Structural Studies with the Glycopeptides from Porcine Pancreatic Ribonuclease

Abstract

Abstract Tryptic glycopeptide fractions GP-1, -2, and -3, representative of the heterosaccharides attached to asparagines-21, -34, and -76, respectively, in porcine pancreatic ribonuclease were subjected to oxidation with periodic acid, either alone or in combination with successive, controlled hydrolyses with carefully purified glycosidases. The enzymes used were α-mannosidase from jack beans, β-mannosidase from the hepatopancreas of Busycotypus, β-galactosidase and β-Nacetylglucosaminidase from Diplococcus pneumoniae, β-N-acetylglucosaminidase from Aspergillus niger, and neuraminidase from Vibrio cholerae. The progress of these degradative procedures was monitored by gas-liquid chromatography of the per-trimethylsilyl derivatives of methyl glycosides produced after acid-catalyzed methanolysis. The following were the principal findings and conclusions. (a) Of the 6 mannose residues in GP-2, four are α-linked, two are β-linked. These distinct types of residue occur to mutual exclusion in separate side chains attached to an N-acetylglucosamine disaccharide core unit, itself glycosidically linked to asparagine. (b) By the successive action of neuraminidase, β-galactosidase and β-N-acetylglucosaminidase on GP-1 and GP-3 fractions, peptido-heptasaccharides were obtained which contained identical heterosaccharide moieties composed of 3 residues each of mannose and N-acetylglucosamine and 1 residue of fucose. By further degradation of these peptido-heptasaccharides a peptidotetrasaccharide was obtained with the following partial structure. Fuc(1 → 3,4)βGlcNAc(1 → 3)αMan(1 → 3,4)GlcNAc → Asn (c) The heterogeneity of GP-1 and GP-3 fractions may be attributed to variation in the extent to which the common heptasaccharide core moiety in these fractions is substituted with β(1 → 6)-linked N-acetylglucosamine-containing side chains which terminate in sialylgalactose units. (d) The results suggest that during biosynthesis differentiation of the two different types of heterosaccharide side chains in porcine ribonuclease occurs as the 2nd carbohydrate residue is attached to an aspartamido-2-acetamido-1,2-dideoxyglucopyranose unit in the peptide chain.