO2002085922A2 (en) | 2001-04-23 | 2002-04-11 | Proteins and nucleic acids encoding same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2443770A1 true CA2443770A1 (en) | 2002-10-31 |
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Family Applications (1)
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| CA002443770A Abandoned CA2443770A1 (en) | 2001-04-23 | 2002-04-11 | Proteins and nucleic acids encoding same |
Country Status (3)
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| EP (1) | EP1383533A4 (en) |
| CA (1) | CA2443770A1 (en) |
| WO (1) | WO2002085922A2 (en) |
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| JP4503801B2 (en) * | 2000-09-08 | 2010-07-14 | 独立行政法人科学技術振興機構 | Novel human cancer / testis antigen and its gene |
| DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
| US7294704B2 (en) | 2003-08-15 | 2007-11-13 | Diadexus, Inc. | Pro108 antibody compositions and methods of use and use of Pro108 to assess cancer risk |
| PT1954718E (en) | 2005-11-30 | 2014-12-16 | Abbvie Inc | Anti-a globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies |
| DK1976877T4 (en) | 2005-11-30 | 2017-01-16 | Abbvie Inc | Monoclonal antibodies to amyloid beta protein and uses thereof |
| US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
| WO2008104386A2 (en) | 2007-02-27 | 2008-09-04 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
| MX360403B (en) | 2010-04-15 | 2018-10-31 | Abbvie Inc | Amyloid-beta binding proteins. |
| EP2603524A1 (en) | 2010-08-14 | 2013-06-19 | AbbVie Inc. | Amyloid-beta binding proteins |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004507202A (en) * | 1999-03-31 | 2004-03-11 | キュラジェン コーポレイション | Nucleic acid containing an open reading frame encoding a polypeptide; "ORFX" |
| AU5489900A (en) * | 1999-06-14 | 2001-01-02 | Zymogenetics Inc. | Helical cytokine zalpha33 |
| EP1383892A2 (en) * | 2000-06-30 | 2004-01-28 | Incyte Genomics, Inc. | Human extracellular matrix and cell adhesion polypeptides |
-
2002
- 2002-04-11 CA CA002443770A patent/CA2443770A1/en not_active Abandoned
- 2002-04-11 WO PCT/US2002/011634 patent/WO2002085922A2/en not_active Application Discontinuation
- 2002-04-11 EP EP02723842A patent/EP1383533A4/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO2002085922A3 (en) | 2003-05-30 |
| WO2002085922A2 (en) | 2002-10-31 |
| EP1383533A4 (en) | 2006-11-02 |
| EP1383533A2 (en) | 2004-01-28 |
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