Towards the structural characterization of the cysteine rich N terminal domain of IA-2

LAURA N.F. SOSA; MARTÍN E. NOGUERA; VALERIA RISSO; EDGARDO POSKUS; MARIO R. ERMÁCORA ; MARÍA E. PRIMO

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

SAD, CeBEM, Latin American Protein Society

IA-2 is a major autoantigen in insulin-dependent diabetes mellitus, and it has been found involved in several crucial steps of insulin production and secretion. Moreover, this protein has been recognized as an important constituent of regulated secretory pathways in various neuroendocrine cells. IA-2 is a type I transmembrane protein that is localized on the secretory granule membrane. The cytosolic region is a protein-tyrosine phosphatase rendered inactive during evolution. The extra cellular region is processed by furin-like convertases yielding the mature receptor and a N-terminal cysteine‑rich domain (ntcrIA-2). The ulterior fate of the latter is unknown but it might be secreted upon exocytosis jointly with insulin. The aim of this work was to characterize the biophysical behavior of ntcrIA-2 as the first step toward the elucidation of its biological role. ntcrIA2 was produced as a recombinant protein in E. coli, and its identity was confirmed by ES‑MS. Although it is intractable in many conventional solution conditions, we found a way to refold this fragment from the urea-induced unfolded state. The conformational state of the protein was analyzed by circular dichroism in the far and near UV. Peptide mapping and MALDI- TOF analysis indicated the presence of an intramolecular disulfide bond (cysteine residues 3 and 4). The remaining cysteine residues (1, 2) seem to participate in intermolecular disulfide bridges that allow the formation of covalently stabilized multimers.