Abstract:
Peptides which comprise sequences as shown in Seq ID NO:2 or HisGlyTrpSerTypGlyGlyPheLeu; LeuAspGluAsnValHisPhePhe; GluArgHisSerIleArg and PheValIleGlnGluGluPhe which show peptidase ability and have substrate specificity for at least one of the compounds H-Ala-Pro-pNA, H-Gly-Pro-pNA, H-Gly-Pro-pNA ans H-Arg-Pro-pNA, peptides having sequence ID No:7 are also claimed. Nucleic acids, vectors, antibodies and hybridoma cells are also claimed with reference to the above sequences and there abilities.

Description:
FIELD OF INVENTION  
         [0001]    The invention relates to a dipeptidyl peptidase, to a nucleic acid molecule which encodes it, and to uses of the peptidase.  
         BACKGROUND OF THE INVENTION  
         [0002]    The dipeptidyl peptidase (DPP) IV-like gene family is a family of molecules which have related protein structure and function [1-3]. The gene family includes the following molecules: DPPIV (CD26), dipeptidyl amino-peptidase-like protein 6 (DPP6), dipeptidyl amino-peptidase-like protein 8 (DPP8) and fibroblast activation protein (FAP) [1,2,4,5]. Another possible member is DPPIV-β[6].  
           [0003]    The molecules of the DPPIV-like gene family are serine proteases, they are members of the peptidase family S9b, and together with prolyl endopeptidase (S9a) and acylaminoacyl peptidase (S9c), they are comprised in the prolyl oligopeptidase family[5,7].  
           [0004]    DPPIV and FAP both have similar postproline dipeptidyl amino peptidase activity, however, unlike DPPIV, FAP also has gelatinase activity[8,9].  
           [0005]    DPPIV substrates include chemokines such as RANTES, eotaxin, macrophage-derived chemokine and stromal-cell-derived factor 1; growth factors such as glucagon and glucagon-like peptides 1 and 2; neuropeptides including neuropeptide Y and substance P; and vasoactive peptides[10-12].  
           [0006]    DPPIV and FAP also have non-catalytic activity; DPPIV binds adenosine deaminase, and FAP binds to α 3 β 1 , and α 5 β 1  integrin[13-14].  
           [0007]    In view of the above activities, the DPPIV-like family members are likely to have roles in intestinal and renal handling of proline containing peptides, cell adhesion, peptide metabolism, including metabolism of cytokines, neuropeptides, growth factors and chemokines, and immunological processes, specifically T cell stimulation[3,11,12].  
           [0008]    Consequently, the DPPIV-like family members are likely to be involved in the pathology of disease, including for example, tumour growth and biology, type II diabetes, cirrhosis, autoimmunity, graft rejection and HIV infection[3,15-18].  
           [0009]    Inhibitors of DPPIV have been shown to suppress arthritis, and to prolong cardiac allograft survival in animal models in vivo[19,20]. Some DPPIV inhibitors are reported to inhibit HIV infection[21]. It is anticipated that DPPIV inhibitors will be useful in other therapeutic applications including treating diarrhoea, growth hormone deficiency, lowering glucose levels in non insulin dependent diabetes mellitus and other disorders involving glucose intolerance, enhancing mucosal regeneration and as immunosuppressants[3,21-24].  
           [0010]    There is a need to identify members of the DPPIV-like gene family as this will allow the identification of inhibitor(s) with specificity for particular family member(s), which can then be administered for the purpose of treatment of disease. Alternatively, the identified member may of itself be useful for the treatment of disease.  
         SUMMARY OF THE INVENTION  
         [0011]    The present invention seeks to address the above identified need and in a first aspect provides a peptide which comprises the amino acid sequence shown in SEQ ID NO:2. As described herein, the inventors believe that the peptide is a prolyl oligopeptidase and a dipeptidyl peptidase, because it has substantial and significant homology with the amino acid sequences of DPPIV and DPP8. As homology is observed between DPP8, DPPIV and DPP9, it will be understood that DPP9 has a substrate specificity for at least one of the following compounds: H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA.  
           [0012]    The peptide is homologous with human DPPIV and DPP8, and importantly, identity between the sequences of DPPIV and DPP8 and SEQ ID NO: 2 is observed at the regions of DPPIV and DPP8 containing the catalytic triad residues and the two glutamate residues of the β-propeller domain essential for DPPIV enzyme activity. The observation of amino acid sequence homology means that the peptide which has the amino acid sequence shown in SEQ ID NO:2 is a member of the DPPIV-like gene family. Accordingly the peptide is now named and described herein as DPP9.  
           [0013]    The following sequences of the human DPPIV amino acid sequence are important for the catalytic activity of DPPIV: (i) Trp 617 GlyTrpSerTyrGlyGlyTyrVal; (ii) Ala 707 AspAspAsnValH is Phe; (iii) Glu 738 AspHisGlylleAlaSer; and (iv) Trp 201 ValTyrGluGluGluVal [25-28]. As described herein, the alignment of the following sequences of DPP9: His 833 GlyTrpSerTyrGlyGlyPheLeu; Leu 913 AspGluAsnValHisPhePhe; Glu 944 ArgHisSerIleArg and Phe 350 ValIleGlnGluGluPhe with sequences (i) to (iv) above, respectively, suggests that these sequences of DPP9 are likely to confer the catalytic activity of DPP9. This is also supported by the alignment of DPP9 and DPP8 amino acid sequences. More specifically, DPP8 has substrate specificity for H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA, and shares near identity, with only one position of amino acid difference, in each of the above described sequences of DPP9. Thus, in a second aspect, the invention provides a peptide comprising the following amino acid sequences: HisGlyTrpSerTyrGlyGlyPheLeu; LeuAspGluAsnValHisPhePhe; GluArgHisSerIleArg and PheValIleGlnGluGluPhe; which has the substrate specificity of the sequence shown in SEQ ID NO:2.  
           [0014]    Also described herein, using the GAP sequence alignment algorithm, it is observed that DPP9 has 53% amino acid similarity and 29% amino acid identity with a  C. elegans  protein. Further, as shown herein, a nucleic acid molecule which encodes DPP9, is capable of hybridising specifically with DPP9 sequences derived from non-human species, including rat and mouse. Further, the inventors have isolated and characterised a mouse homologue of human DPP9. Together these data demonstrate that DPP9 is expressed in non-human species. Thus in a third aspect, the invention provides a peptide which has at least 91% amino acid identity with the amino acid sequence shown in SEQ ID NO:2, and which has the substrate specificity of the sequence shown in SEQ ID NO:2. Typically the peptide has the sequence shown in SEQ ID NO:4. Preferably, the amino acid identity is 75%. More preferably, the amino acid identity is 95%. Amino acid identity is calculated using GAP software [GCG Version 8, Genetics Computer Group, Madison, Wis., USA] as described further herein. Typically, the peptide comprises the following sequences: HisGlyTrpSerTyrGlyGlyPheLeu; LeuAspGluAsnValHisPhePhe; GluArgHisSerIleArg and PheValIleGlnGluGluPhe.  
           [0015]    In view of the homology between DPPIV, DPP8 and DPP9 amino acid sequences, it is expected that these sequences will have similar tertiary structure. This means that the tertiary structure of DPP9 is likely to include the seven-blade β-propeller domain and the α/β hydrolase domain of DPPIV. These structures in DPP9 are likely to be conferred by the regions comprising β-propeller, Val 226  to Ala 705 , α/β hydrolase, Ser 706  to Leu 969  and about 70 to 90 residues in the region Ser 136  to Gly 225 . As it is known that the β-propeller domain regulates proteolysis mediated by the catalytic triad in the α/β hydrolase domain of prolyl oligopeptidase, [29] it is expected that truncated forms of DPP9 can be produced, which have the substrate specificity of the sequence shown in SEQ ID NO:2, comprising the regions referred to above (His 833 GlyTrpSerTyrGlyGlyPheLeu; Leu 913 AspGluAsnValH is PhePhe; Glu 944 ArgHisSerIleArg and Phe 350 ValIleGlnGluGluPhe) which confer the catalytic specificity of DPP9. Examples of truncated forms of DPP9 which might be prepared are those in which the region conferring the β-propeller domain and the α/β hydrolase domain are spliced together. Other examples of truncated forms include those that are encoded by splice variants of DPP9 mRNA. Thus although, as described herein, the biochemical characterisation of DPP9 shows that DPP9 consists of 969 amino acids and has a molecular weight of about 110 kDa, it is recognised that truncated forms of DPP9 which have the substrate specificity of the sequence shown in SEQ ID NO:2, may be prepared using standard techniques [30,31]. Thus in a fourth aspect, the invention provides a fragment of the sequence shown in SEQ ID NO: 2, which has the substrate specificity of the sequence shown in SEQ ID NO:2. The inventors believe that a fragment from Ser136 to Leu969 (numbered according to SEQ ID NO:2) would have enzyme activity.  
           [0016]    It is recognised that DPP9 may be fused, or in other words, linked to a further amino acid sequence, to form a fusion protein which has the substrate specificity of the sequence shown in SEQ ID NO:2. An example of a fusion protein is one which comprises the sequence shown in SEQ ID NO:2 which is linked to a further amino acid sequence: a “tag” sequence which consists of an amino acid sequence encoding the V5 epitope and a His tag. An example of another further amino acid sequence which may be linked with DPP9 is a glutathione S transferase (GST) domain [30]. Another example of a further amino acid sequence is a portion of CD8α [8]. Thus in one aspect, the invention provides a fusion protein comprising the amino acid sequence shown in SEQ ID NO:2 linked with a further amino acid sequence, the fusion protein having the substrate specificity of the sequence shown in SEQ ID NO:2.  
           [0017]    It is also recognised that the peptide of the first aspect of the invention may be comprised in a polypeptide, so that the polypeptide has the substrate specificity of DPP9. The polypeptide may be useful, for example, for altering the protease susceptibility of DPP9, when used in in vivo applications. An example of a polypeptide which may be useful in this regard, is albumin. Thus in another embodiment, the peptide of the first aspect is comprised in a polypeptide which has the substrate specificity of DPP9.  
           [0018]    In one aspect, the invention provides a peptide which includes the amino acid sequence shown in SEQ ID NO:7. In one embodiment the peptide consists of the amino acid sequence shown in SEQ ID NO:7.  
           [0019]    As described further herein, the amino acid sequence shown in SEQ ID NO:7, and the amino acid sequences of DPPIV, DPP8 and FAP are homologous. DPPIV, DPP8 and FAP have dipeptidyl peptidase enzymatic activity and have substrate specificity for peptides which contain the di-peptide sequence, Ala-Pro. The inventors note that the amino acid sequence shown in SEQ ID NO:7 contains the catalytic triad, Ser-Asp-His. Accordingly, it is anticipated that the amino acid sequence shown in SEQ ID NO:7 has enzymatic activity in being capable of cleaving a peptide which contains Ala-Pro by hydrolysis of a peptide bond located C-terminal adjacent to proline in the di-peptide sequence.  
           [0020]    In one embodiment, the peptide comprises an amino acid sequence shown in SEQ ID NO:7 which is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro. The capacity of a dipeptidyl peptidase to cleave a peptide bond which is C-terminal adjacent to proline in the di-peptide sequence Ala-Pro can be determined by standard techniques, for example, by observing hydrolysis of a peptide bond which is C-terminal adjacent to proline in the molecule Ala-Pro-p-nitroanilide.  
           [0021]    The inventors recognise that by using standard techniques it is possible to generate a peptide which is a truncated form of the sequence shown in SEQ ID NO:7, which retains the proposed enzymatic activity described above. An example of a truncated form of the amino acid sequence shown in SEQ ID NO:7 which retains the proposed enzymatic activity is a form which includes the catalytic triad, Ser-Asp-His. Thus a truncated form may consist of less than the 831 amino acids shown in SEQ ID NO:7. Accordingly, in a further embodiment, the peptide is a truncated form of the peptide shown in SEQ ID NO:7, which is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro.  
           [0022]    It will be understood that the amino acid sequence shown in SEQ ID NO:7 may be altered by one or more amino acid deletions, substitutions or insertions of that amino acid sequence and yet retain the proposed enzymatic activity described above. It is expected that a peptide which is at least 47% similar to the amino acid sequence of SEQ ID NO:7, or which is at least 27% identical to the amino acid sequence of SEQ ID NO:7, will retain the proposed enzymatic activity described above. The % similarity can be determined by use of the program/algorithm “GAP” which is available from Genetics Computer Group (GCG), Wisconsin. Thus in another embodiment of the first aspect, the peptide has an amino acid sequence which is at least 47% similar to the amino acid sequence shown in SEQ ID NO:7, and is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro.  
           [0023]    As described above, the isolation and characterisation of DPP9 is necessary for identifying inhibitors of DPP9 catalytic activity, which may be useful for the treatment of disease. Accordingly, in a fifth aspect, the invention provides a method of identifying a molecule capable of inhibiting cleavage of a substrate by DPP9, the method comprising the following steps:  
           [0024]    (a) contacting DPP9 with the molecule;  
           [0025]    (b) contacting DPP9 of step (a) with a substrate capable of being cleaved by DPP9, in conditions sufficient for cleavage of the substrate by DPP9; and  
           [0026]    (c) detecting substrate not cleaved by DPP9, to identify that the molecule is capable of inhibiting cleavage of the substrate by DPP9.  
           [0027]    It is recognised that although inhibitors of DPP9 may also inhibit DPPIV and other serine proteases, as described herein, the alignment of the DPP9 amino acid sequence with most closely related molecules, (i.e. DPPIV), reveals that the DPP9 amino acid is distinctive, particularly at the regions controlling substrate specificity. Accordingly, it is expected that it will be possible to identify inhibitors which inhibit DPP9 catalytic activity specifically, which do not inhibit catalytic activity of DPPIV-like gene family members, or other serine proteases. Thus, in a sixth aspect, the invention provides a method of identifying a molecule capable of inhibiting specifically, the cleavage of a substrate by DPP9, the method comprising the following steps:  
           [0028]    (a) contacting DPP9 and a further protease with the molecule;  
           [0029]    (b) contacting DPP9 and the further protease of step (a) with a substrate capable of being cleaved by DPP9 and the further protease, in conditions sufficient for cleavage of the substrate by DPP9 and the further protease; and  
           [0030]    (c) detecting substrate not cleaved by DPP9, but cleaved by the further protease, to identify that the molecule is capable of inhibiting specifically, the cleavage of the substrate by DPP9.  
           [0031]    In a seventh aspect, the invention provides a method of reducing or inhibiting the catalytic activity of DPP9, the method comprising the step of contacting DPP9 with an inhibitor of DPP9 catalytic activity. In view of the homology between DPP9 and DPP8 amino acid sequences, it will be understood that inhibitors of DPPB activity may be useful for inhibiting DPP9 catalytic activity. Examples of inhibitors suitable for use in the seventh aspect are described in [21,32,33]. Other inhibitors useful for inhibiting DPP9 catalytic activity can be identified by the methods of the fifth or sixth aspects of the invention.  
           [0032]    In one embodiment, the catalytic activity of DPP9 is reduced or inhibited in a mammal by administering the inhibitor of DPP9 catalytic activity to the mammal. It is recognised that these inhibitors have been used to reduce or inhibit DPPIV catalytic activity in vivo, and therefore, may also be used for inhibiting DPP9 catalytic activity in vivo. Examples of inhibitors useful for this purpose are disclosed in the following [21,32-34].  
           [0033]    Preferably, the catalytic activity of DPP9 in a mammal is reduced or inhibited in the mammal, for the purpose of treating a disease in the mammal. Diseases which are likely to be treated by an inhibitor of DPP9 catalytic activity are those in which DPPIV-like gene family members are associated [3,10,11,17,21,36], including for example, neoplasia, type II diabetes, cirrhosis, autoimmunity, graft rejection and HIV infection.  
           [0034]    Preferably, the inhibitor for use in the seventh aspect of the invention is one which inhibits the cleavage of a peptide bond C-terminal adjacent to proline. As described herein, examples of these inhibitors are 4-(2-aminoethyl)benzenesulfonylfluoride, aprotinin, benzamidine/HCl, Ala-Pro-Gly, H-Lys-Pro-OH HCl salt and zinc ions, for example, zinc sulfate or zinc chloride. More preferably, the inhibitor is one which specifically inhibits DPP9 catalytic activity, and which does not inhibit the catalytic activity of other serine proteases, including, for example DPPIV, DPP8 or FAP.  
           [0035]    In an eighth aspect, the invention provides a method of cleaving a substrate which comprises contacting the substrate with DPP9 in conditions sufficient for cleavage of the substrate by DPP9, to cleave the substrate. Examples of molecules which can be cleaved by the method are H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA. Molecules which are cleaved by DPPIV including RANTES, eotaxin, macrophage-derived chemokine, stromal-cell-derived factor 1, glucagon and glucagon-like peptides 1 and 2, neuropeptide Y, substance P and vasoactive peptide are also likely to be cleaved by DPP9 [11,12]. In one embodiment, the substrate is cleaved by cleaving a peptide bond C-terminal adjacent to proline in the substrate. The molecules cleaved by DPP9 may have Ala, or Trp, Ser, Gly, Val or Leu in the P1 position, in place of Pro [11,12].  
           [0036]    The inventors have characterised the sequence of a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2. Thus in a tenth aspect, the invention provides a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2.  
           [0037]    In an eleventh aspect, the invention provides a nucleic acid molecule which consists of the sequence shown in SEQ ID NO:1.  
           [0038]    In another aspect, the invention provides a nucleic acid molecule which encodes a peptide comprising the amino acid sequence shown in SEQ ID NO:7.  
           [0039]    The inventors have characterised the nucleotide sequence of the nucleic acid molecule encoding SEQ ID NO:7. The nucleotide sequence of the nucleic acid molecule encoding DPP4-like-2 is shown in SEQ ID NO:8. Thus, in one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8. In another embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:8.  
           [0040]    The inventors recognise that a nucleic acid molecule which has the nucleotide sequence shown in SEQ ID NO:8 could be made by producing only the fragment of the nucleotide sequence which is translated. Thus in an embodiment, the nucleic acid molecule does not contain 5′ or 3′ untranslated nucleotide sequences.  
           [0041]    As described herein, the inventors observed RNA of 4.4 kb and aminor band of 4.8 kb in length which hybridised to a nucleic acid molecule comprising sequence shown in SEQ ID NO:8. It is possible that these mRNA species are splice variants. Thus in another embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8 and which is approximately 4.4 kb or 4.8 kb in length.  
           [0042]    In another embodiment, the nucleic acid molecule is selected from the group of nucleic acid molecules consisting of DPP4-like-2a, DPP4-like-2b and DPP4-like-2c, as shown in FIG. 2.  
           [0043]    In another aspect, the invention provides a nucleic acid molecule having a sequence shown in SEQ ID NO: 3.  
           [0044]    In a twelfth aspect, the invention provides a nucleic acid molecule which is capable of hybridising to a nucleic acid molecule consisting of the sequence shown in SEQ ID NO:1 in stringent conditions, and which encodes a peptide which has the substrate specificity of the sequence shown in SEQ ID NO:2. As shown in the Northern blot analysis described herein, DPP9 mRNA hybridises specifically to the sequence shown in SEQ ID NO:1, after washing in 2×SSC/1.0% SDS at 37° C., or after washing in 0.1×SSC/0.1% SDS at 50° C. “Stringent conditions” are conditions in which the nucleic acid molecule is exposed to 2×SSC/1.0% SDS. Preferably, the nucleic acid molecule is capable of hybridising to a molecule consisting of the sequence shown in SEQ ID NO:1 in high stringent conditions. “High stringent conditions” are conditions in which the nucleic acid molecule is exposed to 0.1×SSC/0.1% SDS at 50° C.  
           [0045]    As described herein, the inventors believe that the gene which encodes DPP9 is located at band p13.3 on human chromosome 19. The location of the DPP9 gene is distinguished from genes encoding other prolyl oligopeptidases, which are located on chromosome 2, at bands 2q24.3 and 2q23, chromosome 7 or chromosome 15q22. Thus in an embodiment, the nucleic acid molecule is one capable of hybridising to a gene which is located at band p13.3 on human chromosome 19.  
           [0046]    It is recognised that a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2, or which comprises the sequence shown in SEQ ID NO:1, could be made by producing the fragment of the sequence which is translated, using standard techniques [30,31]. Thus in an embodiment, the nucleic acid molecule does not contain 5′ or 3′ untranslated sequences.  
           [0047]    In a thirteenth aspect, the invention provides a vector which comprises a nucleic acid molecule of the tenth aspect of the invention. In one embodiment, the vector is capable of replication in a COS-7 cell, CHO cell or 293T cell, or  E. coli . In another embodiment, the vector is selected from the group consisting of % TripleEx, λTripleEx, pGEM-T Easy Vector, pSecTag2Hygro, pet15b, pEE14.HCMV.gs and pcDNA3.1/VS/His.  
           [0048]    In a fourteenth aspect, the invention provides a cell which comprises a vector of the thirteenth aspect of the invention. In one embodiment, the cell is an  E. coli  cell. Preferably, the  E. coli  is MC1061, DH5α, JM109, BL21DE3, pLysS. In another embodiment, the cell is a COS-7, COS-1, 293T or CHO cell.  
           [0049]    In a fifteenth aspect, the invention provides a method for making a peptide of the first aspect of the invention comprising, maintaining a cell according to the fourteenth aspect of the invention in conditions sufficient for expression of the peptide by the cell. The conditions sufficient for expression are described herein. In one embodiment, the method comprises the further step of isolating the peptide.  
           [0050]    In a sixteenth aspect, the invention provides a peptide when produced by the method of the fifteenth aspect.  
           [0051]    In a seventeenth aspect, the invention provides a composition comprising a peptide of the first aspect and a pharmaceutically acceptable carrier.  
           [0052]    In an eighteenth aspect, the invention provides an antibody which is capable of binding a peptide according to the first aspect of the invention. The antibody can be prepared by immunising a subject with purified DPP9 or a fragment thereof according to standard techniques [35]. An antibody may be prepared by immunising with transiently transfected DPP9 +  cells. It is recognised that the antibody is useful for inhibiting activity of DPP9. In one embodiment, the antibody of the eighteenth aspect of the invention is produced by a hybridoma cell.  
           [0053]    In a nineteenth aspect, the invention provides a hybridoma cell which secretes an antibody of the nineteenth aspect. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0054]    [0054]FIG. 1. Nucleotide sequence of DPP8 (SEQ ID NO:5).  
         [0055]    [0055]FIG. 2. Schematic representation of the cloning of human cDNA DPP9.  
         [0056]    [0056]FIG. 3. Schematic representation of the assembly of nucleotide sequences of human cDNA DPP9.  
         [0057]    [0057]FIG. 4. Nucleotide sequence of human cDNA DPP9 (SEQ ID NO:1) and amino acid sequence of human DPP9 (SEQ ID NO:2).  
         [0058]    [0058]FIG. 5. Alignment of human DPP9 amino acid sequences with the amino acid sequence encoded by a predicted open reading frame of GDD.  
         [0059]    [0059]FIG. 6. Alignment of human DPP8, DPP9, DPP4 and FAP amino acid sequences.  
         [0060]    [0060]FIG. 7. Northern blot analysis of human DPP9 RNA.  
         [0061]    [0061]FIG. 8. Alignment of murine (SEQ ID NO:4) and human DPP9 amino acid sequences.  
         [0062]    [0062]FIG. 9. Alignment of murine (SEQ ID NO:3) and human DPP9 cDNA nucleotide sequences.  
         [0063]    [0063]FIG. 10. Northern blot analysis of rat DPP9 RNA.  
         [0064]    [0064]FIG. 11. Detection of DPP9 cDNA in CEM cells.  
         [0065]    [0065]FIG. 12. Detection of murine DPP9 nucleotide sequence.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     EXAMPLES  
       [0066]    General  
         [0067]    Restriction enzymes and other enzymes used in cloning were obtained from Boehringer Mannheim Roche. Standard molecular biology techniques were used unless indicated otherwise.  
         [0068]    DPP9 Cloning  
         [0069]    The nucleotide sequence of DPP8 shown in FIG. 1 was used to search the GenBank database for homologous nucleotide sequences. Nucleotide sequences referenced by GenBank accession numbers AC005594 and AC005783 were detected and named GDD. The GDD nucleotide sequence is 39.5 kb and has 19 predicted exons. The analysis of the predicted exon-intron boundaries in GDD suggests that the predicted open reading frame of GDD is 3.6 kb in length.  
         [0070]    In view of the homology of DPP8 and the GDD nucleotide sequences, we hypothesised the existence of DPPIV-like molecules other than DPP8. We used oligonucleotide primers derived from the nucleotide sequence of GDD and reverse transcription PCR (RT-PCR) to isolate a cDNA encoding DPPIV-like molecules.  
         [0071]    RT-PCR amplification of human liver RNA derived from a pool of 4 patients with autoimmune hepatitis using the primers GDD pr 1F and GDD pr 1R (Table 1) produced a 500 base pair product. This suggested that DPPIV-like molecules are likely to be expressed in liver cells derived from individuals with autoimmune hepatitis and that RNA derived from these cells is likely to be a suitable source for isolating cDNA clones encoding DPPIV-like molecules.  
         [0072]    Primers GDD pr 3F and GDD pr 1R (Table 1) were then used to isolate a cDNA clone encoding a DPP4-like molecule. A 1.6 kb fragment was observed named DPP4-like-2a. Primers GDD pr 15F and GDD pr 7R (Table 1) were then used to isolate a cDNA clone encoding a DPP4-like molecule. A 1.9 kb product was observed and named DPP4-like-2b. As described further herein, the sequence of DPP4-like-2b overlaps with the sequence of DPP4-like-2a.  
         [0073]    The DPP4-like-2a and 2b fragments were gel purified using WIZARD® PCR preps kit and cloned into the pGEM®-T-easy plasmid vector using the EcoRI restriction sites. The ligation reaction was used to transform JM109 competent cells. The plasmid DNA was prepared by miniprep. The inserts were released by EcoRI restriction digestion. The DNA was sequenced in both directions using the M13Forward and M13Reverse sequencing primers. The complete sequence of DPP4-like-2a and 2b fragments was derived by primer walking.  
         [0074]    The nucleotide sequence 5′ adjacent to DPP4-like-2b was obtained by 5′RACE using dC tailing and the gene specific primers GDD GSP1.1 and 2.1 (Table 1). A fragment of 500 base pairs (DPP4-like-2c) was observed. The fragment was gel purified using WIZARD® PCR preps kit and cloned into the pGEM®-T-easy plasmid vector using the EcoRI restriction sites. The ligation reaction was used to transform JM109 competent cells. The plasmid DNA was prepared by miniprep. The inserts were released by EcoRI restriction digestion. The DNA was sequenced in both directions using the M13Forward and M13Reverse sequencing primers.  
         [0075]    We identified further sequences, BE727051 and BE244612, with identity to the 5′ end of DPP9. These were discovered while performing BLASTn with the 5′ end of the DPP9 nucleotide sequence. BE727051 contained further 5′ sequence for DPP9, which was also present in the genomic sequence for DPP9 on chromosome 19p13.3. This was used to design primer DPP9-22F (5′GCCGGCGGGTCCCCTGTGTCCG3′). Primer 22F was used in conjunction with primer GDD3′end (5′GGGCGGGACAAAGTGC CTCACTGG3′) on cDNA made from the human CEM cell line to produce a 3000 bp product as expected FIG. 11.  
         [0076]    Nucleotide Sequence Analysis of DPP4-like-2a, 2b, and 2c Fragments.  
         [0077]    An analysis of the nucleotide sequence of fragments DPP4-like 2a, 2b and 2c with the Sequencher™ version 3.0 computer program (FIG. 3), and the 5′ fragment isolated by primers DPP9-22F and GDD3′end, revealed the nucleotide sequence shown in FIG. 4.  
         [0078]    The predicted amino acid sequence shown in FIG. 4 was compared to a predicted amino acid sequence encoded by a predicted open reading frame of GDD (predicted from the nucleotide sequence referenced by GenBank Accession Nos. AC005594 and AC005783), to determine the relatedness of the nucleotide sequence of FIG. 4 to the nucleotide sequence of the predicted open reading frame of GDD (FIG. 5). Regions of amino acid identity were observed suggesting that there may be regions of nucleotide sequence identity of the predicted open reading frame of GDD and the sequence of FIG. 4. However, as noted in FIG. 5, there are regions of amino acid sequence encoded by the sequence of FIG. 4 and the amino acid sequence encoded by the predicted open reading frame of GDD which are not identical, demonstrating that the nucleotide sequences encoding the predicted open reading frame of GDD and the sequence shown in FIG. 4 are different nucleotide sequences.  
         [0079]    As described further herein, the predicted amino acid sequence encoded by the cDNA sequence shown in FIG. 4 is homologous to the amino acid sequence of DPP8 (FIG. 6). Accordingly, and as a cDNA consisting of the nucleotide sequence shown in FIG. 4 was not known, the sequence shown in FIG. 4 was named cDNA DPP9.  
         [0080]    The predicted amino acid sequence encoded by cDNA DPP9 (called DPP9) is 969 amino acids and is shown in FIG. 4. The alignment of DPP9 and DPP8 amino acid sequences suggests that the nucleotide sequence shown in FIG. 4 may be a partial length clone. Notwithstanding this point, as discussed below, the inventors have found that the alignment of DPP9 amino acid sequence with the amino acid sequences of DPP8, DPP4 and FAP shows that DPP9 comprises sequence necessary for providing enzymolysis and utility. In view of the similarity between DPP9 and DPP8, a full length clone may be of the order of 882 amino acids. A full length clone could be obtained by standard techniques, including for example, the RACE technique using an oligonucleotide primer derived from the 5′ end of cDNA DPP9.  
         [0081]    In view of the homology between the DPP8 and DPP9 amino acid sequences, it is likely that cDNA DPP9 encodes an amino acid sequence which has dipeptidyl peptidase enzymatic activity. Specifically, it is noted that the DPP9 amino acid sequence contains the catalytic triad Ser-Asp-His in the order of a non-classical serine protease as required for the charge relay system. The serine recognition site characteristic of DPP4 and DPP4-like family members, GYSWGG, surrounds the serine residue also suggesting that DPP9 cDNA will encode a DPP4-like enzyme activity.  
         [0082]    Further, DPP9 amino acid sequence also contains the two glutamic acid residues located at positions 205 and 206 in DPPIV. These are believed to be essential for the dipeptidyl peptidase enzymatic activity. By sequence alignment with DPPIV, the residues in DPP8 predicted to play a pivotal role in the pore opening mechanism in Blade 2 of the propeller are E 259 , E 260 . These are equivalent to the residues Glu 205  and Glu 206  in DPPIV which previously have been shown to be essential for DPPIV enzyme activity. A point mutation Glu259Lys was made in DPP8 cDNA using the Quick Change Site directed Mutagenesis Kit (Stratagene, La Jolla). COS-7 cells transfected with wildtype DPP8 cDNA stained positive for H-Ala-Pro4 MbNA enzyme activity while the mutant cDNA gave no staining. Expression of DPP8 protein was demonstrated in COS cells transfected with wildtype and mutant cDNAs by immunostaining with anti-VS mAB. This mAB detects the V5 epitope that has been tagged to the C-terminus of DPP8 protein. Point mutations were made to each of the catalytic residues of DPP8, Ser739A, Asp817Ala and His849Ala, and each of these residues were also determined to be essential for DPP8 enzyme activity. In summary, the residues that have been shown experimentally to be required for enzyme activity in DPPIV and DPP8 are present in the DPP9 amino acid sequence: Glu 354 , Glu 355 , Ser 136 , Asp 914  and His 946 .  
         [0083]    The DPP9 amino acid sequence shows the closest relatedness to DPP8, having 77% amino acid similarity and 60% amino acid identity. The relatedness to DPPIV is 25% amino acid identity and 47% amino acid similarity. The % similarity was determined by use of the program/algorithm “GAP” which is available from Genetics Computer Group (GCG), Wisconsin.  
         [0084]    DPP9 mRNA Expression Studies  
         [0085]    DPP4-like-2a was used to probe a Human Master RNA Blot™ (CLONTECH Laboratories Inc., USA) to study DPP9 tissue expression and the relative levels of DPP9 mRNA expression.  
         [0086]    The DPP4-like-2a fragment hybridised to all tissue mRNA samples on the blot. The hybridisation also indicated high levels of DPP9 expression in most of the tissues samples on the blot (data not shown).  
         [0087]    The DPP4-like-2a fragment was then used to probe two Multiple Tissue Northern Blots™ (CLONTECH Laboratories Inc., USA) to examine the mRNA expression and to determine the size of DPP9 mRNA transcript.  
         [0088]    The autoradiographs of the DPP9 Multiple Tissue Northern blot are shown in FIG. 8. The DPP9 transcript was seen in all tissues examined confirming the results obtained from the Master RNA blot. A single major transcript 4.4 kb in size was seen in all tissues represented on two Blots after 16 hours of exposure. Weak bands could also be seen in some tissues after 6 hours of exposure. The DPP9 transcript was smaller than the 5.1 kb mRNA transcript of DPP8. A minor, very weak transcript 4.8 kb in size was also seen in the spleen, pancreas, peripheral blood leukocytes and heart. The highest mRNA expression was observed in the spleen and heart. Of all tissues examined the thymus had the least DPP9 mRNA expression. The Multiple Tissue Northern Blots were also probed with a β-actin positive control. A 2.0 kb band was seen in all tissues. In addition as expected a 1.8 kb β-actin band was seen in heart and skeletal muscle.  
         [0089]    Rat DPP9 Expression  
         [0090]    A Rat Multiple Tissue Northern Blot (CLONTECH Laboratories, Inc., USA;catalogue #: 7764-1) was hybridised with a human DPP9 radioactively labeled probe, made using Megaprime DNA Labeling kit and [ 32 P] dCTP (Amersham International plc, Amersham, UK). The DPP9 PCR product used to make the probe was generated using Met3F (GGCTGAGAG GAT GGCCACCAC CGGG) as the forward primer and GDD 3′end (GGGCGGGACAAAGTGC CTCACTGG) as the reverse primer. The hybridisation was carried out according to the manufacturers&#39; instructions at 60° C. to detect cross-species hybridisation. After overnight hybridization the blot was washed at room temperature (2×SSC, 0.1% SDS) then at 40° C.(0.1×SSC, 0.1% SDS).  
         [0091]    The human cDNA probe identified two bands in all tissues examined except in testes. A major transcript of 4 kb in size was seen in all tissues except testes. This 4 kb transcript was strongly expressed in the liver, heart and brain. A second weaker transcript 5.5 kb in size was present in all tissues except skeletal muscle and testes. However in the brain the 5.5 kb transcript was expressed at a higher level than the 4.4 kb transcript. In the testes only one transcript approximately 3.5 kb in size was detected. Thus, rat DPP9 mRNA hybridised with a human DPP9 probe indicating significant homology between DPP9 of the two species. The larger 5.5 kb transcript observed may be due to crosshybridisation to rat DPP8.  
         [0092]    Mouse DPP9 Expression  
         [0093]    A Unigene cluster for Mouse DPP9 was identified (UniGene Cluster Mm.33185) by homology to human DPP9. An analysis of expressed sequence tags contained in this cluster and mouse genomic sequence (AC026385) for Chromosome 17 with the Sequencher™ version 3.0 computer program revealed the nucleotide sequence shown in FIG. 9. This 3517 bp cDNA encodes a 869 aa mouse DPP9 protein (missing N-terminus) with 91% amino acid identity and 94% amino acid similarity to human DPP9. The mouse DPP9 amino acid sequence also has the residues required for enzyme activity, Ser, Asp and His and the two Glu residues.  
         [0094]    The primers mgdd-prlF (5′ACCTGGGAGGAAGCACCCCACTGTG3′) and mgdd-pr4R (5′TTCCACCTGGTCCTCAATCTCC3′) were designed from this sequence and used to amplify a 452 bp product as expected from liver mouse cDNA, as described below.  
         [0095]    RNA Preparation  
         [0096]    B57Bl6 mice underwent carbon tetrachloride treatment to induce liver fibrosis. Liver RNA were prepared from snap-frozen tissues using the TRIzol® Reagent and other standard methods.  
         [0097]    cDNA Synthesis  
         [0098]    2 μg of liver RNA was reverse-transcribed using SuperScript II RNase H-Reverse Transcriptase (Gibco BRL).  
         [0099]    PCR  
         [0100]    PCR using mDPP9-1F (ACCTGGGAGGAAGCACCCCACTGTG) as the forward primer and mDPP9-2R (CTCTCCACATGCAGGGCTACAGAC) as the reverse primer was used to synthesise a 550 base pair mouse DPP9 fragment. The PCR products were generated using AmpliTaq Gold® DNA Polymerase. The PCR was performed as follows: denaturation at 95° C. for 10 min, followed by 35 cycles of denaturation at 95° C. for 30 seconds, primer annealing at 60° C. for 30 seconds, and an extension 720 C for 1 min.  
         [0101]    Southern Blot  
         [0102]    DPP9 PCR products from six mice as well as the largest human DPP9 PCR product were run on a 1% agarose gel. The DNA on the gel was then denatured using 0.4 M NaOH and transferred onto a Hybond-N+ membrane (Amersham International plc, Amersham, UK). The largest human DPP9 PCR product was radiolabeled using the Megaprime DNA Labeling kit and [32 P ] dCTP (Amersham International plc, Amersham, UK). Unincorporated label was removed using a NAP column (Pharmacia Biotech, Sweden) and the denatured probe was incubated with the membrane for 2 hours at 60° C. in Express Hybridisation solution (CLONTECH Laboratories, Inc., USA). (FIG. 12). Thus, DPP9 mRNA of appropriate size was detected in fibrotic mouse liver using rt-PCR. Furthermore, the single band of mouse DPP9 cDNA hybridised with a human DPP9 probe indicating significant homology between DPP9 of the two species.  
       REFERENCES  
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         [0113]    11. Natural substrates of dipeptidyl peptidase IV. De Meester I, C Durinx, G Bal, P Proost, S Struyf, F Goossens, K Augustyns &amp; S Scharpé. 2000, in  Cellular Peptidases in Immune Functions and Diseases  II, J Langner &amp; S Ansorge, Editor. Kluwer: New York. p. 67-88.  
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         [0119]    17. Ohtsuki T, H Tsuda &amp; C Morimoto 2000 Good or evil: CD26 and HIV infection  Journal of Dermatological Science  22: 152-60.  
         [0120]    18. Wesley U V, A P Albino, S Tiwari &amp; A N Houghton 1999 A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells  Journal of Experimental Medicine  190: 311-22.  
         [0121]    19. Korom S, I De Meester, T H W Stadlbauer, A Chandraker, M Schaub, M H Sayegh, A Belyaev, A Haemers, S Scharpé &amp; J W Kupiecweglinski 1997 Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients  Transplantation  63: 1495-500.  
         [0122]    20. Tanaka S, T Murakami, H Horikawa, M Sugiura, K Kawashima &amp; T Sugita 1997 Suppression of arthritis by the inhibitors of dipeptidyl peptidase IV  International Journal of Immunopharmacology  19: 15-24.  
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         [0127]    25. David F, AM Bernard, M Pierres &amp; D Marguet 1993 Identification of serine 624, aspartic acid 702, and histidine 734 as the catalytic triad residues of mouse dipeptidyl-peptidase IV (CD26). A member of a novel family of nonclassical serine hydrolases  J Biol Chem  268: 17247-52.  
         [0128]    26. Ogata S, Y Misumi, E Tsuji, N Takami, K Oda &amp; Y Ikehara 1992 Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis  Biochemistry  31: 2582-7.  
         [0129]    27. Dipeptidyl peptidase IV (DPPIV/CD26): biochemistry and control of cell-surface expression. Trugnan G, T Ait-Slimane, F David, L Baricault, T Berbar, C Lenoir &amp; C Sapin. 1997, in  Cell - Surface Peptidases in Health and Disease , A J Kenny &amp; C M Boustead, Editor. BIOS Scientific Publishers: Oxford. p. 203-17.  
         [0130]    28. Steeg C, U Hartwig &amp; B Fleischer 1995 Unchanged signaling capacity of mutant CD26/dipeptidylpeptidase IV molecules devoid of enzymatic activity  Cell Immunol  164: 311-5.  
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         [0132]    30. Ausubel F M, R Brent, R E Kingston, D D Moore, J G Seidman, J A Smith &amp; K Struhl, ed. Current Protocols in Molecular Biology. 1998, John Wiley &amp; Sons: USA.  
         [0133]    31. Molecular cloning: a laboratory manual. Sambrook J, E F Fritsch &amp; T Maniatis. 1989. 2nd ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press.  
         [0134]    32. Augustyns K J L, A M Lambeir, M Borloo, I Demeester, I Vedernikova, G Vanhoof, D Hendriks, S Scharpe &amp; A Haemers 1997 Pyrrolidides—synthesis and structure-activity relationship as inhibitors of dipeptidyl peptidase IV  European Journal of Medicinal Chemistry  32: 301-9.  
         [0135]    33. Stockel-Maschek A, C Mrestani-Klaus, B Stiebitz, H U Demuth &amp; K Neubert 2000 Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases  Biochimica et Biophysica Acta—Protein Structure  &amp;  Molecular Enzymology  1479: 15-31.  
         [0136]    34. Schön, I Born, H U Demuth, J Faust, K Neubert, T Steinmetzer, A Barth &amp; S Ansorge 1991 Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes  Biological Chemistry Hoppe Seyler  372: 305-11.  
         [0137]    35. Coligan J E, A M Kruisbeek, D H Margulies, E M Shevach &amp; W Strober, eds. Current Protocols in Immunology. 1998, John Wiley &amp; Sons: USA.  
         [0138]    36. Fibroblast activation protein. Rettig W J. 1998, in  Handbook of Proteolytic Enzymes , A J Barrett, N D Rawlings &amp; J F Woessner, Editor. Academic Press: San Diego. p. 387-9.  
         [0139]    [0139] 
     
       
       
         1 
         
           
             8  
           
           
             1  
             3000  
             DNA  
             Homo sapiens  
           
            1 

cggcgggtcc cctgtgtccg ccgcggctgt cgtcccccgc tcccgccact tccggggtcg     60 

cagtcccggg catggagccg cgaccgtgag gcgccgctgg acccgggacg acctgcccag    120 

tccggccgcc gccccacgtc ccggtctgtg tcccacgcct gcagctggaa tggaggctct    180 

ctggaccctt tagaaggcac ccctgccctc ctgaggtcag ctgagcggtt aatgcggaag    240 

gttaagaaac tgcgcctgga caaggagaac accggaagtt ggagaagctt ctcgctgaat    300 

tccgaggggg ctgagaggat ggccaccacc gggaccccaa cggccgaccg aggcgacgca    360 

gccgccacag atgacccggc cgcccgcttc caggtgcaga agcactcgtg ggacgggctc    420 

cggagcatca tccacggcag ccgcaagtac tcgggcctca ttgtcaacaa ggcgccccac    480 

gacttccagt ttgtgcagaa gacggatgag tctgggcccc actcccaccg cctctactac    540 

ctgggaatgc catatggcag ccgggagaac tccctcctct actctgagat tcccaagaag    600 

gtccggaaag aggctctgct gctcctgtcc tggaagcaga tgctggatca tttccaggcc    660 

acgccccacc atggggtcta ctctcgggag gaggagctgc tgagggagcg gaaacgcctg    720 

ggggtcttcg gcatcacctc ctacgacttc cacagcgaga gtggcctctt cctcttccag    780 

gccagcaaca gcctcttcca ctgccgcgac ggcggcaaga acggcttcat ggtgtcccct    840 

atgaaaccgc tggaaatcaa gacccagtgc tcagggcccc ggatggaccc caaaatctgc    900 

cctgccgacc ctgccttctt ctccttcaac aataacagcg acctgtgggt ggccaacatc    960 

gagacaggcg aggagcggcg gctgaccttc tgccaccaag gtttatccaa tgtcctggat   1020 

gaccccaagt ctgcgggtgt ggccaccttc gtcatacagg aagagttcga ccgcttcact   1080 

gggtactggt ggtgccccac agcctcctgg gaaggttcag agggcctcaa gacgctgcga   1140 

atcctgtatg aggaagtcga tgagtccgag gtggaggtca ttcacgtccc ctctcctgcg   1200 

ctagaagaaa ggaagacgga ctcgtatcgg taccccagga caggcagcaa gaatcccaag   1260 

attgccttga aactggctga gttccagact gacagccagg gcaagatcgt ctcgacccag   1320 

gagaaggagc tggtgcagcc cttcagctcg ctgttcccga aggtggagta catcgccagg   1380 

gccgggtgga cccgggatgg caaatacgcc tgggccatgt tcctggaccg gccccagcag   1440 

tggctccagc tcgtcctcct ccccccggcc ctgttcatcc cgagcacaga gaatgaggag   1500 

cagcggctag cctctgccag agctgtcccc aggaatgtcc agccgtatgt ggtgtacgag   1560 

gaggtcacca acgtctggat caatgttcat gacatcttct atcccttccc ccaatcagag   1620 

ggagaggacg agctctgctt tctccgcgcc aatgaatgca agaccggctt ctgccatttg   1680 

tacaaagtca ccgccgtttt aaaatcccag ggctacgatt ggagtgagcc cttcagcccc   1740 

ggggaagatg aatttaagtg ccccattaag gaagagattg ctctgaccag cggtgaatgg   1800 

gaggttttgg cgaggcacgg ctccaagatc tgggtcaatg aggagaccaa gctggtgtac   1860 

ttccagggca ccaaggacac gccgctggag caccacctct acgtggtcag ctatgaggcg   1920 

gccggcgaga tcgtacgcct caccacgccc ggcttctccc atagctgctc catgagccag   1980 

aacttcgaca tgttcgtcag ccactacagc agcgtgagca cgccgccctg cgtgcacgtc   2040 

tacaagctga gcggccccga cgacgacccc ctgcacaagc agccccgctt ctgggctagc   2100 

atgatggagg cagccagctg ccccccggat tatgttcctc cagagatctt ccatttccac   2160 

acgcgctcgg atgtgcggct ctacggcatg atctacaagc cccacgcctt gcagccaggg   2220 

aagaagcacc ccaccgtcct ctttgtatat ggaggccccc aggtgcagct ggtgaataac   2280 

tccttcaaag gcatcaagta cttgcggctc aacacactgg cctccctggg ctacgccgtg   2340 

gttgtgattg acggcagggg ctcctgtcag cgagggcttc ggttcgaagg ggccctgaaa   2400 

aaccaaatgg gccaggtgga gatcgaggac caggtggagg gcctgcagtt cgtggccgag   2460 

aagtatggct tcatcgacct gagccgagtt gccatccatg gctggtccta cgggggcttc   2520 

ctctcgctca tggggctaat ccacaagccc caggtgttca aggtggccat cgcgggtgcc   2580 

ccggtcaccg tctggatggc ctacgacaca gggtacactg agcgctacat ggacgtccct   2640 

gagaacaacc agcacggcta tgaggcgggt tccgtggccc tgcacgtgga gaagctgccc   2700 

aatgagccca accgcttgct tatcctccac ggcttcctgg acgaaaacgt gcactttttc   2760 

cacacaaact tcctcgtctc ccaactgatc cgagcaggga aaccttacca gctccagatc   2820 

taccccaacg agagacacag tattcgctgc cccgagtcgg gcgagcacta tgaagtcacg   2880 

ttactgcact ttctacagga atacctctga gcctgcccac cgggagccgc cacatcacag   2940 

cacaagtggc tgcagcctcc gcggggaacc aggcgggagg gactgagtgg cccgcgggcc   3000 

 
           
