Abstract:
A composition and method for the diagnosis of autoimmune hepatitis. The composition, which contains SLA antigens detects soluble liver antigen (SLA) auto-antibodies, which occur in sera of patients suffering from chronic hepatitis.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The present invention relates to a composition and method for diagnosing auto-immune hepatitis, which use an immune reaction to detect soluble liver antigen (“SLA”) auto-antibodies.  
           [0002]    Auto-immune hepatitis (AIH) is a chronic inflammation of the liver that when left untreated leads to cirrhosis, but when treated has a very good prognosis. For this reason, a timely diagnosis is important. It is estimated that 5% of all patients in Western countries who have chronic hepatitis have AIH. At the present time, there is no specific diagnostic test for AIH. Rather the diagnosis for AIH is undertaken by a plurality of diagnosis, such as excluding viral hepatitis, recognizing [hyper] immunoglobulinaemia, recognizing the tissue type (HLA type), and detecting auto-antibodies. Auto-antibodies are found in about 90% of patients with chronic hepatitis, and most of the detectable auto-antibodies are also present, at least in low titers, in other inflammations of the liver as well. In particular, these auto-antibodies are antibodies to nuclear antigens (ANA) and unstriated muscles (SMA), as well as the very rare antibodies to cytochrome p450 (LKM). SLA auto-antibodies were described for the first time in 1987 (Manns M. et al., Lancet 1987; 1:292-4). Tests have shown that SLA auto-antibodies occur in about 25 to 30% of patients having AIH, but hardly ever occur in patients having other diseases, including other auto-immune diseases (Lohse A. W. et al., Z. Gastroenterol 1995;33:527-33). Detecting SLA auto-antibodies therefore provides a significant diagnostic procedure for recognizing AIH.  
           [0003]    The present invention is directed to use and detection of SLA antigens, which are a prerequisite for developing a specific immunoassay for detecting SLA auto-antibodies in patients&#39; sera. Previous methods known in the art could not detect such SLA auto-antibodies in patients&#39; sera. Liver cytokeratins 8 and 18 were described in 1990 as target antigens of the SLA auto-antibodies (Journal of Hepatology, 1990; 11: 232 to 239), however, these findings were never confirmed, and were even refuted (Wies I. et al., Z. Gastroenterol. 1998;36:93).  
         SUMMARY OF THE INVENTION  
         [0004]    The invention is directed to a composition for detecting the presence of SLA auto-antibodies in blood serum, said composition comprising an antigen that is recognized by SLA auto-antibodies. In particular the invention is directed to antigens having amino acid sequence SEQ ID NO. 1, SEQ ID NO. 2, or antigenic derivatives thereof. Another embodiment of the invention is a purified protein or polypeptide recognized by SLA auto-antibodies. In a perferred embodiment of the invention the purified protein has the amino acid sequence SEQ ID NO. 1, SEQ ID NO. 2, or is an antigenic derivative thereof, or is a fusion protein containing either SEQ ID NO. 1, SEQ ID NO 2, or antigenic derivatives thereof. In another aspect of the invention, there is disclosed a cDNA having a nucleotide sequence that codes for an antigen recognized by SLA auto-antibodies in blood serum. In a preferred embodiment of this aspect of the invention the cDNA codes for an antigen having the amino acid sequence SEQ ID NO. 1, SEQ ID NO. 2, or an antigenic derivative thereof.  
           [0005]    Another aspect of the invention is directed to a method of detecting the presence of SLA auto-antibodies in a blood sample by binding the composition of the invention to a matrix, detecting the binding of SLA auto-antibodies bound to the antigens in the composition, and correlating such binding to the presence of SLA auto-antibodies in the sample. Examples of suitable methods in which the present compositions can be used to detect SLA auto-antibodies in blood include immunoassays such as, but not limited to, radioimmunoassay, chemiluninesence immunoassay, immunoblot assay, enzyme assay and inhibition immunoassay.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0006]    The present invention relates to a composition and a method for diagnosing auto-immune hepatitis, by enabling one to detect in the blood serum of a patient, antibodies to two proteins or polypeptides (SLA antigens), which antigens wholly or partially contain the amino acid sequence corresponding to SEQ ID NO. 1 or SEQ ID NO. 2 or antigenic derivatives of the SLA antigens, which are recognized by SLA auto-antibodies. These polypeptides are referred to as SLA-1 and as SLA-2. One embodiment of the invention is directed to synthetic or natural variants of SLA-1 or SLA-2, or sythetic or natural variants of partial or incomplete polypeptides of SLA-1 or SLA-2, which correspond wholly or partially to the amino acid sequences or antigenic derivatives thereof and are likewise recognized by SLA auto-antibodies.  
