EP1124850A1 - 12 proteines humaines secretees - Google Patents

12 proteines humaines secretees

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Publication number
EP1124850A1
EP1124850A1 EP99972222A EP99972222A EP1124850A1 EP 1124850 A1 EP1124850 A1 EP 1124850A1 EP 99972222 A EP99972222 A EP 99972222A EP 99972222 A EP99972222 A EP 99972222A EP 1124850 A1 EP1124850 A1 EP 1124850A1
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EP
European Patent Office
Prior art keywords
seq
polypeptide
regions
amino acid
polynucleotide
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Withdrawn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
EP99972222A
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German (de)
English (en)
Other versions
EP1124850A4 (fr
Inventor
Jian Ni
Steven M. Ruben
Henrik S. Olsen
Paul E. Young
Joseph J. Kenny
Paul A. Moore
Ying-Fei Wei
John M. Greene
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication of EP1124850A1 publication Critical patent/EP1124850A1/fr
Publication of EP1124850A4 publication Critical patent/EP1124850A4/fr
Withdrawn legal-status Critical Current

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    • C12N9/14—Hydrolases (3)
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Definitions

  • This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, and their production.
  • sorting signals are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
  • One type of sorting signal directs a class of proteins to an organelle called the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • the ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus.
  • the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles. Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein.
  • vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space - a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a "linker" holding the protein to the membrane.
  • the present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting disorders and conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such disorders and conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides. Detailed Description Definitions
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
  • a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
  • polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
  • the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • a "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC.
  • the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
  • a representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table XIII, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.
  • the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC.
  • “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in OJx SSC at about 65 degree C.
  • nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
  • blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
  • polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • polypeptides may be branched , for example, as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide oi nucleotide derivative, covalent attachment of a hpid oi hpid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchoi formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, lacemization, selenoylation, sulfation, transfei- RNA mediated addition of amino acids to proteins such as argin
  • SEQ ID NO:X refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in Table XIII
  • a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose- dependence a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25- fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)
  • translation product of this gene shares sequence homology with a protein from Xenopus laevis that is descnbed as upregulated in response to thyroid hormone in tadpoles, and is thought to be important in the tail resorption process during Xenopus laevis metamorphosis (See Proc Natl. Acad. Sci. USA (1996 Mar. 5):93(5): 1924-9, which is herein incorporated by reference).
  • translation product of this gene shares sequence homology with a recently desc ⁇ bed group of proteins, called hedgehog interacting proteins (HIPs) (See International Publication No. WO98/12326, which is herein incorporated by reference).
  • HIPs hedgehog interacting proteins
  • HIPs bind to hedgehog polypeptides such as Shh and Dhh with high affinity (Kd approx. 1 nM).
  • HIPs exhibit spatiallyand temporally restricted expression domains indicative of important roles hedgehog-mediated induction. They regulate differentiation of neuronal cells, regulate survival of differentiated neuronal cells, proliferation of chondrocytes, proliferation of testicular germ line cells and/or expression of patched or hedgehog genes.
  • the biological activity of this polypeptide is assayed by techniques known in the art, otherwise disclosed herein and as desc ⁇ bed International Publication No. WO98/12326, which is herein incorporated by reference.
  • Preferred polypeptides of the invention comprise the following amino acid sequence: MLRTSTPNLCGGLHCRAPWLSSGILCLCLIFLLGQVGLLQGHPQCLDYGPPFQPP LHLEFCSDYESFGCCDQHKDRRIAARYWDIMEYFDLKRHELCGDYIKDILCQEC SPYAAHLYDAENTQTPLRNLPGLCSDYCSAFHSNCHSAISLLTNDRGLQESHGRD GTRFCHLLDLPDKDYCFPNVLRNDYLNRHLGMVAQDPQGCLQLCLSEVANGLR NPVSMVHAGDGTHRFFVAEQVGVVWVYLPDGSRLEQPFLDLKNIVLTTPWIGD ERGFLGLAFHPKFRHNRKFYIYYSCLDKKKVEKIRISEMKVSRADPNKADLKSER VILEIEEPASNHNGGQLLFGLDGYMYIFTGDGGQAGDPFGLFGNAQNKSSLLGK VLRIDVNRAGSHGKRYRVPSDNPFVSEPGAHPAIYAYGI
  • Figures 1A-C show the nucleotide (SEQ ID NO: 11) and deduced amino acid sequence (SEQ ID NO:29) of this protein.
  • Figure 2 shows the regions of similarity between the amino acid sequences of SEQ ID NO:29, the Xenopus laevis tail resorption protein (gi
  • Figure 3 shows an analysis of the amino acid sequence of SEQ ID NO: 29.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • Northern analysis indicates that a 2.5-3.0 kb transcript of this gene is expressed primarily in testes tissue and A549 lung carcinoma tissue, but interestingly is absent from normal lung tissue. This gene is also expressed in osteoarthritis tissue and human fetal tissues.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO:29), which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figures 1A-C (SEQ ID NOJ 1) was obtained by sequencing a cloned cDNA, which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NOJ 1 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 1.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOJ 1.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 570 of SEQ ID NO 11, or the complementary strand thereto, or the cDNA contained in the deposited gene.
  • polynucleotides of the invention encode functional att ⁇ butes of the conesponding piotein
  • Preferred embodiments of the invention in this regard include fragments that comp ⁇ se alpha-helix and alpha-helix forming legions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-iegions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur m the process of initiation of an immune response.
  • Certain preferred regions in these regards are set out in Figure 3, but may, as shown Table I, be represented or identified by using tabular representations of the data presented in Figure 3.
  • the DNA*STAR computer algo ⁇ thm used to generate Figure 3 was used to present the data in Figure 3 in a tabular format (See Table I).
  • the tabular format of the data m Figure 3 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out m Figure 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 1A-C (SEQ ID NO:29).
  • such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-iegions, beta-iegions, and turn-iegions, Kyte-Doohttle hydiophi c regions and Hopp-Woods hydiophobic regions, Eisenbeig alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming legions.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 1A-C, up to the alanine residue at position number 524 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides compnsing the amino acid sequence of residues n 1-524 of Figures 1A-C, where nl is an integer from 1 to 524 corresponding to the position of the amino acid residue in Figures 1A-C (which is identical to the sequence shown as SEQ ID NO:29).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:29 include polypeptides comprising the amino acid sequence of residues: V-2 to P-529; A-3 to P-529; Q-4 to P- 529; D-5 to P-529; P-6 to P-529; Q-7 to P-529; G-8 to P-529; C-9 to P-529; L-10 to P- 529; Q-l l to P-529; L-12 to P-529; C-13 to P-529; L-14 to P-529; S-15 to P-529; E-16 to P-529; V-17 to P-529; A-18 to P-529; N-19 to P-529; G-20 to P-529; L-21 to P-529; R- 22 to P-529; N-23 to P-529; P-24 to P-529; V-25 to P-529; S-26 to P-529; M-27 to P- 529; V-28 to P-529; H-29 to P-529; A-30 to P-529; G-
  • deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein
  • other functional activities e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.)
  • biological activities e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.
  • the ability to induce and or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majo ⁇ ty of the residues of the complete or mature polypeptide are removed from the C-terminus.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 1A-C. up to the glutamine lesidue at position number 7, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comp ⁇ sing the amino acid sequence of residues 1-ml of Figures 1 A-C, where ml is an integer from 7 to 528 corresponding to the position of the amino acid residue in Figures lA-C.
  • polypeptides comp ⁇ sing, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:29 include polypeptides comp ⁇ sing the ammo acid sequence of residues: M-1 to L- 528; M-1 to S-527; M-1 to R-526; M-1 to G-525; M-1 to A-524; M-1 to R-523; M-1 to K-522; M-1 to Q-521; M-1 to E-520; M-1 to A-519; M-1 to S-518; M-1 to P-517; M-1 to R-516; M-1 to M-515; M-1 to R-514; M-1 to G-513; M-1 to S-512; M-1 to H-511; M-1 to S-510; M-1 to K-509; M-1 to L-508; M-1 to S-507; M-1 to K-506; M-1 to R-505; M-1 to R-504
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental disorders, and degenerative disorders; osteoarthritis, and lung cancer.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • bodily fluids e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid
  • another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e.. the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 29 as residues: Asp-52 to Glu-57, Arg-89 to Tyr-95, Asp- 102 to Glu-107, Ser-117 to Ser-128, Glu-137 to Gly-145, Arg-192 to Arg-199, Val-231 to Gly- 243, Val-250 to Glu-256, Arg-312 to Asn-318, Glu-338 to Asp-349, Pro-405 to Lys-417, Thr-423 to Ue-428, Lys-442 to Ser-453, Glu-467 to Ala-475, Thr-478 to Arg-494, Pro- 497 to Arg-526. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 11 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • a- b preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 2595 of SEQ ID NOJ 1, b is an integer of 15 to 2609, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a + 14.
  • TIDE Ten Integrin Domains with EGF homology
  • integrins which are a superfamily of dimeric ab cell-surface glycoproteins that mediate the adhesive functions of many cell types, enabling cells to interact with one another and with the extracellular matrix
  • Eight human integrin b subunits have been described to date, and in combination with the 12 known a subunits form a large family of heterodimeric cell surface receptors that mediate cell adhesion to counter-receptors on neighboring cells, and to ECM proteins (reviewed by Hynes, 1992).
  • polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention.
  • polypeptides of the invention comprise the following amino acid sequence: TSTPPRAVPLPKSSQAAHQRNCNSGWSPGPASLGVRGSVCPAICWWHLS LLPPPSVNPTLQKCSSPGAAQELSMRPPGFRNFLLLASSLLFAGLSAVPQSFSPSLR SWPGAACRLSRAESERRCRAPGQPPGAALCHGRGRCDCGVCICHVTEPGMFFGP LCECHEWVCETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYPTNCDLTKKK SNQMCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEI CGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESKRRCTSPDGKICSNRGTCV CGECTCHDVDPTGDWGDIHGDTCECDERDCRAVYDRYSDDFCSGHGQCNCGR CDCKAGWYGKKCEHPQSCTLSAEESIRKCQGSSDLPCSGRGKCECGKCTCYPPG DRRV
  • EGF-like domain signature 1 and 2 domains which were identified using the ProSite analysis tool (Swiss Institute of Bioinformatics).
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown [1 to 6] to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two- stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet.
  • Subdomains between the conserved cysteines strongly vary in length as shown in the following schematic representation of the EGF-like domain:
  • I I I I I x (4) -C-x( 0 , 48 ) -C-x(3 , 12 ) -C-x( l , 70 ) -C-x( 1. 6) -C-x(2 ) -G-a-x( 0. 21 ) -G-x(2 ) -C-x
  • Preferred polypeptides of the invention comprise the following amino acid sequence: GKCDCGKCKCDQGWYGDACQYPTNCDLTK (SEQ ID NO: 51), GGHGKCYCGNCYCKAGWHGDKCEFQCDIT (SEQ ID NO'52), HGQCNCGRCDCKAGWYGKKCEHPQSCTLS (SEQ ID NO 53), HGTCSCGRCVCERGWFGKLCQHPRKCNMT (SEQ ID NO: 54), GNGICSCGNCECWDGWNGNACEIWLGSEY (SEQ ID NO 55), and ICGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESK (SEQ ID NO 73) Polynucleotides encoding these polypeptides aie also piovided
  • polypeptides comprising the EGF-hke domain signature 1 and 2 domains of the sequence referenced in Table I for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence
  • the additional contiguous ammo acid lesidues is N-teimmal oi C- terminal to the EGF-hke domain signature 1 and 2 domains
  • the additional contiguous amino acid residues is both N-termmal and C-termmal to the EGF- ke domain signature 1 and 2 domains, wherein the total N- and C-termmal contiguous am o acid residues equal the specified number
  • the above preferred polypeptide domain is characteristic of a signature specific to EGF- ke domain 1 and 2 containing proteins Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with EGF-hke containing proteins Such activities are known in the art, some of which are descnbed elsewhere herein. Included in this invention as preferred domains are integrins beta chain cysteine- ⁇ ch domains, which were identified using the ProSite analysis tool (Swiss Institute of Bio formatics).
  • Integnns [7,8] are a large family of cell surface receptors that mediate cell to cell as well as cell to matrix adhesion. Some mteg ⁇ ns recognize the R-G-D sequence m their extracellular matrix protein ligand. Structurally, integnns consist of a dimer of an alpha and a beta chain. Each subunit has a large N-terminal extracellular domain followed by a transmembrane domain and a short C-terminal cytoplasmic region Some receptors share a common beta chain while having different alpha chains All the integrin beta chains contain four repeats of a forty amino acid region in the C-terminal extremity of their extracellular domain. Each of the repeats contains eight cysteines.
  • Preferred polypeptides of the invention comprise the following amino acid sequence: GQPPGAALCHGRGRCDCGVCICHVTEPGMFFGPLC (SEQ ID NO: 74), ETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYP (SEQ ID NO:58), MCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYG (SEQ ID NO:59), IDDETEEICGGHGKCYCGNCYCKAGWHGDKC (SEQ ID NO:60), KRRCTSPDGKICSNRGTCVCGECTCHDVDPTGDW (SEQ ID NO:61), DRYSDDFCSGHGQCNCGRCDCKAGWYGKKCEHPQ (SEQ ID NO:62), CQGSSDLPCSGRGKCECGKCTCYPPGDRRV
  • polypeptides comprising the integrins beta chain cysteine- rich domain of the sequence referenced in Table XIII for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence.
  • the additional contiguous amino acid residues is N-terminal or C- terminal to the integrins beta chain cysteine-rich domain.
  • the additional contiguous amino acid residues is both N-terminal and C-terminal to the integrins beta chain cysteine-rich domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number.
  • the above preferred polypeptide domain is characteristic of a signature specific to integrin proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically those containing an integrins beta chain cysteine-rich domain. Such activities are known in the art, some of which are described elsewhere herein. The following publications were referenced above and are hereby incorporated herein by reference: [ 1] Davis C.G., New Biol.
  • the polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed Ten Integrin Domains with EGF homology ("TIDE"). This identification has been made as a result of amino acid sequence homology to the human integrin beta-8 subunit (See Genbank Accession No. gi
  • Figures 4A-C shows the nucleotide (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO:30) of TIDE.
  • Predicted amino acids from about 1 to about 23 constitute the predicted signal peptide (amino acid residues from about 1 to about 23 in SEQ ID NO:30) and are represented by the underlined amino acid regions; amino acids from about 108 to about 136, from about 195 to about 223, from about 291 to about 319, from about 379 to about 407, and/or from about 465 to about 493 constitute the predicted EGF-like domain signature 1 and 2 domains (amino acids from about 108 to about 136, from about 195 to about 223, from about 291 to about 319, from about 379 to about 407, and/or from about 465 to about 493 in SEQ ID NO:30) and are represented by the double underlined amino acids; and amino acids from about 55 to about 89, from about 97 to about 129, from about 142 to about 175,
  • FIG. 5 shows the regions of similarity between the amino acid sequences of the Ten Integrin Domains with EGF homology (TIDE) protein (SEQ ID NO:30) and the human integrin beta-8 subunit (SEQ ID NO: 67).
  • Figure 6 shows an analysis of the Ten Integrin Domains with EGF homology
  • TIDE amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • a polynucleotide encoding a polypeptide of the present invention is obtained from human osteoblasts, synovial hypoxia tissue, osteoblast and osteoclast, bone marrow stromal cells, umbilical vein, smooth muscle, placenta, and fetal lung.
  • the polynucleotide of this invention was discovered in a human osteoblast II cDNA library. Its translation product has homology to the characteristic integrins beta chain cysteine- rich domains of integrin family members.
  • the polynucleotide contains an open reading frame encoding the TIDE polypeptide of 494 amino acids. TIDE exhibits a high degree of homology at the amino acid level to the human integrin beta-8 subunit (as shown in Figure 5).
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TIDE polypeptide having the amino acid sequence shown in Figures 4A-C (SEQ ID NO:30).
  • the nucleotide sequence shown in Figures 4A-C (SEQ ID NOJ 2) was obtained by sequencing a cloned cDNA (HOHCH55), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 12 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 12.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 12.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • TIDE polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the mature TIDE protein (amino acid residues from about 221 to about 1705 in Figures 4A-C (amino acids from about 221 to about 1705 in SEQ ID NO:30). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define each domain.
  • the polynucleotides of the invention encode functional attributes of TIDE.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TIDE.
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming regions beta-sheet and beta-sheet forming regions
  • turn- regions turn and turn-forming regions
  • coil and coil-forming regions coil and coil-forming regions
  • the data presented in columns VIII, IX, XIII, and XIV of Table II can be used to determine regions of TIDE which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Figure 6 and in Table II include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 4A-C As set out in Figure 6 and in Table II, such preferred regions include Garnier- Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha- regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus- Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TIDE ammo acid sequence shown in Figures 4A-C, up to the leucme residue at position number 489 and polynucleotides encoding such polypeptides
  • the present invention provides polypeptides comprising the am o acid sequence of residues n 1-494 of Figures 4A-C, where nl is an integer from 2 to 489 corresponding to the position of the amino acid residue in Figures 4A-C (which is identical to the sequence shown as SEQ ID NO:30).
  • N-termmal deletions of the TIDE polypeptide can be desc ⁇ bed by the general formula n2-494, where n2 is a number from 2 to 489, corresponding to the position of ammo acid identified in Figures 4A-C
  • N-terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides comp ⁇ sing the amino acid sequence of residues: R-2 to P-494; P-3 to P-494; P-4 to P-494; G-5 to P-494; F-6 to P- 494; R-7 to P-494; N-8 to P-494; F-9 toP-494; L-10 to P-494; L-l 1 to P-494; L-12 to P- 494; A-13 to P-494; S-14 to P-494; S-15 to P-494; L-16 to P-494; L-17
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TIDE polypeptide shown in Figures 4A-C, up to the phenylalanine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 494 corresponding to the position of the amino acid residue in Figures 4A-C Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides comprising the amino acid sequence of residues: M-1 to Y-493; M- 1 to E-492; M-1 to S-491; M-1 to G-490; M-1 to L-489; M-1 to W-488; M-1 toI-487; M- 1 to E-486; M-1 to C-485; M-1 to A-484; M-1 to N-483; M-1 to G-482; M-1 to N-481; M-1 to W-480; M-1 to G-479;M-1 to D-478; M-1 to W-477; M-1 to C-4
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NOJ2 which have been determined from the following related cDNA genes: HLHFV34R (SEQ ID NO:68), HSRDA85R (SEQ ID NO:69), HSRAZ62R (SEQ ID NO:70), HSRDA17R (SEQ ID NO:71), and HSLEC45R (SEQ ID NO:72).
  • translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically the human integrin beta-8 subunit. Such activities are known in the art, some of which are described elsewhere herein.
  • polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response.
  • Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders.
  • the full- length protein should be a secreted protein, based upon homology to the integrin family. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection.
  • polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types.
  • the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers.
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the "Hyperproliferative Disorders" and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
  • Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers.
  • this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein can also be used to gain new insight into the regulation of cellular growth and proliferation.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • integrins which are a superfamily of dimeric ab cell-surface glycoproteins that mediate the adhesive functions of many cell types, enabling cells to interact with one another and with the extracellular matrix (See Genomics 56, 169-178 (1999); all information and references contained within this publication are hereby incorporated herein by reference).
  • the gene encoding the disclosed cDNA is believed to reside on chromosome 13, at locus 13q33. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 13, generally, and particularly at locus 13q33.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of bone and connective tissues, immune and hematopoietic diseases and/or disorders, vascular disorders, and other disorders involving aberrations in cell-surface interactions.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • tissue or cell types e.g. cartilage, bone, vascular, hypoxic tissue, and cancerous and wounded tissues
  • bodily fluids e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid
  • another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 30 as residues: Met-1 to Phe-6, Arg-44 to Arg-52, His-64 to Cys- 69, Tyr-99 to Gln-147, His-158 to Gly-169, Phe-177 to Asp-182, Cys-194 to Cys-202, Gly-213 to Phe-218, Pro-224 to Gly-236, Asp-254 to Trp-261, Asp-263 to Ala-303, Trp- 305 to Cys-316, Lys-326 to Asp-332, Pro-334 to Cys-343, Pro-350 to Asp-370, Thr-407 to Asn-413, Gly-425 to Cys-431, Asp-449 to Asp-459, Gly-472 to Asn-483. Polynucleotides encoding said polypeptides are also provided.
  • tissue distribution and homology to the human integrin beta-8 subunit indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the "Immune Activity” and "infectious disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
  • the expression indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes indicates a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin, indicates the natural gene product is involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T- cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, p
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support.
  • a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 12 amino acid sequences
  • amino acid sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention.
  • such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 2485 of SEQ ID NOJ2, b is an integer of 15 to 2499, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a + 14.
  • RAMP3 calcitonin-receptor-like receptor
  • RAMP2 receptor-activity-modifying proteins
  • RAMP1 is thought to present the receptor at the cell surface as a mature glycoprotein and a Calcitonin-gene-related peptide (CGRP) receptor.
  • CGRP Calcitonin-gene-related peptide
  • RAMP2-tran sported receptors are core-glycosylated and are adrenomedullin receptors.
  • CGRP a 37-amino-acid neuropeptide
  • CGRP a 37-amino-acid neuropeptide
  • its receptors are widely distributed in the body, and it is the most potent endogenous vasodilatory peptide discovered so far (Crit Rev Neurobiol 1997J 1(2-3): 167-239).
  • adrenomedullin Specific binding sites for adrenomedullin were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons and medulla oblongata), suggesting that a novel neurotransmitter/neuromodulator role may exist for adrenomedullin in human brain (Peptides 1997; 18(8): 1125-9).
  • Figures 7A-B show the nucleotide (SEQ ID NO: 13) and deduced amino acid sequence (SEQ ID NO:31 ) of the Intestine derived extracellular protein.
  • Predicted amino acids from about 1 to about 27 constitute the predicted signal peptide (amino acid residues from about 1 to about 27 in SEQ ID NO:31) and are represented by the underlined amino acid regions; and amino acids from about 122 to about 138 constitute the predicted transmembrane domain (amino acid residues from about 122 to about 138 in SEQ ID NO:31) and are represented by the double-underlined amino acids.
  • Figure 8 shows the regions of similarity between the amino acid sequences of the Intestine derived extracellular protein SEQ ID NO:31, and the RAMP3 protein (gi
  • Figure 9 shows an analysis of the amino acid sequence of SEQ ID NO: 31.
  • Alpha, beta, turn and coil regions hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • Northern analysis indicates that a 1.4kb transcript of this gene is primarily expressed in small intestine tissue, and to a lesser extent in colon and prostate tissue.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:31), which was determined by sequencing a cloned cDNA (HTLEW81).
  • the nucleotide sequence shown in Figures 7A-B (SEQ ID NO: 13) was obtained by sequencing a cloned cDNA (HTLEW81), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl rest ⁇ ction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules desc ⁇ bed herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 13 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and p ⁇ meis as discussed heiein Of couise, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments conesponding to most, if not all, of the nucleotide sequence of the deposited cDNA oi as shown in SEQ ID NOJ3
  • a fragment at least 20 nt in length for example, is intended fragments which include 20 or moie contiguous bases from the nucleotide sequence of the deposited cDNA or the nucle
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 10
  • polynucleotides of the invention encode functional attributes of the corresponding protein.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • the data presented in columns VIII, IX, XIII, and XIV of Table IIII can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Certain preferred regions in these regards are set out in Figure 9, but may, as shown in Table III, be represented or identified by using tabular representations of the data presented in Figure 9.
  • the DNA*STAR computer algorithm used to generate Figure 9 (set on the original default parameters) was used to present the data in Figure 9 in a tabular format (See Table III).
  • the tabular format of the data in Figure 9 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 9 and in Table III include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 7A-B.
  • such preferred regions include Garnier- Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha- regions, beta-regions, and turn-regions, Kyte-Doohttle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus- Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • the present invention further provides polypeptides having one oi more residues deleted from the amino terminus of the ammo acid sequence shown in Figures 7A-B, up to the argmine residue at position number 143 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comp ⁇ sing the ammo acid sequence of residues nl-148 of Figures 7A-B, where nl is an integer from 2 to 143 corresponding to the position of the amino acid residue in Figures 7A-B (which is identical to the sequence shown as SEQ ID NO:31).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:31 include polypeptides comp ⁇ sing the amino acid sequence of residues: E-2 to L-148; T-3 to L-148; G-4 toL- 148; A-5 to L-148; L-6 to L-148; R-7 to L-148; R-8 to L-148;P-9 to L-148; Q-10 to L- 148; L-l 1 to L-148; L-12 to L-148; P-13to L-148; L-14 to L-148; L-15 to L-148; L-16 to L-148; L-17 toL-148; L-18 to L-148; C-19 to L-148; G-20 to L-148; G-21 toL-148; C-22 to L-148; P-23 to L-148; R-24 to L-148; A-25 toL-148; G-26 to L-148; G-27 to L-148; C-28 to L-148; N-29 toL-148; E-30 to L-148; T-31
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.)), may still be retained. For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or matuie polypeptide are removed from the C-terminus.
  • biological activities e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the ammo acid sequence of the polypeptide shown in Figures 7A-B, up to the argmine residue at position number 7, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the ammo acid sequence of residues 1-ml of Figures 7A-B, where ml is an integer from 7 to 147 corresponding to the position of the amino acid residue in Figures 7A-B.
  • the invention provides polynucleotides encoding polypeptides compnsing, or alternatively consisting of, the ammo acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:31 include polypeptides comp ⁇ sing the amino acid sequence of residues: M-1 to L-147; M-1 to T- 146;M-1 to D-145; M-1 to T-144; M-1 to R-143; M-1 to K-142; M-1 toS-141; M-1 to R- 140; M-1 to W-139; M-1 to V-138; M-1 to V-137;M-1 to L-136; M-1 to G-135; M-1 to A-134; M-1 to M-133; M-1 toA-132; M-1
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:31 which have been determined from the following related cDNA genes: HLHCH17RA (SEQ ID NO:76), HTOAT51R (SEQ ID NO:77), and/or HBNBO41R (SEQ ID NO:78).
  • the polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 122 - 138 of the amino acid sequence referenced in Table XIII for this gene.
  • a cytoplasmic tail encompassing amino acids 139 to 149 of this protein has also been determined. Based upon these characte ⁇ stics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the t ⁇ ssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal and neurodegenerative diseases and disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the t ⁇ ssue(s) or cell type(s).
  • expression of this gene at significantly highei or lower levels is routinely detected in certain tissues or cell types (e.g.
  • bodily fluids e.g., lymph, serum, plasma, u ⁇ ne, synovial fluid and spmal fluid
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 31 as residues. Ala-5 to Gin- 10, Pro-23 to Cys-28, Arg- 140 to Asp- 145. Polynucleotides encoding said polypeptides are also provided.
  • the tissue dist ⁇ bution and homology to RAMP3 suggest that the translation product of this gene is useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo.
  • tissue dist ⁇ bution in small intestine and colon tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of disorders involving the small intestine. This may include diseases associated with digestion and food absorption, as well as hematopoietic disorders involving the Peyer's patches of the small intestine, or other hematopoietic cells and tissues within the body.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 13 Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available pnor to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • a- b is any integer between 1 to 1325 of SEQ ID NOJ3, b is an integer of 15 to 1339, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13, and where b is greater than or equal to a + 14.
  • the translation product of this gene shares sequence homology with a proteoglycan from Gallus gallus, and this proteoglycan is believed to participate in the osteogenic processes of cartilage ossification (See Genbank Accession No. g ⁇
  • the translation product of this gene is expected to share biological activities with the Gallus gallus proteoglycan polypeptide.
