Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease.
We have investigated the effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. The sensitivity of nonglycosylated RNase A to trypsin and chymotrypsin was compared with three glycosylated species of RNase B which differed with respect to the size of the carbohydrate chain. Two forms of glycosylated RNase B were isolated by concanavalin A-Sepharose affinity chromatography, and each was shown to contain a single carbohydrate chain composed of GlcNAc2Man1 (RNase B") or GlcNAc2Man5-8 (RNase B). A third form (RNase B'), with oligosaccharide composed of GlcNAc2Man4, was prepared by partial digestion of RNase B with alpha-mannosidase. Fully glycosylated RNase B was found to be 6-10 times more resistant to trypsin digestion than nonglycosylated RNase A. RNase B' and B", with intermediate chain sizes, were 3.0- and 1.3-fold more resistant to trypsin digestion than RNase A, respectively. With chymotrypsin, however, differences in rates of digestion were much less marked, with a maximum difference of 3-fold between RNase A and B. In addition, we found that the specificity of the primary trypsin (Arg 33-Asp 34 bond) or chymotrypsin (Tyr 25-Cys 26 bond) cleavage site was not affected by the presence or size of the oligosaccharide chain. These results are consistent with the view that the size of the oligosaccharide chain and its proximity to the primary or rate-limiting cleavage site are important for expression of the carbohydrate protection against proteolytic degradation, which thus appears to be mediated by steric hindrance.
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D007700:Kinetics; D009844:Oligosaccharides; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
D009844:Oligosaccharides; D010446:Peptide Fragments; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D002918:Chymotrypsin; D014357:Trypsin