64,000-M_r 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-M_r protein identified in [₃₅S]methionine biosynthetically labeled pancreatic islet cells. We have attempted to localize this autoantigen to the surface of the β-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 M_r 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-M_r protein, clearly not BSA, was precipitated by diabetic globulins from human islets but not from RINm5F cells labeled with [³⁵]Slmethionine. In addition, a protein of the same size as well as proteins of ∼35,000, 43,000, 140,000, and 200,000 M_r 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-M_r 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 β-cell damage. They also reveal the existence of other possible protein antigens in islet cells. Finally, our results indicate that assays for islet cell surface antibodies via immunofluorescence on cultured cells should be controlled for the presence of BSA antibodies.