Detection of proteolytic cleavages of diabetes-associated protein IA-2β in the pancreas and the brain using novel anti-IA-2β monoclonal antibodies

Abstract

Insulinoma-associated protein (IA)-2β, an inactive member of the protein-tyrosine phosphatase (PTP) family, is a major autoantigen in type-1 diabetes mellitus. IA-2β exists mainly in a 60-kDa form, and is frequently located in the dense-core secretory vesicles of pancreatic β cells. As IA-2β gene-deficient mice exhibit impaired insulin secretions, IA-2β is probably involved in insulin secretions. In the present study, we characterized the major forms of IA-2β in the brain and pancreas of normal and non-obese diabetic (NOD) mice. Novel monoclonal antibodies (mAbs) against IA-2β revealed that this brain protein was of multiple compositions incorporating the 60-, 64-, 67- and 71-kDa forms, which were designated as IA-2β60, IA-2β64, IA-2β67 and IA-2β71, respectively. On the contrary, only the 60-kDa isoform of IA-2β was expressed in the mouse pancreas and in the mouse pancreatic β cell line, MIN6. Sequence analyses revealed that IA-2β60, IA-2β64 and IA-2β71 (brain-derived immunoprecipitated IA-2β isoforms) contained alternative NH 2-termini starting from Glu489, Ala464, and Ser414, respectively, while IA-2β60 (an MIN6-derived immunoprecipitated IA-2β isoform) contained those from Glu489. Consistent with the lack of an NH2-terminal region of IA-2β, the isoforms were recognized by their respective mAbs characterized with different epitope regions. Furthermore, Western blotting and immunohistochemistry demonstrated that NOD mice expressed similar isoforms present in the brains and pancreatic islets of C57BL/6J, BALC/CA and ICR mice, accordingly. Taken together, these results suggest that IA-2β undergoes at least three distinct proteolytic cleavages.