US20030040081A1

US20030040081A1 - Galectin 9 and 10SV polynucleotides

Info

Publication number: US20030040081A1
Application number: US10/235,674
Authority: US
Inventors: Jian Ni; Reiner Gentz; Steven Ruben
Original assignee: Human Genome Sciences Inc
Current assignee: Human Genome Sciences Inc
Priority date: 1996-10-09
Filing date: 2002-09-06
Publication date: 2003-02-27
Also published as: US6468768B1; US20020150970A1; US6027916A

Abstract

The present invention relates to novel galectin 8, 9, 10 and 10SV proteins which are members of the galectin superfamily. In particular, isolated nucleic acid molecules are provided encoding the human galectin 8, 9, 10 and 10SV proteins. Galectin 8, 9, 10 and 10SV polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of galectin 8, 9, 10 or 10SV activity. Also provided are diagnostic and therapeutic methods.

Description

This application claims the benefit of the filing date of provisional application No. 60/028,093 filed on Oct. 9, 1996, which is herein incorporated by reference.[0001]

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel galectins. More specifically, isolated nucleic acid molecules are provided encoding

human galectin

8, 9, 10, or 10SV. Galectin 8, 9, 10 and 10SV polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of

galectin

8, 9, 10, or 10SV activity. Also provided are diagnostic methods for detecting cell growth disorders and therapeutic methods for cell growth disorders, including autoimmune diseases, cancer, and inflammatory diseases.

2. Related Art

Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates. Barondes et al., J. Biol. Chem. 269(33):20807-20810 (1994). Galectins are members of a family of β-galactoside-binding lectins with related amino acid sequences (For review see, Barondes et al., Cell 76:597-598 (1994); Barondes et al., J. Biol. Chem. 269(33):20807-20810 (August 1994)). Galectin 1 (aka. L-14-1, L-14, RL-14.5, galaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, rIML-1) is a homodimer with a subunit molecular mass of 14,500 which is abundant in smooth and skeletal muscle, and is present in many other cell types (Couraud et al., J. Biol. Chem. 264:1310-1316 (1989)). Galectin 2 was originally found in hepatoma and is a homodimer with a subunit molecular weight of 14,650 (Gitt et al., J. Biol. Chem. 267:10601-10606 (1992)). Galectin 3 (aka. Mac-2, EPB, CBP-35, CBP-30, and L-29) is abundant in activated macrophages and epithelial cells and is a monomer with an apparent molecular mass between 26,320 and 30,300 (Cherayil et al., Proc. Natl. Acad. Sci. USA 87: 7324-7326 (1990)). Galectin 4 has a molecular mass of 36,300 and contains two carbohydrate-binding domains within a single polypeptide chain. (Oda et al., J. Biol. Chem. 268.5929-5939 (1993)). Galectins 5 and 6 are mentioned in Barondes et al., Cell 76:597-598 (1994). Human galectin 7 has a molecular mass of 15,073 and is found mainly in stratified squamous epithelium (Madsen et al., J. Biol. Chem. 270(11):5823-5829 (1995)).

Animal lectins, in general, often function in modulating cell-cell and cell-matrix interactions. Galectin 1 has been shown to either promote or inhibit cell adhesion depending upon the cell type in which it is present. Galectin 1 inhibits cell-matrix interactions in skeletal muscle (Cooper et al, J. Cell Biol. 115:1437-1448 (1991)). In other cell types, galectin 1 promotes cell-matrix adhesion possibly by cross-linking cell surface and substrate glycoconjugates (Zhou et al., Arch. Biochem. Biophys. 300:6-17 (1993); Skrincosky et al., Cancer Res. 53:2667-2675 (1993)).

Galectin 1 also participates in regulating cell proliferation (Wells et al., Cell 64:91-97 (1991)) and some immune functions (Offner et al., J. Neuroimmunol. 28:177-184 (1990)). Galectin 1 has been shown to regulate the immune response by mediating apoptosis of T cells (Perillo et al., Nature 378: 736-739 (1995)).

Galectin 3 promotes the growth of cells cultured under restrictive culture conditions (Yang et al., Proc. Natl. Acad. Sci. USA 93:6737-6742 (June 1996)). Galectin 3 expression in cells confers resistance to apoptosis which indicates that Galectin 3 could be a cell death suppressor which interferes in a common pathway of apoptosis. Id.

Accordingly, there is a need in the art for the identification of novel galectins which can serve as useful tools in the development of therapeutics and diagnostics for regulating immune response.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the

galectin

8, 9, 10, or 10SV polypeptide having the amino acid sequence is shown in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:2, 4, 6, and 8, respectively) or the amino acid sequence encoded by the cDNA clones deposited in bacterial hosts as ATCC Deposit Numbers 97732, 97733 and 97734 on Sep. 24, 1996.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of

galectin

8, 9, 10, or 10SV polypeptides or peptides by recombinant techniques.

The invention further provides an

isolated galectin

8, 9, 10, or 10SV polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by

galectin

8, 9, 10, or 10SV, which involves contacting cells which express

galectin

8, 9, 10, or 10SV with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.

In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on

galectin

8, 9, 10, or 10SV binding to the β-galactosidase sugar. In particular, the method involves contacting the β-galactosidase sugar with a

galectin

8, 9, 10, or 10SV polypeptide and a candidate compound and determining whether

galectin

8, 9, 10, or 10SV binding to β-galactosidase sugar is increased or decreased due to the presence of the candidate compound.

The invention provides a diagnostic method useful during diagnosis disorder.

An additional aspect of the invention is related to a method for treating an individual in need of an increased level of

