Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release.
Endo-beta-N-acetylglucosaminidase H (endo H) was found to bring about the complete hydrolysis of dolichyl pyrophosphoryl oligosaccharides. Both glycosylated and unglucosylated polymannose oligosaccharides were released by the enzyme through cleavage of the di-N-acetylchitobiose sequence. The action of the endo H on the oligosaccharide-lipids was facilitated by the inclusion of Triton X-100 (maximal stimulation at concentrations greater than 0.03%) or small amounts of a variety of other detergents; however, sodium dodecyl sulfate (0.1%) was strongly inhibitory. Although incubations were routinely carried out at pH 5.2, the enzyme was noted to be equally effective at pH 6.5 and to retain 75% of its activity toward oligosaccharide-lipid at pH 7.4. While these results broaden the known specificity of the endo H for the aglycon moiety, it was observed that even under optimal conditions the rate of hydrolysis of lipid-linked Glc3Man9GlcNAc2 was substantially slower than that of the same oligosaccharide attached to asparagine in a peptide sequence. The use of endo H, an enzyme which can be obtained free of exoglycosidases, appears to have a number of advantages over mild acid hydrolysis as a tool for cleaving oligosaccharide-lipids. It was found that the latter procedure causes a small but detectable degradation of the sugar chains and, when carried out in the presence of methanol, leads to the release of about 10% of the oligosaccharide as its beta-methyl glycoside. Furthermore, the oligosaccharides released by the endo H can be directly compared to those liberated by this enzyme from glycoproteins; this may prove to be useful in metabolic studies dealing with oligosaccharide-lipid assembly and their involvement in the N-glycosylation of proteins.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000494:pharmacology; D006596:Hexosaminidases / Q000378:metabolism*; D006851:Hydrochloric Acid; D006868:Hydrolysis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D017830:Octoxynol; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D013305:Streptomyces griseus / Q000201:enzymology
D003902:Detergents; D009844:Oligosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C024962:dolichyl diphosphate oligosaccharides; D011092:Polyethylene Glycols; D017830:Octoxynol; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D006851:Hydrochloric Acid