  • Figures 10A-B shows the nucleotide (SEQ ID NOJ4) and deduced amino acid sequence (SEQ ID NO.32) of the retinal specific protein
  • Predicted ammo acids from about 1 to about 21 constitute the predicted signal peptide (amino acid residues from about 1 to about 21 SEQ ID NO 32) and are repiesented by the undei lined amino acid iegions
  • Figure 1 1 shows the regions of similarity between the amino acid sequences of the letinal specific protein SEQ ID NO 32, and the Gallus gallus pioteoglycan (SEQ ID NO 79)
  • Figuie 12 shows an analysis of the ammo acid sequence of SEQ ID NO: 32.
  • the present invention provides isolated nucleic acid molecules comp ⁇ sing a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 10A-B (SEQ ID NO:32), which was determined by sequencing a cloned cDNA (HARAO44).
  • the nucleotide sequence shown in Figures 10A-B (SEQ ID NOJ4) was obtained by sequencing a cloned cDNA (HARAO44), which was deposited on Nov. 17, 1998 at the Ame ⁇ can Type Culture Collection, and given Accession Number 203484
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl rest ⁇ ction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules desc ⁇ bed herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NOJ4 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt length which are useful as diagnostic probes and p ⁇ mers as discussed herein.
  • fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA oi as shown in SEQ ID NO: 14.
  • fragments at least 20 nt in length is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOJ4.
  • “about” includes the particularly recited size, larger or smallei by several (5, 4, 3, 2, oi 1) nucleotides, at either terminus oi at both termini
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251
  • polynucleotides of the invention encode functional att ⁇ butes of the corresponding protein.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil -forming iegions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming regions turn and turn-forming regions
  • turn- regions turn-forming regions
  • coil and coil -forming iegions coil and coil -forming iegions
  • the data presented in columns VIII, IX, XIII, and XIV of Table IV can be used to determine regions of the protein which exhibit a high degiee of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Figure 12 (set on the o ⁇ ginal default parameters) was used to present the data in Figure 12 in a tabular format (See Table IV).
  • the tabular format of the data in Figure 12 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 12 and m Table IV include, but are not limited to, regions of the aforementioned types identified by analysis of the ammo acid sequence set out in Figures 10A-B.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doohttle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the ammo acid sequence shown in Figures 10A-B, up to the proline residue at position number 327 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides composing the ammo acid sequence of residues nl-332 of Figures 10A-B, where nl is an integer from 2 to 327 corresponding to the position of the ammo acid residue in Figures 10A-B (which is identical to the sequence shown as SEQ ID NO:32).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:32 include polypeptides comprising the amino acid sequence of residues: R-2 to T-332; L-3 to T-
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 10A-B, up to the glutamine residue at position number 7, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comp ⁇ sing the ammo acid sequence of residues 1-ml of Figures 10A-B, where ml is an integer from 7 to 331 corresponding to the position of the amino acid residue in Figures 10A-B.
  • polypeptides comp ⁇ sing, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:32 include polypeptides comprising the amino acid sequence of residues: M-1 to F- 331; M-1 to R-330; M-1 to G-329; M-1 to 1-328; M-1 to P-327; M-1 to L-326; M-1 to R- 325; M-1 to P-324; M-1 to L-323; M-1 to C-322; M-1 to F-321; M-lto Y-320; M-1 to A- 319; M-1 to S-318; M-1 to P-317; M-1 to F-316; M-1 to L-315; MJ to S-314; M-l to L- 313; M-lto N-312; M-1 to 1-311; M-1 to P-310; M-l to N-309; M-l to G-308; M-1
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 14 which have been determined from the following related cDNA genes: HARAY79R (SEQ ID NO:80), HARAO44R (SEQ ID NO:81), HARAJ74R (SEQ ID NO:82), HARAO66R (SEQ ID NO:83), HARAN19R (SEQ ID NO:84), and HARAT78R (SEQ ID NO:85).
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to. retinal disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 32 as residues: Leu-22 to Asp-39, Asn-64 to Pro-76, Pro-98 to Thr-111, Pro-291 to Glu-302. Polynucleotides encoding said polypeptides are also provided.
  • the tissue distribution in retinal tissue, and the homology to a Gallus gallus proteoglycan involved in the ossification process indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of disorders of the retina which involve the adhesion of tissues, or the binding of certain proteins to the cell surface.
  • the translation products of this gene are useful for the treatment of retinal disorders such as retinal detachment in individuals suffe ⁇ ng from myopia, or in the treatment of macular degeneration. Furthermore, this gene may serve as a tumor marker for retinoblastomas, or related tumors. Moie generally, the tissue distribution in retinal tissue indicates that The translation pioduct ot this gene is useful for the diagnosis, detection and/or treatment of eye disordeis including blindness, color blindness, impaired vision, short and long sightedness, retinitis pigmentosa, retinitis prohferans, and retinoblastoma, retinochoroiditis, retmopathy and retinoschisis. Based upon the tissue distribution of this protein, antagonists d ⁇ ected against this protein is useful in blocking the activity of this protein Accordingly, prefen-ed are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises one embodiment an antibody specific for the translation product of this gene bound to a solid support.
  • a method of detecting these tumors in an individual which comp ⁇ ses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate hgands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NOJ4 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 1375 of SEQ ID NOJ4, b is an integer of 15 to 1389, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.
  • CD33 The translation product of this gene shares sequence homology with the CD33 protein (See Genbank Accession No. gi
  • the expression pattern of CD33 within the hematopoietic system indicates a potential role in the regulation of myeloid cell differentiation. However, this expression is absent from hematopoietic stem cells.
  • CD33 is expressed in clonogenic leukemia cells in about 90% of patients suffering from acute myeloid leukemia (AML). While about 60-70% of adults suffering from AML experience complete remission due to chemotherapy application, most of these patients will ultimately die of relapsed leukemia. It is believed that, like CD33, the CD33-like protein of the present invention is also expressed by clonogenic leukemia cells from the vast majority of patients with AML. Thus, there is a clear need to identify and isolate nucleic acid molecules encoding additional polypeptides having
  • CD33-like protein activity It is believed that cancerous tissue contains significantly greater amounts of CD33-like protein gene copy number and expresses significantly enhanced levels of CD33-like protein and mRNA encoding the CD33-like protein when compared to a "standard" mammal, i.e.-a mammal of the same species not having the cancer or inflammatory disease. Thus, enhanced levels of the CD33-like protein will be detected in certain bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) from mammals when compared to sera from mammals of the same species not having the cancer or inflammatory disease.
  • bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
  • polynucleotides comprise the following sequences: CGACCCACGCGTCCGCCGCCTTCGGCTTCCCCTTCTGCCAA
  • Figures 13A-C shows the nucleotide (SEQ ID NOJ5) and deduced amino acid sequence (SEQ ID NO:33) of the CD33-like protein.
  • Predicted amino acids from about 1 to about 16 constitute the predicted signal peptide (amino acid residues from about 1 to about 16 in SEQ ID NO:33) and are represented by the underlined amino acid regions; and amino acids from about 496 to about 512 constitute the predicted transmembrane domain (amino acid residues from about 496 to about 512 in SEQ ID NO:33) and are represented by the double-underlined amino acid regions.
  • Figure 14 shows the regions of similarity between the amino acid sequences of the CD33-like protein SEQ ID NO:33, and the CD33L1 protein (gi
  • Figure 15 shows an analysis of the amino acid sequence of SEQ ID NO:33.
  • Alpha, beta, turn and coil regions hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • Northern analysis indicates that this gene is expressed highest in spleen tissue and peripheral blood leukocytes, and to a lesser extent in ovary and lung tissue.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 13A-C (SEQ ID NO:33), which was determined by sequencing a cloned cDNA (HDPCL05).
  • the nucleotide sequence shown in Figures 13A-C (SEQ ID NO: 15) was obtained by sequencing a cloned cDNA (HDPCL05), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 15 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 15.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 15.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • Representative examples of polynucleotide fragments of the invention include. for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150.
  • polynucleotides of the invention encode functional attnbutes of the corresponding piotein
  • Preferred embodiments of the invention in this regaid include ftagments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming iegions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-iegions”), hydrophilic regions, hydrophobic regions, alpha amphipathic iegions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index iegions
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming iegions turn and turn-forming regions
  • coil and coil-forming regions coil and coil-forming regions
  • hydrophilic regions hydrophobic regions
  • alpha amphipathic iegions alpha amphipathic iegions
  • beta amphipathic regions flexible regions
  • the tabular format of the data in Figure 15 is used to easily determine specific boundaries of a preferred region
  • the above-mentioned preferred regions set out in Figure 15 and m Table V include, but are not limited to, regions of the aforementioned types identified by analysis of the ammo acid sequence set out in Figures 13A-C As set out in Figure 15 and in Table V, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic iegions and Hopp-Woods hydiophobic iegions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible iegions, Jameson-Wolf iegions of high antigenic index and Emini surface-forming iegions Even if deletion of one oi more ammo acids fiom the
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 13A-C, up to the alanme residue at position number 634 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the ammo acid sequence of residues nl-639 of Figures 13A-C, where nl is an integer from 2 to 634 corresponding to the position of the amino acid residue in Figures 13A-C (which is identical to the sequence shown as SEQ ID NO 33)
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:33 include polypeptides comprising the amino acid sequence of residues: L-2 to Q-639; L-3 to Q- 639; P-4 to Q-639;L-5 to Q-639; L-6 to Q-639; L-7 to Q-639; S-8 to Q-639; S-9 toQ- 639; L-10 to Q-639; L-l 1 to Q-639; G-12 to Q-639; G-13 toQ-639; S-14 to Q-639; Q-15 to Q-639; A-16 to Q-639; M-17 toQ-639; D-18 to Q-639; G-19 to Q-639; R-20 to Q-639; F-21
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 13A-C, up to the leucine residue at position number 7, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 13A-C, where ml is an integer from 7 to 638 co ⁇ esponding to the position of the amino acid residue in Figures 13A-C
  • M-1 to K-518 M-1 to P-517; M-1 to L-516; M-1 toI-515; M-1 to K-514; M-1 to M-513;
  • M-1 to N-446 M-1 to A-445; M-1 to W-444;M-1 to P-443; M-1 to G-442; M-1 to
  • A-441 M-1 to S-440; M-1 toS-439; M-1 to P-438; M-1 to T-437; M-1 to V-436; M-1 to E-435;M-1 to F-434; M-1 to S-433; M-1 to D-432; M-1 to Q-431; M-1 toS-430; M-1 to
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 15 which have been determined from the following related cDNA genes: HTOFA26R (SEQ ID NO:93), HWAEM43R (SEQ ID NO:94), HDPMQ69R (SEQ ID NO:95), HDPGA09RA (SEQ ID NO:96), HEOMH10R (SEQ ID NO:97), and HFKCT73F (SEQ ID NO:98).
  • the polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 496 - 512 of the amino acid sequence referenced in Table XIII for this gene. Moreover, a cytoplasmic tail encompassing amino acids 513 to 639 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the immune system, in particular the immunodiagnosis of acute leukemias.
  • polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s).
  • tissue or cell types e.g., immune, cancerous and wounded tissues
  • bodily fluids e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid
  • another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
  • Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 33 as residues: Pro-46 to Gly-52, Asn-76 to Val-82, Ser-85 to Phe-90, Gly-94 to Asn-100, Gln-1 11 to Tyr-116, Pro-146 to Leu-155, Ser-188 to Asn-202, Ser-240 to Arg-246.
  • CD33 monoclonal antibodies are important in the immunodiagnosis of AML.
  • CD33 MoABs have been used in preliminary therapeutic trials for purging bone marrow of AML patients, either before transplantation or for diseases resistant to chemotherapy.
  • a method is necessary for purging leukemia cells from the autografts of patients with advanced AML.
  • this method is provided by which bone marrow from an AML patient is obtained by, for example, percutaneous aspirations from the posterior iliac crest, isolating bone marrow mononuclear by Ficoll-hypaque density gradient centrifugation, and incubating with an anti-CD33-like protein MoAB, for example, 3-5 times for 15-30 min. at 4-6 degrees C, followed by incubation with rabbit complement at about 37 degrees C for 30 minutes.
  • the patient is then subject to myeloablative chemotherapy, followed by reinfusion of the treated autologous bone marrow according to standard techniques.
  • myeloablative chemotherapy followed by reinfusion of the treated autologous bone marrow according to standard techniques.
  • clonogenic tumor cells are depleted from the bone marrow while sparing hematopoietic cells necessary for engraftment.
  • the invention provides an in vivo method for selectively killing or inhibiting growth of tumor cells expressing CD33-like protein antigen of the present invention.
  • the method involves administering to the patient an effective amount of an antagonist to inhibit the CD33-like protein receptor signaling pathway.
  • administering such antagonist of the CD33-like protein to a patient may also be useful for treating inflammatory diseases including arthritis and colitis.
  • Antagonists for use in the present invention include polyclonal and monoclonal antibodies raised aginst the CD33-like protein or a fragment thereof, antisense molecules which control gene expression through antisense DNA or RNA or through triple-helix formation, proteins or other compounds which bind the CD33-like protein domains, or soluble forms of the CD33-like protein, such as protein fragments including the extracellular region from the full length receptor, which antagonize CD33-like protein mediated signaling by competing with the cell surface CD33-like protein for binding to CD33 receptor ligands.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 15 Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • a-b preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2281 of SEQ ID NOJ5, b is an integer of 15 to 2295, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a + 14.
  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides.
  • the polypeptide of the present invention has been putatively identified as a CD33 homolog derived from a human primary dendritic cells cDNA library. More particularly, the polypeptide of the present invention has been putatively identified as a human siglec homolog, sometimes hereafter referred to as "CD33-like 3" and/or "siglec 7".
  • the invention also relates to inhibiting the action of such polypeptides.
  • siglecs sialic acid binding Ig-like lectins
  • Ig superfamily a distinct subset of the Ig superfamily, characterised by their sequence similarities and abilities to bind sialic acids in glycoproteins and glycolipid (Crocker, P.R., et al., Glycobiology:8 (1998)).
  • Members of the Ig Superfamily of proteins are defined as molecules that share domains of sequence similarity with the variable or constant domains of antibodies.