galectin

8, 9, 10, or 10SV activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated

galectin

8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of

galectin

8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a

galectin

8, 9, 10, or 10SV antagonist.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequences of [0018] galectin 8. The protein has a deduced molecular weight of about 36 kDa.
FIGS. [0019] 2A-2B shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequences of galectin 9. The protein has a deduced molecular weight of about 34.7 kDa.
FIGS. [0020] 3A-3B shows the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequences of full length galectin 10. The protein has a deduced molecular weight of about 35.7 kDa.
FIGS. [0021] 4A-4B shows the nucleotide (SEQ ID NO:7) and deduced amino acid (SEQ ID NO:8) sequences of a galectin 10 splice variant (galectin 10SV). The protein has a deduced molecular weight of about 22.4 kDa.
FIGS. [0022] 5A-5B shows the regions of similarity between the amino acid sequences of the galectin 8, 9, and 10 proteins and human galectin 2 (SEQ ID NO:9), human galectin 3 (SEQ ID NO:10), rat galectin 4 (SEQ ID NO:11), rat galectin 5 (SEQ ID NO:12), human galectin 7 (SEQ ID NO:13), rat galectin 3 (SEQ ID NO: 14), rat galectin 8 (SEQ ID NO: 15), and human galectin 1 (SEQ ID NO:16).
FIG. 6 shows the regions of similarity between the amino acid sequences of the galectin 10SV protein and the rat RL30 protein (SEQ ID NO: 17). [0023]
FIG. 7 shows a homology comparison between the [0024] galectin 10 and galectin 10SV proteins.
FIGS. 8, 9, [0025] 10, and 11 show an analysis of the galectin 8, 9, 10, and 10SV amino acid sequence, respectively. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the “Antigenic Index-Jameson-Wolf” graph, amino acid residues 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID NO:2), 62-102, 226-259, 197-308 in FIGS. 2A-2B (SEQ ID NO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIGS. 3A-3B (SEQ ID NO:6), and 25-77, 84-105, 129-140, and 156-183 in FIGS. 4A-4B (SEQ ID NO:8) correspond to the shown highly antigenic regions of the galectin 8, 9, 10, or 10SV protein, respectively.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a [0026] galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence shown in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:2, 4, 6, and 8, respectively), which was determined by sequencing a cloned cDNA. The galectin 8, 9, 10, and 10SV proteins of the present invention share sequence homology with other galectins and the rat RL30 protein (FIGS. 5A-5B and 6) (SEQ ID NOs:9-17). The nucleotide sequences shown in FIGS. 1, 2A-2B, and 4A-4B (SEQ ID NO:1, 3, and 7, respectively) were obtained by sequencing the HSIAL77, HTPBR22, and HETAS87 clones, which were deposited on Sep. 24, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852, and given accession numbers 97732, 97733 and 97734, respectively. The deposited clones are contained in the pBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.).
The nucleotide sequence shown in FIGS. [0027] 3A-3B (SEQ ID NO:5), which encodes the fill-length galectin 10 protein, was obtained by sequencing a clone cDNA obtained from a human endometrial tumor library.
Nucleic Acid Molecules [0028]
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. [0029]
Using the information provided herein, such as the nucleotide sequences in FIGS. [0030] 1, 2A-2B, 3A-3B, and 4A-4B a nucleic acid molecule of the present invention encoding a galectin 8, 9, 10, or 10SV, respectively, polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecules described in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B (SEQ ID NO: 1, 3, 5, and 7, respectively) were discovered in cDNA libraries derived from human adult small intestine, human pancreatic tumor, human endometrial tumor and human endometrial tumor, respectively. These genes were also identified in cDNA libraries from the following tissues pancreas, colon, small intestine, brain, bone marrow, kidney, lung, spleen, and testes tissue. Galectin 8 (SEQ ID NO:1) appears to be mainly expressed in cells of the human colon and small intestine.
The determined nucleotide sequences of the [0031] galectin 8, 9, 10, and 10SV cDNAs of FIGS. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:1, 3, 5, and 7) contain open reading frames encoding proteins of 323, 311, 317, and 200 amino acid residues, with an initiation codon at positions 52-54, 16-18, 118-120, and 118-120 of the nucleotide sequences in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:1, 3, 5, and 7), and a deduced molecular weight of about 36, 34.7, 35.7, and 22.4 kDa, respectively. The galectin 8, 9, 10 and 10SV proteins shown in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B respectively (SEQ ID NOs:2, 4, 6, and 8) share homology with other galectins (See, e.g., FIGS. 5A-5B).
As one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, as well as the variability of processing sites for different known proteins, the predicted [0032] galectin 8 and 9 polypeptides encoded by the deposited cDNAs comprise about 323 and 311 amino acids, but may be anywhere in the range of 300-333 amino acids. Similarly, the predicted galectin 10 polypeptide comprises about 317 amino acids, but may be anywhere in the range of 305-329 amino acids. Further, the predicted galectin 10SV polypeptide encoded by the deposited cDNA comprises about 200 amino acids, but may be anywhere in the range of 190-210 amino acids.
Galectin 10SV is believed to be a splice variant of [0033] galectin 10. As used herein the phrase “splice variant” refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences. The term “splice variant” also refers to the proteins encoded by the above cDNA molecules.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. [0034]
By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. [0035]
Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIGS. [0036] 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:1, 3, 5, and 7); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the galectin 8, 9, 10, or 10SV protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 1 which have been determined from the following related cDNA clones: HSIAL77R (SEQ ID NO:18), HGBDK55R (SEQ ID NO:19), HCNAH29R (SEQ ID NO:20), HKCAA85R (SEQ ID NO:21), HCNAI55R (SEQ ID NO:22), HCNAI87R (SEQ ID NO:23), HCNAS74R (SEQ ID NO:24) and HCNAF43R (SEQ ID NO:25). [0037]
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:3 which have been determined from the following related cDNA clones: HMSCP11R (SEQ ID NO:26), HMSEU32R (SEQ ID NO:27), HTPAO71R (SEQ ID NO:28), HJAAV54R (SEQ ID NO:29), HMSEU43R (SEQ ID NO:30), HILBP03R (SEQ ID NO:31), HTPCG81R (SEQ ID NO:32), HTBAA21R (SEQ ID NO:33), and HFXBU26R (SEQ ID NO:34). [0038]
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:5 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HJBAI38R (SEQ ID NO:37), HETAS87R (SEQ ID NO:38), and HETAR45R (SEQ ID NO:39). [0039]
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:7 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HBNAF37R (SEQ ID NO:40), and HETAS87R (SEQ ID NO:38). [0040]
In another aspect, the invention provides isolated nucleic acid molecules encoding the [0041] galectin 8, 9, 10 or 10SV polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit Nos. 97732, 97733 and 97734, respectively, on Sep. 24, 1996. In a further embodiment, nucleic acid molecules are provided encoding the full- length galectin 8, 9, 10, or 10SV polypeptide lacking the N-terminal methionine. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7) or the nucleotide sequence of the galectin 8, 9, or 10SV cDNA contained in the above-described deposited clones, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the galectin 8, 9, 10, or 10SV gene in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIGS. [0042] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NO:1, 3, 5, or 7) is intended fragments at least about 15 nt, 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. Of course larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, or 1115 nt in length of the sequence shown in SEQ ID NO:1 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97732 or as shown in SEQ ID NO:1. Similarly, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, or 1525 nt in length of the sequence shown in SEQ ID NO:3 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97733 or as shown in SEQ ID NO:3. Similarly, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1464 nt in length of the sequence shown in SEQ ID NO:5 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA molecule as shown in SEQ ID NO:5. Further, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, and 1908 nt in length of the sequence shown in SEQ ID NO:7 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97734 or as shown in SEQ ID NO:7. By a fragment at least 20 nt in 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 NOs:1, 3, 5, or 7.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the [0043] galectin 8, 9, 10, or 10SV protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID NO:2), 62-102, 226-259, 197-308 in FIGS. 2A-2B (SEQ ID NO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIGS. 3A-3B (SEQ ID NO:6), and 25-77, 84-105, 129-140, and 156-183 in FIGS. 4A-4B (SEQ ID NO:8). The inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 8, 9, 10, and 10SV proteins. Methods for determining other such epitope-bearing portions of the galectin 8, 9, 10, and 10SV proteins are described in detail below.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit Nos. 97732, 97733 and 97734. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1× SSC at about 65° C. [0044]
By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below. [0045]
By a portion of a polynucleotide of “at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in FIGS. [0046] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7)). Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3′ terminal poly(A) tract of the galectin 8, 9, 10, or 10SV cDNA shown in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B, respectively (SEQ ID NOs:1, 3, 5, or 7)), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, 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).
As indicated, nucleic acid molecules of the present invention which encode a [0047] galectin 8, 9, 10, or 10SV polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding an amino acid leader or secretory sequence, such as a pre-, or pro- or prepro-protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example—ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion proteins include the galectin 8, 9, 10, or 10SV fused to Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the [0048] galectin 8, 9, 10, or 10SV protein. Variants may occur naturally, such as a natural allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the [0049] galectin 8, 9, 10, or 10SV protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the [0050] galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7); (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7), but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Nos. 97732, 97733 or 97734 on Sep. 24, 1996; or (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence encoding a [0051] galectin 8, 9, 10, or 10SV polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the galectin 8, 9, 10, or 10SV polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIGS. [0052] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7) or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482489 (1981)), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGS. [0053] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7) or to the nucleic acid sequence of one of the deposited cDNAs, irrespective of whether they encode a polypeptide having galectin 8, 9, 10, or 10SV activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having galectin 8, 9, 10, or 10SV activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having galectin 8, 9, 10, or 10SV activity include, inter alia, (1) isolating the galectin 8, 9, 10, or 10SV gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the galectin 8, 9, 10, or 10SV gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergarnon Press, New York (1988); and (3) Northern Blot analysis for detecting galectin 8, 9, 10, or 10SV mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGS. [0054] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7) or to the nucleic acid sequence of one of the deposited cDNAs which do, in fact, encode a polypeptide having galectin 8, 9, 10, or 10SV protein activity. By “a polypeptide having galectin 8, 9, 10, or 10SV activity” is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the galectin 8, 9, 10, or 10SV protein of the invention, as measured in a particular biological assay. For example, galectin 8, 9, 10, or 10SV protein activity can be measured using a lactose binding assay.
Lactose binding activity of the expressed [0055] galectin 8, 9, 10, or 10SV is assayed by immunodetection of in situ binding activity to asialofetuin (Sigma) immobilized on nitrocellulose (Amersham) (Madsen et al., J. Biol. Chem. 270(11):5823-5829 (1995)). Thirty μg of asialofetuin dissolved in 3 μl of water is spotted on a 1-cm²strip of nitrocellulose. The nitrocellulose pieces are then placed in a 24-well tissue culture plate and incubated overnight in buffer B (58 mM Na₂HPO₄, 18 mM KH₂PO₄, 75 mM NaCl, 2 mM EDTA, and 3% BSA, pH 7.2) with constant agitation at 22° C. Following incubation, the blocking medium is aspirated and the nitrocellulose pieces are washed three times in buffer A (58 mM Na₂HPO₄, 18 mM KH₂PO₄, 75 mM NaCl, 2 mM EDTA, 4 mM β-mercaptoethanol and 0.2% BSA, pH 7.2). Cell extracts (preferably, COS cells) are prepared containing 1% BSA and either with or without 150 mM lactose (105 μl of primary extract, 15 μl of 10% BSA in buffer A and either 30 μl of 0.75 M lactose in buffer A or 30 μl of buffer A). The immobilized asialofetuin is incubated with the extracts for 2 h and washed 5 times in buffer A. The nitrocellulose pieces are then fixed in 2% formalin in PBS (58 mM Na₂HPO₄, 18 mM KH₂PO₄, 75 mM NaCl, 2 mM EDTA pH 7.2) for 1 hour to prevent loss of bound galectin. Following extensive washing in PBS the pieces were incubated with rabbit anti-galectin 8, 9, 10, or 10SV polyclonal serum diluted 1:100 in PBS for 2 h at 22° C. The pieces are then washed in PBS and incubated with peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following incubation for 2 h at 22° C., the pieces are washed in PBS and the substrate is added. Nitrocellulose pieces are incubated until the color develops and the reaction is stopped by washing in distilled water.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIGS. [0056] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:1, 3, 5, or 7, respectively) will encode “a polypeptide having galectin 8, 9, 10, or 10SV protein activity.” In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having galectin 8, 9, 10, or 10SV protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” [0057] Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.
Vectors and Host Cells [0058]
The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of [0059] galectin 8, 9, 10, or 10SV polypeptides or fragments thereof by recombinant techniques.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. [0060]
The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the [0061] E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in [0062] E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan. [0063]
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., [0064] Basic Methods In Molecular Biology (1986).
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., [0065] Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
The [0066] galectin 8, 9, 10, or 10SV protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
[0067] Galectin 8, 9, and 10 Polypeptides and Fragments
The invention further provides an [0068] isolated galectin 8, 9, 10, or 10SV polypeptide having (1) the amino acid sequence encoded by one of the deposited cDNAs, (2) the amino acid sequence in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively), or (3) the amino acid sequence of a peptide or polypeptide comprising a portion of the above polypeptides.
It will be recognized in the art that some amino acid sequences of the [0069] galectin 8, 9, 10, or 10SV polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the [0070] galectin 8, 9, 10, or 10SV polypeptide which show substantial galectin 8, 9, 10, or 10SV polypeptide activity or which include regions of galectin 8, 9, 10, or 10SV protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).
Thus, the fragment, derivative or analog of the polypeptide of SEQ ID NOs:2, 4, 6, or 8, or that encoded by one of the deposited cDNAs, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. [0071]
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of a [0072] galectin 8, 9, 10, or 10SV protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).

TABLE 1


Conservative Amino Acid Substitutions.

	Aromatic	Phenylalanine
		Tryptophan
		Tyrosine
	Hydrophobic	Leucine
		Isoleucine
		Valine
	Polar	Glutamine
		Asparagine
	Basic	Arginine
		Lysine
		Histidine
	Acidic	Aspartic Acid
		Glutamic Acid
	Small	Alanine
		Serine
		Threonine
		Methionine
		Glycine

Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above and below. Generally speaking, the number of substitutions for any given [0074] galectin 8, 9, 10, or 10SV polypeptide or mutant thereof will not be more than 50, 40, 30, 20, 10, 5, or 3, depending on the objective.
Amino acids in a [0075] galectin 8, 9, 10, or 10SV protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. Sites that are critical for ligand binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).
The polypeptides of the present invention are preferably provided in an isolated form. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of a [0076] galectin 8, 9, 10, or 10SV polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
The polypeptides of the present invention include the polypeptides encoded by the deposited cDNAs; a polypeptide comprising amino acids about 1 to about 323 in SEQ ID NO:2, about 1 to about 311 in SEQ ID NO:4, about 1 to about 317 in SEQ ID NO:6, and about 1 to about 200 in SEQ ID NO:8; a polypeptide comprising amino acids about 2 to about 323 in SEQ ID NO:2, about 2 to about 311 in SEQ ID NO:4, about 2 to about 317 in SEQ ID NO:6 and about 2 to about 200 in SEQ ID NO:8; as well as polypeptides which are at least 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. [0077]
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a [0078] galectin 8, 9, 10, or 10SV polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the galectin 8, 9, 10, or 10SV polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in FIGS. [0079] 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively) or to the amino acid sequence encoded by one of the deposited cDNA clones (ATCC Deposit Numbers 97732, 97733 and 97734) can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. [0080]
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope described herein. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., [0081] Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with predetermined sites on proteins. [0082] Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., [0083] Cell 37:767-778 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about at least 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. [0084]
Non-limiting examples of antigenic polypeptides or peptides that can be used to generate [0085] galectin 8, 9, 10, or 10SV-specific antibodies include: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID NO:2), 62-102, 226-259, 197-308 in FIGS. 2A-2B (SEQ ID NO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIGS. 3A-3B (SEQ ID NO:6), and 25-77, 84-105, 129-140, and 156-183 in FIGS. 4A-4B (SEQ ID NO:8), respectively. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 8, 9, 10, or 10SV protein.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. [0086] Proc. Natl. Acad. Sc. USA 82:5131-5135. This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, [0087] galectin 8, 9, 10, or 10SV polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric galectin 8, 9, 10, or 10SV protein or protein fragment alone (Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).
Diagnosis and Prognosis [0088]
It is believed that certain tissues in mammals with certain diseases (cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases) express significantly altered (enhanced or decreased) levels of the [0089] galectin 8, 9, 10, or 10SV protein and mRNA encoding the galectin 8, 9, 10, or 10SV protein when compared to a corresponding “standard” mammal, i.e., a mammal of the same species not having the disease. Further, it is believed that altered levels of the galectin 8, 9, 10, or 10SV protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with the disease when compared to sera from mammals of the same species not having the disease. Thus, the invention provides a diagnostic method useful during diagnosis, which involves assaying the expression level of the gene encoding the galectin 8, 9, 10, or 10SV protein in mammalian cells or body fluid and comparing the gene expression level with a standard galectin 8, 9, 10, or 10SV gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of the disease.
Where a diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting altered [0090] galectin 8, 9, 10, or 10SV gene expression will experience a worse clinical outcome relative to patients expressing the gene at a normal level.
By “assaying the expression level of the gene encoding the [0091] galectin 8, 9, 10, or 10SV protein” is intended qualitatively or quantitatively measuring or estimating the level of the galectin 8, 9, 10, or 10SV protein or the level of the mRNA encoding the galectin 8, 9, 10, or 10SV protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the galectin 8, 9, 10, or 10SV protein level or mRNA level in a second biological sample).
Preferably, the [0092] galectin 8, 9, 10, or 10SV protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard galectin 8, 9, 10, or 10SV protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer. As will be appreciated in the art, once a standard galectin 8, 9, 10, or 10SV protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains [0093] galectin 8, 9, 10, or 10SV protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted galectin 8, 9, 10, or 10SV protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.
The present invention is useful for detecting diseases in mammals (for example, cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases). In particular the invention is useful during diagnosis of the of following types of cancers in mammals: melanoma, renal astrocytoma, Hodgkin disease, breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans. [0094]
Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, [0095] Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the galectin 8, 9, 10, or 10SV protein are then assayed using any appropriate method. These include Northern blot analysis, (Harada et al., Cell 63:303-312 (1990) S1 nuclease mapping, (Fijita et al., Cell 49:357-367 (1987)) the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al., Technique 2:295-301 (1990), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
Assaying [0096] galectin 8, 9, 10, or 10SV protein levels in a biological sample can antibody-based techniques. For example, galectin 8, 9, 10, or 10SV protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)).
Other antibody-based methods useful for detecting [0097] galectin 8, 9, 10, or 10SV protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine ([0098] ¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (3H), indium (¹¹²In), and technetium (^99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
Therapeutics [0099]
It is to be understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal that expresses [0100] galectin 8, 9, 10, or 10SV.
As noted above, [0101] galectin 8, 9, 10, and 10SV share significant homology with other galectins. Galectin 1 induces apoptosis of T cells and T cell leukemia cell lines. Thus, it is believed by the inventors that galectin 8, 9, 10, and 10SV are active in modulating growth regulatory activities, immunomodulatory activity, cell-cell and cell-substrate interactions, and apoptosis.
The ability of [0102] galectin 8, 9, 10, or 10SV to modulate growth regulatory activity may be therapeutically valuable in the treatment of clinical manifestations of such cell regulatory disorders. Disorders which can be treated include, but should not be limited to, autoimmune disease, cancer (preferably, melanoma, renal, astrocytoma, and Hodgkin disease), inflammatory disease, wound healing, arteriosclerosis, other heart diseases, microbe infection (virus, fungal, bacterial, and parasite), asthma, and allergic diseases.
Given the activities modulated by [0103] galectin 8, 9, 10, and 10SV, it is readily apparent that a substantially altered (increased or decreased) level of expression of galectin 8, 9, 10, or 10SV in an individual compared to the standard or “normal” level produces pathological conditions such as those described above. It will also be appreciated by one of ordinary skill that the galectin 8, 9, 10, or 10SV protein of the invention will exert its modulating activities on any of its target cells. Therefore, it will be appreciated that conditions caused by a decrease in the standard or normal level of galectin 8, 9, 10, or 10SV activity in an individual, can be treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist thereof. Thus, the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of [0104] galectin 8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 8, 9, 10, or 10SV antagonist. Preferred antagonists for use in the present invention are galectin 8, 9, 10, or 10SV-specific antibodies.
Modes of Administration [0105]
It will be appreciated that conditions caused by a decrease in the standard or normal level of [0106] galectin 8, 9, 10, or 10SV activity in an individual, can be treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist thereof. Thus, the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention, particularly a mature form of the galectin 8, 9, 10, or 10SV, effective to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
As a general proposition, the total pharmaceutically effective amount of [0107] galectin 8, 9, 10, or 10SV polypeptide administered parenterally per dose will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the galectin 8, 9, 10, or 10SV polypeptide is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
Pharmaceutical compositions containing the [0108] galectin 8, 9, 10, or 10SV of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By “pharmaceutically acceptable carrier” is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Chromosome Assays [0109]
The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease. [0110]
In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a [0111] galectin 8, 9, 10, or 10SV protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′ untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. [0112]
Fluorescence in situ hybridization (“FISH”) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al., [0113] Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, [0114] Mendelian Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. [0115]
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting. [0116]

EXAMPLES

Example 1

Expression and Purfication of

Galectin

8, 9, 10 and 10SV in E coli

The DNA sequence encoding the [0117] galectin 9 protein in the deposited cDNA clone was amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the galectin 9 protein and to vector sequences 3′ to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5′ and 3′ sequences.
The DNA sequence encoding the [0118] galectin 8 or 10SV protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 8 or 10SV protein and to vector sequences 3′ to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5′ and 3′ sequences.
The cDNA sequence encoding the [0119] galectin 10 protein is amplified from either a human endometrial tumor or human fetal heart cDNA library using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 10 protein and to vector sequences 3′ to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5′ and 3′ sequences.
The 5′ [0120] galectin 8 oligonucleotide primer has the sequence 5′cgc ccATGg CCTATGTCCCCGCACCG 3′ (SEQ ID NO:41) containing the underlined NcoI restriction site and nucleotides 56 to 72 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO: 1).
The 3′ [0121] galectin 8 primer has the sequence 5′ cgc AAG CTT TTAGATC TGGACATAGGAC 3′ (SEQ ID NO:42) containing the underlined HindIII restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO: 1).
The 5′ [0122] galectin 9 oligonucleotide primer has the sequence 5′cgc ccATGg CCTT CAGCGGTTCCCAG 3′ (SEQ ID NO:43) containing the underlined NcoI restriction site and nucleotides 20 to 36 of the galectin 9 protein coding sequence in FIGS. 2A-2B (SEQ ID NO:3).
The 3′ [0123] galectin 9 primer has the sequence 5′cgc AAG CTT CAGGGTT GGAAAGGCTG (SEQ ID NO:44) containing the underlined HindIII restriction site followed by nucleotides complementary to position 1029 to 1045 of the galectin 9 protein coding sequence in FIGS. 2A-2B (SEQ ID NO:3).
The 5′ [0124] galectin 10 and 10SV oligonucleotide primer has the sequence 5′cgc CCATGc TGTTGTCCTTAAACAAC 3′ (SEQ ID NO:45) containing the underlined SphI restriction site and nucleotides 122-138 of the galectin 10 protein coding sequence in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ [0125] galectin 10 primer has the sequence 5′ cgc CTG CAG CACAGAA GCCATTCTG 3′ (SEQ ID NO:46) containing the underlined PstI restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ galectin 10SV primer has the [0126] sequence 5′ CGCCTGCAGCTA TGCAACTTTATAAAATATTCC 3′ (SEQ ID NO:47) containing the underlined PstI restriction site followed by nucleotides complementary to 3′ end of the galectin 10SV protein coding sequence in FIGS. 4A-4B (SEQ ID NO:7).
The restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60 ([0127] galectin 8 and 9) or pQE6 (galectin 10), which are used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^r”) and contains a bacterial origin of replication (“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.
The amplified [0128] galectin 8, 9, 10, or 10SV DNA and the vector pQE60 or pQE6 both are digested with NcoI and HindIII (for galectin 8 and 9) or SphI and PstI (for galectin 10) and the digested DNAs are then ligated together. Insertion of the galectin 8, 9, 10, or 10SV protein DNA into the restricted pQE60 or pQE6 vector placed the galectin 8, 9, 10, or 10SV protein coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of galectin 8, 9, 10, or 10SV protein.
The ligation mixture is transformed into competent [0129] E. coli cells using standard procedures. Such procedures are described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance (“Kan^r”), is used in carrying out the example described herein. This strain, which is only one of many that are suitable for expressing galectin 8, 9, 10, or 10SV protein, is available commercially from Qiagen.
Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis. [0130]
Clones containing the desired constructs are grown overnight (“O/N”) in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml). [0131]
The O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250. The cells are grown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacI repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation and disrupted, by standard methods. Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate-buffered saline (“PBS”), thereby removing the urea, exchanging the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 μ/ml. [0132]

Example 2

Cloning and Expression of

Galectin

8, 9, 10 and 10SV Protein in a Baculovirus Expression System

The cDNA sequence encoding the [0133] full length galectin 8, 9, 10, or 10SV protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ sequences of the gene:
The 5′ [0134] galectin 8 oligonucleotide primer has the sequence 5′cgc CCC GGG GCCTATGTCCCCGCAC 3′ (SEQ ID NO:48) containing the underlined SmaI restriction site and nucleotides 55 to 70 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO: 1).
The 3′ [0135] galectin 8 primer has the sequence 5′ cgc GGT ACC TTAGATCTGG ACATAGGAC 3′=0 (SEQ ID NO:49) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:1).
The 5′ [0136] galectin 9 oligonucleotide primer has the sequence 5′ cgc CCC GGG GCCTTCAGCGGTTCCCAG 3′ (SEQ ID NO:50) containing the underlined SmaI restriction site and nucleotides 19 to 36 of the galectin 9 protein coding sequence in FIGS. 2A-2B (SEQ ID NO:3).
The 3′ [0137] galectin 9 primer has the sequence 5′ cgc GGT ACC CAGGGTTGG AAAGGCTG 3′ (SEQ ID NO:51) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1029 to 1045 of the galectin 9 protein coding sequence in FIGS. 2A-2B (SEQ ID NO:3).
The 5′ [0138] galectin 10 oligonucleotide primer has the sequence 5′ cgc CCC GGG TTGTCCTTAAACAACCTAC 3′ (SEQ ID NO:52) containing the underlined SmaI restriction site and nucleotides 124-142 of the galectin 10 protein coding sequence in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ [0139] galectin 10 primer has the sequence 5′ cgc GGT ACC CACA GAAGCCATTCTG 3′ (SEQ ID NO:53) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ galectin 10SV primer has the [0140] sequence 5′ CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3′ (SEQ ID NO:54) containing the underlined Asp718 restriction site followed by nucleotides complementary to the 3′ end of the galectin 10SV protein coding sequence in FIGS. 4A-4B (SEQ ID NO:7).
An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., [0141] J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” [0142] BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with XbaI and again is purified on a 1% agarose gel. This fragment is designated herein F2.
The vector pA2-GP is used to express the [0143] galectin 8, 9, 10, or 10SV protein in the baculovirus expression system, using standard methods, as described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., [0144] Virology 170:31-39, among others.
The plasmid is digested with the restriction enzyme SmaI and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” [0145] BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein “V2”.
Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. [0146] E. coli HB101 cells are transformed with ligation mix and spread on culture plates. Bacteria are identified that contain the plasmid with the human galectin 8, 9, 10, or 10SV gene by digesting DNA from individual colonies using XbaI and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacgalectin 8, 9, 10, or 10SV.
5 μg of the [0147] plasmid pBacgalectin 8, 9, 10, or 10SV is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBaegalectin 8, 9, 10, or 10SV are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27° C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27° C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). [0148]
Four days after serial dilution, the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C. A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-[0149] galectin 8, 9, 10, or 10SV.
Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-[0150] galectin 8, 9, 10, or 10SV at a multiplicity of infection (“MOI”) of about 2 (about 1 to about 3). Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.

