Abstract
Fifteen different structures of terminal GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase have been identified. These N-linked oligosaccharides were mostly neutral, because sialic acid content was lower than 0.13 mol of sialic acid/mol of sugar chain, and sulfate was not detected. The oligosaccharides were released from pepsin-digested protein by glycoamidase A (from almond) digestion. The reducing ends of the oligosaccharide chains were aminated with a fluorescent reagent, 2-aminopyridine. The resulting mixture of pyridylamino derivatives of the oligosaccharides were separated by high performance liquid chromatography on an ODS-silica column, and 15 oligosaccharides were isolated. The structure of each oligosaccharide fraction was analyzed by two-dimensional sugar mapping, component sugar analysis, high resolution proton nuclear magnetic resonance and methylation analysis. It was found that each N-linked oligosaccharide associated with human urinary kallidinogenase contains unsubstituted GalNAc residues at the nonreducing terminal. These 15 oligosaccharides include 5 biantennary, 7 triantennary, and 3 tetraantennary oligosaccharides.
Highlights
Fifteen different structures of terminal GalNAc-containing N-linkedoligosaccharidesfrom humanurinary kallidinogenase have been identified
We have developed a two-dimensional (2-D)’ sugar mapping technique by HPLC, which is a convenient and powerful method for structural analysis of minutequantities of N linked oligosaccharides (2)
The abbreviations used are: 2-D, two-dimensional; PA, pyridyl aminated HPLC, high performance liquid chromatography; ’H NMR, proton nuclear magnetic resonance; GC-MS, gas chromatography-mass spectroscopy; Gal, D-galactose;Man, D-mannose; Fuc, Lfucose; GlcNAc, N-acetyl-D-glucosamine;GalNAc, N-acetyl-D-galactosamine; HexNAc, N-acetylhexosamine, (PA) oligosaccharides as standard were chromatographed on two different HPLC columns (ODs-silica and amide-silica), and the elution positions of standard oligosaccharideson both columns were plotted as the coordinates on a 2-D sugar map (30)
Summary
The followingmaterials were purchased from the sources indicated Sephadex G-15,Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden); Bio-Gel P-4 (200-400mesh), Bio-Rad sodium cyanoborohydride, Aldrich Chemical Co.; 2-aminopyridine, Wako Pure Chemical Industries (Osaka, Japan). GlcNAc-Man sequences by direct @-N-acetylhexosaminidase @-galactosidaseand @-N-acetylhexosaminidase.Thesedata digestion (Fig. 3) These data indicate that the oligosaccha- indicate that three Galp4GlcNAc branches are bound to the rides obtained from human urinary kallidinogenase have one trimannosyl core in oligosaccharide n.N-Acetylgalactosamine or more Gal-GlcNAc-Manbranches and N-acetylhexosamine residues were found in the outer chains of all 15 oligosaccha-. Monosaccharide Analysis in the Outer Chains of Oligosac- The major peaks, m, p, g, i, v, and r, have been shown to charides b-u-The monosaccharides component linked to tri- bear GalNAc@4GlcNAc-Mansequencesby 'H NMR spectrosmannosyl core 000.1 of each sample oligosaccharide were copy and methylation analysis as described below. The ratio of terminal and phave 2 GlcNAc residues (Table 11)and all cluster in the GalNAc to terminal Gal determined by comparing the peak vicinity of210.4 (a typical biantennary oligosaccharide) on areas with standards was found to be about 1:l.These results the 2-D sugar map (Fig. 2).
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