tailieunhanh - Báo cáo khoa hoc : Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. | IFEBS Journal Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity Melinda Bence and Miklos Sahin-Toth Department of Molecular and CellBiology Boston University Henry M. Goldman Schoolof DentalMedicine Boston MA 02118 USA Keywords Asn-linked glycosylation ER stress misfolding pancreatic chymotrypsin secretion defect serine protease Correspondence M. Sahin-Toth 72 East Concord Street Evans-433 Boston MA 02118 USA Fax 1 617 414 1041 Tel 1 617 414 1070 E-mail miklos@ Received 25 July 2011 revised 30 August 2011 accepted 7 September 2011 doi Human chymotrypsin C CTRC plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked N-linked glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation Asn-Xaa-Ser Thr in human CTRC by mutating the Asn residues to Ser individually or in combination expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 N52S reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells indicating that N-glycosylation is required for folding of human CTRC. Despite its important role Asn52 is poorly conserved in other mammalian CTRC orthologs including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation

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