EP1309686A2 - Proteine slgp et molecules d'acide nucleique et leurs utilisations - Google Patents

Proteine slgp et molecules d'acide nucleique et leurs utilisations

Info

Publication number
EP1309686A2
EP1309686A2 EP01952313A EP01952313A EP1309686A2 EP 1309686 A2 EP1309686 A2 EP 1309686A2 EP 01952313 A EP01952313 A EP 01952313A EP 01952313 A EP01952313 A EP 01952313A EP 1309686 A2 EP1309686 A2 EP 1309686A2
Authority
EP
European Patent Office
Prior art keywords
slgp
seq
ofthe
nucleic acid
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01952313A
Other languages
German (de)
English (en)
Inventor
Fong-Ying Tsai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Millennium Pharmaceuticals Inc
Original Assignee
Millennium Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals Inc filed Critical Millennium Pharmaceuticals Inc
Publication of EP1309686A2 publication Critical patent/EP1309686A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GPCRs G-protein coupled receptors
  • proteins that have seven transmembrane domains. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property ofthe cell.
  • G protein-coupled receptors GPCRs
  • G-proteins and effectors intracellular enzymes and channels which are modulated by G-proteins
  • GPCRs G protein-coupled receptors
  • the GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications or other processes (as opposed to orthologues, the same receptor from different species and homologues, different forms of a receptor isolated from a single organism).
  • the superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the beta2- adrenergic receptor and currently represented by over 200 unique members (reviewed by Dohlman et al, (1991) Annu. Rev. Biochem. 60:653-688); Family II, the recently characterized parathyroid hormone/calcitonin/secretin receptor family (Juppner et al. (1991) Science 254:1024-1026; Lin et al.
  • Drosophila express a photoreceptor-specific protein bride of sevenless (boss), a seven- transmembrane-segment protein which has been extensively studied and does not show evidence of being a GPCR (Hart et al. (1993) Proc. Natl. Acad. Sci. USA 90:5047- 5051 ).
  • the gene frizzled (fz) in Drosophila is also thought to be a protein with seven transmembrane segments. Like boss, fz has not been shown to couple to G-proteins (Vinson et al, Nature 338:263-264 (1989)).
  • G proteins represent a family of heterotrimeric proteins composed of ⁇ , ⁇ and ⁇ subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane domains. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the ⁇ -subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the ⁇ -subunits.
  • the GTP-bound form ofthe ⁇ -subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • second messengers such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • cyclic AMP e.g., by activation of adenylate cyclase
  • diacylglycerol diacylglycerol
  • inositol phosphates inositol phosphates.
  • G proteins are described extensively in Lodish H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995).
  • GPCRs are a major target for drug
  • the present invention is based, at least in part, on the discovery of novel G- protein coupled receptor (GPCR) family members, referred to herein as "SLGP” nucleic acid and protein molecules.
  • GPCR G- protein coupled receptor
  • the human SLGP molecules ofthe invention are also referred to herein as “1983" nucleic acid and protein molecules.
  • the mouse SLGP molecules ofthe invention are also referred to herein as "12231” or “ml983" nucleic acid and protein molecules.
  • this invention provides isolated nucleic acid molecules encoding SLGP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of SLGP-encoding nucleic acids.
  • the present invention is also based at least in part, on the discovery that the discovery that the
  • SLGP molecules ofthe present invention are involved in the modulation of angiogenesis in endothelial cells (e.g., tumor endothelial cells) and that, therefore, they are useful as targets and therapeutic agents for the modulation of endothelial cell (e.g., tumor endothelial cell) proliferation, growth, differentiation, or migration.
  • endothelial cell e.g., tumor endothelial cell
  • the SLGP molecules ofthe present invention are also useful as targets and therapeutic agents for cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • an SLGP nucleic acid molecule ofthe invention is at least 40%, 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length ofthe nucleotide sequence) shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO: 19, or a complement thereof.
  • the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-19 of SEQ ID NOT.
  • the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 2090-2987 of SEQ ID NOT.
  • the nucleic acid molecule includes SEQ ID NO: 19 and nucleotides 1-69 of SEQ ID NO:17.
  • the nucleic acid molecule includes SEQ ID NO: 19 and nucleotides 2139-3952 of SEQ ID NO: 17.
  • the nucleic acid molecule consists ofthe nucleotide sequence shown in SEQ ID NO: 1,3, 17, or 19. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 749 nucleotides ofthe nucleotide sequence of SEQ ID NOT, SEQ ID NO:3, or a complement thereof.
  • an SLGP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NOT 8, or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • an SLGP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NOT 8, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as
  • an isolated nucleic acid molecule encodes the amino acid sequence of human SLGP. In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of mouse SLGP. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, SEQ DI NOT 8, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number . In yet another preferred embodiment, the nucleic acid molecule is at least 3952 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 3952 nucleotides in length and encodes a protein having an SLGP activity (as described herein).
  • nucleic acid molecules preferably SLGP nucleic acid molecules, which specifically detect SLGP nucleic acid molecules relative to nucleic acid molecules encoding non-SLGP proteins.
  • a nucleic acid molecule is at least 1930, 1900-2000, 1700-2200, 1500-2400, 1300-2600, 1100-2800 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NOT or SEQ ID NO: 17, the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-569, 1058-1295, or 2044-2225 of SEQ ID NOT. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-569, 1058-1295, or 2044-2225 of SEQ ID NOT.
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NOT8, or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ ID NO:3, SEQ ID, SEQ ID NOT7, or SEQ ID NOT9 under stringent conditions.
  • Another embodiment ofthe invention provides an isolated nucleic acid molecule which is antisense to an SLGP nucleic acid molecule, e.g., the coding strand of an SLGP nucleic acid molecule.
  • Another aspect ofthe invention provides a vector comprising an SLGP nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector ofthe invention.
  • the invention also provides a method for producing a protein, preferably an SLGP protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, ofthe invention containing a recombinant expression vector, such that the protein is produced.
  • a host cell e.g., a mammalian host cell such as a non-human mammalian cell
  • a recombinant expression vector such that the protein is produced.
  • an SLGP protein includes at least one transmembrane domain.
  • the isolated protein preferably an SLGP protein includes seven transmembrane domains.
  • an SLGP protein includes at least one transmembrane domain and has an amino acid sequence at least about 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%), 85%, 90%), 95%, 98% or more identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 18, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • the SLGP protein includes at least one transmembrane domain and plays a role in the mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (P1P2 inositol 1,4,5-triphosphate (IP3), or adenylate cyclase; the production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA; cell migration; cell differentiation; cell survival; or angiogenesis, e.g.
  • a signal transduction pathway e.g., phosphatidylinositol 4,5-bisphosphate (P1P2 inositol 1,4,5-triphosphate (IP3), or adenylate cyclase
  • P1P2 inositol 1,4,5-triphosphate IP3
  • adenylate cyclase adenylate cyclase
  • an SLGP protein includes at least one transmembrane domain and plays a role in the modulation of angiogenesis (e.g., angiogenesis in tumors).
  • the protein preferably an SLGP protein, includes at least one transmembrane domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO: 17, or SEQ ID NO: 19.
  • the invention features fragments ofthe protein having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) ofthe amino acid sequence of SEQ ID NO:2, SEQ ID NO: 18, or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with the ATCC as Accession Number .
  • the protein preferably an SLGP protein, has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18.
  • the invention features an isolated protein, preferably an SLGP protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 40%, 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to a nucleotide sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT 7, SEQ ID NOT9, or a complement thereof.
  • This invention further features an isolated protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NOT7, SEQ ID NO:19, or a complement thereof.
  • the proteins ofthe present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-SLGP polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind the SLGP proteins ofthe invention.
  • the SLGP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of an SLGP nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an SLGP nucleic acid molecule, protein or polypeptide such that the presence of an SLGP nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of SLGP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of SLGP activity such that the presence of SLGP activity is detected in the biological sample.
  • the invention provides a method for modulating SLGP activity comprising contacting a cell capable of expressing SLGP with an agent that modulates SLGP activity such that SLGP activity in the cell is modulated.
  • the agent inhibits SLGP activity.
  • the agent stimulates SLGP activity.
  • the agent is an antibody that specifically binds to an SLGP protein.
  • the agent modulates expression of SLGP by modulating transcription of an SLGP gene or translation of an SLGP mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an SLGP mRNA or an SLGP gene.
  • the methods ofthe present invention are used to treat a subject having a disorder characterized by aberrant SLGP protein or nucleic acid expression or activity by administering an agent which is an SLGP modulator to the subject.
  • the SLGP modulator is an SLGP protein.
  • the SLGP modulator is an SLGP nucleic acid molecule.
  • the SLGP modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant SLGP protein or nucleic acid expression is a proliferative disorder, e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • a proliferative disorder e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an SLGP protein; (ii) mis-regulation ofthe gene; and (iii) aberrant post-translational modification of an SLGP protein, wherein a wild-type form ofthe gene encodes an protein with an SLGP activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of an SLGP protein, by providing an indicator composition comprising an SLGP protein having SLGP activity, contacting the indicator composition with a test compound, and determining the effect ofthe test compound on SLGP activity in the indicator composition to identify a compound that modulates the activity of an SLGP protein.
  • Figure 1 depicts the cDNA sequence of human SLGP (also referred to herein as 1983).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2987 of SEQ ID NOT.
  • Figure 2 depicts the predicted amino acid sequence of human SLGP.
  • the amino acid sequence corresponds to amino acids 1 to 690 of SEQ ID NO:2.
  • Figures 3 A and 3B depict the coding region ofthe cDNA sequence of human SLGP.
  • the nucleotide sequence corresponds to amino acids 1 to 2070 of SEQ ID NO:3.
  • Figures 4A and 4B depict an alignment ofthe amino acid sequences of human SLGP (SEQ ID NO:2) and human CD 97 (Accession No. U76764, SEQ ID NO: 15). This alignment were generated utilizing the ALIGN program with the following parameter setting: PAM120, gap penalties: -12/-4 (Myers, E. and Miller, W. (1988) "Optimal Alignments in Linear Space" CABIOS 4:11-17).
  • Figures 5A-5F depict an alignment ofthe nucleotide sequences of human SLGP (SEQ ID NO:2) and human CD 97 (Accession No. U76764, SEQ ID NO: 16). This alignment were generated utilizing the ALIGN program with the following parameter setting: PAM120, gap penalties: -12/-4 (Myers, E. and Miller, W. (1988) "Optimal Alignments in Linear Space” CABIOS 4:11-17).
  • Figures 6 A and 6B depict the cDNA sequence of mouse SLGP (also referred to herein as ml983 and 12231).
  • the nucleotide sequence corresponds to nucleic acids 1 to 3,592 of SEQ ID NO.T 7.
  • Figure 7 depicts the predicted amino acid sequence of mouse SLGP.
  • the amino acid sequence corresponds to amino acids 1 to 689 of SEQ ID NO: 18.
  • Figures 8 A and 8B depict the coding region ofthe cDNA sequence of mouse SLGP.
  • the nucleotide sequence corresponds to amino acids 1 to 2067 of SEQ ID NO:19.
  • Figure 9 is a graph depicting the results of a TaqManTM analysis of expression of human SLGP (1983) cDNA in HMVEC.
  • Human SLGP (1983) is up-regulated in tube forming HMVEC and proliferating HMVEC as compared to arresting HMVEC.
  • Figure 10 is a graph depicting human SLGP (1983) expression in HMVEC by transcripional profiling analysis. Human SLGP (1983) is up-regulated in proliferating HMVEC as compared to arresting HMVEC.
  • Figure 11 is a graph depicting TaqManTM analysis of human SLGP (1983) expression in HMVEC. Human SLGP (1983) is up-regulated in proliferating HMVEC.
  • Figure 12 is a graph depicting mouse SLGP (12231 or ml983) expression in VEGF -induced angiogenic xenograft plugs and parental xenografts by transcriptional profiling.
  • Mouse SLGP (12231 or ml983) expression is up-regulated in VEGF-induced angiogenic xenograft plugs.
  • Figure 13 is a graph depicting TaqManTM analysis of mouse SLGP (12231 or ml 983) expression in VEGF-induced angiogenic xenograft plugs and parental plugs.
  • Mouse SLGP (12231 or ml983) expression is up-regulated in VEGF-induced angiogenic xenograft plugs.
  • Figure 14 is a graph depicting TaqManTM analysis of human SLGP (1983) expression in glioblastomas and normal brains.
  • Human SLGP (1983) expression is up- regulated in glioblastomas as compared to normal brains.
  • the present invention is based, at least in part, on the discovery of novel G- protein coupled receptor (GPCR) family members, referred to herein as SLGP protein and nucleic acid molecules.
  • GPCR G- protein coupled receptor
  • the human SLGP molecules are also referred to as “1983” molecules and the mouse SLGP molecules are also referred to as “12231 or “ml983” molecules.
  • the present invention also provides methods and compositions for the diagnosis and treatment of cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • the present invention is also based, at least in part, on the discovery that the novel SLGP molecules ofthe present invention are upregulated in in vitro proliferating and tube forming Human Dermal Microvascular Endothelial Cells (HMVEC) (see Figures 9, 10, and 11), are expressed in endothelial cells of glioblastomas as compared to normal brains (see Figure 14), and are upregulated in VEGF-induced angiogenic xenograft plugs as compared to parental xenografts (see Figures 12 and 13). Therefore, the SLGP molecules ofthe present invention modulate angiogenesis by endothelial cells ' (e.g., tumor endothelial cells).
  • HMVEC Human Dermal Microvascular Endothelial Cells
  • the SLGP molecules ofthe present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes including angiogenesis (e.g., the proliferation, elongation, and migration of endothelial cells, such as endothelial cells in tumors).
  • Angiogenesis is responsible for the formation of new vessels in tumor sites. The new vessels provide the oxygen and nutritional supply to tumors. Therefore, the SLGP modulators ofthe invention can modulate tumor formation and growth by modulating angiogenesis.
  • inhibition ofthe activity of an SLGP molecule can cause decreased angiogenesis, i.e., a decrease in cellular proliferation, elongation, and migration of endothelial cells and, thus, a decrease in the formation of new vessels, and a decrease in the supply of oxygen and nutrition to a tumor. Therefore, the SLGP modulators ofthe invention can be used to treat formation and growth of tumors, e.g., cancer, and other diseases characterized by excessive vessel formation such as arthritis and retinopathy. Additionally, increasing the activity of an SLGP molecule can cause increased angiogenesis and, therefore, increased vessel formation and can, thus, be used in treating diseases characterized by decreased vessel formation, e.g., tissue ischemia. Therefore, the SLGP molecules ofthe present invention are useful as targets and therapeutic agents for the modulation of diseases characterized by decreased angiogenesis, e.g., tissue ischemia, such as myocardial ischemia.
  • the SLGP protein is a GPCR that participates in signaling pathways within cells, e.g., signaling pathways involved in proliferation or differentiation.
  • a signaling pathway refers to the modulation (e.g., the stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the GPCR (SLGP protein).
  • Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (IP3) or adenylate cyclase; polarization ofthe plasma membrane; production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA and angiogenesis, e.g., proliferation, elongation, and migration of endothelial cells (e.g., tumor endothelial cells) to form new vessels (e.g., endothelial tubes); cell differentiation; and cell survival.
  • a signal transduction pathway e.g., phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (IP3) or adenylate cyclase
  • PIP3 phosphatidy
