64,000-Mr autoantigen in type I diabetes. Evidence against its surface location on human islets.
The sera of type I (insulin-dependent) diabetic subjects are reported to contain autoantibodies against a 64,000-Mr protein identified in [35S]methionine biosynthetically labeled pancreatic islet cells. We have attempted to localize this autoantigen to the surface of the beta-cell and to define its properties. Sera from 10 newly diagnosed type I diabetic subjects, including five of the index sera originally used to identify the autoantigen, were shown to specifically precipitate a reduced protein of 67,000 Mr from Triton-solubilized, surface 125I-labeled cultured adult human islet and rat insulinoma (RINm5F) cells but not from fresh rat spleen cells. Further characterization revealed that this protein was bovine serum albumin (BSA) adsorbed to cells from fetal calf serum (FCS)-supplemented culture medium and precipitated by BSA antibodies present in many diabetic sera. No labeled proteins were specifically precipitated when surface 125I-labeled and solubilized human islet or RINm5F cells were precleared with anti-BSA immunoglobulins or when cells were first cultured in human serum. In contrast, a 64,000-Mr protein, clearly not BSA, was precipitated by diabetic globulins from human islets but not from RINm5F cells labeled with [35S]methionine. In addition, a protein of the same size as well as proteins of approximately 35,000, 43,000, 140,000, and 200,000 Mr were specifically precipitated by diabetic globulins from freshly isolated human islets solubilized in Triton X-100 and then labeled with 125I. These findings suggest that the 64,000-Mr antigen is not expressed on the surface of human islet cells, at least in culture, and therefore question its relevance as a target for islet cell surface antibodies in initiating beta-cell damage.(ABSTRACT TRUNCATED AT 250 WORDS)
Colman
P G
PG
Department of Diabetes and Endocrinology, Royal Melbourne Hospital, Victoria, Australia.
Campbell
I L
IL
Kay
T W
TW
Harrison
L C
LC
D000293:Adolescent; D000328:Adult; D001324:Autoantigens / Q000032:analysis*; D002460:Cell Line; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D007515:Islets of Langerhans / Q000276:immunology*; D008297:Male; D008970:Molecular Weight
D001324:Autoantigens; D007457:Iodine Radioisotopes