Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding reagents

Abstract

The ability of almond emulsion peptide:N-glycosidase to remove oligosaccharide chains from intact glycoproteins was studied. Protein conformation appeared to be the main factor affecting carbohydrate removal. In the native state the oligosaccharides of ribonuclease B and the Fab mu fragment derived from immunoglobulin M were completely resistant to the enzyme, indicating that the polypeptide chain restricts access to the site of hydrolysis. Heat denaturation in sodium dodecyl sulfate rendered these glycoproteins susceptible to peptide:N-glycosidase, but perturbation with chaotropic salts provided a more gentle approach, which was as effective as detergent-unfolding and more compatible with the stability of the enzyme. Once exposed by the unfolding reagents, the complex oligosaccharides of Fab mu were released more rapidly than the high mannose chains of ribonuclease B, consistent with their preferential release from small glycopeptides (Plummer, T. H., Jr., and Tarentino, A. L. (1981) J. Biol. Chem. 256, 10243-10246).