  • the SLGP protein interacts with a "G protein" to produce one or more secondary signals in a variety of intracellular signal transduction pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and turnover, in a cell.
  • G proteins represent a family of heterotrimeric proteins composed of ⁇ , ⁇ and ⁇ subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane domains, such as the ligand receptors.
  • a conformational change is transmitted to the G protein, which causes the ⁇ -subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the ⁇ -subunits.
  • the GTP-bound form ofthe ⁇ -subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • second messengers such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates.
  • G proteins examples include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference.
  • PIP2 phosphatidylinositol turnover and metabolism
  • PIP2 is a phospholipid found in the cytosolic leaflet ofthe plasma membrane.
  • IP3 1,2-diacylglycerol
  • DAG 1,2-diacylglycerol
  • IP3 inositol 1,4,5- triphosphate
  • IP3 can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP4), a molecule which can cause calcium entry into the cytoplasm from the extracellular medium.
  • IP4 and IP4 can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP2) and inositol 1,3,4-triphosphate, respectively.
  • IP2 inositol 1,4-biphosphate
  • IP2 inositol 1,3,4-triphosphate
  • Protein kinase C is usually found soluble in the cytoplasm ofthe cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG.
  • the activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB.
  • phosphatidylinositol activity includes an activity of PIP2 or one of its metabolites.
  • cyclic AMP turnover and metabolism includes molecules involved in the turnover and metabolism of cyclic AMP (cAMP) as well as to the activities of these molecules.
  • Cyclic AMP is a second messenger produced in response to ligand induced stimulation of certain G protein coupled receptors.
  • binding of ligand to a ligand receptor can lead to the activation ofthe enzyme adenylate cyclase, which catalyzes the synthesis of cAMP.
  • the newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase.
  • SLGP molecules ofthe present invention are involved in modulation of cellular proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process” includes a process by which a cell e.g., an endothelial cell, increases in number, size, or content; by which a cell develops a specialized set of characteristics which differ from that of other cells; or by which a cell moves closer to or further from a particular location or stimulus (e.g., angiogenesis).
  • cellular proliferation, growth, differentiation, or migration disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; and other diseases which are characterized by increased or deceased angiogenesis, including, but not limited to arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • cardiovascular disorders may also be implicated in cardiovascular disorders, congestive heart failure, or other cardiac cellular processes.
  • cardiovascular disorder includes a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood.
  • a cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction ofthe heart, or an occlusion of a blood vessel, e.g., by a thrombus.
  • disorders include hypertension, atherosclerosis, coronary artery spasm, coronary artery disease, valvular disease, arrhythmias, cardiomyopathies (e.g., dilated cardiomyopathy, idiopathic cardiomyopathy), arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, long-QT syndrome, congestive heart failure, sinus node disfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, myocardial infarction, cardiac hypertrophy, and coronary artery spasm.
  • cardiomyopathies e.g., dilated cardiomyopathy, idiopathic cardiomyopathy
  • arteriosclerosis ischemia reperfusion injury
  • congestive heart failure includes a condition characterized by a diminished capacity ofthe heart to supply the oxygen demands ofthe body. Symptoms and signs of congestive heart failure include diminished blood flow to the various tissues ofthe body, accumulation of excess blood in the various organs, e.g., when the heart is unable to pump out the blood returned to it by the great veins, exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheral edema resulting from left ventricular dysfunction. Congestive heart failure may be- acute or chronic. The manifestation of congestive heart failure usually occurs secondary to a variety of cardiac or systemic disorders that share a temporal or permanent loss of cardiac function.
  • disorders include hypertension, coronary artery disease, valvular disease, and cardiomyopathies, e.g., hypertrophic, dilative, or restrictive cardiomyopathies.
  • Congestive heart failure is described in, for example, Cohn J.N. et al (1998) American Family Physician 57:1901-04, the contents of which are incorporated herein by reference.
  • cardiac cellular processes includes intra-cellular or inter-cellular processes involved in the functioning ofthe heart.
  • Cellular processes involved in the nutrition and maintenance ofthe heart, the development ofthe heart, or the ability ofthe heart to pump blood to the rest ofthe body are intended to be covered by this term. Such processes include, for example, cardiac muscle contraction, distribution and transmission of electrical impulses, and cellular processes involved in the opening and closing ofthe cardiac valves.
  • the term “cardiac cellular processes” further includes processes such as the transcription, translation and post- translational modification of proteins involved in the functioning ofthe heart, e.g., myofilament specific proteins, such as troponin I, troponin T, myosin light chain 1 (MLC1), and ⁇ - actinin.
  • myofilament specific proteins such as troponin I, troponin T, myosin light chain 1 (MLC1), and ⁇ - actinin.
  • novel SLGP molecules ofthe present invention comprise a family of molecules having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules ofthe invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • Members of a family may also have common functional characteristics.
  • GPCRs G protein-coupled receptors
  • the family of G protein-coupled receptors (GPCRs) comprise an N- terminal domain, seven transmembrane domains (also referred to as membrane-spanning domains), six loop domains, and a C-terminal cytoplasmic domain (also referred to as a cytoplasmic tail).
  • GPCRs G protein-coupled receptors
  • Members ofthe SLGP family also share certain conserved amino acid residues, some of which have been determined to be critical to receptor function and/or G protein signaling.
  • GPCRs usually contain the following features: a conserved asparagine residue in the first transmembrane domain; a cysteine residue in the second loop which is believed to form a disulfide bond with a conserved cysteine residue in the fourth loop; a conserved leucine and aspartate residue in the second transmembrane domain; an aspartate-arginine-tyrosine motif (DRY motif) at the interface ofthe third transmembrane domain and the third loop of which the arginine residue is almost invariant (members ofthe rhodopsin subfamily of GPCRs comprise a histidine-arginine-methionine motif (HRM motif) as compared to a DRY motif); a conserved tryptophan and proline residue in the fourth transmembrane domain; and conserved phenylalanine and leucine residues in the seventh transmembrane domain.
  • DRY motif aspartate-arginine-tyrosine motif
  • Table I depicts an alignment ofthe transmembrane domain of 5 GPCRs. The conserved residues described herein are indicated by asterices. An alignment ofthe transmembrane domains of 44 representative GPCRs can be found at http://mgdkkl.nidll.nih.gov:8000/extended.html.
  • thrombin (6. ) human P25116 rhodopsin (19.) human P08100 mlAC (21.) rat P08482 IL-8A (30.) human P25024 octopamine (40.) Drosophila melanogaster P22270
  • thrombin (Accession No. P25116), rhodopsin (Accession No. P08100), mlACh (Accession No. P08482), IL-8A (Accession No. P25024), octopamine (Accession No. P22270), can be found as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, respectively. Accordingly, GPCR-like proteins such as the SLGP proteins ofthe present invention contain a significant number of structural characteristics ofthe GPCR family.
  • the SLGPs ofthe present invention contain conserved cysteines found in the first two loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys490 and cys562 of SEQ ID NO:2).
  • a highly conserved asparagine residue is present (asnl25 in SEQ ID NO:2).
  • SLGP proteins contains a highly conserved leucine (leu 154 of SEQ ID NO:2). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure.
  • a highly conserved asparagine and arginine in the fourth transmembrane domain ofthe SLGP proteins is present (aspl 58 and arg218 of SEQ ID NO:2).
  • proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket.
  • a conserved tyrosine is present in the seventh transmembrane domain of SLGP-2 (tyr647 of SEQ ID NO :2) .
  • the SLGP proteins ofthe present invention contain at least one, two, three, four, five, six, or preferably, seven transmembrane domains.
  • transmembrane domain includes an amino acid sequence of about 15- 40 amino acid residues in length, more preferably, about 15-30 amino acid residues in length, and most preferably about 18-25 amino acid residues in length, which spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an ⁇ -helical structure.
  • At least 50%, 60%, 10%, 80%), 90%>, 95%) or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.
  • Transmembrane domains are described in, for example, Zaelles W.N. et al, (1996) Annual Rev.
  • an SLGP protein ofthe present invention has more than one transmembrane domain, preferably 2, 3, 4, 5, 6, or 7 transmembrane domains.
  • transmembrane domains can be found at about amino acids 433-452, 465-481, 500-524, 533-553, 570-594, 619-635, and 642-666 of SEQ ID NO:2.
  • an SLGP protein ofthe present invention has 7 transmembrane domains.
  • an SLGP is identified based on the presence of at least one Loop domain, also referred to herein as a 'loop'.
  • the term "loop” includes an amino acid sequence having a length of at least about 4, preferably about 5- 10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60- 70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide.
  • loop regions may be located either extracellularly or in the cytoplasm.
  • the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring SLGP or SLGP-like molecule
  • the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring SLGP or SLGP-like molecule.
  • a "cytoplasmic loop” includes an amino acid sequence located within a cell or. within the cytoplasm of a cell.
  • an extracellular loop includes an amino acid sequence located outside of a cell, or extracellularly.
  • loop domains can be found at about amino acid residues 453-464, 482- 499,525-532, 554-569, 595-618, and 636-641 of SEQ ID NO:2.
  • an SLGP is identified based on the presence of a "C-terminal domain", also referred to herein as a C-terminal tail, in the sequence ofthe protein.
  • a "C-terminal domain” includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid residues and is located within a cell or extracellularly. Accordingly, the N-terminal amino acid residue of a "C-terminal domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring SLGP or SLGP-like protein. For example, a C-terminal domain is found at about amino acid residues 667-690 of SEQ ID NO:2.
  • an SLGP is identified based on the presence of an "N- terminal domain", also referred to herein as an N-terminal loop in the amino acid sequence ofthe protein.
  • an "N-terminal domain” includes an amino acid sequence having about 1-500, preferably about 1-400, more preferably about 1-300, more preferably about 1-200, even more preferably about 1-100, and even more preferably about 1-50, " 1-25, or 1-10 amino acid residues in length and is located outside of a cell orintracellularly.
  • the C-terminal amino acid residue of a "N-terminal domain” is adjacent to an N-te ⁇ ninal amino acid residue of a transmembrane domain in a naturally-occurring SLGP or SLGP-like protein.
  • an N-terminal domain is found at about amino acid residues 1-432 of SEQ ID NO:2.
  • an SLGP includes at least one, preferably 6 or 7, transmembrane domains and and/or at least one loop.
  • the SLGP further includes an N-terminal domain and/or a C-terminal domain.
  • the SLGP can include six transmembrane domains, three cytoplasmic loops, and two extracellular loops, or can include six transmembrane domains, three extracellular loops, and 2 cytoplasmic loops.
  • the former embodiment can further include an N-terminal domain.
  • the latter embodiment can further include a C-terminal domain.
  • the SLGP can include seven transmembrane domains, three cytoplasmic loops, and three extracellular loops and can further include an N-terminal domain or a C-terminal domain.
  • an SLGP is identified based on the presence of at least one "7 transmembrane receptor profile", also referred to as a "Secretin family sequence profile", in the protein or corresponding nucleic acid molecule.
  • the term "7 transmembrane receptor profile” includes an amino acid sequence having at least about 50-350, preferably about 100-300, more preferably about 150-275 amino acid residues, or at least about 200-258 amino acids in length and having a bit score for the alignment ofthe sequence to the 7tm_l family Hidden Markov Model (HMM) of at least 20, preferably 20-30, more preferably 30-40, more preferably 40-50, or 50-75 or greater.
  • HMM Hidden Markov Model
  • the 7tm_l family HMM has been assigned the PFAM Accession PF00001 (http://pfam/wustl.edu/).
  • the amino acid sequence ofthe protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
  • the hmmsf program which is available as part ofthe HMMER package of search programs, is a family specific default program for PF00001 and a score of 15 is the default threshold score for determining a hit.
  • a search using the amino acid sequence of SEQ ID NO:2 was performed against the HMM database resulting in the identification of a 7 TM receptor profile in the amino acid sequence of SEQ ID NO:2. The results of the search are set forth below.
  • an SLGP protein is a human SLGP protein having a 7 transmembrane receptor profile at about amino acids 421-678 of SEQ ID O:2.
  • Such a 7 transmembrane receptor profile has the amino acid sequence:
  • SLGP proteins having at least 20-30%, 30-49%, 40-50%, 50-60% homology, preferably about 60-70%>, more preferably about 70-80%, or about 80-90% homology with the 7 transmembrane receptor profile of human SLGP (e.g., SEQ ID NO:2) are within the scope ofthe invention.
  • an SLGP is identified based on the presence of a "EGF-like domain" in the protein or corresponding nucleic acid molecule.
  • the term "EGF-like domain” includes a protein domain having an amino acid sequence of about 55-90, preferably about 60-85, more preferably about 65-80 amino acid residues, or about 70-79 amino acids and having a bit score for the alignment ofthe sequence to the EGF-like domain (HMM) of at least 6, preferably 7-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater.
  • the EGF- like domain HMM has been assigned the PFAM Accession PF00008
  • one or more cysteine residues in the EGF-like domain are conserved among SLGP family members or other proteins containing EGF- like domains (i.e., located in the same or similar position as the cysteine residues in other SLGP family members or other proteins containing EGF-like domains).
  • an "EGF-like domain” has the consensus sequence C-X-C-X(5)- G-X(2)-C, the 3 C's are involved in disulfide bonds; corresponding to SEQ ID NOT 1.
  • an "EGF-like domain” has the consensus sequence C- X-C-X(2)-[GP]-[FYW]-X(4,8)-C, the three C's are involved in disulfide bonds; corresponding to SEQ ID NO: 12.
  • the amino acid sequence ofthe protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
  • the hmmsf program which is available as part ofthe HMMER package of search programs, is a family specific default program for PF00008 and a score of 15 is the default threshold score for determining a hit.
  • the threshold score for detemiining a hit can be lowered (e.g., to 8 bits).
  • a description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et ⁇ /.(1990) Meth.
  • EGF-like domain has the following amino acid sequence:
  • SLGP proteins having at least 50-60%> homology, preferably about 60- 70%), more preferably about 70-80%, or about 80-90%) homology with an EGF-like domain of human SLGP (e.g., SEQ ID NO.T3) are within the scope ofthe invention.
  • an SLGP is identified based on the presence of a "NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain" in the protein or corresponding nucleic acid molecule.
  • NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain includes a protein domain having an amino acid sequence of about 25-55, preferably about 30-50, more preferably about 35-45 amino acid residues, or about 40-43 amino acids and having a bit score for the alignment ofthe sequence to the NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain (HMM) of at least 6, preferably 7-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater.
  • HMM NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain
  • the NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain HMM has been assigned the PFAM Accession PF00420 (http://pfam.wustl.edu/).
  • the amino acid sequence ofthe protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam HMM_search).
  • HMMs e.g., the Pfam database, release 2.1
  • the default parameters http://www.sanger.ac.uk/Software/Pfam HMM_search.
  • the hmmsf program which is available as part ofthe HMMER package of search programs, is a family specific default program for PF00420 and a score of 15 is the default threshold score for determining a hit.
  • the threshold score for determining a hit can be lowered (e.g., to 8 bits).
  • a description ofthe Pfam database can be found in Sonhammer et al (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et ⁇ /.(1990) Meth. Enzymol 183:146-159; Gribskov et ⁇ Z. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et ⁇ /.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et /.(1993) Protein Sci.
  • NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain has the amino acid sequence:
