Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F.

F K Chu

doi:10.1016/s0021-9258(17)42448-3

Abstract

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.

Highlights

From the Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201
Deglycosylation of High MannoseGlycoproteins with Peptide N-Glvcosidase F-To ensure that all high mannose carbohydrate chainswere accessible to peptideN-glycosidase F, both yeastexternalinvertase (Fig. l A, left panel) and RNase B were heated a t 90 "C for 1 min in 0.5% SDS to unfold the polypeptide subunits before incubation with the endoglycosidase
A sample of each unfolded glycoprotein was treated with endo-0-N-acetylglucosaminidaseH to yield the respective products containing oneGlcNAc/cleaved oligosaccharide chain linked to the polypeptide backbone

Summary

Introduction

OliRosacchnride-clrauing Enzyme Rraction-Usually the glycoprotein substrate (nativeor heat-denatured a t 0.5 mg/ml) was incuhated with the respective endo- or exoglycosidases in a volume of 50-100 p1 in the following buffers: peptide N-glycosidase F (50 milliunits/ml) in 0.1 M sodium phosphate, pH 8.6, containing 0.02 M EDTA and Deglycosylation of High MannoseGlycoproteins with Peptide N-Glvcosidase F-To ensure that all high mannose carbohydrate chainswere accessible to peptideN-glycosidase F, both yeastexternalinvertase (Fig. l A , left panel) and RNase B (right panel) were heated a t 90 "C for 1 min in 0.5% SDS to unfold the polypeptide subunits before incubation with the endoglycosidase.

Results

Conclusion