The mechanism of folding of pancreatic ribonucleases is independent of the presence of covalently linked carbohydrate.
The role of asparagine-linked oligosaccharides for the mechanism of protein folding was investigated. We compared the stability and folding kinetics for two sets of pancreatic ribonucleases (RNases) with identical amino acid sequences and differences in glycosylation. First the folding of RNases A (carbohydrate free) and B (a single N-linked oligosaccharide) from bovine pancreas was investigated. The kinetics of refolding were identical under a wide range of conditions. The rate of unfolding by guanidinium chloride was decreased in RNase B. In further experiments the folding of porcine RNase (three carbohydrate chains at Asn-21, -34, and -76) was compared with the corresponding data for the deglycosylated protein. Even for this RNase with almost 40% carbohydrate content the mechanism of refolding is independent of glycosylation. Although the folding mechanism is conserved, the rates of individual steps in folding are decreased about 2-fold upon deglycosylation. We interpret this to originate from a slight destabilization of folding intermediates by carbohydrate depletion. In control experiments with nonglycosylated bovine RNase A it was ascertained that treatment with HF (as used for deglycosylation) did not affect the folding kinetics. The in vitro folding mechanism of glycosylated RNases apparently does not depend on the presence of N-linked oligosaccharide chains. The information for the folding of glycoproteins is contained exclusively in the protein moiety, i.e. in the amino acid sequence. Carbohydrate chains are attached at chain positions which remain solvent exposed. This ensures that the presence of oligosaccharides does not interfere with correct folding of the polypeptide chain.
D000818:Animals; D001216:Asparagine; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000494:pharmacology*; D006031:Glycosylation; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases; D013552:Swine
D002241:Carbohydrates; D006146:Guanidines; D001216:Asparagine; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D019791:Guanidine