Biochemical, biophysical, and functional properties of ICA512/IA?2 RESP18 homology domain

SOSA, LAURA F.; TORKKO, JHUA M.; PRIMO, MA. EVANGELINA; LLOVERA, RAMIRO E.; TOLEDO, PAMELA L.; RIOS, ANTONELLA S.; GONZALEZ FLECHA, F. LUIS; TRABUCCHI, ALDANA; VALDEZ, SILVINA N.; POSKUS, EDGARDO; SOLIMENA, MICHELLE; ERMÁCORA, MARIO R.

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2016 vol. 1864 p. 511 - 522

1570-9639

Background: ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretorygranules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic,exocytosis and recycling of insulin secretory granules, as well as in β-cell proliferation. While several ICA512domains have been characterized, the function and structure of a large portion of its N-terminal extracellular(or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterizationof ICA512?RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrinespecificprotein 18 (RESP18).Methods: Pure recombinant ICA512?RESP18HD was characterized by CD and fluorescence. Its binding to insulinand proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivitywas measured kinetically. Targeting of ΔRESP18HD ICA512?GFP to the membrane of insulinoma cells wasmonitored by immunofluorescence.Results: ICA512?RESP18HDpossesses a strong tendency to aggregate and polymerize via intermolecular disulfideformation, particularly at pH N 4.5. Its cysteine residues are highly susceptible to oxidation forming an intramoleculardisulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. Theregulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD.ICA512?RESP18HD binds with high-affinity to insulin and proinsulin.Conclusions: RESP18HD is required for efficient sorting of ICA512 to secretory granules.General significance: RESP18HD is a key determinant for ICA512 granule targeting.