           [0007]    In a preferred embodiment, the peptides of the invention are fusion proteins. Such fusion proteins can be made by known methods in the art (Maniatis, T. et al.,  Molecular Cloning,  1982: 412-430).  
           [0008]    Another embodiment of the invention is directed to cDNA, which encodes a natural or synthetic variant of one of the SLA antigens SLA-1 or SLA-2, having a nucleotide sequence corresponding to SEQ ID NO. 1 or to SEQ ID NO. 2 or to antigenic derivatives thereof. The cDNAs are present as two spliced variants, the longer of these (SEQ ID NO. 2) having an insertio of 156 nucleotides. SLA-1 and SLA-2 have similiar nucleotide sequences, except that SLA-2 contains a 156 nucleotide insert.  
           [0009]    SLA-positive sera, i.e. SLA auto-antibody containing sera, produces a double band on a Western blot when probed with the SLA antigens. The double band measures 50 kDa, which corresponds to the molecular weight of the SLA antigens, SLA-1 and SLA-2. When SLA-positive serum is incubated with the fusion protein according to the present invention, e.g. SLA-1 or SLA-2 fused to another protein or polypeptide, the double band corresponding to the SLA-antigen is not detected. Incubation of SLA-positive sera with a control fusion protein, which does not contain either SLA-1 or SLA-2 does not eliminate the appearance of a double band on Western blots.  
           [0010]    The cDNA according to the present invention allows for the production of large quantities of the SLA antigens in a recombinant fashion. Further, the concentration of SLA antigens can be measured using known SLA auto-antibody methods.  
           [0011]    In another aspect of the invention there is dislcosed a method for determining the presence of SLA auto-antibodies in a blood sample using the composition of the invention in known assay methods such as, but not limited to, radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), immunoblot assay or enzyme immunoassay (EIA). In these assays, one of the SLA antigens (SLA-1 or SLA-2) is bound to a matrix, such as a microtiter plate or a blot membrane. Then the patient&#39;s serum to be tested is applied to the matrix with the bound SLA antigen. Following incubation, the test serum is washed off. The specific binding of SLA auto-antibodies (in the test serum) to the matrix-bound SLA-antigen is detected and verified by using a secondary antibody (anti-human immunoglobulin or an immunoglobulin subclass, for example) that has been labeled with a tracer. Tracers commonly used include enzyme compounds such as peroxidase or alkaline phosphatase, radioactive compounds, or a chemiluminescing substances. The more SLA auto-antibodies present in the test serum, the more tracer is bound to the matrix. Normal serum from healthy blood donors can be used as a negative control. Additional controls include the use of SLA auto-antibody positive serum that has SLA auto-antibodies present at known various concentrations.  
           [0012]    Alternatively, an inhibition immunoassay can also be performed using the SLA antigens of the present invention. In such an assay the SLA antigens bind to a selected SLA auto-antibody, which has been labeled with a tracer. The amount of binding of the labeled SLA auto-antibody to the SLA antigens is measured as the control. A patient&#39;s sera is then tested against the control. The patient&#39;s serum is added along with the labeled SLA auto-antibodies. SLA auto-antibodies present in the patient&#39;s serum compete with the labeled SLA auto-antibodies in binding to the SLA antigens. Thus, if the patient&#39;s serum contains SLA auto-antibodies, the amount of labeled SLA auto-antibodies present will be less than the control. Therefore the detection of fewer bound labeled SLA auto-antibodies reveals the presence of competing SLA auto-antibodies in the test serum. 
       