             2  
             969  
             PRT  
             Homo sapiens  
           
            2 

Arg Arg Val Pro Cys Val Arg Arg Gly Cys Arg Pro Pro Leu Pro Pro 
1               5                   10                  15 

Leu Pro Gly Ser Gln Ser Arg Ala Trp Ser Arg Asp Arg Glu Ala Pro 
            20                  25                  30 

Leu Asp Pro Gly Arg Pro Ala Gln Ser Gly Arg Arg Pro Thr Ser Arg 
        35                  40                  45 

Ser Val Ser His Ala Cys Ser Trp Asn Gly Gly Ser Leu Asp Pro Leu 
    50                  55                  60 

Glu Gly Thr Pro Ala Leu Leu Arg Ser Ala Glu Arg Leu Met Arg Lys 
65                  70                  75                  80 

Val Lys Lys Leu Arg Leu Asp Lys Glu Asn Thr Gly Ser Trp Arg Ser 
                85                  90                  95 

Phe Ser Leu Asn Ser Glu Gly Ala Glu Arg Met Ala Thr Thr Gly Thr 
            100                 105                 110 

Pro Thr Ala Asp Arg Gly Asp Ala Ala Ala Thr Asp Asp Pro Ala Ala 
        115                 120                 125 

Arg Phe Gln Val Gln Lys His Ser Trp Asp Gly Leu Arg Ser Ile Ile 
    130                 135                 140 

His Gly Ser Arg Lys Tyr Ser Gly Leu Ile Val Asn Lys Ala Pro His 
145                 150                 155                 160 

Asp Phe Gln Phe Val Gln Lys Thr Asp Glu Ser Gly Pro His Ser His 
                165                 170                 175 

Arg Leu Tyr Tyr Leu Gly Met Pro Tyr Gly Ser Arg Glu Asn Ser Leu 
            180                 185                 190 

Leu Tyr Ser Glu Ile Pro Lys Lys Val Arg Lys Glu Ala Leu Leu Leu 
        195                 200                 205 

Leu Ser Trp Lys Gln Met Leu Asp His Phe Gln Ala Thr Pro His His 
    210                 215                 220 

Gly Val Tyr Ser Arg Glu Glu Glu Leu Leu Arg Glu Arg Lys Arg Leu 
225                 230                 235                 240 

Gly Val Phe Gly Ile Thr Ser Tyr Asp Phe His Ser Glu Ser Gly Leu 
                245                 250                 255 

Phe Leu Phe Gln Ala Ser Asn Ser Leu Phe His Cys Arg Asp Gly Gly 
            260                 265                 270 

Lys Asn Gly Phe Met Val Ser Pro Met Lys Pro Leu Glu Ile Lys Thr 
        275                 280                 285 

Gln Cys Ser Gly Pro Arg Met Asp Pro Lys Ile Cys Pro Ala Asp Pro 
    290                 295                 300 

Ala Phe Phe Ser Phe Asn Asn Asn Ser Asp Leu Trp Val Ala Asn Ile 
305                 310                 315                 320 

Glu Thr Gly Glu Glu Arg Arg Leu Thr Phe Cys His Gln Gly Leu Ser 
                325                 330                 335 

Asn Val Leu Asp Asp Pro Lys Ser Ala Gly Val Ala Thr Phe Val Ile 
            340                 345                 350 

Gln Glu Glu Phe Asp Arg Phe Thr Gly Tyr Trp Trp Cys Pro Thr Ala 
        355                 360                 365 

Ser Trp Glu Gly Ser Gln Gly Leu Lys Thr Leu Arg Ile Leu Tyr Glu 
    370                 375                 380 

Glu Val Asp Glu Ser Glu Val Glu Val Ile His Val Pro Ser Pro Ala 
385                 390                 395                 400 

Leu Glu Glu Arg Lys Thr Asp Ser Tyr Arg Tyr Pro Arg Thr Gly Ser 
                405                 410                 415 

Lys Asn Pro Lys Ile Ala Leu Lys Leu Ala Glu Phe Gln Thr Asp Ser 
            420                 425                 430 

Gln Gly Lys Ile Val Ser Thr Gln Glu Lys Glu Leu Val Gln Pro Phe 
        435                 440                 445 

Ser Ser Leu Phe Pro Lys Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr 
    450                 455                 460 

Arg Asp Gly Lys Tyr Ala Trp Ala Met Phe Leu Asp Arg Pro Gln Gln 
465                 470                 475                 480 

Trp Leu Gln Leu Val Leu Leu Pro Pro Ala Leu Phe Ile Pro Ser Thr 
                485                 490                 495 

Glu Asn Glu Glu Gln Arg Leu Ala Ser Ala Arg Ala Val Pro Arg Asn 
            500                 505                 510 

Val Gln Pro Tyr Val Val Tyr Glu Glu Val Thr Asn Val Trp Ile Asn 
        515                 520                 525 

Val His Asp Ile Phe Tyr Pro Phe Pro Gln Ser Glu Gly Glu Asp Glu 
    530                 535                 540 

Leu Cys Phe Leu Arg Ala Asn Glu Cys Lys Thr Gly Phe Cys His Leu 
545                 550                 555                 560 

Tyr Lys Val Thr Ala Val Leu Lys Ser Gln Gly Tyr Asp Trp Ser Glu 
                565                 570                 575 

Pro Phe Ser Pro Gly Glu Asp Glu Phe Lys Cys Pro Ile Lys Glu Glu 
            580                 585                 590 

Ile Ala Leu Thr Ser Gly Glu Trp Glu Val Leu Ala Arg His Gly Ser 
        595                 600                 605 

Lys Ile Trp Val Asn Glu Glu Thr Lys Leu Val Tyr Phe Gln Gly Thr 
    610                 615                 620 

Lys Asp Thr Pro Leu Glu His His Leu Tyr Val Val Ser Tyr Glu Ala 
625                 630                 635                 640 

Ala Gly Glu Ile Val Arg Leu Thr Thr Pro Gly Phe Ser His Ser Cys 
                645                 650                 655 

Ser Met Ser Gln Asn Phe Asp Met Phe Val Ser His Tyr Ser Ser Val 
            660                 665                 670 

Ser Thr Pro Pro Cys Val His Val Tyr Lys Leu Ser Gly Pro Asp Asp 
        675                 680                 685 

Asp Pro Leu His Lys Gln Pro Arg Phe Trp Ala Ser Met Met Glu Ala 
    690                 695                 700 

Ala Ser Cys Pro Pro Asp Tyr Val Pro Pro Glu Ile Phe His Phe His 
705                 710                 715                 720 

Thr Arg Ser Asp Val Arg Leu Tyr Gly Met Ile Tyr Lys Pro His Ala 
                725                 730                 735 

Leu Gln Pro Gly Lys Lys His Pro Thr Val Leu Phe Val Tyr Gly Gly 
            740                 745                 750 

Pro Gln Val Gln Leu Val Asn Asn Ser Phe Lys Gly Ile Lys Tyr Leu 
        755                 760                 765 

Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Ala Val Val Val Ile Asp 
    770                 775                 780 

Gly Arg Gly Ser Cys Gln Arg Gly Leu Arg Phe Glu Gly Ala Leu Lys 
785                 790                 795                 800 

Asn Gln Met Gly Gln Val Glu Ile Glu Asp Gln Val Glu Gly Leu Gln 
                805                 810                 815 

Phe Val Ala Glu Lys Tyr Gly Phe Ile Asp Leu Ser Arg Val Ala Ile 
            820                 825                 830 

His Gly Trp Ser Tyr Gly Gly Phe Leu Ser Leu Met Gly Leu Ile His 
        835                 840                 845 

Lys Pro Gln Val Phe Lys Val Ala Ile Ala Gly Ala Pro Val Thr Val 
    850                 855                 860 

Trp Met Ala Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Asp Val Pro 
865                 870                 875                 880 

Glu Asn Asn Gln His Gly Tyr Glu Ala Gly Ser Val Ala Leu His Val 
                885                 890                 895 

Glu Lys Leu Pro Asn Glu Pro Asn Arg Leu Leu Ile Leu His Gly Phe 
            900                 905                 910 

Leu Asp Glu Asn Val His Phe Phe His Thr Asn Phe Leu Val Ser Gln 
        915                 920                 925 

Leu Ile Arg Ala Gly Lys Pro Tyr Gln Leu Gln Ile Tyr Pro Asn Glu 
    930                 935                 940 

Arg His Ser Ile Arg Cys Pro Glu Ser Gly Glu His Tyr Glu Val Thr 
945                 950                 955                 960 

Leu Leu His Phe Leu Gln Glu Tyr Leu 
                965 

 
           
             3  
             3287  
             DNA  
             Mus musculus  
           
            3 

ccatcacagg agccccagag gatgtgcagc ggggtctccc cagttgagca ggtggccgca     60 

ggggacatgg atgacacggc agcacgcttc tgtgtgcaga agcactcgtg ggatgggctg    120 

cgtagcatta tccacggcag tcgcaagtcc tcgggcctca ttgtcagcaa ggccccccac    180 

gacttccagt ttgtgcagaa gcctgacgag tctggccccc actctcaccg tctctattac    240 

ctcggaatgc cttacggcag ccgtgagaac tccctcctct actccgagat ccccaagaaa    300 

gtgcggaagg aggccctgct gctgctgtcc tggaagcaga tgctggacca cttccaggcc    360 

acaccccacc atggtgtcta ctcccgagag gaggagctac tgcgggagcg caagcgcctg    420 

ggcgtcttcg gaatcacctc ttatgacttc cacagtgaga gcggcctctt cctcttccag    480 

gccagcaata gcctgttcca ctgcagggat ggtggcaaga atggctttat ggtgtccccg    540 

atgaagccac tggagatcaa gactcagtgt tctgggccac gcatggaccc caaaatctgc    600 

cccgcagacc ctgccttctt ttccttcatc aacaacagtg atctgtgggt ggcaaacatc    660 

gagactgggg aggaacggcg gctcaccttc tgtcaccagg gttcagctgg tgtcctggac    720 

aatcccaaat cagcaggcgt ggccaccttt gtcatccagg aggagttcga ccgcttcact    780 

gggtgctggt ggtgccccac ggcctcttgg gaaggctccg aaggtctcaa gacgctgcgc    840 

atcctatatg aggaagtgga cgagtctgaa gtggaggtca ttcatgtgcc ctcccccgcc    900 

ctggaggaga ggaagacgga ctcctaccgc taccccagga caggcagcaa gaaccccaag    960 

attgccctga agctggctga gctccagacg gaccatcagg gcaaaatcgt gtcaagctgc   1020 

gagaaggaac tggtacagcc attcagctcc cttttcccca aagtggagta catcgcccgg   1080 

gctggctgga cacgggacgg caaatatgcc tgggccatgt tcctggaccg tccccagcaa   1140 

cggcttcagc ttgtcctcct gccccctgct ctcttcatcc cggccgttga gagtgaggcc   1200 

cagcggcagg cagctgccag agccgtcccc aagaatgtgc agccctttgt catctatgaa   1260 

gaagtcacca atgtctggat caacgtccac gacatcttcc acccgtttcc tcaggctgag   1320 

ggccagcagg acttttgttt ccttcgtgcc aacgaatgca agactggctt ctgccacctg   1380 

tacagggtca cagtggaact taaaaccaag gactatgact ggacggaacc cctcagccct   1440 

acagaaggtg agtttaagtg ccccatcaag gaggaggtcg ccctgaccag tggcgagtgg   1500 

gaggtcttgt cgaggcatgg ctccaagatc tgggtcaacg agcagacgaa gctggtgtac   1560 

tttcaaggta caaaggacac accgctggaa catcacctct atgtggtcag ctacgagtca   1620 

gcaggcgaga tcgtgcggct caccacgctc ggcttctccc acagctgctc catgagccag   1680 

agcttcgaca tgttcgtgag tcactacagc agtgtgagca cgccaccctg tgtacatgtg   1740 

tacaagctga gcggccccga tgatgaccca ctgcacaagc aaccacgctt ctgggccagc   1800 

atgatggagg cagccaattg ccccccagac tatgtgcccc ctgagatctt ccacttccac   1860 

acccgtgcag acgtgcagct ctacggcatg atctacaagc cacacaccct gcaacctggg   1920 

aggaagcacc ccactgtgct ctttgtctat gggggcccac aggtgcagtt ggtgaacaac   1980 

tcctttaagg gcatcaaata cctgcggcta aatacactgg catccttggg ctatgctgtg   2040 

gtggtgatcg atggtcgggg ctcctgtcag cggggcctgc acttcgaggg ggccctgaaa   2100 

aatcaaatgg gccaggtgga gattgaggac caggtggaag gcttgcagta cgtggctgag   2160 

aagtatggct tcattgactt gagccgagtc gccatccatg gctggtccta cggcggcttc   2220 

ctctcactca tggggctcat ccacaagcca caagtgttca aggtagccat tgcgggcgct   2280 

cctgtcactg tgtggatggc ctatgacaca gggtacacgg aacgatacat ggatgtcccc   2340 

gaaaataacc agcaaggcta tgaggcaggg tctgtagccc tgcatgtgga gaagctgccc   2400 

aatgagccta accgcctgct tatcctccac ggcttcctgg acgagaacgt tcacttcttc   2460 

cacacaaatt tcctggtgtc ccagctgatc cgagcaggaa agccatacca gcttcagatc   2520 

tacccaaacg agagacatag catccgctgc cgcgagtccg gagagcatta cgaggtgacg   2580 

ctgctgcact ttctgcagga acacctgtga cctcagtccc gactcctgac gccaccgctg   2640 

ctcttcttgc gtttttgtaa tcttttcatt tttgaagctt ccaatttgct tgctgctgct   2700 

gctgcctggg ggccaggaca gaggtagtgg cggcccccat gccgccctcc ttgagctggt   2760 

gaggagaagt cgccattgag cacacaacct ccaccagact gccatggccc cgaacctgca   2820 

attccatcct agcgcagaag catgtgcctg ccacctgctg cccctgcaga gtcatgtgtg   2880 

tttgtggtgg gcattttaaa taattattta aaagacagga agtaagcggt accgagcaat   2940 

gaaactgaag gtacagcact gggcgtctgg ggaccccacg ctctcccaac gcccagacta   3000 

tgtggagctg ccaagcccct gtctgggcac ctctgccctg cctgtctgct gcccggatcc   3060 

tcctcactta gcacctaggg gtgtcagggt cgggagtagg acctgtcctg acctcagggt   3120 

tatatatagc ccttccccac tccctcctac gagagttctg gcataaagaa gtaaaaaaaa   3180 

aaaaaaaaaa aacaaacaaa aaaaccaaac cacctctaca tattatggaa agaaaatatt   3240 

tttgtcaatt cttattcttt tataattatg tggtatgtag actcatt                 3287 

 
           
             4  
             869  
             PRT  
             Mus musculus  
           
            4 

Pro Ser Gln Glu Pro Gln Arg Met Cys Ser Gly Val Ser Pro Val Glu 
1               5                   10                  15 

Gln Val Ala Ala Gly Asp Met Asp Asp Thr Ala Ala Arg Phe Cys Val 
            20                  25                  30 

Gln Lys His Ser Trp Asp Gly Leu Arg Ser Ile Ile His Gly Ser Arg 
        35                  40                  45 

Lys Ser Ser Gly Leu Ile Val Ser Lys Ala Pro His Asp Phe Gln Phe 
    50                  55                  60 

Val Gln Lys Pro Asp Glu Ser Gly Pro His Ser His Arg Leu Tyr Tyr 
65                  70                  75                  80 

Leu Gly Met Pro Tyr Gly Ser Arg Glu Asn Ser Leu Leu Tyr Ser Glu 
                85                  90                  95 

Ile Pro Lys Lys Val Arg Lys Glu Ala Leu Leu Leu Leu Ser Trp Lys 
            100                 105                 110 

Gln Met Leu Asp His Phe Gln Ala Thr Pro His His Gly Val Tyr Ser 
        115                 120                 125 

Arg Glu Glu Glu Leu Leu Arg Glu Arg Lys Arg Leu Gly Val Phe Gly 
    130                 135                 140 

Ile Thr Ser Tyr Asp Phe His Ser Glu Ser Gly Leu Phe Leu Phe Gln 
145                 150                 155                 160 

Ala Ser Asn Ser Leu Phe His Cys Arg Asp Gly Gly Lys Asn Gly Phe 
                165                 170                 175 

Met Val Ser Pro Met Lys Pro Leu Glu Ile Lys Thr Gln Cys Ser Gly 
            180                 185                 190 

Pro Arg Met Asp Pro Lys Ile Cys Pro Ala Asp Pro Ala Phe Phe Ser 
        195                 200                 205 

Phe Ile Asn Asn Ser Asp Leu Trp Val Ala Asn Ile Glu Thr Gly Glu 
    210                 215                 220 

Glu Arg Arg Leu Thr Phe Cys His Gln Gly Ser Ala Gly Val Leu Asp 
225                 230                 235                 240 

Asn Pro Lys Ser Ala Gly Val Ala Thr Phe Val Ile Gln Glu Glu Phe 
                245                 250                 255 

Asp Arg Phe Thr Gly Cys Trp Trp Cys Pro Thr Ala Ser Trp Glu Gly 
            260                 265                 270 

Ser Glu Gly Leu Lys Thr Leu Arg Ile Leu Tyr Glu Glu Val Asp Glu 
        275                 280                 285 

Ser Glu Val Glu Val Ile His Val Pro Ser Pro Ala Leu Glu Glu Arg 
    290                 295                 300 

Lys Thr Asp Ser Tyr Arg Tyr Pro Arg Thr Gly Ser Lys Asn Pro Lys 
305                 310                 315                 320 

Ile Ala Leu Lys Leu Ala Glu Leu Gln Thr Asp His Gln Gly Lys Ile 
                325                 330                 335 

Val Ser Ser Cys Glu Lys Glu Leu Val Gln Pro Phe Ser Ser Leu Phe 
            340                 345                 350 

Pro Lys Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Arg Asp Gly Lys 
        355                 360                 365 

Tyr Ala Trp Ala Met Phe Leu Asp Arg Pro Gln Gln Arg Leu Gln Leu 
    370                 375                 380 

Val Leu Leu Pro Pro Ala Leu Phe Ile Pro Ala Val Glu Ser Glu Ala 
385                 390                 395                 400 

Gln Arg Gln Ala Ala Ala Arg Ala Val Pro Lys Asn Val Gln Pro Phe 
                405                 410                 415 

Val Ile Tyr Glu Glu Val Thr Asn Val Trp Ile Asn Val His Asp Ile 
            420                 425                 430 

Phe His Pro Phe Pro Gln Ala Glu Gly Gln Gln Asp Phe Cys Phe Leu 
        435                 440                 445 

Arg Ala Asn Glu Cys Lys Thr Gly Phe Cys His Leu Tyr Arg Val Thr 
    450                 455                 460 

Val Glu Leu Lys Thr Lys Asp Tyr Asp Trp Thr Glu Pro Leu Ser Pro 
465                 470                 475                 480 

Thr Glu Gly Glu Phe Lys Cys Pro Ile Lys Glu Glu Val Ala Leu Thr 
                485                 490                 495 

Ser Gly Glu Trp Glu Val Leu Ser Arg His Gly Ser Lys Ile Trp Val 
            500                 505                 510 

Asn Glu Gln Thr Lys Leu Val Tyr Phe Gln Gly Thr Lys Asp Thr Pro 
        515                 520                 525 

Leu Glu His His Leu Tyr Val Val Ser Tyr Glu Ser Ala Gly Glu Ile 
    530                 535                 540 

Val Arg Leu Thr Thr Leu Gly Phe Ser His Ser Cys Ser Met Ser Gln 
545                 550                 555                 560 

Ser Phe Asp Met Phe Val Ser His Tyr Ser Ser Val Ser Thr Pro Pro 
                565                 570                 575 

Cys Val His Val Tyr Lys Leu Ser Gly Pro Asp Asp Asp Pro Leu His 
            580                 585                 590 

Lys Gln Pro Arg Phe Trp Ala Ser Met Met Glu Ala Ala Asn Cys Pro 
        595                 600                 605 

Pro Asp Tyr Val Pro Pro Glu Ile Phe His Phe His Thr Arg Ala Asp 
    610                 615                 620 

Val Gln Leu Tyr Gly Met Ile Tyr Lys Pro His Thr Leu Gln Pro Gly 
625                 630                 635                 640 

Arg Lys His Pro Thr Val Leu Phe Val Tyr Gly Gly Pro Gln Val Gln 
                645                 650                 655 

Leu Val Asn Asn Ser Phe Lys Gly Ile Lys Tyr Leu Arg Leu Asn Thr 
            660                 665                 670 

Leu Ala Ser Leu Gly Tyr Ala Val Val Val Ile Asp Gly Arg Gly Ser 
        675                 680                 685 

Cys Gln Arg Gly Leu His Phe Glu Gly Ala Leu Lys Asn Gln Met Gly 
    690                 695                 700 

Gln Val Glu Ile Glu Asp Gln Val Glu Gly Leu Gln Tyr Val Ala Glu 
705                 710                 715                 720 

Lys Tyr Gly Phe Ile Asp Leu Ser Arg Val Ala Ile His Gly Trp Ser 
                725                 730                 735 

Tyr Gly Gly Phe Leu Ser Leu Met Gly Leu Ile His Lys Pro Gln Val 
            740                 745                 750 

Phe Lys Val Ala Ile Ala Gly Ala Pro Val Thr Val Trp Met Ala Tyr 
        755                 760                 765 

Asp Thr Gly Tyr Thr Glu Arg Tyr Met Asp Val Pro Glu Asn Asn Gln 
    770                 775                 780 

Gln Gly Tyr Glu Ala Gly Ser Val Ala Leu His Val Glu Lys Leu Pro 
785                 790                 795                 800 

Asn Glu Pro Asn Arg Leu Leu Ile Leu His Gly Phe Leu Asp Glu Asn 
                805                 810                 815 

Val His Phe Phe His Thr Asn Phe Leu Val Ser Gln Leu Ile Arg Ala 
            820                 825                 830 

Gly Lys Pro Tyr Gln Leu Gln Ile Tyr Pro Asn Glu Arg His Ser Ile 
        835                 840                 845 

Arg Cys Arg Glu Ser Gly Glu His Tyr Glu Val Thr Leu Leu His Phe 
    850                 855                 860 