Example 3

Cloning and Expression in Mammalian Cells

Most of the vectors used for the transient expression of the [0151] galectin 8, 9, 10, or 10SV protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.
A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular signals can also be used (e.g., human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human HeLa, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, [0152] Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells. [0153]
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., [0154] Biochem. J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
The expression vectors pC1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 4384470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., [0155] Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene.

Example 3(a)

Cloning and Expression in COS Cells

The expression plasmid, [0156] pgalectin 8, 9, 10, or 10SV HA, is made by cloning a cDNA encoding galectin 8, 9, 10, or 10SV into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (1) an [0157] E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
A DNA fragment encoding the [0158] galectin 8, 9, 10, or 10SV protein and an HA tag fused in frame to its 3′ end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37:767-778 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows. The [0159] galectin 8, 9, 10, or 10SV cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 8, 9, 10, or 10SV in E. coli. To facilitate detection, purification and characterization of the expressed galectin 8, 9, 10, or 10SV, one of the primers contains a hemagglutinin tag (“HA tag”) as described above.
Suitable primers include the following, which are used in this example. The 5′ [0160] galectin 8 primer has the sequence 5′cgc CCC GGG gcc atc ATG GCCTATGTCCCCG 3′ (SEQ ID NO:55) containing the underlined SmaI restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ ID NO: 1).
The 3′ [0161] galectin 8 primer has the sequence 5′ cgc GGT ACC TTAGAT CTGGACATAGGAC 3′ (SEQ ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (SEQ ID NO: 1).
The 5′ [0162] galectin 9 primer has the sequence 5′ cgc CCC GGG gcc atc ATGGCCTTCAGCGGTTC 3′ (SEQ ID NO:57) containing the underlined SmaI restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIGS. 2A-2B (SEQ ID NO:3).
The 3′ [0163] galectin 9 primer has the sequence 5′ cgc GGT ACC CAGGGTT GGAAAGGCTG 3′ (SEQ ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIGS. 2A-2B (SEQ ID NO:3), including the stop codon.
The 5′ [0164] galectin 10 and 10SV primer has the sequence 5′ cgc CCC GGG gcc atc ATGATGTTGTCCTTAAAC 3′ (SEQ ID NO:59) containing the underlined SmaI restriction enzyme site followed by nucleotide sequence 118-135 of FIGS. 3A-3B (SEQ ID NO:5).
The 3′ [0165] galectin 10 primer has the sequence 5′ cgc GGT ACC CACAG AAGCCATTCTG 3′ (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ galectin 10SV primer has the [0166] sequence 5′ CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3′ (SEQ ID NO:54) containing the Asp718 restriction followed by nucleotides complementary to the 3′ end of the galectin 10SV coding sequence set out in FIGS. 4A-4B (SEQ ID NO:7).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with HindIII and XhoI and then ligated. The ligation mixture is transformed into [0167] E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the galectin 8, 9, 10, or 10SV-encoding fragment.
For expression of [0168] recombinant galectin 8, 9, 10, or 10SV, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under conditions for expression of galectin 8, 9, 10, or 10SV by the vector.
Expression of the [0169] galectin 8, 9, 10, or 10SV HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing ³⁵S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.

Example 3(b)

Cloning and Expression in CHO Cells

The vector pC1 is used for the expression of [0170] galectin 8, 9, 10, or 10SV protein. Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually co-amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
Plasmid pC1 contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March [0171] 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, PvuII, and NruI. Behind these cloning sites the plasmid contains translational stop codons in all three reading frames followed by the 3′ intron and the polyadenylation site of the rat preproinsulin gene. Other high efficient promoters can also be used for the expression, e.g., the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate. [0172]
The plasmid pC1 is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel. [0173]
The DNA [0174] sequence encoding galectin 8, 9, or 10SV, ATCC Deposit Nos. 97732, 97733 and 97734, respectively, is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ sequences of the gene. The galectin 10 sequence is similarly amplified from a human endometrial tumor or human fetal heart cDNA library.
The 5′ [0175] galectin 8 primer has the sequence 5′ cgcCCCGGGgccatcATG GCCTATGTCCCCG 3′ (SEQ ID NO:55) containing the underlined SmaI restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ ID NO: 1). Inserted into an expression vector, as described below, the 5′ end of the amplified fragment encoding human galectin 8 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
The 3′ [0176] galectin 8 primer has the sequence 5′ cgc GGT ACC TTAGAT CTGGACATAGGAC 3′ (SEQ ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (SEQ ID NO: 1).
The 5′ [0177] galectin 9 primer has the sequence 5′ cgc CCC GGG gcc atc ATGGCCTTCAGCGGTTC 3′ (SEQ ID NO:57) containing the underlined SmaI restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIGS. 2A-2B (SEQ ID NO:3). Inserted into an expression vector, as described below, the 5′ end of the amplified fragment encoding human galectin 9 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
The 3′ [0178] galectin 9 primer has the sequence 5′ cgc GGT ACC CAGGGTT GGAAAGGCTG 3′ (SEQ ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIGS. 2A-2B (SEQ ID NO:3), including the stop codon.
The 5′ [0179] galectin 10 and 10SV primer has the sequence 5′ cgc CCC GGG gcc atc ATGATGTTGTCCTTAAAC 3′ (SEQ ID NO:59) containing the underlined SmaI restriction enzyme site followed by nucleotide sequence 118-135 of FIGS. 3A-3B (SEQ ID NO:5). Inserted into an expression vector, as described below, the 5′ end of the amplified fragment encoding human galectin 10 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
The 3′ [0180] galectin 10 primer has the sequence 5′ cgcGGTACC CACAG AAGCCATTCTG 3′ (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIGS. 3A-3B (SEQ ID NO:5).
The 3′ galectin 10SV primer has the [0181] sequence 5′ CGCGGTACC CTA TGCAACTTTATAAAATATTCC 3′ (SEQ ID NO:54) containing the Asp718 restriction followed by nucleotides complementary to the 3′ end of the galectin 10SV coding sequence set out in FIGS. 4A-4B (SEQ ID NO:7).
The amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonucleases SmaI and Asp718 and then purified again on a 1% agarose gel. [0182]
The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. [0183] E. coli HB101 cells are then transformed and bacteria identified that contained the plasmid pC1 inserted in the correct orientation using the restriction enzyme SmaI. The sequence of the inserted gene is confirmed by DNA sequencing.
Transfection of CHO-DHFR-Cells [0184]
Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. Five μg of the expression plasmid C1 are cotransfected with 0.5 μg of the plasmid pSVneo using the lipofecting method (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure is repeated until clones grow at a concentration of 100 μM. [0185]
The expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE. [0186]

