Abstract
How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to alpha-Man, Neu5Acalpha2,6Galbeta1,4Glc, and alpha-galactose sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing alpha-Man or alpha-NeuAc but not to those possessing beta-galactose or beta-GlcNAc residue. Enzyme-linked immunosorbent assay (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 10(10) m(-1), which was the highest among the glycoproteins examined, and that alpha-galactosidase treatment decreased it to 10(5) m(-1). PMSF-treated trypsin bound to other glycoproteins, including ovomucoid, a trypsin inhibitor, with the affinity constants of 10(8)-10(5) 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.
Highlights
Trypsin is a principal pancreatic serine protease that plays a key role in digestion in the duodenum by activating zymogens and degrading dietary proteins
This study demonstrates that mammalian pancreatic trypsin commonly binds to glycoproteins possessing N-linked glycans by carbohydrate-specific interaction
Trypsin bound to glycoproteins possessing N-glycans with very high affinity, reaching 1010-106 MϪ1, whereas it did not bind to bovine submaxillary gland mucin (BSM) (Fig. 4 and Table 1)
Summary
Materials—Porcine pancreatic trypsin (PPT), N-␣-benzoyl-L-arginine ethyl ester (BAEE), N-␣-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPA), soybean trypsin inhibitor, bovine serum albumin (BSA), 3,3Ј-diaminobenzidine tetrahydrochloride, methyl-␣-Dmannoside, and mannitol were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Bovine pancreatic trypsin (BPT), bovine submaxillary gland mucin (BSM), human holotransferrin, fetuin from fetal calf serum, hen ovomucoid, human orosomucoid, bovine thyroglobulin, streptavidin-biotinylated horseradish peroxidase complex (ABC complex), and 4-nitrophenyl phosphate magnesium salt were purchased from Sigma. Sugar-biotinylated polyacrylamide probes (sugar-BP probes) were purchased from Lectinity Holdings, Inc. Moscow,
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