Rat monoclonal autoantibody to pancreatic islet cells recognizes a sugar sequence in paragloboside

The BB rat is an experimental model of spontaneously occurring diabetes mellitus (DM). To investigate the autoimmune pathogenesis of DM, spleen cells of newly diagnosed diabetic BB rats were fused with mouse myeloma cells. Hybridomas were screened by indirect immunofluorescence (IF) and by 51Cr release assays using the RINm5F rat insulinoma cell line. One clone (E5C2) produced an IgM antibody cytotoxic for RINm5F cells but not other rat cells. By IF, neuraminidase (NASE) increased binding of E5C2 to RINm5F cells (10% to 80%). E5C2 bound specifically to normal rat and human pancreatic islets, but only after NASE treatment. Using direct immunostaining of thin-layer chromatography plates, E5C2 bound to glycolipids isolated from various tissues. With purified glycolipids E5C2 bound only to paragloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-cer) and higher polylactosamine containing structures. Periodate treatment or substitution of paragloboside (PG) with other sugars abolished antigenicity. By hapten-inhibition assays lactose (Galβ1-4G1c) and Galβ1-4GlcNAc inhibited binding but Galβ1-3GlcNAc was ineffective. PG or its sialylated derivatives were not found in RINm5F cells. However, glycoproteins were found on Western blots after NASE treatment. Lactose also inhibited this E5C2 binding. Similar autoantibodies may play a role in the pathogenesis of DM