The Release of Intact Oligosaccharides from Specific Glycoproteins by Endo-β-N-acetylglucosaminidase H

Abstract

Abstract Endo-β-N-acetylglucosaminidase H, purified to homogeneity from cultural filtrates of Streptomyces griseus (Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811–817) was tested for its capacity to hydrolyze oligosaccharides from various glycoproteins. A complete and rapid release of oligosaccharide was effected from sulfitolyzed ovalbumin, bovine pancreatic deoxyribonuclease A, and ribonuclease B, and an invertase from Saccharomyces cerevisiae. These glycoproteins all contain a neutral oligosaccharide that is attached to the protein by a glycosyl asparagine bond and has the following composition: Asn-(GlcNAc)2(Man)x(GlcNAc)y. Analysis of the hydrolysis products revealed that the N-acetylglucosamine residue of di-N-acetylchitobiose distal to the asparagine is on the reducing end of the oligosaccharide, whereas the proximal N-acetylglucosamine remains with the protein. Neither the enzymatic properties nor the circular dichroic patterns of ribonuclease B and deoxyribonuclease A were altered significantly by the removal of their respective oligosaccharides. However, the elution of ribonuclease B from IRC-50 was changed, as was the electrophoretic mobility of deoxyribonuclease. Treatment of proteins that contain both neutral and acidic oligosaccharides, such as thyroglobulin, immunoglobulin M, and porcine ribonuclease, resulted in the release of the neutral chains only. A marked increase in the release of oligosaccharides from immunoglobulin M was effected by 2-mercaptoethanol. Removal of the neutral oligosaccharides did not appear to affect the immunological properties of this immunoglobulin.