title: Detection of proteolytic cleavages of diabetes-associated protein IA-2 beta in the pancreas and the brain using novel anti-IA-2 beta monoclonal antibodies.
abstract: Insulinoma-associated protein (IA)-2 beta, an inactive member of the protein-tyrosine phosphatase (PTP) family, is a major autoantigen in type-1 diabetes mellitus. IA-2 beta exists mainly in a 60-kDa form, and is frequently located in the dense-core secretory vesicles of pancreatic beta cells. As IA-2 beta gene-deficient mice exhibit impaired insulin secretions, IA-2 beta is probably involved in insulin secretions. In the present study, we characterized the major forms of IA-2 beta in the brain and pancreas of normal and non-obese diabetic (NOD) mice. Novel monoclonal antibodies (mAbs) against IA-2 beta revealed that this brain protein was of multiple compositions incorporating the 60-, 64-, 67- and 71-kDa forms, which were designated as IA-2 beta 60, IA-2 beta 64, IA-2 beta 67 and IA-2 beta 71, respectively. On the contrary, only the 60-kDa isoform of IA-2 beta was expressed in the mouse pancreas and in the mouse pancreatic beta cell line, MIN6. Sequence analyses revealed that IA-2 beta 60, IA-2 beta 64 and IA-2 beta 71 (brain-derived immunoprecipitated IA-2 beta isoforms) contained alternative NH2- termini starting from Glu489, Ala464, and Ser414, respectively, while IA-2 beta 60 (an MIN6-derived immunoprecipitated IA-2 beta isoform) contained those from Glu489. Consistent with the lack of an NH2-terminal region of IA-2 beta, 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-2 beta undergoes at least three distinct proteolytic cleavages.
authors.affiliation: Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Tomiko
authors.initials: T
authors.lastname: Kawakami
authors.forename: Keiichi
authors.initials: K
authors.lastname: Saeki
authors.forename: Natsumi
authors.initials: N
authors.lastname: Takeyama
authors.forename: Guoying
authors.initials: G
authors.lastname: Wu
authors.forename: Akikazu
authors.initials: A
authors.lastname: Sakudo
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hayashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Onodera
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001324:Autoantigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D016688:Mice, Inbred NOD; D008819:Mice, Nude; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011499:Protein Processing, Post-Translational; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516863:Ptprn protein, mouse; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8