Microbial endo-beta-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins.

The activity and substrate specificity of endo-beta-N-acetylglucosaminidase [glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosamino-hydrolase, EC 3.2.1.96] obtained from Mucor hiemalis (Endo-M) was compared with that of the enzyme obtained from Flavobacterium meningosepticum (Endo-F), which is the only enzyme available that acts on the complex oligosaccharides of asparagine-linked sugar chains in glycoproteins. They showed almost the same activities toward DNS-ovalbumin glycopeptide containing high-mannose and hybrid asparagine-linked oligosaccharides. However, Endo-M showed high activity towards DNS-asialotransferrin and DNS-transferrin glycopeptides, which contain complex biantennary oligosaccharides. Endo-M could weakly act even on DNS-asialofetuin glycopeptide containing complex triantennary oligosaccharides, while Endo-F could not. SDS-denatured asialotransferrin was deglycosylated by both enzymes in the presence of non-ionic detergent (NP-40) and EDTA, and the deglycosylated protein migrated to a lower molecular weight position than asialotransferrin on SDS-PAGE. However, even in the absence of detergent, Endo-M deglycosylated native asialotransferrin and transferrin. Deglycosylation of asialotransferrin was confirmed by means of Con A-Sepharose 4B column chromatography and SDS-PAGE.