  • SLGP proteins having at least 50-60% homology, preferably about 60- 70%, more preferably about 70-80%, or about 80-90% homology with a NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain of human SLGP (e.g., SEQ ID NO:14) are within the scope of the invention.
  • an SLGP protein includes at least an EGF-like domain. In another embodiment, an SLGP protein includes at least an NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment, an SLGP protein includes at least a 7 transmembrane receptor profile. In another embodiment, an SLGP protein includes an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment, an SLGP protein includes an EGF-like domain and a 7 transmembrane receptor profile.
  • an SLGP protein in another embodiment, includes an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain, and a 7 transmembrane receptor profile. In another embodiment, an SLGP protein includes an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and a 7 transmembrane receptor profile. In another embodiment, an SLGP protein is human SLGP which includes an EGF-like domain having about amino acids 22-100 of SEQ ID NO:2.
  • an SLGP protein is human SLGP which includes an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain having about amino acids 475-517 of SEQ ID NO:2.
  • an SLGP protein is human SLGP which includes a 7 transmembrane receptor profile having about amino acids 421-678 of SEQ ID NO:2.
  • an SLGP protein is human SLGP which includes a an EGF-like domain having about amino acids 22- 100 of SEQ ID NO:2, an NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain having about amino acids 475- 517 of SEQ ID NO:2, and a 7 transmembrane receptor profile having about amino acids 421-678 of SEQ ID NO:2.
  • Preferred SLGP molecules ofthe present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18.
  • the term "sufficiently homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second • amino acid or nucleotide sequences share common structural domains and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least about 50%> homology, preferably 60%) homology, more preferably 70%-80%, and even more preferably 90-95%> homology across the amino acid sequences ofthe domains and contain at least one and preferably two structural domains, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.
  • an "SLGP activity”, “biological activity of SLGP” o. "functional activity of SLGP”, refers to an activity exerted by an SLGP protein, polypeptide or nucleic acid molecule on an SLGP responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • an SLGP activity is a direct activity, such as an association with a SLGP-target molecule.
  • a "target molecule” or “binding partner” is a molecule with which an SLGP protein binds or interacts in nature, sucl that SLGP-mediated function is achieved.
  • An SLGP target molecule can be a non-SLGP molecule or an SLGP protein or polypeptide ofthe present invention.
  • an SLGP target molecule is an SLGP ligand.
  • an SLGP activity i an indirect activity, such as a cellular signaling activity mediated by interaction ofthe SLGP protein with an SLGP ligand.
  • an SLGP activity is at least one or more ofthe following activities: (i) interaction of an SLGP protein with soluble SLGP ligand (e.g., CD55); (ii) interaction of an SLGP protein with a membrane-bound non-SLGP protein; (iii) interaction of an SLGP protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); (iv) indirect interaction of an SLGP protein with an intracellular protein (e.g., a downstream signal transduction molecule); and (v) modulation of cellular proliferation, growth, differentiation, or migration.
  • soluble SLGP ligand e.g., CD55
  • interaction of an SLGP protein with a membrane-bound non-SLGP protein e.g., an intracellular enzyme or signal transduction molecule
  • an intracellular protein e.g., an intracellular enzyme or signal transduction molecule
  • indirect interaction of an SLGP protein with an intracellular protein e.g., a downstream signal transduction
  • an SLGP activity is at least one or more ofthe following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of activation in a cell expressing an SLGP protein exposure to alpha- latrotoxin); (3) regulation of inflammation; or (4) modulation of angiogenesis (e.g., proliferation, elongation, and migration of endothelial cells (e.g. tumor endothelial cells), to form new vessels).
  • angiogenesis e.g., proliferation, elongation, and migration of endothelial cells (e.g. tumor endothelial cells), to form new vessels.
  • SLGP proteins have at least one transmembrane domain and an SLGP activity.
  • an SLGP protein has a 7 transmembrane receptor profile and an SLGP activity.
  • an SLGP protein has an EGF-like domain and an SLGP activity.
  • an SLGP protein has an NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity.
  • an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, and SLGP activity.
  • an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity.
  • an SLGP protein has a 7 transmembrane receptor profile and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity.
  • an SLGP protein has an EGF-like domain and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity.
  • an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, an SLGP activity, and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18.
  • the nucleotide sequence ofthe isolated human SLGP cDNA and the predicted amino acid sequence ofthe human SLGP polypeptide are shown in Figure 1 and in SEQ ID NOs.T and 2, respectively.
  • the human SLGP cDNA which is approximately 2987 nucleotides in length, encodes a protein which is approximately 690 amino acid residues in length.
  • the nucleotide sequence ofthe isolated mouse SLGP cDNA and the predicted amino acid sequence ofthe mouse SLGP polypeptide are shown in Figures 6A-B and 7 and in SEQ ID NOs:17 and 18, respectively.
  • the mouse SLGP cDNA which is approximately 3952 nucleotides in length, encodes a protein which is approximately 689 amino acid residues in length.
  • Plasmids containing the nucleotide sequence encoding human and mouse SLGP were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on __ and assigned Accession Numbers and . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • nucleic acid molecules that encode SLGP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify SLGP-encoding nucleic acids (e.g., SLGP mRNA) and fragments for use as PCR primers for the amplification or mutation of SLGP nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs ofthe DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from chromosomal DNA, e.g., other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated SLGP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • 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 substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOT or SEQ ID NO: 17, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • nucleic acid sequence of SEQ ID NOT, SEQ ID NO: 17 the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • SLGP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO : 1 , SEQ ID NO : 17, the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe
  • DNA insert ofthe plasmid deposited with ATCC as Accession Number can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NOT, SEQ ID NOT 7, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • a nucleic acid ofthe 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.
  • oligonucleotides corresponding to SLGP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 17. The sequence of SEQ ID NO corresponds to the human SLGP cDNA.
  • This cDNA comprises sequences encoding the human SLGP protein (i.e., "the coding region”, from nucleotides 19-2090, as well as 5' untranslated sequences (nucleotides 1-19), and 3' untranslated sequences (nucleotides 2090-2987).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NOT (e.g., nucleotides 19- 2090, corresponding to SEQ ID NO:3.
  • the sequence of SEQ ID NO: 17 corresponds to the mouse SLGP cDNA.
  • This cDNA comprises sequences encoding the mouse SLGP protein (i.e., "the coding region”, from nucleotides 70-2139, as well as 5' untranslated sequences (nucleotides 1-69), and 3' untranslated sequences (nucleotides 2140-3952).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO.T7 (e.g., nucleotides 70-2139, corresponding to SEQ ID NO.T9).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NOT9, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO , SEQ ID NO:3, SEQ ID NOT7, SEQ ID NOT9, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , such that it can hybridize to the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , thereby forming a stable duplex.
  • an isolated nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at least about 40%, 42%), 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire length ofthe nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the entire length ofthe nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • nucleic acid molecule of the invention can comprise only a portion ofthe nucleic acid sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • the nucleotide sequence determined from the cloning ofthe SLGP gene allows for the generation of probes and primers designed for use in identifying and/or cloning other SLGP family members, as well as SLGP homologues from other species.
  • 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 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NOT9, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • SEQ ID NO: 17 or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or of a naturally occurring allelic variant or mutant of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or of a naturally occurring allelic variant or mutant of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is greater than 749, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • Probes based on the SLGP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • 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 tissue which misexpress an SLGP protein, such as by measuring a level of an SLGP- encoding nucleic acid in a sample of cells from a subject e.g., detecting SLGP mRNA levels or determining whether a genomic SLGP gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of an SLGP protein" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NOT, SEQ ID NO: 17, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , which encodes a polypeptide having an SLGP biological activity (the biological activities of the SLGP proteins have previously been described), expressing the encoded portion of the SLGP protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion ofthe SLGP protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 18.
  • gene and “recombinant gene” refer to nucleic acid molecules isolated from chromosomal DNA, which include an open reading frame encoding an SLGP protein, preferably a mammalian SLGP protein.
  • a gene includes coding DNA sequences, non- coding regulatory sequences, and introns.
  • a gene refers to an isolated nucleic acid molecule, as defined herein.
  • Allelic variants of human SLGP include both functional and non-functional SLGP proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants ofthe human SLGP protein that maintain the ability to bind an SLGP ligand and/or modulate programmed cell death.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or SEQ ID NO: 18 or substitution, deletion or insertion of non-critical residues in non- critical regions ofthe protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants ofthe human SLGP protein that do not have the ability to either bind an SLGP ligand and/or modulate programmed cell death.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation ofthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO:18 or a substitution, insertion or deletion in critical residues or critical regions.
  • the present invention further provides non-human orthologues ofthe human SLGP protein.
  • Orthologues ofthe human SLGP protein are proteins that are isolated from non-human organisms and possess the same SLGP ligand binding and/or modulation of programmed cell death capabilities ofthe human SLGP protein.
  • Orthologues ofthe human SLGP protein can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:2.
  • nucleic acid molecules encoding other GPCR family members e.g., other SLGP family members
  • SLGP family members e.g., other SLGP family members
  • nucleic acid molecules encoding other GPCR family members e.g., other SLGP family members
  • SEQ ID NO:3 SEQ ID NO:17
  • SEQ ID NO: 19 nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • another SLGP cDNA can be identified based on the nucleotide sequence of human SLGP.
  • nucleic acid molecules encoding SLGP proteins from different species and which, thus, have a nucleotide sequence which differs from the SLGP sequences of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe SLGP cDNAs ofthe invention can be isolated based on their homology to the SLGP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NO: 19, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • the nucleic acid is at least 30, 50, 100, 150, 200, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 nucleotides in length.
  • 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.
  • the conditions are such that sequences at least about 70%>, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 , SEQ ID NO: 17, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with
  • ATCC as Accession Number corresponds to a naturally-occurring nucleic acid molecule.
  • 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).
  • allelic variants of the SLGP sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NO: 19, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , thereby leading to changes in the amino acid sequence ofthe encoded SLGP proteins, without altering the functional ability ofthe SLGP proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NOT9, or the nucleotide sequence of • the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of SLGP (e.g., the sequence of SEQ ID NO:2 or SEQ ID NO: 18) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the SLGP proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the SLGP proteins ofthe present invention and other members ofthe GPCR families are not likely to be amenable to alteration.
  • nucleic acid molecules encoding SLGP proteins that contain changes in amino acid residues that are not essential for activity. Such SLGP proteins differ in amino acid sequence from SEQ ID NO:2 or SEQ ID NO: 18, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 25%), 28%), 30%>, 35%>, 40%>, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2 or SEQ ID NO:18.
  • An isolated nucleic acid molecule encoding an SLGP protein homologous to the protein of SEQ ID NO:2 or SEQ ID NO: 18 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NOT9, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NO.T9, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • 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 in the art.
  • 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
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in an SLGP protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an SLGP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for SLGP biological activity to identify mutants that retain activity.
  • SEQ ID NO SEQ ID NO:3, SEQ ID NO: 17, SEQ ID NOT 9, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number
  • the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.
  • a mutant SLGP protein can be assayed for the ability to affect the (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of activation in a cell expressing an SLGP protein; or (3) modulation of angiogenesis in endothelial cells (e.g., tumor endothelial cells).
  • an antisense nucleic acid comprises a nucleotide sequence which 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. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire SLGP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding SLGP.
  • the term "coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues- (e.g., the coding region of human SLGP corresponds to SEQ ID NO:3 and the coding region of mouse SLGP corresponds to SEQ ID NOT9).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding SLGP.
  • 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).
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of SLGP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of SLGP mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SLGP 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 ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methyIcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5
  • 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 ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an SLGP protein to thereby inhibit expression ofthe 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 which binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • 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 which 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 intracellular concentrations ofthe antisense 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.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave SLGP mRNA transcripts to thereby inhibit translation of SLGP mRNA.
  • a ribozyme having specificity for an SLGP-encoding nucleic acid can be designed based upon the nucleotide sequence of an SLGP cDNA disclosed herein (i.e., SEQ ID NOT or SEQ ID NOT7).
  • SEQ ID NOT a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an SLGP-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,116,742.
  • SLGP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • SLGP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe SLGP (e.g., the SLGP promoter and/or enhancers) to form triple helical structures that prevent transcription of the SLGP gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe SLGP e.g., the SLGP promoter and/or enhancers
  • SLGP promoter and/or enhancers nucleotide sequences complementary to the regulatory region ofthe SLGP
  • the SLGP nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids 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 B. et al (1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.