  • Ig superfamily proteins consist of multiple tandem Ig-like domains connected to other domains, such as Fn-III repeat domains (Vaughn, D.E., and P.J. Bjorkman, Neuron, 16:261-73 (1996)).
  • Fn-III repeat domains Vaughn, D.E., and P.J. Bjorkman, Neuron, 16:261-73 (1996).
  • traditional Ig-like domains can be identified by the presence of two cysteine residues separated by approximately 55-75 amino acid residues, and an "invariant" tryptophan residue located 10-15 residues C-terminal to the first of the two conserved cysteine residues. The two conserved cysteine residues are thought to be involved in disulfide bonding to form the folded Ig structures (Vaughn, D.E., (1996)).
  • Ig-like domains further share a common folding pattern, that of a sandwich or fold structure of two b-sheets consisting of antiparallel b-strands containing 5-10 amino acids (Huang, Z., et al., Biopolymers, 43:367-82 (1997)).
  • Ig-hke domains are divided, based upon sequence and structural similarities, into four classifications known as Cl, C2, 1 and V-hke domains.
  • Ig-like domains The functional determinants of the Ig-like domains are presented on the faces of b-sheets or the loop regions of the Ig-fold Accordingly, protem-protem interactions can occur either between the faces of the b-sheets, or the loop regions of the Ig-fold (Huang, Z., ( 1998)). These Ig-like domains are involved in mediating a diversity of biological functions such as mtermolecular binding and protem-protem homophihc oi heterophihc interactions. Thus, Ig-like domains play an integial role in facilitating the activities of proteins of the Ig superfamily.
  • the group curcently comprises s ⁇ aloadhes ⁇ n/s ⁇ glec-1, CD22/s ⁇ glec-2, CD33/s ⁇ glec-3, myehn associated glycoprotein (MAG/s ⁇ glec-4), siglecs- 5, -6 and -7 (Crocker, P.R., et al., EMBO J., 13.4490-503 (1994); Sgroi, D., et al., J Biol.
  • Each of these proteins has an extracellular region made up of a membrane distal V-set domain followed by varying numbers of C2 set domains which range from 16 in sialoadhesin to 1 in CD33.
  • the sialic acid binding site has been mapped to the V-set domain and for sialoadhesin it has been further characte ⁇ sed at the molecular level by X-ray crystallography 11 (Nath, D., et al., J Biol. Chem., 270:26184-91 (1995); van der Merwe, P.A., et al., J. Biol.
  • CD22 is present only on mature B cells
  • sialoadhesin is on macrophage subsets
  • CD33 is a marker of early committed myeloid progenitor cells
  • siglec-5 is expressed by monocytes and mature neutrophils
  • siglec-6 is on B cells
  • siglec-7 is expressed by NK cells and monocytes (Dorken, B., et al., J. Immunology, 136:4470-79 (1986); Crocker, P.R., et al., J. Exp. Med., 164:1862- 75 (1986); Peiper, S.C., et al., In Leukocyte Typing IV. Oxford University Press, Oxford.
  • Proposed functions include cell-cell interactions through recognition of sialylated glycoconjugates on other cells.
  • cell-cell adhesion mediated by siglecs can be modulated by cis-interactions with sialic acids present in the host plasma membrane.
  • the cytoplasmic tails of CD33 and siglecs-5, -6 and -7 have two well- conserved tyrosine-based motifs that are similar to well-characterised signaling motifs in other leukocyte receptors (Gergely, J., et al., Immun. Lett., 68:3-15 (1999)).
  • both tyrosine residues can be phosphorylated by src-like kinase(s) and, in the case of the membrane proximal tyrosine, this leads to subsequent recruitment of the tyrosine phosphatases, SHP-1 and SHP-2 (Falco, M., et al., J. Exp.
  • siglec proteins Although structurally related, such proteins may possess diverse and multifaceted functions in a variety of cell and tissue types.
  • inventive purified siglec proteins are research tools useful for the identification, characterization and purification of cell signaling molecules.
  • siglecs permits the development of a range of derivatives, agonists and antagonists at the nucleic acid and protein levels which in turn have applications in the treatment and diagnosis of a range of conditions such as cancer, inflammation, neurological disorders and immunological disorders, amongst many other conditions.
  • the polypeptide of the present invention has been putatively identified as a member of the siglec family and has been termed CD33-like 3. This identification has been made as a result of amino acid sequence homology to the human cd3311 (See Genbank Accession No. gi
  • Figures 16A-B show the nucleotide (SEQ ID NO: 16) and deduced amino acid sequence (SEQ ID NO:34) of CD33-like 3.
  • Predicted amino acids from about 1 to about 18 constitute the predicted signal peptide (amino acid residues from about 1 to about 18 in SEQ ID NO:34) and are represented by the underlined amino acid regions; and amino acids from about 360 to about 376 constitute the predicted transmembrane domain
  • FIG. 17 shows the regions of similarity between the amino acid sequences of the CD33-like 3 protein (SEQ ID NO:34) and the human CD33L1 protein (SEQ ID NO:99).
  • Figure 18 shows an analysis of the CD33-like 3 amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • a polynucleotide encoding a polypeptide of the present invention is obtained from human NK cells, T-cells, primary dendritic cells, placenta, spleen, primary breast cancer, gall bladder, apoptotic t-cells, macrophage. and chronic lymphocytic leukemia spleen.
  • the polynucleotide of this invention was discovered in a human primary dendritic cell cDNA library.
  • CD33-like 3 has a transmembrane domain (the transmembrane domains comprise amino acids from about 360 to about 376 of SEQ ID NO:34; which correspond to amino acids from about 360 to about 376 of Figures 16A-B ).
  • the polynucleotide contains an open reading frame encoding the CD33-like 3 polypeptide of 467 amino acids.
  • CD33-like 3 exhibits a high degree of homology at the amino acid level to the human CD33L1 (as shown in Figure 18).
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CD33-like 3 polypeptide having the amino acid sequence shown in Figures 16A-B (SEQ ID NO:34).
  • the nucleotide sequence shown in Figures 16A-B (SEQ ID NO: 16) was obtained by sequencing a cloned cDNA (HDPUW68), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 16 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 16.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 16.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • CD33-like 3 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide compnsing or alternatively, consisting of, the transmembrane domain (amino acid residues from about 360 to about 376 in Figures 16A-B (ammo acids from about 360 to about 376 in SEQ ID NO:34).
  • the polynucleotides of the invention encode functional att ⁇ butes of CD33-hke 3
  • Preferred embodiments of the invention in this regaid include fiagments that compnse alpha-helix and alpha-helix forming iegions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming iegions ("coil-iegions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of CD33-hke 3
  • the data presented in columns VIII, IX, XIII, and XIV of Table VI can be used to determine regions of CD33-l ⁇ ke 3 which exhibit a high degree of potential for antigenic
  • the above-mentioned preferred regions set out in Figure 18 and Table VI include, but aie not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 16A-B As set out in Figure 18 and in Table VI, such preferred iegions include Gargori-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-iegions, Kyte-Doolittle hydrophilic iegions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic iegions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emmi surface-forming regions Even if deletion of one or more amino acids from the N-termmus of a protein results m modification of loss of one or more biological functions of the protein, othei functional activities (e.g., biological activities, ability to
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the CD33-hke 3 amino acid sequence shown in Figures 16A-B , up to the glutamic acid residue at position number 462 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues nl-467 of Figures 16A-B , where nl is an integer from 2 to 462 corresponding to the position of the amino acid residue in Figures 16A-B (which is identical to the sequence shown as SEQ ID NO:34).
  • N-terminal deletions of the CD33-like 3 polypeptide can be described by the general formula n2-467, where n2 is a number from 2 to 462, co ⁇ 'esponding to the position of amino acid identified in Figures 16A-B .
  • N-terminal deletions of the CD33-like 3 polypeptide of the invention shown as SEQ ID NO:34 include polypeptides comprising the amino acid sequence of residues: L-2 to K-467; L-3 to K-467; L-4 to K-467; L-5 to K-467; L-6 to K-467; L-7 to K-467; P-8 to K-467; L-9 to K-467; L-10 toK-467; W-l 1 to K-467; G-l 2 to K-467; R-l 3 to K-467; E-l 4 to K-467; R-15 to K-467; V-16 to K-467; E-17 to K-467; G-18 to K-467;Q-19 to K-467; K-20 to K-467; S-21 to K-467; N-22 to K-467; R-23 to K-467; K-24 to K-467; D
  • CD33-like 3 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six CD33-like 3 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the CD33-like 3 polypeptide shown in Figures 16A-B , up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1. where ml is an integer from 6 to 467 corresponding to the position of the amino acid residue in Figures 16A-B .
  • polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the CD33- like 3 polypeptide of the invention shown as SEQ ID NO:34 include polypeptides comprising the amino acid sequence of residues: M-1 to P-466; M-1 to 1-465; M-1 to K- 464; M-1 to 1-463; M-1 to E-462; M-1 to S-461; M-1 to Y-460; M-1 toE-459; M-1 to N- 458; M-1 to N-457; M-1 to T-456; M-1 to A-455; M-1 to E-454; M-1 to Q-453; M-1 to G-452; M-1 to S-451; M-1 toL-450; M-1 to D-449; M-1 to Q-448; M-1 to P-447; M-1 to E-446; M-1 to G-445; M-1 to K-444; M-1 to H-443; M-1 to F-442;
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 16 which have been determined from the following related cDNA genes: HGBAY02R (SEQ ID NO: 100) and HLYBY62R (SEQ ID NOJ01). Based on the sequence similarity to the human CD33L1, translation product of this gene is expected to share at least some biological activities with CD33 proteins, and specifically myeloid modulatory proteins and/or siglec proteins. Such activities are known in the art, some of which are described elsewhere herein.
  • polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response.
  • Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders.
  • the full- length protein should be a secreted protein, based upon homology to the CD33 family. Therefore, it is secreted into serum, u ⁇ ne, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection.
  • expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancel
  • Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems
  • Polynucleotides and polypeptides of the invention may also be involved m the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.).
  • inflammatory conditions e.g., inflammatory bowel syndrome, diverticulitis, etc.
  • the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers This gene is expressed predominantly on NK cells, and to a lesser extent on T- cells. Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the t ⁇ ssue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, immune disorders and cancer, as well as the immunodiagnosis of acute leukemias.
  • polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the t ⁇ ssue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g. immune, cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 34 as residues: Gly-12 to Tyr-26, Val-52 to Asp-59, Gln-88 to Asp-93, Arg-124 to Asn-129, His-193 to Arg-198, Gln-207 to Thr-213, Gln-338 to Arg- 346, Ser-378 to Ala-384.
  • Polynucleotides encoding said polypeptides are also provided.
  • NK cells are bone-marrow derived granular lymphocytes that play an important role in natural immunity to infectious diseases and have the capacity to kill certain virally-infected cells and tumor cells that have down-regulated MHC Class-I antigen expression.
  • the killing and proinflammatory activities of NK cells are regulated through a variety of cell surface receptors that can mediate either activity or inhibitory signals.
  • the best understood receptors are those that recognize MHC Class I molecules at the cell surface and deliver a negative signal, thereby protecting normal host cells from cytotoxicity.
  • KIRs killer cell Ig-like receptors
  • Representative uses are described in the "Immune Activity” and "infectious disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes indicates a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • lymphoid ongm indicates the natural gene product is involved in immune functions. Therefore it would also be useful as an agent for immunological disorders including arthntis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophi a, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-veisus-host diseases, or autoimmunity disordeis, such as autoimmune infertility, lense tissue injury, demyehnation.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophi a, psoriasis, hypers
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of va ⁇ ous blood lineages, and in the differentiation and/or proliferation of va ⁇ ous cell types
  • antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comp ⁇ ses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, addition to its use as a nut ⁇ tional supplement Protein, as well as, antibodies directed against the protein may show utility as a tumor markei and/or immunotheiapy targets for the above listed tissues
  • polynucleotide sequences such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 16 and may have been publicly available p ⁇ or to conception of the present invention Pieferably, such related polynucleotides aie specifically excluded fiom the scope of the present invention To list eveiy l elated sequence is cumbersome Accordingly, preferably excluded from the piesent invention are one or more polynucleotides comprising a nucleotide sequence described by the geneial formula of a- b, where a is any integer between 1 to 1734 of SEQ ID NO 16, b is an integer of 15 to 1748, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 16, and where b is greater than or equal to a + 14
  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides
  • the polypeptide of the present invention has been putatively identified as a human integ ⁇ n alpha 11 homolog derived from a human osteoblast II cDNA library More particularly, the polypeptide of the present invention has been putatively identified as a human integ ⁇ n alpha 11-subun ⁇ t homolog, sometimes hereafter referred to as "integnn alpha 11", "integrin alpha 11- subunit", “al l”, “Al l-subunit", and/or "Integ ⁇ n al l-subunit".
  • the invention also relates to inhibiting the action of such polypeptides.
  • the integnns are a large family of cell adhesion molecules consisting of noncovalently associated ab heterodimers.
  • a novel human tegnn a -subunit cDNA designated al 1
  • the al 1 cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellulai domain that contains an I-domain of 207 lesidues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 ammo acids
  • the deduced al l piotein shows the typical structuial features of tegnn a-subunits and is similai to a distinct gioup of a-subunits fiom collagen-b dmg integnns However, it differs from most integrin a-chams by an incompletetely preserved cytoplasmic GFFKR motif
  • the human ITGA11 gene was located to bands q22 3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase al l mRNA was also detectable in ovary and small intestine.
  • All vertebrate cells express members of the integ ⁇ n family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation ⁇ Hynes, 92; Springer, 92 ⁇ .
  • Integrins are structurally homologous heterodime ⁇ c type-I membrane glycoproteins formed by the noncovalent association of one of eight b -subunits with one of the 17 different a-subunits desc ⁇ bed to date, resulting in at least 22 different ab complexes
  • Their binding specificities for cellular and extracellular hgands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell ⁇ Diamond and Sp ⁇ nger, 94 ⁇
  • the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a b -propeller domain ⁇ Corbi et al., 1987; Springer, 1997 ⁇ .
