HETEROGENEITY OF GLYCANS AT EACH N-GLYCOSYLATION SITE OF HORSERADISH-PEROXIDASE

The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) w ere studied by methylation linkage analysis, exoglycosidase degradatio n, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS). Over 90% of the predicted tryptic peptides and glycopeptides of HRPc could be identified in the unfractionated d igest.]Four glycans, namely (Xyl)Man(3)(Fuc)GlcNAc(2) (major species), (Xyl)Man(2)(Fuc)GlcNAc(2), (Xyl)Man(3)GlcNAc(2), and Man(3)(Fuc)GlcNA c(2) (minor species), were observed at all of the N-glycosylation site s and account for greater than 95% of the carbohydrate. Other members of this glycan family, namely (Xyl)(x)Man(m)(Fuc)(f) GlcNAc(2) (x = 0 or 1, f = 0 or 1, m = 4, 5, 6, or 7), account for the rest of the glyc ans. Only traces of high mannose-type glycans were detected in HRPc. T wo sites, namely those at Asn-57 and Asn-267, were found to be more he terogeneous than the sites at Asn-13, Asn-158, Asn-186, 198 (doubly gl ycosylated peptide), Asn-214, and Asn-255. Two of the glycopeptides we re observed as part of disulfide-linked species. MALDITOFMS confirmed the N-glycosylation sites previously reported [K.G. Welinder, fur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogenei ty of the glycan pool at each site. (C) 1998 Elsevier Science Ltd. All rights reserved.