The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2.
Major targets for autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) include tryptic fragments with a molecular mass of 37 kDa and/or 40 kDa of a pancreatic islet cell antigen of unknown identity. The assay identifying autoantibodies against the 37/40-kDa antigen in human sera is based on the immunoprecipitation of 35S-labeled rat insulinoma cell proteins with sera from IDDM patients, followed by limited trypsin digestion of the immunoprecipitated material. To identify cDNA clones coding for the 37/40-kDa antigen, we have screened a cDNA expression library from rat insulinoma cells with a serum from an IDDM patient that precipitated the 37/40-kDa antigen in our assay. Among the cDNA products that reacted with the IDDM serum, we identified one cDNA clone whose open reading frame encodes a protein with a predicted mass of 105 kDa that we termed "ICA105" for 105-kDa islet cell antibody. The deduced amino acid sequence has high homology to a recently cloned putative tyrosine phosphatase IA-2 from human and mouse cDNA libraries. Translation of the cDNA in vitro results in a polypeptide with the expected molecular mass of 105 kDa. The evidence that ICA105 is indeed the precursor of the 37/40-kDa tryptic fragments is based on the following three results: (i) Sera from IDDM patients containing autoantibodies to the 37/40-kDa antigen precipitate the in vitro translated polypeptide, whereas sera from healthy subjects as well as sera from IDDM patients not reactive with the 37/40-kDa antigen do not precipitate the cDNA product. (ii) Immunoprecipitation of the in vitro translated protein with sera containing autoantibodies to the 37/40-kDa antigen followed by limited trypsin digestion of the precipitated proteins results in a 40-kDa polypeptide. (iii) The protein derived from our cDNA but not from an unrelated control cDNA clone can block immunoprecipitation of the 37/40-kDa antigen from a labeled rat insulinoma cell extract. The availability of the cloned 37/40-kDa antigen should facilitate the identification of individuals at risk of IDDM with increased accuracy. Furthermore, the identification of the 37/40-kDa antigen as the putative tyrosine phosphatase IA-2 is of relevance in elucidating the role of this antigen in the development of IDDM.
Passini
N
N
Roche Milano Ricerche, Italy.
Larigan
J D
JD
Genovese
S
S
Appella
E
E
Sinigaglia
F
F
Rogge
L
L
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000096:biosynthesis / Q000097:blood*; D001324:Autoantigens / Q000096:biosynthesis / Q000097:blood*; D001483:Base Sequence; D002460:Cell Line; D002648:Child; D003001:Cloning, Molecular; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000235:genetics / Q000276:immunology*; D005190:Family; D005260:Female; D006801:Humans; D007340:Insulinoma; D007515:Islets of Langerhans / Q000276:immunology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010190:Pancreatic Neoplasms; D011236:Prediabetic State / Q000097:blood / Q000276:immunology*; D011237:Predictive Value of Tests; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000097:blood* / Q000276:immunology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D001323:Autoantibodies; D001324:Autoantigens; D011994:Recombinant Proteins; C024211:islet cell antibody; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases