Biochemical Characterization of Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain Expressed in Pichia pastoris

SAMUS, S. IVÁN; NOGUERA, MARTÍN E.; FERREYRA, RAUL G.; ERMÁCORA, MARIO R.

Villa Carlos Paz, Córdoba

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica (SAB)

Human Receptor-Type Protein-Tyrosine Phosphatase ICA512 (or IA-2) is a transmembrane protein located in secretory granules of neuroendocrine cells. Identified as one of the main antigens of autoimmune diabetes, it is associated with protein secretion processes1. During insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The structures of the intracellular pseudocatalytic and extracellular mature domains are known, but the transmembrane domain and several other parts of the receptor are poorly characterized. Previously, we solved the structure of the mature ectodomain ME ICA512 (residues 449 to 575) and identified potential dimerization interfaces involved in the regulation of the secretion2, 3. Furthermore, ICA512 is a glycosylated protein and molecular modeling studies suggest that the positioning of the sugar residues imposes steric restraints on the type of dimerization interface. To address this question, we express ME ICA512 in Pichia pastoris. The protein secreted to the culture medium was purified and characterized by mass spectrometry and size exclusion chromatography. The implications of these findings for biological activity will be discussed.