  • the three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association ⁇ Diamond and Springer, 94 ⁇ .
  • the al, a2, a 10, aD, aE, aL, aM and aX-subunits contain an approximately 200 amino acid I- domain inserted between the second and third repeat that is not present in other a-chains ⁇ Larson et al., 1989 ⁇ .
  • I-domains have been shown to independently bind the ligands of the parent integrin heterodimer ⁇ Kamata and Takada, 1994; Randi and Hogg, 1994 ⁇ .
  • the a3, a5-8, allb and aV-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the a4-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated ⁇ Hemler et al., 90 ⁇ .
  • Additional a-subunit variants are generated by alternative splicing of primary transcripts ⁇ Ziober et al., 93; Delwel et al., 95; Leung et al., 98 ⁇ .
  • the extracellular domains of a-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif ⁇ Sastry and Horwitz, 1993 ⁇ .
  • the cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states ⁇ Williams et al., 1994 ⁇ .
  • polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed integrin alpha 11 subunit ("al 1 "). This identification has been made as a result of amino acid sequence homology to the human integrin alpha 1 subunit (See Genbank Accession No. gi
  • Figures 19A-F show the nucleotide (SEQ ID NO: 17) and deduced amino acid sequence (SEQ ID NO:35) of al 1.
  • Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:35) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or ammo acids from about 1145 to about 1161 in SEQ ID NO:35) and are represented by the double underlined ammo acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:35) and are represented by the bold amino acids
  • Figure 20 shows the regions of similarity between the ammo acid sequences of the mtegnn alpha 11 subunit (al l) protein (SEQ ID NO:35) and the human integrin alpha 1 subunit (SEQ ID NO: 103)
  • Figure 21 shows an analysis of the integrin alpha 1 1 subunit (al l) ammo acid sequence Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity, amphipathic regions; flexible regions; antigenic index and surface probability are shown
  • a polynucleotide encoding a polypeptide of the present invention is obtained from human ovary ,small intestine, fetal heart, fetal brain, large intestine, osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow tissues and cells.
  • the polynucleotide of this invention was discovered in a human osteoblast II cDNA library Its translation product has homology to the characteristic immunoglobulin and major histocompatibility complex protein domain of integrin family members.
  • al 1 has transmembrane domains (the transmembrane domains comprise amino acids 666 - 682 and/or 1145 - 1161 of SEQ ID NO:35; which correspond to amino acids 666 - 682 and/or 1145 - 1161 of Figures 19A-F) with strong conservation between other members of the integ ⁇ n family.
  • the polynucleotide contains an open reading frame encoding the al 1 polypeptide of 1189 amino acids.
  • the present invention exhibits a high degree of homology at the ammo acid level to the human integ ⁇ n alpha 1 subunit (as shown in Figure 20)
  • Preferred polypeptides of the invention comp ⁇ se the following amino acid sequence: TNGYQKTGDVYKCPVIHGNCTKLNLGRVTLSNV (SEQ ID NO: 102). Polynucleotides encoding these polypeptides are also provided.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the al 1 polypeptide having the ammo acid sequence shown m Figures 19A-F (SEQ ID NO:35)
  • the nucleotide sequence shown in Figures 19A-F (SEQ ID NO 35) was obtained by sequencing a cloned cDNA (HOHBY69), which was deposited on Novembei 17 at the American Type Culture Collection, and given Accession Numbei 203484
  • the present invention is further directed to fragments of the isolated nucleic acid molecules descnbed herein
  • a fiagment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 17 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and p ⁇ mers as discussed herein.
  • fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown SEQ ID NO 17.
  • a fragment at least 20 nt m length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 17.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • al 1 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, any one of the transmembrane domains (amino acid residues from about 666 to about 682 and/or 1145 to about 1161 in Figures 19A-F (amino acids from about 666 to about 682 and/or 1145 to about 1161 in SEQ ID NO:35), in addition to the immunoglobulin and major histocompatibility complex protein domain (amino acid residues from about 64 to about 96 in Figures 19A-F (amino acids from about 64 to about 96 in SEQ ID NO:35).
  • a polypeptide comprising or alternatively, consisting of, any one of the transmembrane domains (amino acid residues from about 666 to about 682 and/or 1145 to about 1161 in Figures 19A-F (amino acids from about 666 to about 682 and/or 1145 to about
  • polynucleotides of the invention encode functional attributes of al l.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of the present invention.
  • the data presented in columns VIII, IX, XIII, and XIV of Table VII can be used to determine iegions of al 1 which exhibit a high degiee of potential for antigenicity Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/oi XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occui in the process of initiation of an immune response
  • Figuie 21 (set on the onginal default parameters) was used to present the data in Figure 21 in a tabular format (See Table VII).
  • the tabular format of the data in Figure 21 is used to easily determine specific bounda ⁇ es of a preferred region
  • the above-mentioned preferred regions set out in Figure 21 and in Table VII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 19A-F.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multime ⁇ ze, etc.) may still be retained.
  • other functional activities e.g., biological activities, ability to multime ⁇ ze, etc.
  • the ability of shortened al 1 mutems to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majonty of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • the present invention further provides polypeptides having one oi more residues deleted from the amino terminus of the al 1 ammo acid sequence shown in Figures 19A-F, up to the threonine residue at position number 1184 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides compnsmg the ammo acid sequence of residues n 1-1189 of Figures 19A-F, where nl is an integer from 2 to 1184 corresponding to the position of the amino acid residue in Figures 19A-F (which is identical to the sequence shown as SEQ ID NO:35).
  • N-terminal deletions of the al l polypeptide can be desc ⁇ bed by the general formula n2-l 189, where n2 is a number from 2 to 1184, corresponding to the position of amino acid identified in Figure 19.
  • N-termmal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides comp ⁇ sing the amino acid sequence of residues: N-terminal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides comp ⁇ smg the amino acid sequence of residues: D-2 to E-l 189; L-3 to E-l 189; P-4 to E-l 189; R-5 toE-1189; G-6 to E-l 189; L-7 to fil l 89; V-8 to E-l 189; V-9 to E-l 189;A- 10 to E-l 189; W-l l to E-l 189; A- 12 to E-l 189; L-13 to E-1189;S-14 to E-1189; L-15 to E-1189; W-16 to E-1189; P-17 to E-1 189;G-18 to E-1189; F-19 to E-l 189; T-20 to E-l 189; D-21 to E-l 189
  • deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein
  • other functional activities e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained.
  • biological activities e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.
  • the ability of the shortened al 1 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the al 1 polypeptide shown in Figures 19A-F, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 19A-F, where ml is an integer from 6 to 1189 corresponding to the position of the amino acid residue in Figures 19A-F.
  • polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides comprising the amino acid sequence of residues: M-1 to L-l 188; M-1 to V-l 187; M-1 to K-1186;M-1 to P-l 185; M-1 to T-l 184; M-1 to P-l 183; M-1 to D-1182; M-l to L-1181; M-l to G-1180; M-l toP-1179; M-l to E-1178; M-l to R-l 177; M-1 to R-l 176; M-1 to R-l 175; M-1 to R-l 174; M-1 to A-l 173; M-lto S-l 172; M-1 to R-l 171; M-1 to F-1170; M-1 to F-1169; M-1 to G-l 168; M-1
  • M-1 to K-282 M-1 to E-281; M-1 to L-280; M-1 to D-279; M-1 to P-278; M-1 to S- 277; M-1 to D-276; M-1 toH-275; M-1 to S-274; M-1 to E-273; M-1 to G-272; M-1 to D-271; M-1 to T-270; M-1 to 1-269; M-1 to V-268;M-1 to 1-267; M-1 to M-266; M-1 to V-265; M-1 to K-264; M-1 to K-263; M-1 to A-262; M-1 to G-261; M-lto K-260; M-1 to R-259; M-1 to G-258; M-1 to G-257; M-1 to K-256; M-1 to Q-255; M-1 to F-254; M- 1 toA-253; M-1 to E-252; M-1 to S-251; M-1 to R-250; M-1 to A-249; M-1 to F-248; M- 1 to
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NOJ 7 which have been determined from the following related cDNA genes: HEEAB54R (SEQ ID NOJ04), HRDAF83R (SEQ ID NO: 105), HOUBC62R (SEQ ID NO: 106), HCDBI19R (SEQ ID NO: 107), HOHCU94R (SEQ ID NOJ08), HOACC13R (SEQ ID NO: 109), HCDAP21R (SEQ ID NO: 110), HNHHA34R (SEQ ID NO: 11 1), HOHEA75R (SEQ ID NO: 112) and HNGEL59R (SEQ ID NOJ 13).
  • polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response.
  • Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders.
  • the full- length protein should be a secreted protein, based upon homology to the integrin family.
  • polypeptides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types.
  • the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticuhtis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarc oma cells, and other cancers.
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions.
  • Representative uses are desc ⁇ bed in the "Hyperpro ferative Disorders" and "Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
  • Dysregulation of apoptosis can result in mapprop ⁇ ate suppression of cell death, as occurs in the development of some cancers, or m failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification.
  • the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein can also be used to gain new insight into the regulation of cellular growth and proliferation.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement.
  • Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. This gene is expressed almost exclusively in osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, and to a lesser extent in osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the skeletal system, connective tissues, and immune and hematpoietic diseases and/or disorders.
  • polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g.
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 35 as residues: Phe-23 to Arg-31, Leu-62 to Asp-72, Val-96 to Asp-101, Thr-111 to Asn-116, Glu-128 to Thr-135, Val-142 to Ser-149, Asn-217 to Val- 222, GIu-233 to Arg-241, Gly-272 to Leu-280, Gln-286 to Thr-293, Tyr-303 to Ue-308, Gly-354 to Thr-360, Glu-408 to Lys-419, Glu-508 to Lys-514, Arg-521 to Val-526, Gly- 529 to Phe-542, Asp-551 to Tyr-557, Thr-587 to Thr-593, His-656 to Asp-665, Met-697 to Arg-705, Asp-709 to Thr-716, Glu-755 to Gly-760, Asn-779 to His-786, Leu-810
  • the tissue distribution in osteoblasts and homology to integrin alpha subunit 10 indicates that the protein products of this gene are useful for the treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis and treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spmal deformation, and specific joint abnormalities as well as chondrodysplasias (ie.
  • connective tissues e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation
  • various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spmal deformation, and specific joint abnormalities as well
  • polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukope a, thrombocytopenia oi leukemia since stromal cells aie important in the production of cells of hematopoietic lineages
  • hematopoietic related disorders such as anemia, pancytopenia, leukope a, thrombocytopenia oi leukemia since stromal cells aie important in the production of cells of hematopoietic lineages
  • tissue distnbution m bone marrow cells Integnns play pivotal roles in cell migration, inflammation, proliferation, and cellulai mfiltiation
  • the present invention is expected to share at least some of these activities Representative uses are desc ⁇ bed in the "Immune Activity" and "infectious disease" sections below, in Example 1
  • this gene product may have commercial utility in the expansion of stem cells and committed progenitors of vanous blood lineages, and in the differentiation and or proliferation of va ⁇ ous cell types.
  • antagonists directed against this protein is useful blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comp ⁇ ses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4981 of SEQ ID NO: 17, b is an integer of 15 to 4995, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a + 14.
  • the present invention relates to three novel peptidoglycan recognition binding proteins expressed by keratinocytes, wound-healing tissues and chondrosarcoma tissue. More specifically, isolated nucleic acid molecules are provided encoding a human peptidoglycan recognition protein-related protein, sometimes referred to herein as "human tag7" or “tag7” or “htag7". Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of activities of the tag7 protein, polypeptides and diagnostic methods for detecting tag7 gene expression.
  • Peptidoglycan as well as Lipopolysacchande (LPS) is a surface component of many bactena which illicit a wide range of physiological and immune responses in humans.
  • peptidoglycan has been shown to manifest itself clinically by reproducing most of the symptoms of bacterial infection, including fevei , acute-phase response, inflammation, septic shock, leukocytosis, sleepiness, malaise, abcess formation, and arthritis (see Dziarski et al., JBC, 273 (15): 8680 (1998)).
  • peptidoglycan i.e.- the specific stereoisomers or analogs of muramyl dipeptide, N-acetylglucosammyl-beta(l-4)-N-acteylmuramyl tetrapeptides, etc.
  • peptidoglycan i.e.- the specific stereoisomers or analogs of muramyl dipeptide, N-acetylglucosammyl-beta(l-4)-N-acteylmuramyl tetrapeptides, etc.
  • lipopolysacchande binding protein exists that was discovered as a trace plasma protein (See Schumann et al., Science, 249(4975): 1429 (1990)). It is thought that one of the modes of action by which this lipopolysacchande binding protein functions is by forming high-affinity complexes with lipopolysacchande, that then bind to macrophages and monocytes, inducing the secretion of tumor necrosis factor.
  • Dziarski and Gupta (See Dziarski et al., JBC, 269(3): 2100 (1994)) demonstrated that a 70kDa receptor protein present on the surface of mouse lymphocytes served to bind hepa ⁇ n, hepa ⁇ noids, bactenal poteichoic acids, peptidoglycan, and popolysaccha ⁇ des.
  • Dziarski et al. demonstrated that the CD14, a glycosylphosphatidyl ositol-linked protein present on the surface of macrophage and polymorphonuclear leukocytes, bound peptidoglycan and lipopolysacchande.
  • CD 14 for lipopolysacchande was significantly increased in the presence of a LPS-binding protein present in plasma. It is thought that the LPS-binding protein functions as a transfer molecule, whereby it binds LPS and presents it to the CD14 receptor (See Dziarski et al., JBC, 273(15): 8680 (1998)).
  • Yoshida et al. isolated a peptidoglycan binding protein from the hemolymph of the Silkworm, Bombyx mo ⁇ , using column chromatography This protein was found to have a very specific affinity for peptidoglycan (See Yoshida et al., JBC, 271(23) 13854 (1996))
  • Kang et al. recently cloned a peptidoglycan binding protein from the moth Trichoplusia ni.
  • the peptidoglycan binding protein was shown to bind strongly to insoluble peptidoglycan (See Kang etal., PNAS, 95(17). 10078 (1998)).
  • the peptidoglycan binding protein was upiegulated by a bacterial infection m T. ni.
  • the insect immune system is regarded as a model foi innate immunity.
  • Kang et al weie able to gene both mouse and human homologs of the T ni peptidoglycan binding protein.
  • peptidoglycan binding proteins shared regions of homology, as well as foui conserved cysteine residues which may function in the tertiary structure of the protein, possibly in helping to form binding domains.
  • peptidoglycan is an integral component of bactenal cell walls, and that it induces many physiological responses from cytokine secretion to inflammation and macrophage activation, it appears as if this family of proteins is a ubiquitous group involved in the binding and recognition of peptidoglycan, the presentation of antigens (e.g., cell wall components, etc.), and the activation of the immune system, such as the secretion of cytokines, such as TNF.
  • TNF is noted for its pro-mflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J.S. et al., J. Immunol. 136:1680 (1986)), increased adherence of neutrophils and lymphocytes (Pober, J.S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166: 1390 (1987)). Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A.
  • TNF is an important mediator of the cachexia in cancer, infectious pathology, and in other catabohc states. TNF is thought to play a cential role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H.R. et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p.463-466 (1989); Simpson, S. Q. et al., Cnt. Care Clin. 5:27-47 (1989)), including fevei , malaise, anorexia, and cachexia.
  • Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kombluth, S.K. et al., J. Immunol. 137:2585-2591 (1986)) and other cytokines. Because TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-denved cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H.R. et al., N. Eng. J. Med. 318:1481-1486 (1988)).
  • Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 123: 162-170 (1988)). Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al., Lancet 1 :355-357 (1987); Hammerle, A.F. et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al., Cnt. Care Med. 17:489-497 (1989); Calandra, T. et al., J.
  • Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above.
  • Antibodies to a "modulator" matenal which was characterized as cachectin (later found to be identical to TNF) were disclosed by Cerami et al. (EPO Patent Publication 0,212,489, March 4, 1987) Such antibodies were said to be useful in diagnostic immunoassays and in therapy of shock in bacterial infections.
  • Rubin et al. (EPO Patent Publication 0,218,868, April 22, 1987) disclosed monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such antibodies, and the use of such antibodies in immunoassay of TNF. Yone et al.
  • Such novel proteins could be useful in augmenting the immune system m such areas as immune recognition, antigen presentation, and immune system activation.
  • Antibodies or antagonists directed against these proteins is useful in reducing or eliminating disorders associated with TNF and TNF- ke cytokines, such as endotoxic shock and auto-immune disorders, for example.
  • polypeptide of the present invention has been putatively identified as a member of the novel peptidoglycan recognition binding protein family and has been termed human tag7. This identification has been made as a result of amino acid sequence homology to the mouse tag7 (See Genbank Accession No. emb
  • Figure 34 shows the nucleotide (SEQ ID NO: 18) and deduced ammo acid sequence (SEQ ID NO:36) of htag7.
  • Predicted amino acids from about 1 to about 21 constitute the predicted signal peptide (amino acid residues from about 1 to about 21 in SEQ ID NO 36) and are represented by the underlined amino acid regions; and amino acids from about 34 to about 117 constitute the fileicted PGRP-hke domain (ammo acids from about 34 to about 117 in SEQ ID NO:36) and are repiesented by the double underlined amino acids
  • Figuie 35 shows the regions of similarity between the amino acid sequences of the htag7 piotein (SEQ ID NO 36) and the mouse tag7 protein (SEQ ID NOJ 14)
  • Figure 36 shows an analysis of the htag7 amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown
  • a polynucleotide encoding a polypeptide of the present invention is obtained from human chondrosarcoma cells, bone marrow, and neutrophils.
  • the polynucleotide of this invention was discovered in a human chondrosarcoma cDNA library.
  • htag7 has a PGRP domain (the PGRP domain comprise amino acids from about 34 to about 117 of SEQ ID NO:36; which correspond to amino acids from about 34 to about 117 of Figure 34).
  • the polynucleotide contains an open reading frame encoding the htag7 polypeptide of 198 amino acids.
  • htag7 exhibits a high degree of homology at the amino acid level to the mouse tag7 (as shown in Figure 35).
  • the present invention provides isolated nucleic acid molecules compnsmg a polynucleotide encoding the htag7 polypeptide having the ammo acid sequence shown in Figure 34 (SEQ ID NO:36).
  • SEQ ID NO:36 The nucleotide sequence shown in Figure 34 (SEQ ID NO:36).
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 18 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 8.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 18.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • htag7 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 726, and from about 130 to about 379 of SEQ ID NO: 18, or the complementary strand thereto, or the cDNA contained in the deposited gene.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide compnsmg or alternatively, consisting of, the PGRP-hke domain (amino acid residues from about 34 to about 117 m Figure 34 (amino acids from about 34 to about 117 in SEQ ID NO:36) Since the location of these domains have been predicted by computer analysis, one of oidinaiy skill would appieciate that the ammo acid lesidues constituting these domains may vary slightly (e g , by about 1 to 15 amino acid lesidues) depending on the c ⁇ teiia used to define each domain
  • nucleic acid molecules of the present invention which encode a htag7 polypeptide may include, but aie not limited to
  • Pieferred embodiments of the invention in this regard include fragments that compnse alpha-helix and alpha-helix forming regions ("alpha-regions”), beta-sheet and beta-sheet forming regions ("beta-regions”), turn and turn-forming regions ("turn- regions”), coil and coil-formmg regions ("coil-iegions”), hydiophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of htag7.
  • the data presented in columns VIII, IX, XIII, and XIV of Table XII can be used to determine regions of htag7 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Certain preferred regions in these regards are set out in Figure 36, but may, as shown in Table XII, be represented or identified by using tabular representations of the data presented in Figure 36.
  • the DNA*STAR computer algorithm used to generate Figure 36 (set on the original default parameters) was used to present the data in Figure 36 in a tabular format (See Table XII).
  • the tabular format of the data in Figure 36 is used to easily determine specific boundaries of a prefen-ed region.
  • the above-mentioned preferred regions set out in Figure 36 and in Table XII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 34.
  • such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the htag7 ammo acid sequence shown in Figure 34, up to the proline residue at position number 191 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides compnsmg the amino acid sequence of residues nl- 196 of Figure 34 , where nl is an integer from 2 to 191 co ⁇ espond ⁇ ng to the position of the amino acid residue in Figure 34 (which is identical to the sequence shown as SEQ ID NO:36).
  • N-terminal deletions of the htag7 polypeptide can be descnbed by the general formula n2-196, where n2 is a number from 2 to 191, corresponding to the position of ammo acid identified in Figure 34.
  • N-terminal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the amino acid sequence of residues:
  • N-termmal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the ammo acid sequence of residues: S-2 to P-196; R-3 to P-196; R-4 to P-196; S-5 to P-196; M-6 to P-196; L-7 to P-196; L-8 to P- 196; A-9 to P-196; W-10 to P-196, A-U to P-196; L-12 to P-196; P-13 to P-196; S-14 to P-196; L-15 to P-196; L-16 to P-196; R
  • C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities ) may still be retained.
  • other functional activities e.g., biological activities
  • the ability of the shortened htag7 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majo ⁇ ty of the residues of the complete or mature polypeptide are removed from the C-termmus.
  • the present invention furthei provides polypeptides having one oi more residues deleted from the carboxy terminus of the amino acid sequence of the htag7 polypeptide shown in Figure 34 , up to the methionine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 196 corresponding to the position of the ammo acid residue in Figure 34 .
  • polypeptides comprising, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the amino acid sequence of residues: M-1 to S-195; M-1 to R- 194; M-1 to Y-193; M-1 to H-192; M-1 to P-191; M-1 to W-190; M-1 to N-189; M-1 to Q-188; M-1 to 1-187; M-1 to L-186; M-1 to HI 85; M-1 to Y-184; M-1 to L-183; M-1 to Q-182; M-1 to N-181; M-1 to G-180; M-1 to P-179; M-1 to S-178; M-1 to L-177; M-1 to T-176; M-1 to R-175; M-1 to Q-174; M-1 to V-173; M-1 to D-172; M-1 to R-171;
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:36 which have been determined from the following related cDNA genes: HBMTB79R (SEQ ID NOJ 15) and HCDDP40R (SEQ ID NOJ 16).
  • polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response.
  • Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders.
  • the full-length protein should be a secreted protein, based upon homology to the tag7 protein.
  • this protein is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer.
  • Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.).
  • inflammatory conditions e.g., inflammatory bowel syndrome, diverticulitis, etc.
  • the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers.
  • the translation product of this gene shares sequence homology with Tag7, which is a mouse cytokine that, m soluble form, triggers apoptosis in mouse L929 cells in vitro
  • the translation product of this gene also shaies sequence homology with antimicrobial BGP-A, a bovine antimiciobial peptide from bovine neutrophils.
  • Preferred polypeptides of this invention comprise residues 184 to 196 shown in SEQ ID NO: 36 This polypeptide is believed to be the active mature form of the translation product of
  • This gene is expressed primarily in bone marrow and to a lesser extent in human chondrosarcoma and neutrophils.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the t ⁇ ssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, infections, cancer, and disordeis of the immune system.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the t ⁇ ssue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 36 as residues: Ala-63 to Asn-68, Ala-71 to Gln-81, Tyr-135 to Thr- 141, Leu- 167 to Gin- 174, Pro-191 to Pro- 196. Polynucleotides encoding said polypeptides are also provided.
  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides.
  • the polypeptide of the present invention has been putatively identified as a human butyrophilin homolog derived from a human testes tumor cDNA library.
  • the polypeptide of the present invention is sometimes hereafter referred to as "Butyrophlin and B7-like IgG superfamily receptor", and/or "BBIR II".
  • the invention also relates to inhibiting the action of such polypeptides.
  • Butyrophilin is a glycoprotein of the immunoglobulin superfamily that is secreted in association with the milk-fat-globule membrane from mammary epithelial cells.
  • the butyrophilin gene appears to have evolved from a subset of genes in the immunoglobulin superfamily and genes encoding the B30.2 domain, which is conserved in a family of zinc-finger proteins.
  • expression analysis of butyrophilin genes has shown that butyrophilin expression increases during lactation in conjunction with an increase in milk fat content.
  • the polypeptide of the present invention has been putatively identified as a member of the milk fat globule membrane glycoprotein family, and more particularly the butyrophilin family, and has been termed Butyrophlin and B7-like IgG superfamily receptor ("BBIR II"). This identification has been made as a result of amino acid sequence homology to the bovine butyrophilin precursor (See Genbank Accession No. gi
  • Preferred polypeptides of the invention comprise the following nucleic acid sequence: ACATCCATGGCTCTAATGCTCAGTTTGGTTCTGAGTCTCCTCAAGCTGGGATC AGGGCAGTGGCAGGTGTTTGGGCCAGACAAGCCTGTCCAGGCCTTGGTGGGG GAGGACGCAGCATTCTCCTGTTTCCTGTCTCCTAAGACCAATGCAGAGGCCA TGGAAGTGCGGTTCTTCAGGGGCCAGTTCTCTAGCGTGGTCCACCTCTACAG GGACGGGAAGGACCAGCCATTTATGCAGATGCCACAGTATCAAGGCAGGAC AAA ACTGGTGAAGGATTCTATTGCGGAGGGGCGCATCTCTCTGAGGCTGGA A AACATTACTGTGTTGGATGCTGGCCTCTATGGGTGCAGGATTAGTTCCCAGTC TTACTACCAGAAGGCCATCTGGGAGCTACAGGTGTCAGCACTGGGCTCAGTT CCTCTCATTTCCATCACGGGATATGTTGATAGAGACATCCAGCTACTCTGTCA GTCC
  • Figures 22A-D show the nucleotide (SEQ ID NO: 19) and deduced amino acid sequence (SEQ ID NO:37) of BBIR II. Predicted amino acids from about 1 to about 17 constitute the predicted signal peptide (amino acid residues from about 1 to about 17 in SEQ ID NO:37) and are represented by the underlined amino acid regions.
  • Figure 23 shows the regions of similarity between the amino acid sequences of the Butyrophlin and B7-like IgG superfamily receptor (BBIR II) protein (SEQ ID NO:37) and the bovine butyrophilin precursor (SEQ ID NO: 121)
  • Figure 24 shows an analysis of the integrin alpha 11 subunit (BBIR II) amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • a polynucleotide encoding a polypeptide of the present invention is obtained from human small intestine, colon tumor, and human testes tumor cells and tissues.
  • the polynucleotide of this invention was discovered in a human testes tumor cDNA library. Its translation product has homology to the B30.2-like domain which is characteristic of proteins containing zinc-binding B-box motifs, and particularly for butyrophilin family members.
  • the polynucleotide contains an open reading frame encoding the BBIR II polypeptide of 318 amino acids. BBIR II exhibits a high degree of homology at the amino acid level to the bovine butyrophilin precursor (as shown in Figure 23).
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the BBIR II polypeptide having the amino acid sequence shown in Figures 22A-D (SEQ ID NO: 37).
  • the nucleotide sequence shown in Figures 22A-D (SEQ ID NO: 19) was obtained by sequencing a cloned cDNA (HTTDB46), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 19 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 9.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 19.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • Representative examples of BBIR II polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200.
  • 1700 from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000. from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350, from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2500, from about 2501 to about 2550, from about 2551 to about 2600, from about 2601 to about 2650, from about 2651 to about 2700, from about 2701 to about 2750, from about 2751 to about 2800, from about 2801 to about 2850, from about 2851 to about 2900, from about 2901 to about 2950, from about 2951 to about 3000, from about 3001 to about 3050, from about 3051 to about 3059 of SEQ ID NO: 19, or
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the mature BBIR II protein (amino acid residues from about 18 to about 318 in Figures 22A-D (amino acids from about 18 to about 318 in SEQ ID NO:37). Since the location of this form of the protein has been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define this location.
  • the polynucleotides of the invention encode functional attributes of BBIR II.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of BBIR II.