  • PNAs of SLGP nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of SLGP nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
  • PNAs of SLGP 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.
  • PNA-DNA chimeras of SLGP nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), 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 (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1.996) supra and Finn P.J. et ⁇ l. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • 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 as a between the PNA and the 5' end of DNA (Mag, M. et ⁇ l. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a step wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et ⁇ l. (1996) supra).
  • modified nucleoside analogs e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • 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. US. 86:6553-6556; Lemaitre et al (1981) Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • SLGP molecules ofthe present invention are in the screening for SLGP ligands (e.g., surrogate ligands) and/or SLGP modulators, it is intended that the following are also within the scope ofthe present invention: isolated nucleic acids which encode and SLGP ligands or SLGP modulators, probes and/or primers useful for identifying SLGP ligands or SLGP modulators based on the sequences of nucleic acids which encode and SLGP ligands or SLGP modulators, isolated nucleic acid molecules which are complementary or antisense to the sequences of nucleic acids which encode and SLGP ligands or SLGP modulators, isolated nucleic acid molecules which are at least about 60-65%>, preferably at least about 70-75%>, more preferable at least about 80-85%), and even more preferably at least about 90-95%> or more homologous to the sequences of nucleic acids which encode and SLGP ligands
  • One aspect ofthe invention pertains to isolated SLGP proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-SLGP antibodies.
  • native SLGP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • SLGP proteins are produced by recombinant DNA techniques.
  • Alternative to recombinant expression, an SLGP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the SLGP 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 SLGP protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of SLGP protein having less than about 30%) (by dry weight) of non-SLGP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20%) of non-SLGP protein, still more preferably less than about 10%> of non-SLGP protein, and most preferably less than about 5%> non- SLGP protein.
  • a contaminating protein also referred to herein as a "contaminating protein”
  • the SLGP 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%> ofthe volume ofthe protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of SLGP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of SLGP protein having less than about 30% (by dry weight) of chemical precursors or non-SLGP chemicals, more preferably less than about 20%> chemical precursors or non-SLGP chemicals, still more preferably less than about 10% chemical precursors or non-SLGP chemicals, and most preferably less than about 5% chemical precursors or non-SLGP chemicals.
  • Biologically active portions of an SLGP protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe SLGP protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 18, which include less amino acids than the full length SLGP proteins, and exhibit at least one activity of an SLGP protein.
  • biologically active portions comprise a domain or motif with at least one activity ofthe SLGP protein.
  • a biologically active portion of an SLGP protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of an SLGP protein comprises at least a transmembrane domain. In another embodiment, a biologically active portion of an SLGP protein comprises at least one 7 transmembrane receptor profile. In another embodiment, a biologically active portion of an SLGP protein comprises at least an EGF-like domain. In another embodiment, a biologically active portion of an SLGP protein comprises at least an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L ⁇ domain. In another embodiment a biologically active portion of an SLGP protein comprises at least a 7 transmembrane receptor profile and an EGF-like domain.
  • a biologically active portion of an SLGP protein comprises at least a 7 transmembrane receptor profile and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment a biologically active portion of an SLGP protein comprises at least a an EGF-like domain and an NADH- ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment a biologically active portion of an SLGP protein comprises at least a 7 transmembrane receptor profile, an EGF-like domain and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain.
  • a preferred biologically active portion of an SLGP protein ofthe present invention may contain at least one ofthe above-identified structural domains and/or profiles.
  • a more preferred biologically active portion of an SLGP protein may contain at least two ofthe above-identified structural domains and/or profiles.
  • other biologically active portions, in which other regions ofthe protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native SLGP protein.
  • the SLGP protein has an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 18.
  • the SLGP protein is substantially homologous to SEQ ID NO:2 or SEQ ID NO: 18, and retains the functional activity ofthe protein of SEQ ID NO:2 or SEQ ID NO: 18, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the SLGP protein is a protein which comprises an amino acid sequence at least about 25%, 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2 or SEQ ID NO: 18.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%>, preferably at least 40%>, more preferably at least 50%>, even more preferably at least 60%>, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence (e.g., when aligning a second sequence to the SLGP amino acid sequence of SEQ ID NO:2 having 689 amino acid residues, at least 100, 200, preferably at least 300, more preferably at least 400, even more preferably at least 500, and even more preferably at least 600, 650 or 689 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”
  • the percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences ofthe present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • an SLGP "chimeric protein” or “fusion protein” comprises an SLGP polypeptide operatively linked to a non-SLGP polypeptide.
  • a "SLGP polypeptide” refers to a polypeptide having an amino acid sequence corresponding to SLGP
  • a non- SLGP polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SLGP protein, e.g., a protein which is different from the SLGP protein and which is derived from the same or a different organism.
  • an SLGP fusion protein the SLGP polypeptide can correspond to all or a portion of an SLGP protein.
  • an SLGP fusion protein comprises at least one biologically active portion of an SLGP protein.
  • an SLGP fusion protein comprises at least two biologically active portions of an SLGP protein.
  • the term "operatively linked" is intended to indicate that the SLGP polypeptide and the non- SLGP polypeptide are fused in-frame to each other.
  • the non-SLGP polypeptide can be fused to the N-terminus or C-terminus ofthe SLGP polypeptide.
  • the fusion protein is a GST-SLGP fusion protein in which the SLGP sequences are fused to the C-terminus ofthe GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant SLGP.
  • the fusion protein is an SLGP protein containing a heterologous signal sequence at its N-terminus.
  • a native SLGP signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of SLGP can be increased through use of a heterologous signal sequence.
  • the SLGP fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the SLGP fusion proteins can be used to affect the bioavailability of an SLGP substrate. Use of SLGP fusion proteins may be useful therapeutically for the treatment of SLGP-related disorders (e.g., paroxysmal nocturnal hemoglobinuria). Moreover, the SLGP-fusion proteins ofthe invention can be used as immunogens to produce anti-SLGP antibodies in a subject, to purify SLGP ligands and in screening assays to identify molecules which inhibit the interaction of SLGP with an SLGP ligand.
  • SLGP-fusion proteins ofthe invention can be used as immunogens to produce anti-SLGP antibodies in a subject, to purify SLGP ligands and in screening assays to identify molecules which inhibit the interaction of SLGP with an SLGP ligand.
  • the SLGP-fusion proteins ofthe invention can be used as immunogens to produce anti-SLGP antibodies in a subject, to purify SLGP ligands and in screening assays to identify molecules which inhibit the interaction of SLGP with an SLGP substrate.
  • an SLGP chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example 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.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric • gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric • gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An SLGP- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SLGP protein.
  • the present invention also pertains to variants of the SLGP proteins which function as either SLGP agonists (mimetics) or as SLGP antagonists.
  • Variants ofthe SLGP proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of an SLGP protein.
  • An agonist ofthe SLGP proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of an SLGP protein.
  • An antagonist of an SLGP protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe SLGP protein by, for example, competitively inhibiting the protease activity of an SLGP protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe SLGP protein.
  • variants of an SLGP protein which function as either SLGP agonists (mimetics) or as SLGP antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an SLGP protein for SLGP protein agonist or antagonist activity.
  • a variegated library of SLGP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of SLGP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SLGP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SLGP sequences therein.
  • a degenerate set of potential SLGP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SLGP sequences therein.
  • methods which can be used to produce libraries of potential SLGP 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.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential SLGP sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al (1983) Nucleic Acid Res. 11:477.
  • libraries of fragments of an SLGP protein coding sequence can be used to generate a variegated population of SLGP fragments for screening and subsequent selection of variants of an SLGP protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an SLGP 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 which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes ofthe SLGP protein.
  • REM Recursive ensemble mutagenesis
  • cell based assays can be exploited to analyze a variegated SLGP library.
  • a library of expression vectors can be transfected into a cell line which ordinarily synthesizes SLGP.
  • the transfected cells are then cultured such that a particular mutant SLGP is expressed and the effect of expression ofthe mutant on SLGP activity in the cell can be detected, e.g., by any of a number of activity assays for native SLGP protein.
  • Plasmid DNA can then be recovered from the cells which score for modulated SLGP activity, and the individual clones further characterized.
  • An isolated SLGP protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind SLGP using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length SLGP protein can be used or, alternatively, the invention provides antigenic peptide fragments of SLGP for use as immunogens.
  • the antigenic peptide of SLGP comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 18 and encompasses an epitope of SLGP such that an antibody raised against the peptide forms a specific immune complex with SLGP.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of SLGP that are located on the surface ofthe protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • An SLGP immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed SLGP protein or a chemically synthesized SLGP polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic SLGP preparation induces a polyclonal anti-SLGP antibody response. Accordingly, another aspect ofthe invention pertains to anti-SLGP antibodies.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as SLGP.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind SLGP.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of SLGP.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular SLGP protein with which it immunoreacts.
  • Polyclonal anti-SLGP antibodies can be prepared as described above by immunizing a suitable subject with an SLGP immunogen.
  • the anti-SLGP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized SLGP.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against SLGP can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J. Biol Chem .255:4980-83; Yeh et al (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody ofthe invention are detected by screening the hybridoma culture supernatants for antibodies that bind STGP, e.g., using a standard ELISA assay.
  • a monoclonal anti-SLGP antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with SLGP to thereby isolate immunoglobulin library members that bind SLGP.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al U.S.
  • recombinant anti-SLGP antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science
  • Patent 5,225,539 Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol 141:4053-4060.
  • An anti-SLGP antibody (e.g., monoclonal antibody) can be used to isolate SLGP by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-SLGP antibody can facilitate the purification of natural SLGP from cells and of recombinantly produced SLGP expressed in host cells.
  • an anti-SLGP antibody can be used to detect SLGP protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe SLGP protein.
  • Anti-SLGP 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.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I,
  • SLGP molecules ofthe present invention are in the screening for SLGP ligands (e.g., surrogate ligands) and/or SLGP modulators, it is intended that the following are also within the scope ofthe present invention: "isolated” or “purified” SLGP ligands or SLGP modulators, biologically-active portions of SLGP ligands or SLGP modulators, chimeric or fusion proteins comprising all or a portion of an SLGP ligand or SLGP modulator, and antibodies comprising all or a portion of an SLGP ligand or SLGP modulator.
  • vectors preferably expression vectors, containing a nucleic acid encoding an SLGP protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector 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).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • 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".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • 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 ofthe invention comprise a nucleic acid of the invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe 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).
  • 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, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression ofthe 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 ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors ofthe 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., SLGP proteins, mutant forms of SLGP proteins, fusion proteins, etc.).
  • the recombinant expression vectors ofthe invention can be designed for expression of SLGP proteins in prokaryotic or eukaryotic cells.
  • SLGP proteins can be expressed in bacterial cells such as E. 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, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • SLGP activity assays e.g., direct assays or competitive assays described in detail below
  • an SLGP fusion protein expressed in a retro viral expression vector ofthe present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter.
  • the SLGP expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al. , (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • SLGP proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect 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).
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al (1987) EMBO J. 6: 187- 195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression ofthe 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.
  • 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. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (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 which allows for expression (by transcription ofthe DNA molecule) of an RNA molecule which is antisense to SLGP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which 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.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect ofthe invention pertains to host cells into which an SLGP nucleic acid molecule ofthe invention is introduced, e.g., an SLGP nucleic acid molecule within a recombinant expression vector or an SLGP nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site ofthe host cell's genome.
  • 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 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 ofthe term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an SLGP 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).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • 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.
  • 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, D ⁇ A ⁇ -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, NY, 1989), and other laboratory manuals.
  • 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.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SLGP protein 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 ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an SLGP protein.
