Novel carbohydrate-binding activity of pancreatic trypsins to N-linked glycans of glycoproteins

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概要

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• How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to α-Man, Neu5Acα2,6Galβ1,4Glc, and α-Gal sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing α-Man or α-NeuAc but not to those possessing β-Gal or β-GlcNAc residue. ELISA showed that trypsin binds to six kinds of biotinylated glycoproteins possessing high mannose-type and complex type N-glycans but not to bovine submaxillary mucin, which possesses only O-glycans. Further, the binding of trypsin to glycoproteins was differentially changed by treatments with sequential exoglycosidases, endoglycosidase H, or N-glycosidase F. Quantitative kinetic studies indicated that PMSF-treated trypsin binds with bovine thyroglobulin with the affinity constant of 1010 M-1, which was the highest among the glycoproteins examined, and that α-galactosidase treatment decreased it to 105 M-1. PMSF-treated trypsin bound to other glycoproteins including ovomucoid, a trypsin inhibitor, with the affinity constants of 108-105 mol-1 and were markedly changed by glycosidase treatments in manners consistent with the sugar-binding specificities suggested by ELISA. Thus, the binding site for glycans was shown to be distinct from the catalytic site, allowing trypsin to function as an uncompetitive activator in the hydrolysis of a synthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment with PMSF or soybean trypsin inhibitor. The results indicate the presence of an allosteric regulatory site on trypsin that sugar-specifically interacts with glycoproteins in addition to the proteolytic catalytic site.

• The American Society for Biochemistry and Molecular Biologyの論文
• 2006-03-31

著者

• Ogawa Haruko

  Department Of Chemistry Faculty Of Science Ochanomizu University

• Ogawa Haruko

  Department Of Advanced Biosciences Graduate School Of Humanities And Sciences Ochanomizu University

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