Leu Gln Glu His Leu 
865 

 
           
             5  
             3120  
             DNA  
             Homo sapiens  
           
            5 

aagtgctaaa gcctccgagg ccaaggccgc tgctactgcc gccgctgctt cttagtgccg     60 

cgttcgccgc ctgggttgtc accggcgccg ccgccgagga agccactgca accaggaccg    120 

gagtggaggc ggcgcagcat gaagcggcgc aggcccgctc catagcgcac gtcgggacgg    180 

tccgggcggg gccgggggga aggaaaatgc aacatggcag cagcaatgga aacagaacag    240 

ctgggtgttg agatatttga aactgcggac tgtgaggaga atattgaatc acaggatcgg    300 

cctaaattgg agccttttta tgttgagcgg tattcctgga gtcagcttaa aaagctgctt    360 

gccgatacca gaaaatatca tggctacatg atggctaagg caccacatga tttcatgttt    420 

gtgaagagga atgatccaga tggacctcat tcagacagaa tctattacct tgccatgtct    480 

ggtgagaaca gagaaaatac actgttttat tctgaaattc ccaaaactat caatagagca    540 

gcagtcttaa tgctctcttg gaagcctctt ttggatcttt ttcaggcaac actggactat    600 

ggaatgtatt ctcgagaaga agaactatta agagaaagaa aacgcattgg aacagtcgga    660 

attgcttctt acgattatca ccaaggaagt ggaacatttc tgtttcaagc cggtagtgga    720 

atttatcacg taaaagatgg agggccacaa ggatttacgc aacaaccttt aaggcccaat    780 

ctagtggaaa ctagttgtcc caacatacgg atggatccaa aattatgccc cgctgatcca    840 

gactggattg cttttataca tagcaacgat atttggatat ctaacatcgt aaccagagaa    900 

gaaaggagac tcacttatgt gcacaatgag ctagccaaca tggaagaaga tgccagatca    960 

gctggagtcg ctacctttgt tctccaagaa gaatttgata gatattctgg ctattggtgg   1020 

tgtccaaaag ctgaaacaac tcccagtggt ggtaaaattc ttagaattct atatgaagaa   1080 

aatgatgaat ctgaggtgga aattattcat gttacatccc ctatgttgga aacaaggagg   1140 

gcagattcat tccgttatcc taaaacaggt acagcaaatc ctaaagtcac ttttaagatg   1200 

tcagaaataa tgattgatgc tgaaggaagg atcatagatg tcatagataa ggaactaatt   1260 

caaccttttg agattctatt tgaaggagtt gaatatattg ccagagctgg atggactcct   1320 

gagggaaaat atgcttggtc catcctacta gatcgctccc agactcgcct acagatagtg   1380 

ttgatctcac ctgaattatt tatcccagta gaagatgatg ttatggaaag gcagagactc   1440 

attgagtcag tgcctgattc tgtgacgcca ctaattatct atgaagaaac aacagacatc   1500 

tggataaata tccatgacat ctttcatgtt tttccccaaa gtcacgaaga ggaaattgag   1560 

tttatttttg cctctgaatg caaaacaggt ttccgtcatt tatacaaaat tacatctatt   1620 

ttaaaggaaa gcaaatataa acgatccagt ggtgggctgc ctgctccaag tgatttcaag   1680 

tgtcctatca aagaggagat agcaattacc agtggtgaat gggaagttct tggccggcat   1740 

ggatctaata tccaagttga tgaagtcaga aggctggtat attttgaagg caccaaagac   1800 

tcccctttag agcatcacct gtacgtagtc agttacgtaa atcctggaga ggtgacaagg   1860 

ctgactgacc gtggctactc acattcttgc tgcatcagtc agcactgtga cttctttata   1920 

agtaagtata gtaaccagaa gaatccacac tgtgtgtccc tttacaagct atcaagtcct   1980 

gaagatgacc caacttgcaa aacaaaggaa ttttgggcca ccattttgga ttcagcaggt   2040 

cctcttcctg actatactcc tccagaaatt ttctcttttg aaagtactac tggatttaca   2100 

ttgtatggga tgctctacaa gcctcatgat ctacagcctg gaaagaaata tcctactgtg   2160 

ctgttcatat atggtggtcc tcaggtgcag ttggtgaata atcggtttaa aggagtcaag   2220 

tatttccgct tgaataccct agcctctcta ggttatgtgg ttgtagtgat agacaacagg   2280 

ggatcctgtc accgagggct taaatttgaa ggcgccttta aatataaaat gggtcaaata   2340 

gaaattgacg atcaggtgga aggactccaa tatctagctt ctcgatatga tttcattgac   2400 

ttagatcgtg tgggcatcca cggctggtcc tatggaggat acctctccct gatggcatta   2460 

atgcagaggt cagatatctt cagggttgct attgctgggg ccccagtcac tctgtggatc   2520 

ttctatgata caggatacac ggaacgttat atgggtcacc ctgaccagaa tgaacagggc   2580 

tattacttag gatctgtggc catgcaagca gaaaagttcc cctctgaacc aaatcgttta   2640 

ctgctcttac atggtttcct ggatgagaat gtccattttg cacataccag tatattactg   2700 

agttttttag tgagggctgg aaagccatat gatttacaga tctatcctca ggagagacac   2760 

agcataagag ttcctgaatc gggagaacat tatgaactgc atcttttgca ctaccttcaa   2820 

gaaaaccttg gatcacgtat tgctgctcta aaagtgatat aattttgacc tgtgtagaac   2880 

tctctggtat acactggcta tttaaccaaa tgaggaggtt taatcaacag aaaacacaga   2940 

attgatcatc acattttgat acctgccatg taacatctac tcctgaaaat aaatgtggtg   3000 

ccatgcaggg gtctacggtt tgtggtagta atctaatacc ttaaccccac atgctcaaaa   3060 

tcaaatgata catattcctg agagacccag caataccata agaattacta aaaaaaaaaa   3120 

 
           
             6  
             882  
             PRT  
             Homo sapiens  
           
            6 

Met Ala Ala Ala Met Glu Thr Glu Gln Leu Gly Val Glu Ile Phe Glu 
1               5                   10                  15 

Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro Lys Leu 
            20                  25                  30 

Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu Lys Lys Leu 
        35                  40                  45 

Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met Ala Lys Ala Pro 
    50                  55                  60 

His Asp Phe Met Phe Val Lys Arg Asn Asp Pro Asp Gly Pro His Ser 
65                  70                  75                  80 

Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly Glu Asn Arg Glu Asn Thr 
                85                  90                  95 

Leu Phe Tyr Ser Glu Ile Pro Lys Thr Ile Asn Arg Ala Ala Val Leu 
            100                 105                 110 

Met Leu Ser Trp Lys Pro Leu Leu Asp Leu Phe Gln Ala Thr Leu Asp 
        115                 120                 125 

Tyr Gly Met Tyr Ser Arg Glu Glu Glu Leu Leu Arg Glu Arg Lys Arg 
    130                 135                 140 

Ile Gly Thr Val Gly Ile Ala Ser Tyr Asp Tyr His Gln Gly Ser Gly 
145                 150                 155                 160 

Thr Phe Leu Phe Gln Ala Gly Ser Gly Ile Tyr His Val Lys Asp Gly 
                165                 170                 175 

Gly Pro Gln Gly Phe Thr Gln Gln Pro Leu Arg Pro Asn Leu Val Glu 
            180                 185                 190 

Thr Ser Cys Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp 
        195                 200                 205 

Pro Asp Trp Ile Ala Phe Ile His Ser Asn Asp Ile Trp Ile Ser Asn 
    210                 215                 220 

Ile Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn Glu Leu 
225                 230                 235                 240 

Ala Asn Met Glu Glu Asp Ala Arg Ser Ala Gly Val Ala Thr Phe Val 
                245                 250                 255 

Leu Gln Glu Glu Phe Asp Arg Tyr Ser Gly Tyr Trp Trp Cys Pro Lys 
            260                 265                 270 

Ala Glu Thr Thr Pro Ser Gly Gly Lys Ile Leu Arg Ile Leu Tyr Glu 
        275                 280                 285 

Glu Asn Asp Glu Ser Glu Val Glu Ile Ile His Val Thr Ser Pro Met 
    290                 295                 300 

Leu Glu Thr Arg Arg Ala Asp Ser Phe Arg Tyr Pro Lys Thr Gly Thr 
305                 310                 315                 320 

Ala Asn Pro Lys Val Thr Phe Lys Met Ser Glu Ile Met Ile Asp Ala 
                325                 330                 335 

Glu Gly Arg Ile Ile Asp Val Ile Asp Lys Glu Leu Ile Gln Pro Phe 
            340                 345                 350 

Glu Ile Leu Phe Glu Gly Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr 
        355                 360                 365 

Pro Glu Gly Lys Tyr Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr 
    370                 375                 380 

Arg Leu Gln Ile Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val Glu 
385                 390                 395                 400 

Asp Asp Val Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser 
                405                 410                 415 

Val Thr Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn 
            420                 425                 430 

Ile His Asp Ile Phe His Val Phe Pro Gln Ser His Glu Glu Glu Ile 
        435                 440                 445 

Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr 
    450                 455                 460 

Lys Ile Thr Ser Ile Leu Lys Glu Ser Lys Tyr Lys Arg Ser Ser Gly 
465                 470                 475                 480 

Gly Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Ile Lys Glu Glu Ile 
                485                 490                 495 

Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg His Gly Ser Asn 
            500                 505                 510 

Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu Gly Thr Lys 
        515                 520                 525 

Asp Ser Pro Leu Glu His His Leu Tyr Val Val Ser Tyr Val Asn Pro 
    530                 535                 540 

Gly Glu Val Thr Arg Leu Thr Asp Arg Gly Tyr Ser His Ser Cys Cys 
545                 550                 555                 560 

Ile Ser Gln His Cys Asp Phe Phe Ile Ser Lys Tyr Ser Asn Gln Lys 
                565                 570                 575 

Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser Ser Pro Glu Asp Asp 
            580                 585                 590 

Pro Thr Cys Lys Thr Lys Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala 
        595                 600                 605 

Gly Pro Leu Pro Asp Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser 
    610                 615                 620 

Thr Thr Gly Phe Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu 
625                 630                 635                 640 

Gln Pro Gly Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro 
                645                 650                 655 

Gln Val Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg 
            660                 665                 670 

Leu Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn 
        675                 680                 685 

Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr 
    690                 695                 700 

Lys Met Gly Gln Ile Glu Ile Asp Asp Gln Val Glu Gly Leu Gln Tyr 
705                 710                 715                 720 

Leu Ala Ser Arg Tyr Asp Phe Ile Asp Leu Asp Arg Val Gly Ile His 
                725                 730                 735 

Gly Trp Ser Tyr Gly Gly Tyr Leu Ser Leu Met Ala Leu Met Gln Arg 
            740                 745                 750 

Ser Asp Ile Phe Arg Val Ala Ile Ala Gly Ala Pro Val Thr Leu Trp 
        755                 760                 765 

Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His Pro Asp 
    770                 775                 780 

Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met Gln Ala Glu 
785                 790                 795                 800 

Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu His Gly Phe Leu 
                805                 810                 815 

Asp Glu Asn Val His Phe Ala His Thr Ser Ile Leu Leu Ser Phe Leu 
            820                 825                 830 

Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln Ile Tyr Pro Gln Glu Arg 
        835                 840                 845 

His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu Leu His Leu 
    850                 855                 860 

Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile Ala Ala Leu Lys 
865                 870                 875                 880 

Val Ile 

 
           
             7  
             830  
             PRT  
             Homo sapiens  
           
            7 

Leu Arg Ser Ile Ile His Gly Ser Arg Lys Tyr Ser Gly Leu Ile Val 
1               5                   10                  15 

Asn Lys Ala Pro His Asp Phe Gln Phe Val Gln Lys Thr Asp Glu Ser 
            20                  25                  30 