  • the invention further provides methods for producing an SLGP protein using the host cells ofthe invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SLGP protein has been introduced) in a suitable medium such that an SLGP protein is produced.
  • the method further comprises isolating an SLGP protein from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce nonhuman transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which SLGP-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous SLGP sequences have been introduced into their genome or homologous recombinant animals in which endogenous SLGP sequences have been altered.
  • Such animals are useful for studying the function and/or activity of an SLGP - and for identifying and/or evaluating modulators of SLGP activity.
  • 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 ofthe cells ofthe 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 which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the.transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous SLGP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell of the animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing an SLGP- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the SLGP cDNA sequence of SEQ ID NOT can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human SLGP gene such as a mouse or rat SLGP gene, can be used as a transgene.
  • an SLGP gene homologue such as another GPCR family member, can be isolated based on hybridization to the SLGP cDNA sequences of SEQ ID NOT, SEQ ID NO:3, SEQ ID NOT7, SEQ ID NOT9, or the DNA insert ofthe plasmid deposited with ATCC as Accession Number (described further in subsection I above) 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 sequence(s) can be operably linked to an SLGP transgene to direct expression of an SLGP protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of an SLGP transgene in its genome and/or expression of SLGP mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an SLGP protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an SLGP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SLGP gene.
  • the SLGP gene can be a human gene (e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non-human homologue of a human SLGP gene (e.g., the cDNA of SEQ ID NOT9).
  • the mouse SLGP gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous SLGP gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous SLGP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous SLGP 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 ofthe, endogenous SLGP protein).
  • the altered portion ofthe SLGP gene is flanked at its 5' and 3' ends by additional nucleic acid sequence ofthe SLGP gene to allow for homologous recombination to occur between the exogenous SLGP gene carried by the homologous recombination nucleic acid molecule and an endogenous SLGP gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking STGP nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced SLGP gene has homologously recombined with the endogenous SLGP gene are selected (see e.g., Li, E. et al.
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical
  • 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 ofthe animal contain the homologously recombined DNA by germline transmission ofthe transgene.
  • Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression ofthe transgene.
  • One example of such a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe 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 ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813.
  • a cell e.g., a somatic cell
  • 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 ofthe animal from which the cell, e.g., the somatic cell, is isolated.
  • a cell e.g., an embryonic stem cell, from the inner cell mass of a developing embryo can be transformed with a preferred transgene.
  • a cell, e.g., a somatic cell, from cell culture line can be transformed with a preferred transgene and induced to exit the growth cycle and enter G 0 phase.
  • the cell can then be fused, e.g., through the use of electrical pulses, to an enucleated mammalian oocyte.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone ofthe animal from which the nuclear donor cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier 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. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (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; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • 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
  • 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.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene 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 ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., an SLGP protein or anti-SLGP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., an SLGP protein or anti-SLGP antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe 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 pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated 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 carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe 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.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • 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.
  • 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.
  • 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.
  • Dosage unit form 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 ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50%> ofthe population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
  • LD50/ED50 Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method ofthe invention, the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the nucleic acid molecules ofthe 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 U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation ofthe 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.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • an SLGP protein ofthe invention has one or more of the following activities: (i) interaction of an SLGP protein with soluble SLGP ligand (e.g., CD55); (ii) interaction of an SLGP protein with a membrane-bound non-SLGP protein; (iii) interaction of an SLGP protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); (iv) indirect interaction of an SLGP protein with an intracellular protein (e.g., a downstream signal transduction molecule); and (v) modulation of cellular proliferation, migration, and growth and thus can be used in, for example, (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of activation in a cell expressing an SLGP protein exposure to alpha-latrotoxin); (3) regulation of inflammation; or (4) modulation of angiogenesis (e.g., proliferation, elongation, and migration of endothelial cells (e.g. tumor endo
  • the isolated nucleic acid molecules ofthe invention can be used, for example, to express SLGP protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect SLGP mRNA (e.g., in a biological sample) or a genetic alteration in an SLGP gene, and to modulate SLGP activity, as described further below.
  • the SLGP proteins can be used to treat disorders characterized by insufficient or excessive production of an SLGP protein and/or SLGP ligand (e.g., cancer and diseases characterized by increased or decreased angiogenesis such as, for example arthritis, retinal and optic disk neovascularization, and tissue ischemia , such as myocardial ischemia).
  • the SLGP proteins can be used to screen drugs or compounds which modulate the SLGP activity as well as to treat disorders characterized by insufficient or excessive production of SLGP protein or production of SLGP protein forms which have decreased or aberrant activity compared to SLGP wild type protein (e.g., cellular proliferation, growth, differentiation, or migration disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • the anti-SLGP antibodies ofthe invention can be used to detect and isolate SLGP proteins, regulate the bioavailability of SLGP proteins, and modulate SLGP activity.
  • 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) which bind to SLGP proteins, or have a stimulatory or inhibitory effect on, for example, SLGP expression or SLGP activity (e.g., angiogenesis).
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to SLGP proteins, or have a stimulatory or inhibitory effect on, for example, SLGP expression or SLGP activity (e.g., angiogenesis).
  • Compounds identified using the assays described herein may be useful for treating diseases associated with aberrant angiogenesis (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an SLGP protein or polypeptide or biologically active portion thereof.
  • the test compounds ofthe present invention can be obtained using any ofthe 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 (Lam, K.S. (1991) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses an SLGP protein on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to the SLGP protein determined.
  • the cell for example, can be of mammalian origin or a yeast cell. Determining the ability ofthe test compound to bind to an SLGP protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the SLGP protein can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • 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.
  • a microphysiometer can be used to detect the interaction of a test compound with an SLGP protein without the labeling of either the test compound or the receptor. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing an SLGP target molecule (e.g., an SLGP substrate) with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe SLGP target molecule. Determining the ability ofthe test compound to modulate the activity of an SLGP target molecule can be accomplished, for example, by determining the ability ofthe SLGP protein to bind to or interact with the SLGP target molecule.
  • an assay is a cell-based assay comprising contacting cells expressing an SLGP molecule with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) an SLGP activity, e.g., cellular proliferation, migration, growth or differentiation, and formation of vessels in endothelial tissues (e.g., angiogenesis).
  • the cells can be of mammalian origin, e.g., an endothelial cell.
  • Cellular models may also be used to identify modulators of SLGP activity (e.g., modulators of angiogenesis) and to determine the ability of test compounds to modulate the activity of an SLGP target molecule.
  • Cellular models for the study of angiogenesis include models of endothelial cell differentiation on Matrigel (Baatout, S. et al. (1996) Rom. J. Intern. Med. 34:263-269; Benelli, R et al. (1999) Int. J. Biol. Markers 14:243- 246), the culture of microvessel fragments in physiological gels (Hoying, JB et al. (1996) In Vitro Cell Dev. Biol. Anim. 32: 409-419; US Patent No.
  • the assay comprises contacting a cell which expresses an SLGP protein or biologically active portion thereof, on the cell surface with an SLGP ligand, to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with the SLGP protein or biologically active portion thereof, wherein determining the ability ofthe test compound to interact with the SLGP protein or biologically active portion thereof, comprises determining the ability ofthe test compound to preferentially bind to the SLGP protein or biologically active portion thereof, as compared to the ability ofthe SLGP ligand to bind to the SLGP protein or biologically active portion thereof.
  • Determining the ability ofthe SLGP ligand or SLGP modulator to bind to or interact with an SLGP protein or biologically active portion thereof can be accomplished by one ofthe methods described above for determining direct binding.
  • determining the ability ofthe SLGP ligand or modulator to bind to or interact with an SLGP protein or biologically active portion thereof can be accomplished by determining the activity of an SLGP protein or of a downstream SLGP target molecule.
  • the target molecule can be a cellular second messenger, and the activity ofthe target molecule can be determined by detecting induction ofthe target (i.e.
  • a reporter gene comprising an SLGP-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, a cellular proliferation, growth, differentiation, or migration response.
  • the present invention involves a method of identifying a compound which modulates the activity of an SLGP protein, comprising contacting a cell which expresses an SLGP protein with a test compound, determining the ability of the test compound to modulate the activity the SLGP protein (e.g., angiogenesis), and identifying the compound as a modulator of SLGP activity.
  • the present invention involves a method of identifying a compound which modulates the activity of an SLGP protein, comprising contacting a cell which expresses an SLGP protein with a test compound, determining the ability ofthe test compound to modulate the activity of a downstream SLGP target molecule, and identifying the compound as a modulator of SLGP activity.
  • an assay ofthe present invention is a cell-free assay in which an SLGP protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the SLGP protein or biologically active portion thereof is determined. Binding ofthe test compound to the SLGP protein can be determined either directly or indirectly as described above. Binding ofthe test compound to the SLGP protein can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • BIOA Biomolecular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcoreTM). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • the assay includes contacting the SLGP protein or biologically active portion thereof with a known ligand which binds SLGP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an SLGP protein, wherein determining the ability ofthe test compound to interact with an SLGP protein comprises determining the ability ofthe test compound to preferentially bind to SLGP or biologically active portion thereof as compared to the known ligand.
  • the assay is a cell-free assay in which an SLGP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity ofthe SLGP protein or biologically active portion thereof is determined.
  • the cell-free assay involves contacting an SLGP protein or biologically active portion thereof with a known ligand which binds the SLGP protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with the SLGP protein, wherein determining the ability ofthe test compound to interact with the SLGP protein comprises determining the ability ofthe test compound to preferentially bind to or modulate the activity of an SLGP target molecule, as compared to the known ligand.
  • the cell-free assays ofthe present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g. SLGP proteins or biologically active portions thereof or SLGP proteins).
  • isolated proteins e.g. SLGP proteins or biologically active portions thereof or SLGP proteins.
  • a membrane-bound form an isolated protein e.g., an SLGP protein
  • non-ionic detergents such as n-oc
  • binding of a test compound to an SLGP protein, or interaction of an SLGP 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 microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutathione-S -transferase/ SLGP fusion proteins or glutathione-S- transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or SLGP protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre 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 above. Alternatively, the complexes can be dissociated from the matrix, and the level of SLGP binding or activity determined using standard techniques.
  • an SLGP protein or an SLGP target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated SLGP protein or target molecules can be prepared from biotin-NHS (N-
  • hydroxy-succinimide using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with SLGP protein or target molecules but which do not interfere with binding ofthe SLGP protein to its target molecule can be derivatized to the wells ofthe plate, and unbound target or SLGP protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the SLGP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the SLGP protein or target molecule.
  • modulators of SLGP expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of SLGP mRNA or protein in the cell is determined. The level of expression of SLGP mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of SLGP mRNA or protein in the absence ofthe candidate compound.
  • the candidate compound can then be identified as a modulator of SLGP expression based on this comparison. For example, when expression of SLGP mRNA or protein is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of SLGP mRNA or protein expression. Alternatively, when expression of SLGP mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of SLGP mRNA or protein expression.
  • the level of SLGP mRNA or protein expression in the cells can be determined by methods described herein for detecting SLGP mRNA or protein.
  • the SLGP proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No.
  • SLGP-binding proteins proteins which bind to or interact with SLGP
  • SLGP-binding proteins proteins which bind to or interact with SLGP
  • Such SLGP-binding proteins are also likely to be involved in the propagation of signals by the SLGP proteins as, for example, downstream elements of an SLGP-mediated signaling pathway.
  • SLGP-binding proteins are likely to be cell-surface molecules associated with non-SLGP expressing cells, wherein such SLGP-binding proteins are involved in chemoattraction.
  • 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 an SLGP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a known transcription factor e.g., GAL-4
  • 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 ofthe known transcription factor. If the "bait” and the “prey” proteins are able to interact, in vivo, forming an SLGP- dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an SLGP modulating agent, an antisense SLGP nucleic acid molecule, an SLGP-specific antibody, or an SLGP-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • Models for studying angiogenesis in vivo include, for example, tumor cell- induced angiogenesis and tumor metastasis (Hoffman, RM (1998-99) Cancer Metastasis Rev. 17:271-277; Holash, J et al. (1999) Oncogene 18:5356-5362; Li, CY et al. (2000) J. Natl Cancer Inst. 92:143-147), matrix induced angiogenesis (US Patent No. 5,382,514), the disc angiogenesis system (Kowalski, j. et al.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, 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. These applications are described in the subsections below.
