The glycans of horseradish peroxidase

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprotein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligosaccharides of HRPc were investigated by a combination of High pH Anion-Exchange Chromatography with Pulsed Amperometric Detection, methylation analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The structure of the major oligosaccharide released by digestion with glycopeptidase A, accounting for between 75 and 80% of the total, was confirmed to be alpha-Man-(1 --> 6)[alpha-Man-(1 --> 3)][beta-Xyl-(1 --> 2)]-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4)[alpha-Fuc-(1 --> 3)]-GlcNAc. Most of the remaining oligosaccharides were found to belong to the (Xyl)(x)Man(m)(Fuc)(f)GlcNAc(2) (m = 2, 4, 5, 6; f = 0 or 1, x = 0 or 1) family. Less than 5% of the oligosaccharides were of the Man(m)GlcNAc(2) (m = 4 to 7) type. Methylation analysis of holo- and apo-HRPc and its tryptic glycopeptides support the structures proposed for the oligosaccharides. Furthermore, methylation analysis of the tryptic glycopeptides provides evidence for the heterogeneity of the oligosaccharides occurring at each of the N-linked sites. (C) 1996 Elsevier Science Ltd.