Example 4

Tissue Distribution of Protein Expression

Northern blot analysis is carried out to examine [0187] galectin 8, 9, 10, or 10SV gene expression in human tissues, using methods described by, among others, Sambrook et al., cited above. A cDNA probe containing the entire nucleotide sequence of the galectin 8, 9, 10, or 10SV protein (SEQ ID NO:1, 3, 5, or 7, respectively) is labeled with ³²P using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for galectin 8, 9, 10, or 10SV mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) are obtained from Clontech and are examined with labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70° C. overnight, and films developed according to standard procedures. [0188]
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. [0189]
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. [0190]
The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. [0191]
1 60 1 1138 DNA Homo sapiens CDS (52)..(1020) 1 ttcggcacga gagctcttct cacaggacca gccactagcg cacctcgagc g atg gcc 57 Met Ala 1 tat gtc ccc gca ccg ggc tac cag ccc acc tac aac ccg acg ctg cct 105 Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr Leu Pro 5 10 15 tac tac cag ccc atc ccg ggc ggg ctc aac gtg gga atg tct gtt tac 153 Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser Val Tyr 20 25 30 atc caa gga gtg gcc agc gag cac atg aag cgg ttc ttc gtg aac ttt 201 Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val Asn Phe 35 40 45 50 gtg gtt ggg cag gat ccg ggc tca gac gtc gcc ttc cac ttc aat ccg 249 Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe Asn Pro 55 60 65 cgg ttt gac ggc tgg gac aag gtg gtc ttc aac acg ttg cag ggc ggg 297 Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln Gly Gly 70 75 80 aag tgg ggc agc gag gag agg aag agg agc atg ccc ttc aaa aag ggt 345 Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys Lys Gly 85 90 95 gcc gcc ttt gag ctg gtc ttc ata gtc ctg gct gag cac tac aag gtg 393 Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr Lys Val 100 105 110 gtg gta aat gga aat ccc ttc tat gag tac ggg cac cgg ctt ccc cta 441 Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu Pro Leu 115 120 125 130 cag atg gtc acc cac ctg caa gtg gat ggg gat ctg caa ctt caa tca 489 Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu Gln Ser 135 140 145 atc aac ttc atc gga ggc cag ccc ctc cgg ccc cag gga ccc ccg atg 537 Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro Pro Met 150 155 160 atg cca cct tac cct ggt ccc gga cat tgc cat caa cag ctg aac agc 585 Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu Asn Ser 165 170 175 ctg ccc acc atg gaa gga ccc cca acc ttc aac ccg cct gtg cca tat 633 Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val Pro Tyr 180 185 190 ttc ggg agg ctg caa gga ggg ctc aca gct cga aga acc atc atc atc 681 Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile Ile Ile 195 200 205 21 0 aag ggc tat gtg cct ccc aca ggc aag agc ttt gct atc aac ttc aag 729 Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn Phe Lys 215 220 225 gtg ggc tcc tca ggg gac ata gct ctg cac att aat ccc cgc atg ggc 777 Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg Met Gly 230 235 240 aac ggt acc gtg gtc cgg aac agc ctt ctg aat ggc tcg tgg gga tcc 825 Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp Gly Ser 245 250 255 gag gag aag aag atc acc cac aac cca ttt ggt ccc gga cag ttc ttt 873 Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln Phe Phe 260 265 270 gat ctg tcc att cgc tgt ggc ttg gat cgc ttc aag gtt tac gcc aat 921 Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr Ala Asn 275 280 285 290 ggc cag cac ctc ttt gac ttt gcc cat cgc ctc tcg gcc ttc cag agg 969 Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe Gln Arg 295 300 305 gtg gac aca ttg gaa atc cag ggt gat gtc acc ttg tcc tat gtc cag 1017 Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr Val Gln 310 315 320 atc taatctattc ctggggccat aactcatggg aaaacagaat tatcccctag 1070 Ile gactcctttc taagccccta ataaaatgtc tgagggtgtc tcatgaaaaa aaaaaaaaaa 1130 aaaaaaaa 1138 2 323 PRT Homo sapiens 2 Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr 1 5 10 15 Leu Pro Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser 20 25 30 Val Tyr Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val 35 40 45 Asn Phe Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe 50 55 60 Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln 65 70 75 80 Gly Gly Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys 85 90 95 Lys Gly Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr 100 105 110 Lys Val Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125 Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu 130 135 140 Gln Ser Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro 145 150 155 160 Pro Met Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu 165 170 175 Asn Ser Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val 180 185 190 Pro Tyr Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile 195 200 205 Ile Ile Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn 210 215 220 Phe Lys Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg 225 230 235 240 Met Gly Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp 245 250 255 Gly Ser Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln 260 265 270 Phe Phe Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr 275 280 285 Ala Asn Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe 290 295 300 Gln Arg Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr 305 310 315 320 Val Gln Ile 3 1545 DNA Homo sapiens CDS (16)..(948) 3 agaggcggcg gagag atg gcc ttc agc ggt tcc cag gct ccc tac ctg agt 51 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser 1 5 10 cca gct gtc ccc ttt tct ggg act att caa gga ggt ctc cag gac gga 99 Pro Ala Val Pro Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly 15 20 25 ctt cag atc act gtc aat ggg acc gtt ctc agc tcc agt gga acc agg 147 Leu Gln Ile Thr Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg 30 35 40 ttt gct gtg aac ttt cag act ggc ttc agt gga aat gac att gcc ttc 195 Phe Ala Val Asn Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe 45 50 55 60 cac ttc aac cct cgg ttt gaa gat gga ggg tac gtg gtg tgc aac acg 243 His Phe Asn Pro Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr 65 70 75 agg cag aac gga agc tgg ggg ccc gag gag agg aag aca cac atg cct 291 Arg Gln Asn Gly Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro 80 85 90 ttc cag aag ggg atg ccc ttt gac ctc tgc ttc ctg gtg cag agc tca 339 Phe Gln Lys Gly Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser 95 100 105 gat ttc aag gtg atg gtg aac ggg atc ctc ttc gtg cag tac ttc cac 387 Asp Phe Lys Val Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His 110 115 120 cgc gtg ccc ttc cac cgt gtg gac acc atc tcc gtc aat ggc tct gtg 435 Arg Val Pro Phe His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val 125 130 135 140 cag ctg tcc tac atc agc ttc cag acc cag aca gtc atc cac aca gtg 483 Gln Leu Ser Tyr Ile Ser Phe Gln Thr Gln Thr Val Ile His Thr Val 145 150 155 cag agc gcc cct gga cag atg ttc tct act ccc gcc atc cca cct atg 531 Gln Ser Ala Pro Gly Gln Met Phe Ser Thr Pro Ala Ile Pro Pro Met 160 165 170 atg tac ccc cac ccc gcc tat ccg atg cct ttc atc acc acc att ctg 579 Met Tyr Pro His Pro Ala Tyr Pro Met Pro Phe Ile Thr Thr Ile Leu 175 180 185 gga ggg ctg tac cca tcc aag tcc atc ctc ctg tca ggc act gtc ctg 627 Gly Gly Leu Tyr Pro Ser Lys Ser Ile Leu Leu Ser Gly Thr Val Leu 190 195 200 ccc agt gct cag agg ttc cac atc aac ctg tgc tct ggg aac cac atc 675 Pro Ser Ala Gln Arg Phe His Ile Asn Leu Cys Ser Gly Asn His Ile 205 210 215 220 gcc ttc cac ctg aac ccc cgt ttt gat gag aat gct gtg gtc cgc aac 723 Ala Phe His Leu Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn 225 230 235 acc cag atc gac aac tcc tgg ggg tct gag gag cga agt ctg ccc cga 771 Thr Gln Ile Asp Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg 240 245 250 aaa atg ccc ttc gtc cgt ggc cag agc ttc tca gtg tgg atc ttg tgt 819 Lys Met Pro Phe Val Arg Gly Gln Ser Phe Ser Val Trp Ile Leu Cys 255 260 265 gaa gct cac tgc ctc aag gtg gcc gtg gat ggt cag cac ctg ttt gaa 867 Glu Ala His Cys Leu Lys Val Ala Val Asp Gly Gln His Leu Phe Glu 270 275 280 tac tac cat cgc ctg agg aac ctg ccc acc atc aac aga ctg gaa gtg 915 Tyr Tyr His Arg Leu Arg Asn Leu Pro Thr Ile Asn Arg Leu Glu Val 285 290 295 300 ggg ggc gac atc cag ctg acc cat gtg cag aca taggcggctt cctggccctg 968 Gly Gly Asp Ile Gln Leu Thr His Val Gln Thr 305 310 gggccggggg ctggggtgtg gggcagtctg ggtcctctca tcatccccac ttcccaggcc 1028 cagcctttcc aaccctgcct gggatctggg ctttaatgca gaggccatgt ccttgtctgg 1088 tcctgcttct ggctacagcc accctggaac ggagaaggca gctgacgggg attgccttcc 1148 tcagccgcag cagcacctgg ggctccagct gctggaaatc ctaccatccc aggaggcagg 1208 cacagccagg gagaggggag gagtgggcag tgaagatgaa gccccatgct cagtcccctc 1268 ccatccccca cgcagctcca ccccagtccc aagccaccag ctgtctgctc ctggtgggag 1328 gtggcctcct cagcccctcc tctctgacct ttaacctcac tctcaccttg caccgtgcac 1388 caacccttca cccctcctgg aaagcaggcc tgatggcttc ccactggcct ccaccacctg 1448 accagagtgt tctcttcaga ggactggctc ctttcccagt gtccttaaaa taaagaaatg 1508 aaaatgcttg ttggcaaaaa aaaaaaaaaa aaaaaaa 1545 4 311 PRT Homo sapiens 4 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15 Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30 Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45 Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60 Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly 65 70 75 80 Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95 Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110 Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125 His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140 Ile Ser Phe Gln Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro 145 150 155 160 Gly Gln Met Phe Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His 165 170 175 Pro Ala Tyr Pro Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr 180 185 190 Pro Ser Lys Ser Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln 195 200 205 Arg Phe His Ile Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu 210 215 220 Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp 225 230 235 240 Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe 245 250 255 Val Arg Gly Gln Ser Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys 260 265 270 Leu Lys Val Ala Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg 275 280 285 Leu Arg Asn Leu Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile 290 295 300 Gln Leu Thr His Val Gln Thr 305 310 5 1479 DNA Homo sapiens CDS (118)..(1068) 5 acaccagtct ttggggccag tgcctcagtt tcaatccagg taacctttaa atgaaacttg 60 cctaaaatct taggtcatac acagaagaga ctccaatcga caagaagctg gaaaaga 117 atg atg ttg tcc tta aac aac cta cag aat atc atc tat aac ccg gta 165 Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val 1 5 10 15 atc ccg ttt gtt ggc acc att cct gat cag ctg gat cct gga act ttg 213 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 att gtg ata cgt ggg cat gtt cct agt gac gca gac aga ttc cag gtg 261 Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 gat ctg cag aat ggc agc agt gtg aaa cct cga gcc gat gtg gcc ttt 309 Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att gtt tgc aat act 357 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc tat gac acg cct 405 Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 ttc aaa aga gaa aag tct ttt gag atc gtg att atg gtg cta aag gac 453 Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 aaa ttc cag gtg gct gta aat gga aaa cat act ctg ctc tat ggc cac 501 Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125 agg atc ggc cca gag aaa ata gac act ctg ggc att tat ggc aaa gtg 549 Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val 130 135 140 aat att cac tca att ggt ttt agc ttc agc tcg gac tta caa agt acc 597 Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr 145 150 155 160 caa gca tct agt ctg gaa ctg aca gag ata gtt aga gaa aat gtt cca 645 Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Val Arg Glu Asn Val Pro 165 170 17 5 aag tct ggc acg ccc cag ctt agc ctg cca ttc gct gca agg ttg aac 693 Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn 180 185 190 acc ccc atg ggc cct gga cga act gtc gtc gtt aaa gga gaa gtg aat 741 Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn 195 200 205 gca aat gcc aaa agc ttt aat gtt gac cta cta gca gga aaa tca aag 789 Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys 210 215 220 gat att gct cta cac ttg aac cca cgc ctg aat att aaa gca ttt gtg 837 Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val 225 230 235 240 aga aat tct ttt ctt caa gag tcc tgg gga gaa gaa gag aga aat att 885 Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile 245 250 25 5 acc gct ttc cca ttt agt cct ggg atg tac ttt gag atg ata att tat 933 Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr 260 265 270 tgt gat gtt aga gaa ttc aag gtt gca gta aat ggc gta cac agc ctg 981 Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu 275 280 285 gag tac aaa cac aga ttt aaa gag ctc agc agt att gac acg ctg gaa 1029 Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu 290 295 300 att aat gga gac atc cac tta ctg gaa gta agg agc tgg tagcctacct 1078 Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp 305 310 315 acacagctgc tacaaaaacc aaaatacaga atggcttctg tgatactggc cttgctgaaa 1138 cgcatctcac tgtcattcta ttgtttatat tgttaaaatg agcttgtgca ccattaggtc 1198 ctgctgggtg ttctcagtcc ttgccatgaa gtatggtggt gtctagcact gaatggggaa 1258 actgggggca gcaacactta tagccagtta aagccactct gccctctctc ctactttggc 1318 tgactcttca agaatgccat tcaacaagta tttatggagt cctactatat acagtagcta 1378 acatgtattg agcacagatt tttttggtaa acctgtgagg gctagggtat atccttggga 1438 acaaaccaga atgtcctgtc ccttgaaaaa aaaaaaaaaa a 1479 6 317 PRT Homo sapiens 6 Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val 1 5 10 15 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125 Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val 130 135 140 Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr 145 150 155 160 Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Val Arg Glu Asn Val Pro 165 170 175 Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn 180 185 190 Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn 195 200 205 Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys 210 215 220 Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val 225 230 235 240 Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile 245 250 255 Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr 260 265 270 Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu 275 280 285 Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu 290 295 300 Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp 305 310 315 7 1936 DNA Homo sapiens CDS (118)..(717) 7 acaccagtct ttggggccag tgcctcagtt tcaatccagg taacctttaa atgaaacttg 60 cctaaaatct taggtcatac acagaagaga ctccaatcga caagaagctg gaaaaga 117 atg atg ttg tcc tta aac aac cta cag aat atc atc tat aac ccg gta 165 Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val 1 5 10 15 atc ccg ttt gtt ggc acc att cct gat cag ctg gat cct gga act ttg 213 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 att gtg ata cgt ggg cat gtt cct agt gac gca gac aga ttc cag gtg 261 Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 gat ctg cag aat ggc agc agc atg aaa cct cga gcc gat gtg gcc ttt 309 Asp Leu Gln Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att gtt tgc aat act 357 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc tat gac acg cct 405 Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 ttc aaa aga gaa aag tct ttt gag atc gtg att atg gtg ctg aag gac 453 Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 aaa ttc cag gtg gct gta aat gga aaa cat act ctg ctc tat ggc cac 501 Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125 agg atc ggc cca gag aaa ata gac act ctg ggc att tat ggc aaa gtg 549 Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val 130 135 140 aat att cac tca att ggt ttt agc ttc agc tcg gac tta caa agt acc 597 Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr 145 150 155 160 caa gca tct agt ctg gaa ctg aca gag ata agt aga gaa aat gtt cca 645 Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro 165 170 175 aag tct ggc acg ccc cag ctt gtg agt att ttt gcc tgg gtt att tca 693 Lys Ser Gly Thr Pro Gln Leu Val Ser Ile Phe Ala Trp Val Ile Ser 180 185 190 tgt gga ata ttt tat aaa gtt gca tagaaaatga acagtttaaa ccgtggaggg 747 Cys Gly Ile Phe Tyr Lys Val Ala 195 200 cagcttcatt cattccattc cttactgtag aactgtttcc ctacagccta gtaatagagg 807 aggagacatt tctaaaatcg cacccagaac tgtctacacc aagagcaaag attcgactgt 867 caatcacact ttgacttgca ccaaaatacc acctatgaac tatgtgtcaa agggtttgaa 927 gagcaccaaa ttttcttaac tctatataaa aattaagttg taatgagctg ttacgagtaa 987 cctgtatcca caatagaggc ccaaagcagc cccctctgca tttgtgtgcc gtccctggac 1047 ggattcgaga gtcaaccagg cctgcctctg agccatttct gtgtatttcc tcagcacctc 1107 cctgcttggc tgcttcccct tcaggcagaa cacagtactg cctcagaccc caggcacagg 1167 gggccttcct ggcgtgtttc actcatacag agggcatcgg gtcccaccct gtcactcatt 1227 tcatcgtcta aaatgtaatc atgtgtgttt gcttcgagcc agggacagtg ctgctgcagg 1287 ggacccagct gggaccaagg cagactgtct ctcccctcct gggatttaca gggtcatggc 1347 tctgaaacat tccgtagtgt tctttggaca cgagttttcc ctggagatcg ctttctgcag 1407 gctcttggtc ctgactgtgg cttcttttca gaggctgcca tttcgctgca aggttgaaca 1467 cccccatggg ccctggacga actgtcgtcg ttaaaggaga agtgaatgca aatgccaaaa 1527 gctttaatgt tgacctacta gcaggaaaat caaaggatat tgctctacac ttgaacccac 1587 gcctgaatat taaagcattt gtaagaaatt cttttcttca ggagtcctgg ggagaagaag 1647 agagaaatat tacctctttc ccatttagtc ctgggatgta ctttgagatg ataatttatt 1707 gtgatgttag agaattcaag gttgcagtaa atggcgtaca cagcctggag tacaaacaca 1767 gatttaaaga gctcagcagt attgacacgc tggaaattaa tggagacatc cacttactgg 1827 aagtaaggag ctggtagcct acctacacag ctgctacaaa aaccaaaata cagaatggct 1887 tctgtgatac tggccttgct gaaacgcaaa aaaaaaaaaa aaaaaaaaa 1936 8 200 PRT Homo sapiens 8 Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val 1 5 10 15 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 Asp Leu Gln Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125 Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val 130 135 140 Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr 145 150 155 160 Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro 165 170 175 Lys Ser Gly Thr Pro Gln Leu Val Ser Ile Phe Ala Trp Val Ile Ser 180 185 190 Cys Gly Ile Phe Tyr Lys Val Ala 195 200 9 132 PRT Homo sapiens 9 Met Thr Gly Glu Leu Glu Val Lys Asn Met Asp Met Lys Pro Gly Ser 1 5 10 15 Thr Leu Lys Ile Thr Gly Ser Ile Ala Asp Gly Thr Asp Gly Phe Val 20 25 30 Ile Asn Leu Gly Gln Gly Thr Asp Lys Leu Asn Leu His Phe Asn Pro 35 40 45 Arg Phe Ser Glu Ser Thr Ile Val Cys Asn Ser Leu Asp Gly Ser Asn 50 55 60 Trp Gly Gln Glu Gln Arg Glu Asp His Leu Cys Phe Ser Pro Gly Ser 65 70 75 80 Glu Val Lys Phe Thr Val Thr Phe Glu Ser Asp Lys Phe Lys Val Lys 85 90 95 Leu Pro Asp Gly His Glu Leu Thr Phe Pro Asn Arg Leu Gly His Ser 100 105 110 His Leu Ser Tyr Leu Ser Val Arg Gly Gly Phe Asn Met Ser Ser Phe 115 120 125 Lys Leu Lys Glu 130 10 250 PRT Homo sapiens 10 Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 25 30 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35 40 45 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr His 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro 65 70 75 80 Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser 85 90 95 Ala Pro Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160 Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His 195 200 205 Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215 220 Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile 245 250 11 324 PRT Rat 11 Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr 1 5 10 15 Leu Pro Tyr Lys Arg Pro Ile Pro Gly Gly Leu Ser Val Gly Met Ser 20 25 30 Ile Tyr Ile Gln Gly Ile Ala Lys Asp Asn Met Arg Arg Phe His Val 35 40 45 Asn Phe Ala Val Gly Gln Asp Glu Gly Ala Asp Ile Ala Phe His Phe 50 55 60 Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Met Gln 65 70 75 80 Ser Gly Gln Trp Gly Lys Glu Glu Lys Lys Lys Ser Met Pro Phe Gln 85 90 95 Lys Gly His His Phe Glu Leu Val Phe Met Val Met Ser Glu His Tyr 100 105 110 Lys Val Val Val Asn Gly Thr Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125 Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Glu Leu 130 135 140 Gln Ser Ile Asn Phe Leu Gly Gly Gln Pro Ala Ala Ser Gln Tyr Pro 145 150 155 160 Gly Thr Met Thr Ile Pro Ala Tyr Pro Ser Ala Gly Tyr Asn Pro Pro 165 170 175 Gln Met Asn Ser Leu Pro Val Met Ala Gly Pro Pro Ile Phe Asn Pro 180 185 190 Pro Val Pro Tyr Val Gly Thr Leu Gln Gly Gly Leu Thr Ala Arg Arg 195 200 205 Thr Ile Ile Ile Lys Gly Tyr Val Leu Pro Thr Ala Lys Asn Leu Ile 210 215 220 Ile Asn Phe Lys Val Gly Ser Thr Gly Asp Ile Ala Phe His Met Asn 225 230 235 240 Pro Arg Ile Gly Asp Cys Val Val Arg Asn Ser Tyr Met Asn Gly Ser 245 250 255 Trp Gly Ser Glu Glu Arg Lys Ile Pro Tyr Asn Pro Phe Gly Ala Gly 260 265 270 Gln Phe Phe Asp Leu Ser Ile Arg Cys Gly Thr Asp Arg Phe Lys Val 275 280 285 Phe Ala Asn Gly Gln His Leu Phe Asp Phe Ser His Arg Phe Gln Ala 290 295 300 Phe Gln Arg Val Asp Met Leu Glu Ile Lys Gly Asp Ile Thr Leu Ser 305 310 315 320 Tyr Val Gln Ile 12 145 PRT Rat 12 Met Ser Ser Phe Ser Thr Gln Thr Pro Tyr Pro Asn Leu Ala Val Pro 1 5 10 15 Phe Phe Thr Ser Ile Pro Asn Gly Leu Tyr Pro Ser Lys Ser Ile Val 20 25 30 Ile Ser Gly Val Val Leu Ser Asp Ala Lys Arg Phe Gln Ile Asn Leu 35 40 45 Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro Arg Phe Asp Glu 50 55 60 Asn Ala Val Val Arg Asn Thr Gln Ile Asn Asn Ser Trp Gly Pro Glu 65 70 75 80 Glu Arg Ser Leu Pro Gly Ser Met Pro Phe Ser Arg Gly Gln Arg Phe 85 90 95 Ser Val Trp Ile Leu Cys Glu Gly His Cys Phe Lys Val Ala Val Asp 100 105 110 Gly Gln His Ile Cys Glu Tyr Ser His Arg Leu Met Asn Leu Pro Asp 115 120 125 Ile Asn Thr Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val Glu 130 135 140 Thr 145 13 136 PRT Homo sapiens 13 Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro 1 5 10 15 Gly Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg 20 25 30 Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala 35 40 45 Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser 50 55 60 Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro 65 70 75 80 Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp 85 90 95 Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His 100 105 110 Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115 120 125 Gln Leu Asp Ser Val Arg Ile Phe 130 135 14 262 PRT Rat 14 Met Ala Asp Gly Phe Ser Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala 20 25 30 Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln Ala 35 40 45 Pro Pro Gly Gly Tyr Pro Gly Gln Ala Pro Pro Ser Ala Tyr Pro Gly 50 55 60 Pro Thr Gly Pro Ser Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr 65 70 75 80 Pro Gly Pro Thr Ala Pro Gly Ala Phe Pro Gly Gln Pro Gly Gly Pro 85 90 95 Gly Ala Tyr Pro Ser Ala Pro Gly Ala Tyr Pro Ser Ala Pro Gly Ala 100 105 110 Tyr Pro Ala Thr Gly Pro Phe Gly Ala Pro Thr Gly Pro Leu Thr Val 115 120 125 Pro Tyr Asp Met Pro Leu Pro Gly Gly Val Met Pro Arg Met Leu Ile 130 135 140 Thr Ile Ile Gly Thr Val Lys Pro Asn Ala Asn Ser Ile Thr Leu Asn 145 150 155 160 Phe Lys Lys Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg Phe Asn 165 170 175 Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp Asn Asn 180 185 190 Trp Gly Arg Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu Ser Gly Lys 195 200 205 Pro Phe Lys Ile Gln Val Leu Val Glu Ala Asp His Phe Lys Val Ala 210 215 220 Val Asn Asp Val His Leu Leu Gln Tyr Asn His Arg Met Lys Asn Leu 225 230 235 240 Arg Glu Ile Ser Gln Leu Gly Ile Ile Gly Asp Ile Thr Leu Thr Ser 245 250 255 Ala Ser His Ala Met Ile 260 15 316 PRT Rat 15 Met Leu Ser Leu Ser Asn Leu Gln Asn Ile Ile Tyr Asn Pro Thr Ile 1 5 10 15 Pro Tyr Val Ser Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30 Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45 Phe Gln His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Thr His Asp Met Pro Phe 85 90 95 Arg Lys Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asn Lys 100 105 110 Phe His Val Ala Val Asn Gly Lys His Ile Leu Leu Tyr Ala His Arg 115 120 125 Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Ile Phe Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu 145 150 155 160 Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Lys Glu Asn Ile Gln Lys 165 170 175 Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190 Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr 195 200 205 Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ala Val 290 295 300 Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp 305 310 315 16 135 PRT Homo sapiens 16 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys 50 55 60 Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn 85 90 95 Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Asp 130 135 17 316 PRT Rat 17 Met Leu Ser Leu Ser Asn Leu Gln Asn Ile Ile Tyr Asn Pro Thr Ile 1 5 10 15 Pro Tyr Val Ser Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30 Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45 