  • this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe SLGP nucleotide sequences, described herein, can be used to map the location ofthe SLGP genes on a chromosome. The mapping ofthe SLGP sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • SLGP genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the SLGP nucleotide sequences. Computer analysis ofthe SLGP sequences can be used to predict 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 SLGP 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 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. (D'Eustachio P.
  • 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 SLGP nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
  • Other mapping strategies which can similarly be used to map a 9o, lp, or lv sequence to its chromosome include in situ hybridization (described in Fan, Y.
  • FISH Fluorescence in situ hybridization
  • 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).
  • 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 ofthe genes actually are preferred for mapping pu ⁇ oses. 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 ofthe sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the SLGP gene can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe 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 polymo ⁇ hisms.
  • the SLGP sequences ofthe present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • 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. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences ofthe present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the SLGP nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends ofthe 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 ofthe present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the SLGP nucleotide sequences ofthe invention uniquely represent portions ofthe 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.
  • Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses.
  • noncoding sequences of SEQ ID NOT can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from SLGP nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification ofthe individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology.
  • Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification ofthe origin ofthe biological sample.
  • sequences ofthe present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NOT are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the SLGP nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NOT, having a length of at least 20 bases, preferably at least 30 bases.
  • the SLGP nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., HMVEC tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such SLGP probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., SLGP primers or probes can be used to screen tissue culture for contamination (i. e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trails are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining SLGP protein and/or nucleic acid expression as well as SLGP activity in the context of a biological sample (e.g., blood, serum, cells, (e.g., endothelial cells, including endothelial cells in tumors), and 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 SLGP expression or activity (e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • a biological sample e.g., blood, serum, cells, (e.g.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with SLGP protein, nucleic acid expression or activity. For example, mutations in an SLGP gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with SLGP protein, nucleic acid expression or activity.
  • Another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of SLGP in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of SLGP protein or nucleic acid 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 SLGP protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes SLGP protein such that the presence of SLGP protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting SLGP protein or nucleic acid e.g., mRNA, genomic DNA
  • a preferred agent for detecting SLGP mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to SLGP mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length SLGP nucleic acid, such as the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 17, or a fragment or portion of an SLGP nucleic acid 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 SLGP mRNA or genomic DNA.
  • a full-length SLGP nucleic acid such as the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 17, or a fragment or portion of an SLGP nucleic acid 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 SLGP mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • a preferred agent for detecting SLGP protein is an antibody capable of binding to SLGP 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')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe 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.
  • 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 ofthe invention can be used to detect SLGP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of SLGP mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of SLGP protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of SLGP genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of SLGP protein include introducing into a subject a labeled anti-SLGP antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting SLGP protein, mRNA, or genomic DNA, such that the presence of SLGP protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of SLGP protein, mRNA or genomic DNA in the control sample with the presence of SLGP protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of SLGP in a biological sample can comprise a labeled compound or agent capable of detecting SLGP protein or mRNA in a biological sample; means for determining the amount of SLGP in the sample; and means for comparing the amount of SLGP 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 SLGP protein or nucleic acid.
  • 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 SLGP expression or activity.
  • aberrant includes an SLGP expression or activity which deviates from the wild type SLGP expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant SLGP expression or activity is intended to include the cases in which a mutation in the SLGP gene causes the SLGP gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional SLGP protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with an SLGP ligand or one which interacts with a non-SLGP ligand.
  • the assays described herein can be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant SLGP expression or activity, e.g., a cellular proliferation, growth, differentiation, or migration disorder such as cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • a disease or disorder associated with aberrant SLGP expression or activity e.g., a cellular proliferation, growth, differentiation, or migration disorder such as cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with SLGP protein, nucleic acid expression or activity such as a cellular proliferation, growth, differentiation, or migration disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a cellular proliferation, growth, differentiation, or migration disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • the present invention provides a method for identifying a disease or disorder associated with aberrant SLGP expression or activity in which a test sample is obtained from a subject and SLGP protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of SLGP protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant SLGP expression or activity (e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • a disease or disorder associated with aberrant SLGP expression or activity e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample (e.g., endothelial cells or tumor endothelial cells), or tissue.
  • 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 SLGP expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • a disorder such as a cellular proliferation, growth, differentiation, or migration disorder
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia myocardial ischemia).
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant SLGP expression or activity in which a test sample is obtained and SLGP protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of SLGP protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant SLGP expression or activity (e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • a disorder associated with aberrant SLGP expression or activity e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)
  • the methods ofthe invention can also be used to detect genetic alterations
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an SLGP -protein, or the mis-expression ofthe SLGP gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an SLGP gene; 2) an addition of one or more nucleotides to an SLGP gene; 3) a substitution of one or more nucleotides of an SLGP gene, 4) a chromosomal rearrangement of an SLGP gene; 5) an alteration in the level of a messenger RNA transcript of an SLGP gene, 6) aberrant modification of an SLGP gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of an SLGP gene, 8) a non- wild type level of an SLGP-protein, 9) allelic loss of an SLGP gene, and 10) inappropriate post-translational modification of an SLGP-protein.
  • a preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.
  • detection ofthe alteration 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.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • 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 ofthe sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an SLGP gene under conditions such that hybridization and amplification ofthe SLGP-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • primers which specifically hybridize to an SLGP gene under conditions such that hybridization and amplification ofthe SLGP-gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size ofthe amplification product and comparing the length to a control sample.
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for
  • mutations in an SLGP gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • 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.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in SLGP 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 (Cronin, M.T. et al. (1996) Human Mutation 1: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753- 759).
  • genetic mutations in SLGP can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • 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 step 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.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the SLGP gene and detect mutations by comparing the sequence ofthe sample SLGP with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.
  • RNA/RNA or RNA/DNA heteroduplexes methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.
  • Myers et al. (1985) Science 230:1242 methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type SLGP sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • 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 ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • 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 SLGP cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an SLGP sequence e.g., a wild-type SLGP sequence
  • 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, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in SLGP genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (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.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end ofthe 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 prepackaged 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 SLGP gene.
  • any cell type or tissue in which SLGP is expressed may be utilized in the prognostic assays described herein.
  • SLGP protein e.g., a cellular proliferation, growth, differentiation, or migration process, e.g., angiogenesis
  • agents e.g., drugs, compounds
  • angiogenesis a cellular proliferation, growth, differentiation, or migration process, e.g., angiogenesis
  • an agent determined by a screening assay as described herein to increase SLGP gene expression, protein levels, or upregulate SLGP activity can be monitored in clinical trials of subjects exhibiting decreased SLGP gene expression, protein levels, or downregulated SLGP activity.
  • the effectiveness of an agent determined by a screening assay to decrease SLGP gene expression, protein levels, or downregulate SLGP activity can be monitored in clinical trails of subjects exhibiting increased SLGP gene expression, protein levels, or upregulated SLGP activity.
  • the expression or activity of an SLGP gene, and preferably, other genes that have been implicated in, for example, a cellular proliferation, growth, differentiation, or migration disorder e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia
  • tissue ischemia such as myocardial ischemia
  • genes including SLGP, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates SLGP activity (e.g., identified in a- screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • SLGP activity e.g., identified in a- screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of SLGP and other genes implicated in the cellular proliferation, growth, differentiation, or migration disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia), respectively.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of SLGP or other genes.
  • the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment ofthe individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample (e.g., a tumor sample) from a subject prior to administration ofthe agent; (ii) detecting the level of expression of an SLGP protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples (e.g., a tumor samples) from the subject; (iv) detecting the level of expression or activity ofthe SLGP protein, mRNA, or genomic DNA in the post-administration samples (e.g., a tumor samples); (v) comparing the level of expression or activity ofthe SLGP protein, mRNA, or genomic DNA in the preadministration sample (e.g., a tumor sample
  • an agent
  • increased administration ofthe agent may be desirable to increase the expression or activity of SLGP to higher levels than detected, i.e., to increase the effectiveness ofthe agent.
  • decreased administration ofthe agent may be desirable to decrease expression or activity of SLGP to lower levels than detected, i.e. to decrease the effectiveness ofthe agent.
  • SLGP expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observable phenotypic response (e.g., angiogenesis).
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant SLGP expression or activity, e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • a disorder associated with aberrant SLGP expression or activity e.g., a cellular proliferation, growth, differentiation, or migration disorder, (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).
  • Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the pu ⁇ ose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting the disease or disorder, the symptoms of disease or disorder or the predisposition toward a disease or disorder.
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or “drug response genotype”.)
  • a patient's drug response phenotype e.g., a patient's "drug response phenotype", or “drug response genotype”.
  • another aspect ofthe invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the SLGP molecules ofthe present invention or SLGP modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant SLGP expression or activity, by administering to the subject an SLGP or an agent which modulates SLGP expression or at least one SLGP activity (e.g., modulation of a cellular proliferation, growth, differentiation, or migration process (e.g., angiogenesis).
  • an SLGP or an agent which modulates SLGP expression or at least one SLGP activity e.g., modulation of a cellular proliferation, growth, differentiation, or migration process (e.g., angiogenesis).
  • Subjects at risk for a disease which is caused or contributed to by aberrant SLGP expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe SLGP aberrancy, such that a disease or disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia) is prevented or, alternatively, delayed in its progression.
  • a disease or disorder e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia
  • tissue ischemia such as myocardial ischemia
  • an SLGP, SLGP agonist or SLGP antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • the modulatory method ofthe invention involves contacting a cell with an SLGP molecule ofthe present invention such that the activity of an SLGP is modulated.
  • the modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of SLGP protein activity associated with the cell.
  • An agent that modulates SLGP protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an SLGP protein (e.g., CD55), an SLGP antibody, an SLGP agonist or antagonist, a peptidomimetic of an SLGP agonist or antagonist, or other small molecule.
  • the agent stimulates one or more SLGP activities. Examples of such stimulatory agents include active SLGP protein and a nucleic acid molecule encoding SLGP that has been introduced into the cell.
  • the agent inhibits one or more SLGP activities. Examples of such inhibitory agents include antisense SLGP nucleic acid molecules and anti-SLGP antibodies.
  • modulatory methods can be performed in vitro (e.g., by culturing the cells, e.g., tumor endothelial cells, with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an SLGP protein or nucleic acid molecule (a cellular proliferation, growth, differentiation, or migration disorder (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) SLGP expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering an SLGP protein or nucleic acid molecule as therapy to compensate for reduced or aberrant SLGP expression or activity. Stimulation of SLGP activity is desirable in situations in which SLGP is abnormally downregulated and/or in which increased SLGP activity is likely to have a beneficial effect.
