title: Heterogeneity of glycans at each N-glycosylation site of horseradish peroxidase.
abstract: The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) were studied by methylation linkage analysis, exoglycosidase degradation, 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 digest. Four glycans, namely (Xyl)Man3(Fuc)GlcNAc2 (major species), (Xyl)Man2(Fuc)GlcNAc2, (Xyl)Man3GlcNAc2, and Man3(Fuc)GlcNAc2 (minor species), were observed at all of the N-glycosylation sites and account for greater than 95% of the carbohydrate. Other members of this glycan family, namely (Xyl)xManm(Fuc)f GlcNAc2 (x = 0 or 1, f = 0 or 1, m = 4, 5, 6, or 7), account for the rest of the glycans. Only traces of high mannose-type glycans were detected in HRPc. Two sites, namely those at Asn-57 and Asn-267, were found to be more heterogeneous than the sites at Asn-13, Asn-158, Asn-186, 198 (doubly glycosylated peptide), Asn-214, and Asn-255. Two of the glycopeptides were observed as part of disulfide-linked species. MALDITOFMS confirmed the N-glycosylation sites previously reported [K.G. Welinder, Eur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogeneity of the glycan pool at each site.
authors.affiliation: Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Gray
authors.forename: B Y
authors.initials: BY
authors.lastname: Yang
authors.forename: R
authors.initials: R
authors.lastname: Montgomery
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D007527:Isoenzymes; D011134:Polysaccharides; D006735:Horseradish Peroxidase