Gly Pro His Ser His Arg Leu Tyr Tyr Leu Gly Met Pro Tyr Gly Ser 
        35                  40                  45 

Arg Glu Asn Ser Leu Leu Tyr Ser Glu Ile Pro Lys Lys Val Arg Lys 
    50                  55                  60 

Glu Ala Leu Leu Leu Leu Ser Trp Lys Gln Met Leu Asp His Phe Gln 
65                  70                  75                  80 

Ala Thr Pro His His Gly Val Tyr Ser Arg Glu Glu Glu Leu Leu Arg 
                85                  90                  95 

Glu Arg Lys Arg Leu Gly Val Phe Gly Ile Thr Ser Tyr Asp Phe His 
            100                 105                 110 

Ser Glu Ser Gly Leu Phe Leu Phe Gln Ala Ser Asn Ser Leu Phe His 
        115                 120                 125 

Cys Arg Asp Gly Gly Lys Asn Gly Phe Met Val Ser Pro Met Lys Pro 
    130                 135                 140 

Leu Glu Ile Lys Thr Gln Cys Ser Gly Pro Arg Met Asp Pro Lys Ile 
145                 150                 155                 160 

Cys Pro Ala Asp Pro Ala Phe Phe Ser Phe Asn Asn Asn Ser Asp Leu 
                165                 170                 175 

Trp Val Ala Asn Ile Glu Thr Gly Glu Glu Arg Arg Leu Thr Phe Cys 
            180                 185                 190 

His Gln Gly Leu Ser Asn Val Leu Asp Asp Pro Lys Ser Ala Gly Val 
        195                 200                 205 

Ala Thr Phe Val Ile Gln Glu Glu Phe Asp Arg Phe Thr Gly Tyr Trp 
    210                 215                 220 

Trp Cys Pro Thr Ala Ser Trp Glu Gly Ser Gln Gly Leu Lys Thr Leu 
225                 230                 235                 240 

Arg Ile Leu Tyr Glu Glu Val Asp Glu Ser Glu Val Glu Val Ile His 
                245                 250                 255 

Val Pro Ser Pro Ala Leu Glu Glu Arg Lys Thr Asp Ser Tyr Arg Tyr 
            260                 265                 270 

Pro Arg Thr Gly Ser Lys Asn Pro Lys Ile Ala Leu Lys Leu Ala Glu 
        275                 280                 285 

Phe Gln Thr Asp Ser Gln Gly Lys Ile Val Ser Thr Gln Glu Lys Glu 
    290                 295                 300 

Leu Val Gln Pro Phe Ser Ser Leu Phe Pro Lys Val Glu Tyr Ile Ala 
305                 310                 315                 320 

Arg Ala Gly Trp Thr Arg Asp Gly Lys Tyr Ala Trp Ala Met Phe Leu 
                325                 330                 335 

Asp Arg Pro Gln Gln Trp Leu Gln Leu Val Leu Leu Pro Pro Ala Leu 
            340                 345                 350 

Phe Ile Pro Ser Thr Glu Asn Glu Glu Gln Arg Leu Ala Ser Ala Arg 
        355                 360                 365 

Ala Val Pro Arg Asn Val Gln Pro Tyr Val Val Tyr Glu Glu Val Thr 
    370                 375                 380 

Asn Val Trp Ile Asn Val His Asp Ile Phe Tyr Pro Phe Pro Gln Ser 
385                 390                 395                 400 

Glu Gly Glu Asp Glu Leu Cys Phe Leu Arg Ala Asn Glu Cys Lys Thr 
                405                 410                 415 

Gly Phe Cys His Leu Tyr Lys Val Thr Ala Val Leu Lys Ser Gln Gly 
            420                 425                 430 

Tyr Asp Trp Ser Glu Pro Phe Ser Pro Gly Glu Asp Glu Phe Lys Cys 
        435                 440                 445 

Pro Ile Lys Glu Glu Ile Ala Leu Thr Ser Gly Glu Trp Glu Val Leu 
    450                 455                 460 

Ala Arg His Gly Ser Lys Ile Trp Val Asn Glu Glu Thr Lys Leu Val 
465                 470                 475                 480 

Tyr Phe Gln Gly Thr Lys Asp Thr Pro Leu Glu His His Leu Tyr Val 
                485                 490                 495 

Val Ser Tyr Glu Ala Ala Gly Glu Ile Val Arg Leu Thr Thr Pro Gly 
            500                 505                 510 

Phe Ser His Ser Cys Ser Met Ser Gln Asn Phe Asp Met Phe Val Ser 
        515                 520                 525 

His Tyr Ser Ser Val Ser Thr Pro Pro Cys Val His Val Tyr Lys Leu 
    530                 535                 540 

Ser Gly Pro Asp Asp Asp Pro Leu His Lys Gln Pro Arg Phe Trp Ala 
545                 550                 555                 560 

Ser Met Met Glu Ala Ala Ser Cys Pro Pro Asp Tyr Val Pro Pro Glu 
                565                 570                 575 

Ile Phe His Phe His Thr Arg Ser Asp Val Arg Leu Tyr Gly Met Ile 
            580                 585                 590 

Tyr Lys Pro His Ala Leu Gln Pro Gly Lys Lys His Pro Thr Val Leu 
        595                 600                 605 

Phe Val Tyr Gly Gly Pro Gln Val Gln Leu Val Asn Asn Ser Phe Lys 
    610                 615                 620 

Gly Ile Lys Tyr Leu Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Ala 
625                 630                 635                 640 

Val Val Val Ile Asp Gly Arg Gly Ser Cys Gln Arg Gly Leu Arg Phe 
                645                 650                 655 

Glu Gly Ala Leu Lys Asn Gln Met Gly Gln Val Glu Ile Glu Asp Gln 
            660                 665                 670 

Val Glu Gly Leu Gln Phe Val Ala Glu Lys Tyr Gly Phe Ile Asp Leu 
        675                 680                 685 

Ser Arg Val Ala Ile His Gly Trp Ser Tyr Gly Gly Phe Leu Ser Leu 
    690                 695                 700 

Met Gly Leu Ile His Lys Pro Gln Val Phe Lys Val Ala Ile Ala Gly 
705                 710                 715                 720 

Ala Pro Val Thr Val Trp Met Ala Tyr Asp Thr Gly Tyr Thr Glu Arg 
                725                 730                 735 

Tyr Met Asp Val Pro Glu Asn Asn Gln His Gly Tyr Glu Ala Gly Ser 
            740                 745                 750 

Val Ala Leu His Val Glu Lys Leu Pro Asn Glu Pro Asn Arg Leu Leu 
        755                 760                 765 

Ile Leu His Gly Phe Leu Asp Glu Asn Val His Phe Phe His Thr Asn 
    770                 775                 780 

Phe Leu Val Ser Gln Leu Ile Arg Ala Gly Lys Pro Tyr Gln Leu Gln 
785                 790                 795                 800 

Ile Tyr Pro Asn Glu Arg His Ser Ile Arg Cys Pro Glu Ser Gly Glu 
                805                 810                 815 

His Tyr Glu Val Thr Leu Leu His Phe Leu Gln Glu Tyr Leu 
            820                 825                 830 

 
           
             8  
             2495  
             DNA  
             Homo sapiens  
           
            8 

ctccggagca tcatccacgg cagccgcaag tactcgggcc tcattgtcaa caaggcgccc     60 

cacgacttcc agtttgtgca gaagacggat gagtctgggc cccactccca ccgcctctac    120 

tacctgggaa tgccatatgg cagccgggag aactccctcc tctactctga gattcccaag    180 

aaggtccgga aagaggctct gctgctcctg tcctggaagc agatgctgga tcatttccag    240 

gccacgcccc accatggggt ctactctcgg gaggaggagc tgctgaggga gcggaaacgc    300 

ctgggggtct tcggcatcac ctcctacgac ttccacagcg agagtggcct cttcctcttc    360 

caggccagca acagcctctt ccactgccgc gacggcggca agaacggctt catggtgtcc    420 

cctatgaaac cgctggaaat caagacccag tgctcagggc cccggatgga ccccaaaatc    480 

tgccctgccg accctgcctt cttctccttc aacaataaca gcgacctgtg ggtggccaac    540 

atcgagacag gcgaggagcg gcggctgacc ttctgccacc aaggtttatc caatgtcctg    600 

gatgacccca agtctgcggg tgtggccacc ttcgtcatac aggaagagtt cgaccgcttc    660 

actgggtact ggtggtgccc cacagcctcc tgggaaggtt cagagggcct caagacgctg    720 

cgaatcctgt atgaggaagt cgatgagtcc gaggtggagg tcattcacgt cccctctcct    780 

gcgctagaag aaaggaagac ggactcgtat cggtacccca ggacaggcag caagaatccc    840 

aagattgcct tgaaactggc tgagttccag actgacagcc agggcaagat cgtctcgacc    900 

caggagaagg agctggtgca gcccttcagc tcgctgttcc cgaaggtgga gtacatcgcc    960 

agggccgggt ggacccggga tggcaaatac gcctgggcca tgttcctgga ccggccccag   1020 

cagtggctcc agctcgtcct cctccccccg gccctgttca tcccgagcac agagaatgag   1080 

gagcagcggc tagcctctgc cagagctgtc cccaggaatg tccagccgta tgtggtgtac   1140 

gaggaggtca ccaacgtctg gatcaatgtt catgacatct tctatccctt cccccaatca   1200 

gagggagagg acgagctctg ctttctccgc gccaatgaat gcaagaccgg cttctgccat   1260 

ttgtacaaag tcaccgccgt tttaaaatcc cagggctacg attggagtga gcccttcagc   1320 

cccggggaag atgaatttaa gtgccccatt aaggaagaga ttgctctgac cagcggtgaa   1380 

tgggaggttt tggcgaggca cggctccaag atctgggtca atgaggagac caagctggtg   1440 

tacttccagg gcaccaagga cacgccgctg gagcaccacc tctacgtggt cagctatgag   1500 

gcggccggcg agatcgtacg cctcaccacg cccggcttct cccatagctg ctccatgagc   1560 

cagaacttcg acatgttcgt cagccactac agcagcgtga gcacgccgcc ctgcgtgcac   1620 

gtctacaagc tgagcggccc cgacgacgac cccctgcaca agcagccccg cttctgggct   1680 

agcatgatgg aggcagccag ctgccccccg gattatgttc ctccagagat cttccatttc   1740 

cacacgcgct cggatgtgcg gctctacggc atgatctaca agccccacgc cttgcagcca   1800 

gggaagaagc accccaccgt cctctttgta tatggaggcc cccaggtgca gctggtgaat   1860 

aactccttca aaggcatcaa gtacttgcgg ctcaacacac tggcctccct gggctacgcc   1920 

gtggttgtga ttgacggcag gggctcctgt cagcgagggc ttcggttcga aggggccctg   1980 

aaaaaccaaa tgggccaggt ggagatcgag gaccaggtgg agggcctgca gttcgtggcc   2040 

gagaagtatg gcttcatcga cctgagccga gttgccatcc atggctggtc ctacgggggc   2100 

ttcctctcgc tcatggggct aatccacaag ccccaggtgt tcaaggtggc catcgcgggt   2160 

gccccggtca ccgtctggat ggcctacgac acagggtaca ctgagcgcta catggacgtc   2220 

cctgagaaca accagcacgg ctatgaggcg ggttccgtgg ccctgcacgt ggagaagctg   2280 

cccaatgagc ccaaccgctt gcttatcctc cacggcttcc tggacgaaaa cgtgcacttt   2340 

ttccacacaa acttcctcgt ctcccaactg atccgagcag ggaaacctta ccagctccag   2400 

atctacccca acgagagaca cagtattcgc tgccccgagt cgggcgagca ctatgaagtc   2460 

acgttactgc actttctaca ggaatacctc tgagc                              2495