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming regions turn and turn-forming regions
  • turn- regions turn and turn-forming regions
  • coil and coil-forming regions coil and coil-forming regions
  • hydrophilic regions hydrophobic regions
  • alpha amphipathic regions alpha amphipathic regions
  • beta amphipathic regions flexible regions
  • surface-forming regions and high antigenic index regions of BBIR II The data representing the structural or functional attributes of BB
  • the data presented in columns VIII, IX, XIII, and XIV of Table VIII can be used to determine regions of BBIR II which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response. Certain preferred regions in these regards are set out in Figure 24, but may. as shown in Table VIII, be represented or identified by using tabular representations of the data presented in Figure 24.
  • the DNA*STAR computer algorithm used to generate Figure 24 was used to present the data in Figure 24 in a tabular format (See Table VIII).
  • the tabular format of the data in Figure 24 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 24 and in Table VIII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 22A-D.
  • such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, etc.
  • the ability of shortened BBIR II muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the BBIR II amino acid sequence shown in Figures 22A-D, up to the cystein residue at position number 313 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 1-318 of Figures 22A-D, where nl is an integer from 2 to 313 corresponding to the position of the amino acid residue in Figures 22A-D (which is identical to the sequence shown as SEQ ID NO:37).
  • N-terminal deletions of the BBIR II polypeptide can be described by the general formula n2-318, where n2 is a number from 2 to 313, corresponding to the position of amino acid identified in Figures 22A-D.
  • N-terminal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues:
  • N-terminal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues: A-2 to T-318; L-3 to T-318; M-4 to T- 318; L-5 to T-318; S-6to T-318; L-7 to T-318; V-8 to T-318; L-9 to T-318; S-10 to T- 318; L-l 1 to T-318; L-12 to T-318; K-13 toT-318; L-14 to T-318; G-15 to T-318; S-16 to T-318; G
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results m modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained. For example the ability of the shortened BBIR II mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majo ⁇ ty of the residues of the complete or mature polypeptide aie removed from the C-termmus.
  • biological activities e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.
  • a particulai polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by loutine methods desc ⁇ bed herein and otherwise known in the art. It is not unlikely that an BBIR II mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six BBIR II amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the ammo acid sequence of the BBIR II polypeptide shown in Figures 22A-D, up to the serine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the ammo acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 318 corresponding to the position of the amino acid residue in Figures 22A-D.
  • polypeptides comprising, or alternatively consisting of, the ammo acid sequence of C-termmal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues M- 1 to P-317; M-1 to F-316; M-1 to L-315;M-1 to A-314; M-1 to C-313; M-1 to P-312; M- 1 to S-311; M-l to P-310; M-l to F-309; MJ to S-308; M-lto W-307; M-1 to P-306; M- 1 to N-305; M-1 to P-304; M-1 to G-303; M-1 to K-302; M-1 to K-301; M-1 toL-300; M-1 to T-299; M-1 to T-298; M-1 to S-297; M-1 to G-296; M-1 to G-295; M-1 to S-294;
  • M-1 to R-227 M-1 to W-226; M-1 to D-225; M-1 to G-224; M-1 toI-223; M-1 to
  • M-1 to R-162 M-1 to P-161; M-1 toF-160; M-1 to W-159; M-1 to G-158; M-1 to S-157; M-1 to S-156; M-1 to Q-155; M-1 to C-154; M-1 toL-153; M-1 to L-152; M-1 to
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 19 which have been determined from the following related cDNA genes: HTTDB46R (SEQ ID NO: 122), and HSIEA44R- (SEQ ID NO: 123). Based on the sequence simila ⁇ ty to the bovin butyrophilin precursor, translation product of this gene is expected to share at least some biological activities with B30.2- like domain containing proteins, and specifically butyrophilin proteins.
  • polynucleotides and polypeptides of the invention are also useful for modulating the diffei en nation of normal and malignant cells, modulating the proliferation and/oi differentiation of cancer and neoplastic cells, and regulation of cell giowth and differentiation
  • Polynucleotides and polypeptides of the invention may represent a diagnostic marker for breast diseases and/or disoideis, in addition to disorders of secretory organs and tissues (which include, testicular and gastrointestinal disordeis, particularly those cells which serve secretory functions for seminal fluid oi gastrointestinal hormones, and disorders of the mucosal membranes of such cells and tissues, etc.).
  • the full-length protein should be a secreted protein, based upon homology to the butyrophilin family of proteins. Therefore, it is secreted into milk, serum, unne, seminal fluid, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of breast disorders (i.e , breast cancer, breast dysfunction, etc.) this protein would provide a convenient diagnostic for eaily detection of such disorders In addition, expression of this gene product may also be linked to the progression of breast diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of breast cancer.
  • Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of secretory cells and tissues. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions. Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in cancers.
  • the invention including agonists and/or antagonists thereof, is useful in modulating the nutritional value of milk, its caloric content, its fat content, and may conceivably be useful in mediating the adaption of breast secretory function as a delivery vehicle for therapeutics (i.e., transgenic breast secretory tissue for transferring therapeutically active proteins to infants).
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the "Hyperproliferative Disorders" and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
  • Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers.
  • this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein can also be used to gain new insight into the regulation of cellular growth and proliferation.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • This gene is expressed primarily in small intestine, colon tumor, and to a lesser extent in human testes tumor cells.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal diseases and/or disorders, in addition to lactation disorders, and tumors of the testes.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 37 as residues: Tyr-67 to Pro-74, Ser-117 to Gln-123, Pro-161 to Met-185, Gly-224 to His-242, Thr-299 to Trp-307. Polynucleotides encoding said polypeptides are also provided.
  • the translation product of this gene contains a serine protease motif and accordingly is believed to possess serine protease activity. Assays for determining such activity are well known in the art. Preferred polypeptides of this invention possess such activity. Included in this invention as preferred domains are serine protease histidine active site domains, which were identified using the ProSite analysis tool (Swiss Institute of Bioinformatics). The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen- bonded to a histidine, which itself is hydrogen-bonded to a serine.
  • Consensus pattern [LIVM]-[ST]-A-[STAG]-H-C , H is the active site residue.
  • Preferred polypeptides of the invention comprise the following amino acid sequence: GTLVAEKHVLTAAHCIHDGKTYVKGTQ (SEQ ID NO: 124). Polynucleotides encoding these polypeptides are also provided.
  • polypeptides comprising the serine protease histidine active site domain of the sequence referenced in Table XIII for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence.
  • the additional contiguous amino acid residues is N-terminal or C- terminal to the serine protease histidine active site domain.
  • the additional contiguous amino acid residues is both N-terminal and C-terminal to the serine protease histidine active site domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number.
  • the above preferred polypeptide domain is characteristic of a signature specific to serine protease proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with serine proteases. Such activities are known in the art, some of which are described elsewhere herein.
  • polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention.
  • polypeptides of the invention comprise the following amino acid sequence: GTRGQ AWEPRALSRRPHLSERRSEPRPGRAARRGTVLGMAGIPGLLFLLFF
  • a preferred polypeptide variant of the invention comprises the following amino acid sequence:
  • Figures 25 A-B show the nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:38) of the present invention.
  • Predicted amino acids from about 1 to about 19 constitute the predicted signal peptide (amino acid residues from about 1 to about 19 in SEQ ID NO:38) and are represented by the underlined amino acid regions;
  • amino acids from about 162 to about 188 constitutes the predicted serine protease histidine active site domain (amino acids residues from about 162 to about 188 in SEQ ID NO:38) and are represented by the double underlined amino acid regions;
  • amino acid residue 175 (amino acid residue 175 in SEQ ID NO:38) constitutes the predicted histidine active site residue and is represented by the bold amino acid.
  • Figure 26 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:38, and the Human Pancreatic Elastase 2 protein (gi
  • Figure 27 shows an analysis of the amino acid sequence of SEQ ID NO:38.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • Northern analysis indicates that this gene is expressed highest in HUVEC, HUVEC +LPS, smooth muscle, fibroblasts, present in heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon and weakly in PBLs.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 25 A-B (SEQ ID NO:38), which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figures 25 A-B was obtained by sequencing a cloned cDNA (HUSAQ05), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:20 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:20.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:20.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251
  • polynucleotides of the invention encode functional attributes of the corresponding protein.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming regions turn and turn-forming regions
  • turn- regions turn and turn-forming regions
  • coil and coil-forming regions coil and coil-forming regions
  • the data presented in columns VIII, IX, XIII, and XIV of Table IX can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Certain preferred regions in these regards are set out in Figure 27, but may, as shown in Table IX, be represented or identified by using tabular representations of the data presented in Figure 27.
  • the DNA*STAR computer algorithm used to generate Figure 27 (set on the original default parameters) was used to present the data in Figure 27 in a tabular format (See Table IX).
  • the tabular format of the data in Figure 27 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 27 and in Table IX include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 25A-B, up to the aspartic acid residue at position number 370 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues nl-375 of Figures 25A-B, where nl is an integer from 2 to 370 corresponding to the position of the amino acid residue in Figures 25A-B (which is identical to the sequence shown as SEQ ID NO:38).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:38 include polypeptides comprising the amino acid sequence of residues: A-2 to V-375; G-3 to V-375; 1-4 to V-375; P-5 to V-375; G-6 to V-375; L-7 to V-375; L-8 to V-375; F-9 to V-375; L-10 to V-375; L-l 1 to V-375; F-12 to V-375; F-13 to V-375; L-14 to V-375; L- 15 to V-375; C-16 to V-375; A- 17 to V-375; V-l 8 to V-375; G-19 to V-375; Q-20 to V- 375; V-21 to V-375; S-22 to V-375; P-23 to V-375; Y-24 to V-375; S-25 to V-375; A-26 to V-375; P-27 to V-375; W-28 to V-375; K-29 to V-375; P-30 to V-375; T
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 25A-B, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 25A-B, where ml is an integer from 6 to 375 corresponding to the position of the amino acid residue in Figures 25A-B.
  • polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:38 include polypeptides comprising the amino acid sequence of residues: M-1 to G-374; M-1 to I- 373; M-1 to S-372; M-1 to 1-371; M-1 to D-370; M-1 to P-369; M-1 to 1-368; M-1 to Y- 367; M-1 to Q-366; M-1 to L-365; M-1 to P-364; M-1 to T-363; M-1 to 1-362; M-1 to E- 361; M-1 to S-360; M-1 to C-359; M-1 to G-358; M-1 to R-357; M-1 to T-356; M-1 to F-355; M-1 to E-354; M-1 to Q-353; M-1 to P-352; M-1 to S-351; M-1 to G-350; M-1 to D-3
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:20 which have been determined from the following related cDNA genes: HFKCF40F (SEQ ID NO.128), HSRDF26R
  • HAQBJl lR (SEQ ID NO: 132), HAFBB 1 IR (SEQ ID NO: 133), HOEFO85R (SEQ ID NO: 133), HAQBJl lR (SEQ ID NO: 132), HAFBB 1 IR (SEQ ID NO: 133), HOEFO85R (SEQ ID NO: 133), HOEFO85R (SEQ ID NO: 133).
  • polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 12.
  • This gene is expressed primarily in endothelial cells, fibroblasts, smooth muscle, and osteoblasts, and to a lesser extent in brain, heart, placental tissues, lung, and many other tissues.
  • the transcript is present in HUVEC, HUVEC +LPS, smooth muscle, fibroblasts; present in heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon and weakly in
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of vascularized tissues.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • bodily fluids e.g., lymph, seminal, fluid, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid
  • tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 38 as residues: Pro-67 to Thr-73, Pro-76 to Gln-83, Asn-93 to Thr-99, His-115 to Arg-128, His-178 to Lys-189, Pro-197 to Ala-212, Val-224 to Trp- 233, Lys-253 to Lys-259, Ser-280 to Asn-289, Asp-296 to Tyr-302, Gln-308 to Ala-315, Arg-327 to Lys-335, Asp-349 to Gly-358. Polynucleotides encoding said polypeptides are also provided.
  • tissue distribution in the vascularized endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of diseases of vascularized tissues, such as atherosclerosis, ataxia malabsortion, and hyperlipidemia. These and other factors often result in other cardiovascular disease.
  • translation product of this gene is useful for the treatment of wounds, and may facilitate the wound healing process.
  • the protein is useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis.
  • antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support.
  • a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:20 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • a- b is any integer between 1 to 1685 of SEQ ID NO:20
  • b is an integer of 15 to 1699
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20
  • b is greater than or equal to a + 14.
  • the translation product of this gene shares sequence homology with Cytotoxic- Regulatory T-Cell Associated Molecule (CRTAM) protein, which is thought to be important in the regulation of celluar physiology, development, differentiation or function of various cell types, including haematopoietic cells and various T-cell progenitors. See for example, PCT publication WO 96/34102 incorporated herein by reference in its entirety.
  • CTAM Cytotoxic- Regulatory T-Cell Associated Molecule
  • the protein product of this gene also shares homology with the thymocyte activation and developmental protein and the class-I MHC-restricted T cell associated molecule (See Genbank Accession Nos.
  • the translation product of this gene is expected to share at least some biological activities with T-cell modulatory proteins. Such activities are known in the art, some of which are described elsewhere herein.
  • Preferred polypeptides of the invention comprise the following amino acid sequence: MASVVLPSGSQCAAAAAAAAPPGLRLRLLLLLFSAAALIPTGDGQNLFTKDVTVI EGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSL TNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCT AMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVIC QVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQ PVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDY MLYVYDPPTTIPPPTTTTTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDHAVIGGVVAV VVFAMLCLLIILGRYFARHKGTYFTHEA
  • polypeptide of this latter embodiment has been determined to have a transmembrane domain at about amino acid position 379 - 395 of the amino acid sequence referenced in Table XIII for this gene. Moreover, a cytoplasmic tail encompassing amino acids 396 to 442 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.
  • Preferred polynucleotides comprise the following sequence: ATGGCGAGTGTAGTGC
  • Figures 28A-B shows the nucleotide (SEQ ID NO:21) and deduced amino acid sequence (SEQ ID NO:39) of the present invention.
  • Predicted amino acids from about 1 to about 44 constitute the predicted signal peptide (amino acid residues from about 1 to about 44 in SEQ ID NO:39) and are represented by the underlined amino acid regions.
  • Figure 29 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:39, the human poliovirus receptor protein (gi
  • Figure 30 shows an analysis of the amino acid sequence of SEQ ID NO:39.
  • Alpha, beta, turn and coil regions hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 28A-B (SEQ ID NO:39), which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figures 28A-B (SEQ ID NO:21) was obtained by sequencing a cloned cDNA (HOUDJ81), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:21 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein-
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:2l.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:2L
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or altematively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 1 1 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 12
  • polynucleotides of the invention encode functional attributes of the corresponding protein.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • the data presented in columns VIII, IX, XIII, and XIV of Table X can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Figure 30 (set on the original default parameters) was used to present the data in Figure 30 in a tabular format (See Table X).
  • the tabular format of the data in Figure 30 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 30 and in Table X include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 28A-B.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions. Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, etc.
  • the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 28A-B, up to the threonine residue at position number 359 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 1-364 of Figures 28A-B, where nl is an integer from 2 to 359 corresponding to the position of the amino acid residue in Figures 28A-B (which is identical to the sequence shown as SEQ ID NO:39).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:39 include polypeptides comprising the amino acid sequence of residues: A-2 to R-364; S-3 to R- 364; V-4 to R-364; V-5 to R-364; L-6 to R-364; P-7 to R-364; S-8 to R-364; G-9 to R- 364; S-10 to R-364; Q-l 1 to R-364; C-12 to R-364; A-13 to R-364; A-14 to R-364; A-15 to R-364; A- 16 to R-364; A- 17 to R-364; A- 18 to R-364; A- 19 to R-364; A-20 to R-364; P-21 to R-364; P-22 to R-364; G-23 to R-364; L-24 to R-364; R-25 to R-364; L-26 to R- 364; R-27 to R-364; L-28 to R-364; L-29 to R-364; L-30 to R-364;
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 28A-B, up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 28A-B, where ml is an integer from 6 to 364 con-esponding to the position of the amino acid residue in Figures 28 A-B.
  • polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:39 include polypeptides comprising the amino acid sequence of residues: M-1 to A-363; M-1 to R- 362; M-1 to S-361; M-1 to D-360; M-1 to T-359; M-1 to 1-358; M-1 to 1-357; M-1 to T- 356; M-1 to L-355; M-1 to 1-354; M-1 to T-353; M-1 to T-352; M-1 to T-351; M-1 to T- 350; M-1 to T-349; M-1 to T-348; M-1 to T-347; M-1 to T-346; M-1 to T-345; M-1 to T- 344; M-1 to T-343; M-1 to T-342; M-1 to T-341; M-1 to P-340; M-1 to P-339; M-1 to P- 339; M-1 to P- 3
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:21 which have been determined from the following related cDNA genes: HSQFJ92R (SEQ ID NO: 139), HFLAB18F (SEQ ID NO: 140), HAQBH82R (SEQ ID NO: 141), HLHTMIOR (SEQ ID NO: 142), and HLHAL65R (SEQ ID NO: 143).
  • This gene is expressed pnma ⁇ ly in immune system related tissues such as ulcerative colitis, rejected kidney tissues, and to a lesser extent in thymus and bone marrow.
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the t ⁇ ssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and hematopoietic diseases and/or disordeis, particulaily ulcerative colitis and rejected organs.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the t ⁇ ssue(s) or cell type(s) Foi a number of disorders of the above tissues or cells, particularly of the immune system, expiession of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g transplanted kidney, immune, hematopoeitic, renal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder
  • tissues or cell types e.g transplanted kidney, immune, hematopoeitic, renal, and cancerous and wounded tissues
  • bodily fluids e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 39 as residues: Gly-42 to Phe-48, Val-66 to Asp-71, Asn-78 to Thr-83, Asp-88 to Arg-96, Tyr- 127 to Tyr-135, Lys-181 to Trp- 195, H ⁇ s-210 to Gly-215, Leu-303 to Thr-310, Thr-341 to Thr-350. Polynucleotides encoding said polypeptides are also provided.
  • tissue dist ⁇ bution p ⁇ ma ⁇ ly in immune cells and tissues combined with the homology to the CRT AM, thymocyte activation and developmental protein, the class-I MHC-rest ⁇ cted T cell associated molecule protein, and the pohvirus receptor, indicates that the protein products of this gene are useful for the regulation of celluar physiology, development, differentiation or function of va ⁇ ous cell types, including haematopoietic cells and particularly T-cell progenitors. Representative uses are descnbed in the "Immune Activity" and "infectious disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
  • the proteins can be used to develop products for the diagnosis and treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g. cancers, or degenerative conditions.
  • the physiology or development of a cell can be modulated by contacting the cell with an agonist or antagonist (i.e. an anti- CRTAM-like peptide antibody).
  • an agonist or antagonist i.e. an anti- CRTAM-like peptide antibody.
  • the CRTAM- like polypeptides of the present invention include treatment of ulcerative colitis, organ rejection and other immune system related disorders. Agonists or antagonists may treat or prevent such disorders as ulcerative colitis and rejected organs, such as kidney.
  • antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
  • kits for detecting tumors in which expression of this protein occurs comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support.
  • a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid.
  • the antibody is bound to a solid support and the bodily fluid is serum.
  • the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 1506 of SEQ ID NO:21, b is an integer of 15 to 1520, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a + 14.
  • Figure 31 shows the nucleotide (SEQ ID NO:22) and deduced amino acid sequence (SEQ ID NO:40) of the present invention. Predicted amino acids from about 1 to about 23 constitute the predicted signal peptide (amino acid residues from about 1 to about 23 in SEQ ID NO:40) and are represented by the underlined amino acid regions.
  • Figure 32 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:40 and the human FAP protein (gi
  • Figure 33 shows an analysis of the amino acid sequence of SEQ ID NO:40.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figure 31 (SEQ ID NO:40), which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figure 31 (SEQ ID NO:22) was obtained by sequencing a cloned cDNA (HPWCM76), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484.
  • the deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:22 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:22.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:22.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 807 of SEQ ID NO:22, or the complementary strand thereto, or the cDNA contained in the deposited gene.
  • polynucleotides of the invention encode functional attributes of the corresponding protein.
  • Prefen-ed embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions.
  • alpha-regions alpha-helix and alpha-helix forming regions
  • beta-sheet and beta-sheet forming regions beta-sheet and beta-sheet forming regions
  • turn- regions turn and turn-forming regions
  • coil and coil-forming regions coil and coil-forming regions
  • the data presented in columns VIII, IX, XIII, and XIV of Table XI can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • Certain preferred regions in these regards are set out in Figure 33, but may, as shown in Table XI, be represented or identified by using tabular representations of the data presented in Figure 33.
  • the DNA*STAR computer algorithm used to generate Figure 33 (set on the original default parameters) was used to present the data in Figure 33 in a tabular format (See Table XI).
  • the tabular format of the data in Figure 33 is used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in Figure 33 and in Table XI include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 31.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • deletion of one or more amino acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, etc.
  • the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figure 31, up to the arginine residue at position number 61 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues nl-66 of Figure 31, where nl is an integer from 2 to 61 corresponding to the position of the amino acid residue in Figure 31 (which is identical to the sequence shown as SEQ ID NO:40).
  • N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:40 include polypeptides comprising the amino acid sequence of residues: S-2 to N-66; S-3 to N-66; S-4 to N-66; S-5 to N-66; L-6 to N-66; K-7 to N-66; H-8 to N-66; L-9 to N-66; L-10 to N-66; C-11 to N-66; M-12 to N-66; A-13 to N-66; L-14 to N-66; S-15 to N-66; W-16 to N-66; F-17 to N-66; S-18 to N-66; S-l 9 to N-66; F-20 to N-66; 1-21 to N-66; S-22 to N-66; G-23 to N-66; E-24 to N-66; T-25 to N-66; S-26 to N-66; F-27 to N-66; S-28 to N-66; L-29 to N-66; L-30 to N- 66; N-31 to N-
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figure 31, up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 31, where ml is an integer from 6 to 66 corresponding to the position of the amino acid residue in Figure 31.
  • polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:40 include polypeptides comprising the amino acid sequence of residues: M-1 to E-65; M-1 to W- 64; M-1 to P-63; M-1 to F-62; M-1 to R-61; M-1 to M-60; M-1 to S-59; M-1 to N-58; M-1 to C-57; M-1 to S-56; M-1 to F-55; M-1 to P-54; M-1 to D-53; M-1 to L-52; M-1 to 1-51; M-1 to S-50: M-1 to C-49; M-1 to Q-48; M-1 to V-47; M-1 to S-46; M-1 to F-45; M-1 to C-44; M-1 to C-43; M-1 to C-42; M-1 to R-41 ; M-1 to S-40; M-1 to S
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:22 which have been determined from the following related cDNA genes: HPWCM76R (SEQ ID NO: 147).
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammation of the prostate, or related tissues.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the t ⁇ ssue(s) or cell type(s).
  • expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g.
  • prostate cancerous and wounded tissues
  • bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
  • another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disordei .
  • the tissue dist ⁇ bution in prostate BPH tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of inflammatory conditions which result in an enlargement of the prostate, or related tissues. Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g.
  • this gene product is useful in the treatment of male infertility and/or impotence.
  • This gene product is also useful assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents.
  • the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer.
  • the testes are also a site of active gene expression of transcnpts that is expressed, particularly at low levels, m other tissues of the body.
  • this gene product is expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
  • antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene. Also provided is a kit for detecting tumors in which expression of this protein occurs.
  • Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support
  • a method of detecting these tumors in an individual which composes a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid
  • the antibody is bound to a solid support and the bodily fluid is serum
  • the homology to the FAP protein indicates that the protein product of this gene is useful in treating, detecting, and/or preventing iron metabolism disorders, particularly those resulting in high oxidative states, tissue damage, athersclerosis, free radical damage, vascular disordeis, iron binding protein dysfunction, nitnc oxide synthase dysfunction or aberration, vasodilation disorders, and tissue edema Based on the sequence similarity, the translation product of this gene is expected
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO 22 and may have been publicly available p ⁇ or to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention.
  • a- b a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 793 of SEQ ID NO:22, b is an integer of 15 to 807, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a + 14.
  • Trp 345 A T 3.43 -1.29 * F 1.30 6.68
  • Lys 442 A A 0.81 -0.84 * F 0.90 1.25

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Abstract

La présente invention concerne 12 protéines humaines sécrétées ainsi que des acides nucléiques qui ont été isolés et qui contiennent les régions codant les gènes codant ces protéines. L'invention concerne également des vecteurs, des cellules hôtes, des anticorps et des procédés à recombinaison permettant la production de ces protéines humaines sécrétées. L'invention concerne enfin des techniques de diagnostic et des traitements thérapeutiques permettant de diagnostiquer et de traiter des troubles en relation avec ces protéines humaines sécrétées.
EP99972222A 1998-10-28 1999-10-27 12 proteines humaines secretees Withdrawn EP1124850A4 (fr)

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US10597198P 1998-10-28 1998-10-28
US105971P 1998-10-28
PCT/US1999/025031 WO2000029435A1 (fr) 1998-10-28 1999-10-27 12 proteines humaines secretees

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US20020137890A1 (en) * 1997-03-31 2002-09-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
NZ510356A (en) 1998-08-07 2004-12-24 Immunex Corp Molecules designated LDCAM
US20030055231A1 (en) 1998-10-28 2003-03-20 Jian Ni 12 human secreted proteins
AU3128000A (en) * 1998-12-23 2000-07-31 Human Genome Sciences, Inc. Peptidoglycan recognition proteins
SE9902056D0 (sv) * 1999-06-03 1999-06-03 Active Biotech Ab An integrin heterodimer and an alpha subunit thereof
EP1067182A3 (fr) * 1999-07-08 2001-11-21 Helix Research Institute Proteine secretoire ou proteine de membrane
US7129338B1 (en) 1999-07-08 2006-10-31 Research Association For Biotechnology Secretory protein or membrane protein
WO2001081414A2 (fr) * 2000-04-27 2001-11-01 Millennium Pharmaceuticals, Inc. Nouvelle sous-unite $g(a) d'integrine et utilisations correspondantes
EP1339871B1 (fr) * 2000-08-15 2008-10-15 The Johns Hopkins University School Of Medicine Diagnostic et traitement des troubles associes aux suppresseurs de tumeur
IL149851A0 (en) * 2002-05-26 2002-11-10 Yeda Res & Dev Resistin binding proteins, their preparation and use
DE10254601A1 (de) * 2002-11-22 2004-06-03 Ganymed Pharmaceuticals Ag Differentiell in Tumoren exprimierte Genprodukte und deren Verwendung
AU2015242979B2 (en) * 2002-11-22 2016-12-01 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof
EP2133362B1 (fr) 2003-07-25 2012-04-18 Amgen, Inc Procédés rélatifs aux LDCAM et CRTAM
EP2069793B9 (fr) 2006-08-29 2017-08-16 Oxford BioTherapeutics Ltd Identification d'une protéine associée à un carcinome hépatocellulaire, à un glioblastome et à un cancer du poumon
KR101769160B1 (ko) 2009-03-05 2017-08-17 옥스포드 바이오테라퓨틱스 리미티드 Ｃａｄｍ１에 특이적인 완전 인간 항체
CN113481185B (zh) * 2021-08-05 2022-12-02 云南师范大学 一种耐盐β-半乳糖苷酶GalNC2-13及其制备方法和应用
CN116082456B (zh) * 2022-11-07 2023-08-01 优睿赛思(武汉)生物科技有限公司 一种突变型信号肽及含有该突变型信号肽的重组载体和应用

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SAKAGUCHI M: "Eukaryotic protein secretion." CURRENT OPINION IN BIOTECHNOLOGY. OCT 1997, vol. 8, no. 5, October 1997 (1997-10), pages 595-601, XP002326585 ISSN: 0958-1669 *
See also references of WO0029435A1 *

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