Phe Gln His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Thr His Asp Met Pro Phe 85 90 95 Arg Lys Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asn Lys 100 105 110 Phe His Val Ala Val Asn Gly Lys His Ile Leu Leu Tyr Ala His Arg 115 120 125 Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Ile Phe Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu 145 150 155 160 Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Lys Glu Asn Ile Gln Lys 165 170 175 Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190 Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr 195 200 205 Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ala Val 290 295 300 Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp 305 310 315 18 499 DNA Homo sapiens misc_feature (21) n is A, C, T, or G 18 aattcggcac gagagctctt ntcacaggac cagccactag cgcanctcga gcgatggcct 60 atgtccccgc accgggctac cagcccacct acaacccgac gctgccttac taccagccca 120 tcccgggcgg gctcaacgtg ggaatgtctg tttacatcca aggagtggcc agcgagcaca 180 tgaagcggtt cttcgtgaac tttgtggttg ggcaggatcc gggctcagac gtcgccttcc 240 acttcaatcc gcggtttgac ggctgggaca aggtggtctt caacacgttg cagggcggga 300 agtggggcag cgaggagagg aagaggagca tgcccttcaa aaagggtgcc gcctttgagc 360 ttggtcttca tagtcctngg ttgagcacta caaggtngtn gtaaatggaa tccctctatg 420 antaggggac cgntttccct anaattgtaa ccanctnnaa ttgatgggnn tcaattaatn 480 atcaattatt ggnggcanc 499 19 391 DNA Homo sapiens misc_feature (175) n is A, C, T, or G 19 agtggatggg gatctgcaac ttcaatcaat caacttcatc ggaggccagc ccctccggcc 60 ccagggaccc ccgatgatgc caccttaccc tggtcccgga cattgccatc aacagctgaa 120 cagcctgccc accatggaag gacccccaac cttcaacccg cctgtgccat atttngggag 180 gctgcaagga gggctcacag ctcgaagaac catcatcatc aagggctatg tgcctcccac 240 aggcaagagc tttgctatca acttcaaggt gggctcctca ggggacatag ctctgcacat 300 taatccccgc atgggcaacg gtaccgtggt ccggaacagc cttcttgaat ggttcgtggg 360 gttncgagga gaagaagntc acccacaacc c 391 20 423 DNA Homo sapiens misc_feature (309) n is A, C, T, or G 20 ccggccccag ggacccccga tgatgccacc ttaccctggt cccggacatt gccatcaaca 60 gctgaacagc ctgcccacca tggaaggacc cccaaccttc aacccgcctg tgccatattt 120 cgggaggctg caaggagggc tcacagctcg aagaaccatc atcatcaagg gctatgtgcc 180 tcccacaggc aagagctttg ctatcaactt caaggtgggc tcctcagggg acatagctct 240 gcacattaat ccccgcatgg gcaacggtac cgtggtccgg aacagncttc tgaatggctc 300 gtggggatnc gaggagaagg aaggtcancc acaanccatt ttgtnccgga canttttttt 360 natctgtcca nttggttgtg gtttggatcg tttcaaggtt taaggcaatg gccagaactt 420 ttt 423 21 434 DNA Homo sapiens misc_feature (266) n is A, C, T, or G 21 aattcggcac gagcacaggc aagagctttg ctatcaactt caaggtgggc tcctcagggg 60 acatagctct gcacattaat ccccgcatgg gcaacggtac cgtggtccgg aacagccttc 120 tgaatggctc gtggggatcc gaggagaaga agatcaccca caacccattt ggtcccggac 180 agttctttga tctgtccatt cgctgtggct tggatcgctt caaggtttac ggcaatggcc 240 agcacctctt tgactttgcc catcgnctct cggccttcca gagggtggac anattngaaa 300 tccagggtga tgtcaacttg tcctatgtcc agatctaatc ttattcctgg ggccataatt 360 catgggaaac agattatncn ctagggttct tttttaggcc ctaataaaat gtcttagggg 420 ggtaaaaaaa aaaa 434 22 354 DNA Homo sapiens misc_feature (238) n is A, C, T, or G 22 cttcaatccg cggtttgacg gctgggacaa ggtggtcttc aacacgttgc agggcgggaa 60 gtggggcagc gaggagagga agaggagcat gcccttcaaa aagggtgccg cctttaagct 120 ggtcttcata gtcctggctg agcactacaa ggtggtggta aatggaaatc ccttctatga 180 gtacgggcac cggcttcccc tacagatggt cacccacctg caagtggatg gggatctnca 240 acttcaatca atcaacttca tcgggaggnc agcccntccg gccccaggga cccccgatga 300 tgccacctta ccctggtncc ggacattggc catcagcagt tgaacagctg tcca 354 23 329 DNA Homo sapiens misc_feature (244) n is A, C, T, or G 23 gtggtccgga acagccttct gaatggctcg tggggatccg aggagaagaa gatcacccac 60 aacccatttg gtcccggaca gttctttgat ctgtccattc gctgtggctt ggatcgcttc 120 aaggtttacg ccaatggcca gcacctcttt gactttgccc atcgcctctc ggccttccag 180 agggtggaca cattggaaat ccagggtgat gtcaccttgt cctatgtcca gatctaatct 240 attnctgggg ccataactca tgggaaaaca gaattatccc ctaggactcc tttctaaagc 300 ccnctaataa aaangtctga gggtgtctc 329 24 229 DNA Homo sapiens misc_feature (186) n is A, C, T, or G 24 gcgggctcaa cgtgggaatg tctgtttaca tccaaggagt ggccagcgag cacatgaagc 60 ggttcttcgt gaactttgtg gttgggcagg atccgggctc agacgtcgcc ttccacttca 120 atccgcggtt tgacggctgg gacaaggtgg tcttcaacac gttgcagggc gggaagtggg 180 gcagcnagga gaggaagagg agcatgccct tcaaaaaggg tgccgcctt 229 25 194 DNA Homo sapiens misc_feature (102) n is A, C, T, or G 25 ggaagaggag catgcccttc aaaaagggtg ccgcctttaa cctggtnttc atagtcctgg 60 ctgagcacta caaggtggtg gtaaatggaa atcccttcta tnagtacggg caccggcttc 120 ccctacagat ggtcacccac ctgcaagtgg atggggatct gcaacttcat tcattcaact 180 tcatcggagg ccag 194 26 499 DNA Homo sapiens misc_feature (142) n is A, C, T, or G 26 aattccgttc tctactcccg ccatcccacc tataatgtac ccccaccccg cctatccaat 60 gcctttaatc accaccattc tgggagggct gtacccatcc aagtccatcc tcctgtaagg 120 cacttgcctg cccagtgctc anaggttcca catcaacctg tgctctggga aaccacatcg 180 ccttccacct gnaacccccg ttttgaatga gaatgctgtg gtccgcaaca cccagatnga 240 caactcctgg gggtctgagg agcgaagtgt gccccgaaaa atgcccttgg tncgtggcca 300 gaggttntna ggtggatctt gtgtgaagtt caatgngtnc aagtgggcct ggatggtnag 360 nantgtttgn atnattannc tgggnttgng gnaactgngc aannttnaac agatngnagt 420 tgggggggng anantcagnt gnaccgtttt gnagnnatag ggggntttnt tggccttggg 480 ggggggggtt ggggttttg 499 27 376 DNA Homo sapiens misc_feature (21) n is A, C, T, or G 27 cttttgccaa caagcatttt natttcttta ttttaaggac actgggaaag gagccagtcc 60 cctgaagaga acactctggt caggtggtgg aggccagtgg gaagccatca ggcctgcttt 120 ccaggagggg tgaagggttg gtgcacggtg caaggtgaga gtgaaggtta aaggtcagag 180 aggaggggct gaggaggcca ccttccacca ggagcagaca gctggtggct tgggaactgg 240 ggtggagctg cgtgggggat gggaagggga ctgagcatgg ggcttcatct tncactgccc 300 actcctgccc tcttccctgg ctgtgcctgc ctncctggga tggtagggtt tccancantt 360 ggaggcccca ngtgct 376 28 282 DNA Homo sapiens misc_feature (51) n is A, C, T, or G 28 ttcagatcac tgtcaatggg accgttctca gctccagtgg aaccaggttt nctgtgaact 60 ttcagactgg cttcagtgga aataacattg ccttccactt caaccctcgg tttgaagatg 120 gagggtacgt ggtgtgcaca gnaggcagaa cggaagctgg gggcccgagg agaggaagac 180 acacatgcct ttccagaagg ggatgccctt taacctctgc ttcctggtgc agagctcaga 240 tttcaaggtg atggtgaacg ggatcctctt cgtgcagtac tt 282 29 274 DNA Homo sapiens misc_feature (26) n is A, C, T, or G 29 gtgcagagcg cccctggaca gatgtnctct actcccgcca tcccacctat gatgtacccc 60 caccccgcct atccgatgcc tttnaacacc accattctgg gagggctgta cccatccaag 120 atccatcctc ctgtcaggca ctgtcctgcc cagtgctcag aggttccaca tcaacctgtg 180 ctctgggaac cacatcgcct tccacctgaa cccccgtttt gatgagaatg ctgtggtccg 240 caacacccag atcgacaaat tcctgggggg tctt 274 30 342 DNA Homo sapiens misc_feature (21) n is A, C, T, or G 30 cttttgccaa caagcatttt natttcttta ttttaaggac actgggaaag gagccagtcc 60 cctgaagaga acactctggt caggtggtgg aggccagtgg gaagccatca ggcctgcttt 120 ccaggagggg tgaagggttg gtgcacggtg caaggtgaga gtnaaggtta aaggtcagag 180 aggaggggct gaggaggcca ccttccacca ggagcagaca gctggtggct tgggaactgg 240 ggtgggagct gtcgtngggg gatggnaagg ggactgagcc atgggggctt tcatcttnca 300 ctgcccactc ctgccctttt ccctggtttg tgnctgncct tc 342 31 246 DNA Homo sapiens misc_feature (23) n is A, C, T, or G 31 cctgcttctg gctacagcca ccntggaacg gagaaggcag ctgacgggga ttgccttcnt 60 cagccgcagc agcacctggg gctccagctg ctggaatcnt accatcccag gaggcaggca 120 cagccaggga gaggggagga gtgggcagtg aagatnaagc cccatgctca gtcccctccc 180 atcccccacg cagctccacc ccagttccaa gncaccagct gtctgctcct ggtgggaggt 240 ggcctc 246 32 228 DNA Homo sapiens misc_feature (5) n is A, C, T, or G 32 ggcanagcag aggtgtggat cttntntaaa gctcactgcc tcaaggtggc cgtggatggt 60 cagcacctgt ttaaatacta ccatcgcctg aggaacctgc ccaccatcaa cagactggga 120 gtggggggcg aacatccagc tgacccatgt gcagacatag gcggcttcct ggccctgggg 180 cgggggctna gntttggggn agtctgggtc ctntaatnat ccncantt 228 33 161 DNA Homo sapiens misc_feature (32) n is A, C, T, or G 33 ttccctctac aaaggacttc ctagtgggtg tnaaaggcag cggtggccac anaggcggcg 60 gagagatggc cttcagcggt tcccaggctc cctacctgag tccagctgtc cccttttttg 120 ggactattca aggaggtctc caggacggac ttcagatcac t 161 34 306 DNA Homo sapiens misc_feature (33)..(34) n is A, C, T, or G 34 ctctgtgcag ctgtcctaca tcagcttcca ggnnagactg tccacctggc accggtncca 60 ggggcgggga atgcggggng nagcgtagtt gatactgaag ncnctgatgg gtggggcnna 120 agncanatct cctnacccag gtcactctgg gggacaacct ctggcttccc tgtcccagta 180 cctggctgnc nacttctcct ctgtgaactc tganccctcc ttctgtgttt actgtctctg 240 tccggaacaa ctgccttggt ctcccagant gctcaggtga ccctttnttn tttcnaccct 300 tcaatt 306 35 449 DNA Homo sapiens misc_feature (89) n is A, C, T, or G 35 ctcatacaga gggcatcggg tcccaccctg tcactcattt catcgtctaa aatgtaatca 60 tgagtgtttg cttcgagcca gggacagtnc tgctgcaggg gacccagctg ggaccaaggc 120 agactgtctc tcccctcctg ggatttacag ggtcatggct ctgaaacatt ctgtagtgtt 180 ctttgaacac gagttttccc tggagatcgc tttctgcagg cctcttggtc ctgactgtgg 240 cttcttttca gagcctgcca ttcgctgcaa ggttgaacan ccccatgggc cctgggacga 300 actgtcgtcg ttaaaaggag aagtgaatgc aaatgnccaa aaagctttta atgtttgacc 360 tactagcagg aaatcaaagg gtattgcntc ttacaattgn acccaggctg aatattaaag 420 cattttaaag aattcttttt cttcaggag 449 36 265 DNA Homo sapiens misc_feature (48) n is A, C, T, or G 36 ttcaatcctc gtttcaaaag ggccggctgc attgtttgca atactttnat aaatgaaaaa 60 tggggacggg aagagatcac ctatgacacg cctttcaaaa gagaaaagtc ttttnagatc 120 gtaattatgg tgctgaagga caaattccag gtggctgtaa atggaaaaca tactctgctc 180 tatggccaca ggatcggccc agagaaaata gacactctgg gcatttatgg caaagtgaat 240 attcactcaa ttggttttag cttca 265 37 353 DNA Homo sapiens misc_feature (323) n is A, C, T, or G 37 aagccactct gccctctctc ctactttggc tgactcttca agaatgccat tcaacaagta 60 tttatggagt acctactata atacagtagc taacatgtat tgagcacaga ttttttttgg 120 taaaactgtg aggagctagg atatatactt ggtgaaacaa accagtatgt tccctgttct 180 cttgagcttc gactcttctg tgctctattg ctgcgcactg ctttttctac aggcattaca 240 tcaactccta aggggtcctc tggggattag ttaagcagct atttaaatca cccgaaggac 300 acttaattta cagatgacac aantcctttc cccagtgatt caactgttca taa 353 38 234 DNA Homo sapiens misc_feature (12) n is A, C, T, or G 38 gaaacaccag tntttggggc cagtncctca ntttcaatcc aggtaacctt taantgaaac 60 ttgcctaaaa tnttaggtca tacacagaag agactccaat cgacaagaag ctggaaaaga 120 atgatgttgt ccttaaacaa cctacagant atcatctata acccggtaat cccgtttntt 180 ggcaccattc ctgatcagct ggatcctgga actttgattg taatacgtgg gcat 234 39 344 DNA Homo sapiens misc_feature (45) n is A, C, T, or G 39 acacgctgga aattaatgga gacatccact tactggaagt aaggngntgg tagcctacct 60 acacagctgc tacaaaaacc aaaatacaga atggcttctg tgatactggc cttgctgaaa 120 cgcatctcac tgtcattcta ttgtttatat tgttaaaatg agcttgtgca ccattaggtc 180 ctgctgggtg ttctcagtcc ttgccatgaa gtatggtggt gtctagcact gaatggggaa 240 actgggggca gcaacactta tagccagtta aagccactct gccctctctc ctactttggg 300 ctgactcttc aagaatgcca ttcaacaagt atttatgggg tacc 344 40 502 DNA Homo sapiens misc_feature (10) n is A, C, T, or G 40 aattcggcan agcttcaaac ctttgagaca tagttcatag gtggtatttt ggtgcaagtc 60 aaagtgtgat ngacagtcga atntttgctc ttggtgtaga cagttctggg tgcgatttta 120 gaaatgtctg ctcctctatt actaggctgt ngggaaacag ttctacagta aggaatggaa 180 tganatgaag ctgccctcca cggtttaaac tgttcatttt ctatgcaact ttataaaata 240 ttccacatga antaacccag gcaaaaatac ttcacaggct ggggggcgtg gccaganctt 300 tgggaaccta ttgggaaaag gaaaccaaan cacancaatg tttagaaggg ggaaggattt 360 ttagtttatn aatntgaagt nttgggnngt tgctgaggct gaggcctggg ccggnggctt 420 ggggattgtt tccnggttnc cactctggtg nggnnttncc ngggcagttg ggtgntttta 480 tgacgggatt ggtattgtgt tg 502 41 26 DNA synthetic construct 41 cgcccatggc ctatgtcccc gcaccg 26 42 28 DNA synthetic construct 42 cgcaagcttt tagatctgga cataggac 28 43 26 DNA synthetic construct 43 cgcccatggc cttcagcggt tcccag 26 44 26 DNA synthetic construct 44 cgcaagcttc agggttggaa aggctg 26 45 26 DNA synthetic construct 45 cgcccatgct gttgtcctta aacaac 26 46 25 DNA synthetic construct 46 cgcctgcagc acagaagcca ttctg 25 47 33 DNA synthetic construct 47 cgcctgcagc tatgcaactt tataaaatat tcc 33 48 25 DNA synthetic construct 48 cgccccgggg cctatgtccc cgcac 25 49 28 DNA synthetic construct 49 cgcggtacct tagatctgga cataggac 28 50 27 DNA synthetic construct 50 cgccccgggg ccttcagcgg ttcccag 27 51 26 DNA synthetic construct 51 cgcggtaccc agggttggaa aggctg 26 52 28 DNA synthetic construct 52 cgccccgggt tgtccttaaa caacctac 28 53 25 DNA synthetic construct 53 cgcggtaccc acagaagcca ttctg 25 54 33 DNA synthetic construct 54 cgcggtaccc tatgcaactt tataaaatat tcc 33 55 31 DNA synthetic construct 55 cgccccgggg ccatcatggc ctatgtcccc g 31 56 28 DNA synthetic construct 56 cgcggtacct tagatctgga cataggac 28 57 32 DNA synthetic construct 57 cgccccgggg ccatcatggc cttcagcggt tc 32 58 26 DNA synthetic construct 58 cgcggtaccc agggttggaa aggctg 26 59 33 DNA synthetic construct 59 cgccccgggg ccatcatgat gttgtcctta aac 33 60 25 DNA synthetic construct 60 cgcggtaccc acagaagcca ttctg 25

Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 323 in SEQ ID NO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about 1 to about 200 in SEQ ID NO:8;

(b) a nucleotide sequence encoding a polypeptide comprising amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about 311 in SEQ ID NO:4, from about 2 to about 317 in SEQ ID NO:6, or from about 2 to about 200 in SEQ ID NO:8;

(c) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97732, 97733 or 97734;

(d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).

2. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), or (d) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

3. An isolated nucleic acid fragment of the polynucleotide of claim 1, wherein said fragment is selected from the group consisting of:

(a) a nucleotide sequence comprising at least 520 contiguous nucleotides of SEQ ID NO: 1;

(b) a nucleotide sequence comprising at least 460 contiguous nucleotides of SEQ ID NO:3; and

(c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b).

4. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.

5. A recombinant vector produced by the method of claim 4.

6. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 5 into a host cell.

7. A recombinant host cell produced by the method of claim 6.

8. A recombinant method for producing a galectin 8, 9, 10 or 10SV polypeptide, comprising culturing the recombinant host cell of claim 7 under conditions such that said polypeptide is expressed and recovering said polypeptide.

9. An isolated galectin 8, 9, 10, or 10SV polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) amino acids from about 1 to about 323 in SEQ ID NO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about 1 to about 200 in SEQ ID NO:8;

(b) amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about 311 in SEQ ID NO:4, from about 2 to about 317 in SEQ ID NO:6, or from about 2 to about 200 in SEQ ID NO:8;

(c) the amino acid sequence of the galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97732, 97733 or 97734; and

(d) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), or (c).

10. An isolated antibody that binds specifically to a galectin 8, 9, 10, or 10SV polypeptide of claim 9.

11. An isolated nucleic acid molecule comprising a polynucleotide encoding a galectin 8, 9, 10, or 10SV polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has a sequence selected from the group consisting of:

12. An isolated galectin 8, 9, 10, or 10SV polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has a sequence selected from the group consisting of:

13. A method of detecting a galectin 8, 9, 10, or 10SV polypeptide in a sample, comprising:

a) contacting said sample with an antibody according to claim 10, under conditions such that immunocomplexes form, and

b) detecting the presence of said antibody bound to said polypeptide.

15. A method of treatment of a cell growth disorder in a mammal, comprising administering a therapeutically effective amount of the polypeptide of claim 9 to said mammal.

16. The method of claim 15, wherein said disorder is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases.