Endo-β-N-acetylglucosaminidase Acting on Carbohydrate Moieties of Glycoproteins

Abstract

Abstract A purified ovalbumin glycopeptide, Asn-(GlcNAc)2(Man)5 was [14C]acetylated in the asparagine moiety. Using the [14C]acetylated glycopeptide as a substrate, an endo-β-N-acetylglucosaminidase was purified 2400-fold from the culture fluid of Diplococcus pneumoniae. The enzyme preparation was practically free from various exoglycosidases and proteases. The enzyme cleaved the di-N-acetylchitobiose structure of the [14C]acetylated glycopeptide, yielding equimolar amounts of [14C]acetyl-Asn-GlcNAc and (Man)5(GlcNAc). The enzyme had a pH optimum of 6.5, with a Km of 0.25 mm and with a Vmax of 4.67 µmoles per mg of protein per min toward the substrate. A mouse myeloma IgG glycopeptide and a bovine IgG glycopeptide was hydrolyzed by the enzyme, only after treatment with β-galactosidase and β-N-acetylglucosaminidase. In both cases the products of the enzymic action were a fucose-containing glycopeptide fragment and a mannose-containing oligosaccharide whose reducing end was N-acetylglucosamine. The enzyme had a strict specificity with respect to the structures of oligomannosyl cores of glycopeptides. Thus, the enzyme hydrolyzed only 20% of glycopeptide mixtures from ovalbumin. Furthermore, the enzyme hydrolyzed porcine thyroglobulin glycopeptides, Unit B (sialic acid containing heteropolysaccharide unit) which had been treated with neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase, but did not hydrolyze calf thyroglobulin glycopeptides, Unit A (mannose-N-acetylglucosamine unit).