    
    
     EXAMPLE 1  
       [0013]    Detecting SLA Auto-Antibodies in the ELISA Test Using the Recombinant SLA Antigen (SEQ ID No. 1)  
         [0014]    The recombinant SLA antigen was purified using the his-tag. 50 μg of the SLA antigen in 50 μl phosphate buffered saline (“PBS”) (pH 7.0) was placed in a microtiter plate and left over night. After pipetting off and washing the microtiter plate, a post-coating was carried out for one hour using a 1% Bovine Serum Albumin (“BSA”)/PBS solution at room temperature. 50 μl each of normal sera, patients&#39; sera, and control sera were diluted 1:100 with PBS. 50 μl of each diluted serum was incubated for 30 minutes at room temperature on its respective microtiter plate. The plates were then washed 1-5 times and a peroxidase-labeled secondary antibody, an antihuman immunoglobulin, diluted 1:8000 was then added and allowed to incubate. After washing out the excess secondary antibody, ABTS solution [55 mg of 2 2′-azidodi-3-ethyl-benzthiazine-6-sulphonic acid (Serva, Heidelberg) in 5 ml 0.01 M K 2  HPO 4  (pH 6.0), containing 50 μl H 2 0 2 , diluted in a 1:300 proportion in PBS] was added. The optical density of the microtiter plates was measured with a Titertec Multiscan MC (Flow Laboratories, Meckenheim). Table 1 reveals that all of the patients&#39; sera containing SLA auto-bodies exhibited distinctly elevated values of light absorption, as compared to normal serum, and serum of patients who suffered from other liver diseases.  
                           TABLE 1                                   Serum   OD                           normal serum 1   0.476           normal serum 1   0.471           SLA-serum 1   1.863           SLA-serum 2   1.995           SLA-serum 3   1.791           SLA-serum 4   1.867           AMA-pos. serum (PBC)   0.615           LKM-pos. serum (hep. C)   0.769                                  
 
         [0015]    [0015] 
     
       
       
         1 
         
           
             4  
           
           
             1  
             1335  
             DNA  
             Homo sapiens  
             
               CDS  
               (1)...(1335)  
             
           
            1 

tcg cgg cgg gag agc ggc tgg tgt cgc cgg ctt acg tgc ggc agg gct       48 
Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
 1               5                   10                  15 

gtg agg ccc gcc gct cgc atg agc acc tca tac ggc tgc ttc tgg aga       96 
Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
             20                  25                  30 

agg ttc att cat ggc att gga cga tcc ggt gat att tct gct gtg caa      144 
Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile Ser Ala Val Gln 
         35                  40                  45 

cca aaa gct gca ggc tct agc ctt ttg aac aaa att acc aat tct ttg      192 
Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile Thr Asn Ser Leu 
     50                  55                  60 

gtc ctg gac att ata aag ctg gct ggt gtc cat aca gta gcc aac tgc      240 
Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr Val Ala Asn Cys 
 65                  70                  75                  80 

ttt gta gtt cct atg gca act ggt atg agt cta act ctg tgt ttc tta      288 
Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr Leu Cys Phe Leu 
                 85                  90                  95 

aca tta cga cac aaa aga cca aag gca aag tat att ata tgg cca cga      336 
Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile Ile Trp Pro Arg 
            100                 105                 110 

ata gac cag aag tcc tgc ttt aaa tcc atg atc act gca ggt ttt gag      384 
Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr Ala Gly Phe Glu 
        115                 120                 125 

cct gtg gtg ata gaa aat gtt ttg gaa ggt gac gag ctg cgt aca gac      432 
Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu Leu Arg Thr Asp 
    130                 135                 140 

ctg aaa gca gtg gag gct aaa gtc cag gaa ctt ggg cct gat tgc att      480 
Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly Pro Asp Cys Ile 
145                 150                 155                 160 

ctg tgt att cat tct act aca tcc tgt ttt gct cca agg gtg cct gat      528 
Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro Arg Val Pro Asp 
                165                 170                 175 

aga tta gaa gaa ctg gct gtg att tgt gct aat tat gac att cca cat      576 
Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr Asp Ile Pro His 
            180                 185                 190 

ata gtt aat aat gct tat gga gtg cag tct tca aag tgt atg cat ctc      624 
Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys Cys Met His Leu 
        195                 200                 205 

att cag cag ggg gct cga gtt ggt aga ata gat gct ttt gtt cag agc      672 
Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala Phe Val Gln Ser 
    210                 215                 220 

ttg gac aaa aat ttt atg gtt cca gta ggt ggt gct ata att gct ggc      720 
Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala Ile Ile Ala Gly 
225                 230                 235                 240 

ttt aat gat tca ttc att cag gaa atc agc aag atg tat cca gga aga      768 
Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met Tyr Pro Gly Arg 
                245                 250                 255 

gct tca gct tca cct tct tta gat gtc ctt att act tta ttg tca ctt      816 
Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr Leu Leu Ser Leu 
            260                 265                 270 

gga tca aat ggc tat aag aag cta cta aaa gaa aga aag gaa atg ttt      864 
Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg Lys Glu Met Phe 
        275                 280                 285 

tca tat ttg tcc aac caa ata aag aag ttg tca gaa gcc tac aat gaa      912 
Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu Ala Tyr Asn Glu 
    290                 295                 300 

aga ctg ttg cat aca cct cac aat ccc ata tct tta gct atg aca ctt      960 
Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu Ala Met Thr Leu 
305                 310                 315                 320 

aaa aca cta gat gaa cac cgt gac aaa gct gtc act cag ctt ggc tcg     1008 
Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr Gln Leu Gly Ser 
                325                 330                 335 

atg ctt ttt acc aaa cag gtt tct gga gcc agg gtt gtg cct ctt ggg     1056 
Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val Val Pro Leu Gly 
            340                 345                 350 

tcc atg caa act gtg agt ggc tat act ttc aga ggc ttt atg tca cat     1104 
Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly Phe Met Ser His 
        355                 360                 365 

aca aat aat tac cct tgt gct tac ctc aat gct gca tca gcc atc gga     1152 
Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala Ser Ala Ile Gly 
    370                 375                 380 

atg aar atg cag gat gtg gac ctg ttc ata aac ara ctt gac agg tgt     1200 
Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa Leu Asp Arg Cys 
385                 390                 395                 400 

tta aag gca gta aga aaa gaa cga agt aaa gag agt gat gac aat tat     1248 
Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser Asp Asp Asn Tyr 
                405                 410                 415 

gac aaa act gaa rat gtg gat att gaa gaa atg gct tta aaa cta gat     1296 
Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala Leu Lys Leu Asp 
            420                 425                 430 

aat gta ctt ctt gac aca tac cag gat gct tct tca tga                 1335 
Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser  * 
        435                 440 

 
           
             2  
             444  
             PRT  
             Homo sapiens  
             
               VARIANT  
               (1)...(444)  
               Xaa = Any Amino Acid  
             
           
            2 

Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
 1               5                  10                  15 

Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
            20                  25                  30 

Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile Ser Ala Val Gln 
        35                  40                  45 

Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile Thr Asn Ser Leu 
    50                  55                  60 

Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr Val Ala Asn Cys 
65                  70                  75                  80 

Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr Leu Cys Phe Leu 
                85                  90                  95 

Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile Ile Trp Pro Arg 
            100                 105                 110 

Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr Ala Gly Phe Glu 
        115                 120                 125 

Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu Leu Arg Thr Asp 
    130                 135                 140 

Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly Pro Asp Cys Ile 
145                 150                 155                 160 

Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro Arg Val Pro Asp 
                165                 170                 175 

Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr Asp Ile Pro His 
            180                 185                 190 

Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys Cys Met His Leu 
        195                 200                 205 

Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala Phe Val Gln Ser 
    210                 215                 220 

Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala Ile Ile Ala Gly 
225                 230                 235                 240 

Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met Tyr Pro Gly Arg 
                245                 250                 255 

Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr Leu Leu Ser Leu 
            260                 265                 270 

Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg Lys Glu Met Phe 
        275                 280                 285 

Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu Ala Tyr Asn Glu 
    290                 295                 300 

Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu Ala Met Thr Leu 
305                 310                 315                 320 

Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr Gln Leu Gly Ser 
                325                 330                 335 

Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val Val Pro Leu Gly 
            340                 345                 350 

Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly Phe Met Ser His 
        355                 360                 365 

Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala Ser Ala Ile Gly 
    370                 375                 380 

Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa Leu Asp Arg Cys 
385                 390                 395                 400 

Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser Asp Asp Asn Tyr 
                405                 410                 415 

Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala Leu Lys Leu Asp 
            420                 425                 430 

Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
        435                 440 

 
           
             3  
             1491  
             DNA  
             Homo sapiens  
             
               CDS  
               (1)...(1491)  
             
             
               misc_feature  
               (1)...(1491)  
               n = A,T,C or G  
             
           
            3 

tcg cgg cgg gag agc ggc tgg tgt cgc cgg ctt acg tgc ggc agg gct       48 
Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
 1               5                   10                  15 

gtg agg ccc gcc gct cgc atg agc acc tca tac ggc tgc ttc tgg aga       96 
Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
             20                  25                  30 

agg gcw nnn tgt cca aag aat ggc tgg gat gaa agt aca ctt gaa ctc      144 
Arg Xaa Xaa Cys Pro Lys Asn Gly Trp Asp Glu Ser Thr Leu Glu Leu 
         35                  40                  45 

ttt tta cat gaa ctt gca atc atg gac agc aac aat ttc tta ggc aat      192 
Phe Leu His Glu Leu Ala Ile Met Asp Ser Asn Asn Phe Leu Gly Asn 
     50                  55                  60 

tgt ggt gtg gga gaa agg gaa ggg aga gtg gca tcc gca ctg gtt gct      240 
Cys Gly Val Gly Glu Arg Glu Gly Arg Val Ala Ser Ala Leu Val Ala 
 65                  70                  75                  80 

cgt cgt cat tac agg ttc att cat ggc att gga cga tcc ggt gat att      288 
Arg Arg His Tyr Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile 
                 85                  90                  95 

tct gct gtg caa cca aaa gct gca ggc tct agc ctt ttg aac aaa att      336 
Ser Ala Val Gln Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile 
            100                 105                 110 

acc aat tct ttg gtc ctg gac att ata aag ctg gct ggt gtc cat aca      384 
Thr Asn Ser Leu Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr 
        115                 120                 125 

gta gcc aac tgc ttt gta gtt cct atg gca act ggt atg agt cta act      432 
Val Ala Asn Cys Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr 
    130                 135                 140 

ctg tgt ttc tta aca tta cga cac aaa aga cca aag gca aag tat att      480 
Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile 
145                 150                 155                 160 

ata tgg cca cga ata gac cag aag tcc tgc ttt aaa tcc atg atc act      528 
Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr 
                165                 170                 175 

gca ggt ttt gag cct gtg gtg ata gaa aat gtt ttg gaa ggt gac gag      576 
Ala Gly Phe Glu Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu 
            180                 185                 190 

ctg cgt aca gac ctg aaa gca gtg gag gct aaa gtc cag gaa ctt ggg      624 
Leu Arg Thr Asp Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly 
        195                 200                 205 

cct gat tgc att ctg tgt att cat tct act aca tcc tgt ttt gct cca      672 
Pro Asp Cys Ile Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro 
    210                 215                 220 

agg gtg cct gat aga tta gaa gaa ctg gct gtg att tgt gct aat tat      720 
Arg Val Pro Asp Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr 
225                 230                 235                 240 

gac att cca cat ata gtt aat aat gct tat gga gtg cag tct tca aag      768 
Asp Ile Pro His Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys 
                245                 250                 255 

tgt atg cat ctc att cag cag ggg gct cga gtt ggt aga ata gat gct      816 
Cys Met His Leu Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala 
            260                 265                 270 

ttt gtt cag agc ttg gac aaa aat ttt atg gtt cca gta ggt ggt gct      864 
Phe Val Gln Ser Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala 
        275                 280                 285 

ata att gct ggc ttt aat gat tca ttc att cag gaa atc agc aag atg      912 
Ile Ile Ala Gly Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met 
    290                 295                 300 

tat cca gga aga gct tca gct tca cct tct tta gat gtc ctt att act      960 
Tyr Pro Gly Arg Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr 
305                 310                 315                 320 

tta ttg tca ctt gga tca aat ggc tat aag aag cta cta aaa gaa aga     1008 
Leu Leu Ser Leu Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg 
                325                 330                 335 

aag gaa atg ttt tca tat ttg tcc aac caa ata aag aag ttg tca gaa     1056 
Lys Glu Met Phe Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu 
            340                 345                 350 

gcc tac aat gaa aga ctg ttg cat aca cct cac aat ccc ata tct tta     1104 
Ala Tyr Asn Glu Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu 
        355                 360                 365 

gct atg aca ctt aaa aca cta gat gaa cac cgt gac aaa gct gtc act     1152 
Ala Met Thr Leu Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr 
    370                 375                 380 

cag ctt ggc tcg atg ctt ttt acc aaa cag gtt tct gga gcc agg gtt     1200 
Gln Leu Gly Ser Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val 
385                 390                 395                 400 

gtg cct ctt ggg tcc atg caa act gtg agt ggc tat act ttc aga ggc     1248 
Val Pro Leu Gly Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly 
                405                 410                 415 

ttt atg tca cat aca aat aat tac cct tgt gct tac ctc aat gct gca     1296 
Phe Met Ser His Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala 
            420                 425                 430 

tca gcc atc gga atg aar atg cag gat gtg gac ctg ttc ata aac ara     1344 
Ser Ala Ile Gly Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa 
        435                 440                 445 

ctt gac agg tgt tta aag gca gta aga aaa gaa cga agt aaa gag agt     1392 
Leu Asp Arg Cys Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser 
    450                 455                 460 

gat gac aat tat gac aaa act gaa rat gtg gat att gaa gaa atg gct     1440 
Asp Asp Asn Tyr Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala 
465                 470                 475                 480 

tta aaa cta gat aat gta ctt ctt gac aca tac cag gat gct tct tca     1488 
Leu Lys Leu Asp Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
                485                 490                 495 

tga                                                                 1491 
 * 

 
           
             4  
             496  
             PRT  
             Homo sapiens  
             
               VARIANT  
               (1)...(496)  
               Xaa = Any Amino Acid  
             
           
            4 

Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
 1               5                  10                  15 

Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
            20                  25                  30 

Arg Xaa Xaa Cys Pro Lys Asn Gly Trp Asp Glu Ser Thr Leu Glu Leu 
        35                  40                  45 

Phe Leu His Glu Leu Ala Ile Met Asp Ser Asn Asn Phe Leu Gly Asn 
    50                  55                  60 

Cys Gly Val Gly Glu Arg Glu Gly Arg Val Ala Ser Ala Leu Val Ala 
65                  70                  75                  80 

Arg Arg His Tyr Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile 
                85                  90                  95 

Ser Ala Val Gln Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile 
            100                 105                 110 

Thr Asn Ser Leu Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr 
        115                 120                 125 

Val Ala Asn Cys Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr 
    130                 135                 140 

Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile 
145                 150                 155                 160 

Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr 
                165                 170                 175 

Ala Gly Phe Glu Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu 
            180                 185                 190 

Leu Arg Thr Asp Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly 
        195                 200                 205 

Pro Asp Cys Ile Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro 
    210                 215                 220 

Arg Val Pro Asp Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr 
225                 230                 235                 240 

Asp Ile Pro His Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys 
                245                 250                 255 

Cys Met His Leu Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala 
            260                 265                 270 

Phe Val Gln Ser Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala 
        275                 280                 285 

Ile Ile Ala Gly Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met 
    290                 295                 300 

Tyr Pro Gly Arg Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr 
305                 310                 315                 320 

Leu Leu Ser Leu Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg 
                325                 330                 335 

Lys Glu Met Phe Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu 
            340                 345                 350 

Ala Tyr Asn Glu Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu 
        355                 360                 365 

Ala Met Thr Leu Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr 
    370                 375                 380 

Gln Leu Gly Ser Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val 
385                 390                 395                 400 

Val Pro Leu Gly Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly 
                405                 410                 415 

Phe Met Ser His Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala 
            420                 425                 430 

Ser Ala Ile Gly Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa 
        435                 440                 445 

Leu Asp Arg Cys Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser 
    450                 455                 460 

Asp Asp Asn Tyr Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala 
465                 470                 475                 480 

Leu Lys Leu Asp Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
                485                 490                 495