Peptide mapping and characterisation of glycation patterns of the glima 38antigen recognised by autoantibodies in Type I diabetic patients

Aims/hypothesis. Glima 38 is an N-glycated neuroendocrine membrane protein of M-r 38 000, which is recognised by autoantibodies in approximately 20 % of patients with Type I (insulin-dependent) diabetes mellitus. The aim of t his study was to characterise the carbohydrate moiety and generate peptide maps of glima 38. Methods. Sera of high immunoreactivity to glima 38 were used to isolate 35- S methionine-labelled protein from beta TC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidas es. The protein moiety was subjected to digestion by proteases to define pe ptide maps. Results. The autoreactive epitopes in glima 38 recognised by Type I diabeti c sera are conformational and independent of the carbohydrate moiety. Inhib ition of N-glycation of glima 38 in vivo, shows a protein core of M-r 22 00 0 in both pancreatic beta-(beta TC3) and neuronal (GT1.7) cell lines. The c arbohydrate moieties in the two cell types are distinct but contain a simil ar amount of terminal sialic acid residues and at least five oligosaccharid e chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. En doproteinase treatment of the M-r 22 000 core protein results in peptides o f M-r 4500 and M-r 20 000 with Lys-C, and peptides of M-r 4 000 and M-r 11 000-12 000 with Glu-C/V8 and Asp-N proteases. Conclusion/interpretation. The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purifi cation.