  • SLGP inhibition is desirable in situations in which SLGP is abnormally upregulated and/or in which decreased SLGP activity is likely to have a beneficial effect (e.g., cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • a beneficial effect e.g., cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)).
  • SLGP molecules ofthe present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on SLGP activity (e.g., SLGP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)) associated with aberrant SLGP activity.
  • disorders e.g., cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia)
  • pharmacogenomics i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • pharmacogenomics i.e., the study ofthe relationship between an individual'
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an SLGP molecule or SLGP modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an SLGP molecule or SLGP modulator.
  • 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, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11) :983- 985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • a genome-wide association relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNP single nucleotide polymo ⁇ hisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome.
  • treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., an SLGP protein or SLGP protein ofthe present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • the discovery of genetic polymo ⁇ hisms of drag metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymo ⁇ hisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for C YP2D6 is highly polymo ⁇ hic 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 drag 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 mo ⁇ hine. 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 CYP2D6 gene amplification.
  • a method termed the "gene expression profiling” can be utilized to identify genes that predict drug response.
  • the gene expression of an animal dosed with a drug e.g., an SLGP molecule or SLGP modulator ofthe present invention
  • a drug e.g., an SLGP molecule or SLGP modulator ofthe present invention
  • Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. 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 SLGP molecule or SLGP modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
  • SLGP sequence information refers to any nucleotide and/or amino acid sequence information particular to the SLGP molecules ofthe present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymo ⁇ hisms (SNPs), epitope sequences, and the like.
  • information "related to" said SLGP sequence information includes detection ofthe presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymo ⁇ hism, etc.), determination ofthe level of a sequence (e.g., detection of a level of expression, for example, a quantative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like.
  • electronic apparatus readable media refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus.
  • Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media.
  • the medium is adapted or configured for having recorded thereon SLGP sequence information ofthe present invention.
  • the term "electronic apparatus" is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
  • Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • networks including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet
  • electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like
  • PDAs personal digital assistants
  • recorded refers to a process for storing or encoding information on the electronic apparatus readable medium.
  • Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media to generate manufactures comprising the SLGP sequence information.
  • sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms.
  • a database application such as DB2, Sybase, Oracle, or the like
  • Any number of data processor structuring formats e.g., text file or database
  • SLGP sequence information By providing SLGP sequence information in readable form, one can routinely access the sequence information for a variety of pu ⁇ oses.
  • sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
  • Search means are used to identify fragments or regions ofthe sequences ofthe invention which match a particular target sequence or target motif.
  • the present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a SLGP- associated disease or disorder or a pre-disposition to a SLGP-associated disease or disorder, wherein the method comprises the steps of determining SLGP sequence information associated with the subject and based on the SLGP sequence information, determining whether the subject has a SLGP -associated disease or disorder or a pre-disposition to a SLGP- associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a SLGP-associated disease or disorder or a pre-disposition to a disease associated with a SLGP wherein the method comprises the steps of determining SLGP sequence information associated with the subject, and based on the SLGP sequence information, determining whether the subject has a SLGP -associated disease or disorder or a pre-disposition to a SLGP-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.
  • the present invention also provides in a network, a method for determining whether a subject has a SLGP-associated disease or disorder or a pre-disposition to a SLGP associated disease or disorder associated with SLGP, said method comprising the steps of receiving SLGP sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to SLGP and/or a SLGP-associated disease or disorder, and based on one or more ofthe phenotypic information, the SLGP information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a SLGP-associated disease or disorder or a pre-disposition to a SLGP-associated disease or disorder (e.g., a cellular proliferation, growth, differentiation, or migration disorder).
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the present invention also provides a business method for determining whether a subject has a SLGP-associated disease or disorder or a pre-disposition to a SLGP- associated disease or disorder, said method comprising the steps of receiving information related to SLGP (e.g. , sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to SLGP and/or related to a SLGP-associated disease or disorder, and based on one or more ofthe phenotypic information, the SLGP information, and the acquired information, determining whether the subject has a SLGP- associated disease or disorder or a pre-disposition to a SLGP-associated disease or disorder.
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the invention also includes an array comprising a SLGP sequence ofthe present invention.
  • the array can be used to assay expression of one or more genes in the array.
  • the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array.
  • tissue specificity of genes in the array.
  • up to about 7600 genes can be simultaneously assayed for expression, one of which can be SLGP.
  • the invention allows the quantitation of gene expression.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue.
  • tissue can be perturbed and the effect on gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell • type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis ofthe undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level.
  • the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a SLGP-associated disease or disorder, progression of SLGP-associated disease or disorder, and processes, such a cellular transformation associated with the SLGP-associated disease or disorder.
  • the array is also useful for ascertaining the effect ofthe expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of SLGP expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells.
  • This provides a battery of genes (e.g., including SLGP) that could serve as a molecular target for diagnosis or therapeutic intervention.
  • CDNAS In this example, the identification and characterization ofthe gene encoding human SLGP (also referred to as "Fcl ⁇ rb021h09”) is described.
  • a program termed 'signal sequence trapping' was utilized to analyze the sequences of several cDNAs of a cDNA library derived from bronchial epithelial cells which had been stimulated with the cytokine, TNF ⁇ .
  • This analysis identified a human clone having an insert of approximately 3 kb containing a protein-encoding sequence of approximately 2987 nucleotides capable of encoding approximately 690 amino acids of SLGP (e.g., the starting methionine through residue 690 of, for example, SEQ ID NO:2).
  • the nucleotide sequence encoding the human SLGP protein is shown in Figure 1 and is set forth as SEQ ID NO .
  • the full length protein encoded by this nucleic acid is comprised of about 690 amino acids and has the amino acid sequence shown in Figure 1 and set forth as SEQ ID NO:2.
  • the coding portion (open reading frame) of SEQ ID NOT is set forth as SEQ ID NO:3.
  • BLAST search Altschul et al. (1990) J. Mol. Biol. 215:403 ofthe nucleotide sequence of human SLGP has revealed that SLGP is significantly similar to a protein identified as human CD 97 (Accession No. U76764) and to a protein identified as rat latrophilin (Accession Nos. U78105, U72487).
  • the SLGP proteins ofthe present invention contain a significant number of structural characteristics ofthe GPCR family. For instance, the SLGPs ofthe present invention contain conserved cysteines found in the first 2 loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys490 and cys562 of SEQ ID NO:2). A highly conserved asparagine residue is present (asnl25 in SEQ ID NO:2). SLGP proteins contains a highly conserved leucine (leul 54 of SEQ ID NO:2). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure.
  • a highly conserved asparagine and arginine in the fourth transmembrane domain ofthe SLGP proteins is present (aspl 58 and arg218 of SEQ ID NO:2).
  • a highly conserved proline is present (pro307 of SEQ ID NO:2).
  • Proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket.
  • a conserved tyrosine is present in the seventh ' transmembrane domain of SLGP-2 (tyr647 of SEQ ID NO:2).
  • the SLGP family of proteins like the Secretin family of proteins, are referred to herein as G protein-coupled receptor-like proteins.
  • SLGP is predicted to contain the following sites: N-glycosylation site at residues 15-18, residues 21-24, residues 64-67, residues 74-77, residues 127-130, residues 177- 180, residues 188-191, residues 249-252, residues 381-384, and at residues 395-398 of SEQ ID NO:2; Glycosaminoglycan attachment site at residues 49-52 of SEQ ID NO:2; cAMP- and cGMP-dependent protein kinase phosphorylation sites at residues 360-363 of SEQ ID NO:2; Protein kinase C phosphorylation sites at residues 135-137, residues 181-183, residues 233-235, residues 358-360, residues 363-365, residues 400-402, residues 457-459, residues 485-487, residues 558-560, and residues 667-669 of SEQ ID NO:2; Casein kinase II phosphorylation sites
  • This Example describes the tissue distribution of SLGP mRNA, as determined by Northern blot hybridization. Northern blot hybridizations with the various RNA samples were performed
  • SLGP cDNA This example describes the tissue distribution of human and mouse SLGP cDNA, as determined using the TaqManTM procedure.
  • the TaqmanTM procedure is a quantitative, real-time PCR-based approach to detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cDNA is generated from the samples of interest and serves as the starting materials for PCR amplification.
  • a gene-specific oligonucleotide probe (complementary to the region being amplified) is included in the reaction (i.e., the TaqmanTM probe).
  • the TaqManTM probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end ofthe probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy- 4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7- dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N',N'- tetramethylrhodamine) at the 3' end ofthe probe.
  • a fluorescent reporter dye covalently linked to the 5' end ofthe probe
  • TET 6-carboxy- 4,7,2',7'-tetrachlorofluorescein
  • JOE 6-carboxy-4,5-dichloro-2,7- dimethoxyfluorescein
  • VIC a quencher dye
  • cleavage ofthe probe separates the reporter dye and the quencher dye, resulting in increased fluorescence ofthe reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites. The 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization ofthe strand continues.
  • Results indicate that Human SLGP is highest in endothelial cells, heart tissue and skeletal muscle.
  • Human SLGP is upregulated in tube forming Human Microvascular Endothelial Cells (HMVEC) and in proliferating HMVEC as compared to arresting HMVEC. Human SLGP is also upregulated in glioblastomas as compared to normal brain. As shown in Figure 13, Mouse SLGP (ml 983) is upregulated in VEGF-induced angiogenic xenograft plugs as compared to parental plugs.
  • HMVEC Human Microvascular Endothelial Cells
  • Taqman analysis was also performed on a mouse ischemic hindlimb panel (day 6), indicating increased expression in ischemic versus sham limbs for 3 or 6 pairs with increased VE-cadherin (endothelial marker) expression, which indicates a role for SLGP in the regulation of angiogenesis.
  • tissue distribution of human SLGP as determined using in situ hybridization analysis.
  • tissues e.g. brain and glioblastoma tissues
  • in situ analysis tissues, e.g. brain and glioblastoma tissues, were first frozen on dry ice.
  • Ten-micrometer-thick sections ofthe tissues were postfixed with 4%o formaldehyde in DEPC-treated IX phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0).
  • Hybridizations were performed with 35s-radiolabeled (5 X 10 cpm/ml) cRNA probes. Probes were incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1%> sodium thiosulfate for 18 hours at 55°C.
  • SDS sodium dodecyl sulfate
  • slides were washed with 2X SSC. Sections were then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with lO ⁇ g of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides were then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA
  • results of in situ hybridization also show focal expression of Human SLGP in non-necrotic/inflammatory infiltrate areas in mouse ischemic hindlimb, with a pattern resembling that of platelet endothelial cell adhesion molecule (PEC AM). Furthermore, results indicate that Human SLGP is also expressed at high levels in the aortic root of ApoE mice in endothelial cells on the valvular leaflet.
  • the ApoE model is a well-characterized animal model of atherosclerosis, indicating a role for SLGP in cardiovascular disease, e.g., atherosclerosis.
  • EXPRESSION This example describes the expression of human and mouse SLGP as determined by transcriptional profiling experiments. Expression of human SLGP in proliferating HMVEC and arresting HMVEC was analyzed by transcriptional profiling. As shown in Figure 10, expression of human SLGP is up-regulated in proliferating HMVEC as compared to arresting HMVEC.
  • mouse SLGP expression is up-regulated in VEGF-induced angiogenic xenograft plugs as compared to parental xenografts.
  • SLGP is expressed as a recombinant glutathione-S-transferase
  • GST GST fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • SLGP is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • Expression ofthe GST-SLGP fusion protein in PEB199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates ofthe induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.
  • EXAMPLE 6 EXPRESSION OF RECOMBINANT SLGP PROTEIN
  • the pcDNA/Amp vector by Invitrogen Co ⁇ oration (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire SLGP protein and an HA tag (Wilson et al. (1984) Cell 31:161) or a FLAG tag fused in-frame to its 3' end ofthe fragment is cloned into the polylinker region ofthe vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.
  • the SLGP DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe SLGP coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides ofthe SLGP coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the SLGP gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence ofthe correct fragment. COS cells are subsequently transfected with the SLGP-pcDNA/Amp plasmid
  • DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression ofthe SLGP polypeptide is detected by radiolabelling (35 S -methionine or ⁇ S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D.
  • HA specific monoclonal antibody A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988
  • the cells are labeled for 8 hours with 35s-methionine (or 35s-cysteine).
  • the culture media are then collected and the cells are lysed using detergents (RIP A buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA-specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the SLGP coding sequence is cloned directly into the polylinker ofthe pCDN A/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression ofthe SLGP polypeptide is detected by radiolabelling and immunoprecipitation using an SLGP-specific monoclonal antibody.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur des molécules d'acide nucléique isolées appelées molécules SLGP qui codent de nouveaux éléments de la famille GPCR. L'invention porte également sur des molécules d'acide nucléique antisens, des vecteurs d'expression recombinés contenant les molécules d'acide nucléique SLGP, des cellules hôtes dans lesquelles ont été introduits les vecteurs d'expression et des animaux transgéniques dans lesquels un gène SLGP a été introduit ou rompu. L'invention porte en outre sur des protéines SLGP isolées, des protéines de fusion, des peptides antigéniques et des anticorps anti-SLGP. Des méthodes diagnostiques utilisant les compositions de l'invention sont également décrites.
EP01952313A 2000-06-30 2001-06-29 Proteine slgp et molecules d'acide nucleique et leurs utilisations Withdrawn EP1309686A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US608921 1996-02-29
US60892100A 2000-06-30 2000-06-30
PCT/US2001/020751 WO2002002602A2 (fr) 2000-06-30 2001-06-29 Nouvelle proteine slgp et nouvelles molecules d'acide nucleique et leurs utilisations

Publications (1)

Publication Number Publication Date
EP1309686A2 true EP1309686A2 (fr) 2003-05-14

Family

ID=24438633

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01952313A Withdrawn EP1309686A2 (fr) 2000-06-30 2001-06-29 Proteine slgp et molecules d'acide nucleique et leurs utilisations

Country Status (3)

Country Link
EP (1) EP1309686A2 (fr)
AU (1) AU2001273077A1 (fr)
WO (1) WO2002002602A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ503343A (en) * 1997-09-17 2002-09-27 Genentech Inc Secreted and transmembrane polypeptides and use to induce apoptosis of tumour cells
JP2002525117A (ja) * 1998-09-30 2002-08-13 ミレニアム・フアーマシユーチカルズ・インコーポレーテツド Ｓｌｇｐタンパク質及び核酸分子並びにそれらの使用
AU2600800A (en) * 1999-03-08 2000-09-28 Genentech Inc. Promotion or inhibition of angiogenesis and cardiovascularization
WO2001060999A1 (fr) * 2000-02-14 2001-08-23 Imclone Systems Incorporated Sequences d'acides nucleiques caracteristiques de cellules souches hematopoietiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0202602A3 *

Also Published As

Publication number Publication date
WO2002002602A9 (fr) 2003-03-06
AU2001273077A1 (en) 2002-01-14
WO2002002602A3 (fr) 2002-08-22
WO2002002602A2 (fr) 2002-01-10

Similar Documents

Publication Publication Date Title
EP1131433A1 (fr) Nouveaux elements de la famille de proteines receptrices de capsicine/vanilloide, et utilisations de ceux-ci
US8133689B2 (en) Members of the capsaicin/vanilloid receptor family of proteins and uses thereof
EP1100877A1 (fr) Nouvelles molecules de canaux potassium et leurs utilisations
US20060275870A1 (en) Novel G-protein coupled receptors and uses therefor
WO2000052164A2 (fr) Nouvelles molecules du canal potassique et leur utilisation
WO2000042170A1 (fr) Molecules d'acide nucleique et de proteine lrsg et leurs utilisations
US20020173636A1 (en) 66784, a novel human potassium channel and uses therefor
AU4101599A (en) Novel secreted and membrane-associated proteins and uses therefor
WO2002038767A2 (fr) Nouvelles proteine recepteur couplee a une proteine g et molecules d'acides nucleiques, et utilisations associees
US20030087343A1 (en) Novel SLGP nucleic acid molecules and uses therefor
US6204013B1 (en) MSP-5 nucleic acid molecules and uses therefor
EP1285064A2 (fr) Nouveaux recepteurs couples a la proteine g et utilisations de ceux-ci
WO2001083523A2 (fr) Nouvelles proteines et molecules d'acide nucleique stmst et utilisations correspondantes
US6756212B1 (en) Isolated proteins and nucleic acid molecules having homology to the NIP2 protein and uses thereof
US20020004193A1 (en) Novel MP-7 protein and nucleic acid molecules and uses therefor
US20030119147A1 (en) Novel SLGP protein and nucleic acid molecules and uses therefor
WO2002002602A2 (fr) Nouvelle proteine slgp et nouvelles molecules d'acide nucleique et leurs utilisations
WO2001000878A2 (fr) Nouveau membre de la famille des facteurs de croissance des cellules endotheliales vasculaires et son utilisation
EP1792913A1 (fr) Nouvelles protéines secretées et associées à la membrane et leur utilization
WO2000043514A9 (fr) Canal ionique detectant les acides et ses utilisations
WO2000017236A2 (fr) Proteines reliees a l'apoptose et utilisations associees

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030129

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20030710

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040914