Structures of the N‐linked oligosaccharides on porcine plasma vitronectin

Abstract

The structures of N-linked oligosaccharides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine, and the pyridylaminooligosaccharides were fractionated by anion-exchange and reverse-phase HPLC. Nine major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo-forms were analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 'H-NMR spectroscopy.The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N-acetyllactosamine type, to which 1-3 mol sialic acids was linked. Sialic acids were linked predominantly through a2-6 linkages, although a2-3 linkages were also present, and fucose was linked to the innermost N-acetylglucosamine through an a1 -6 linkage. It was found that every pyridylamino-oligosaccharide population contained N-glycolylneuraminic acid and N-acetylneuraminic acid in a molar ratio of 1 : 2-9, and that N-glycolylneuraminic acids were located predominantly on the Man al-6 a m .Vitronectin (VN) is a cell-adhesive glycoprotein and is involved in physiological processes, such as cell-substrate attachment, modulation of the immune system and the blood coagulation system. Its functions are based on its ability to interact with various molecules [l, 21. Previously we purified six animal plasma VN [3]. The carbohydrate contents of the six VN were 10-20% (by mass) and partial characterization by means of a lectin-binding assay indicated that N-linked oligosaccharides are present on all species, and the amount of 0-linked oligosaccharides varied with the species. Recently, the molecular-mass diversity was extensively studied for 14 animal VN, all of which were glycoproteins [4]. The complete primary structures of the human [5,6], rabbit [7], and mouse [8] VN deduced from their cDNA sequences exhibit high similarity throughout the molecule, including the complete conservation of three potential N-glycosylation sites, with one additional potential site in mouse VN.In this study, detailed structural analysis is reported of Nlinked oligosaccharides on porcine VN (pVN) from plasma, which contains both N-acetylneuraminic acid (Neu5 Ac) and N-glycolylneuraminic acid (Neu5Gc) as sialic acid components. We also describe a novel approach involving Salmonella sialidase as a probe for the determination of sialyl a2-3 linkages.Correspondence to H. Ogawa, Department of Chemistry, Faculty of Science, Ochanomizu University, Tokyo 112, Japan Abbreviations. VN, vitronectin; pVN, porcine VN; Neu5 Ac, Nacetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid.Enzymes. N-Glycosidase F (EC 3.5.1.52) ; a-chymotrypsin (EC 3.4.21 .l); a-galactosidase (EC 3.2.1.23); P-N-acetylhexos...

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing TerminalN-Acetylneuraminic Acid α2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans

“…Further confirmation of each PA-oligosaccharide was carried out by exoglycosidases (␤-galactosidase, ␤-N-acetylhexosaminidase, and ␣-L-fucosidase) under conditions as described previously (27,28) to analyze the asialo-structure on the sugar map. The ␣2,3-linkage of NeuAc was determined as previously described (30,31).…”

Structures of the N‐Linked Oligosaccharides on Human Plasma Vitronectin

“…The sialyl linkages to the terminal Gal were mainly of the a2-6 type, while sialyl a2-3 linkages were also found on the PI-4-branching lactosamine of a1 -3-linked mannose on trisialotriantennary oligosaccharides, and on the branch of the al-6 arm on disialodiantennary oligosaccharides. On comparison of the results obtained for hVN with those for pVN [13], the most remarkable difference between hVN and pVN was the amount of fucose linked to the innermost N-acetylglucosamine by an al-6 linkage: more than 84% of the oligosaccharides on pVN were of the fucosyl type, while only 7.9% were on hVN. These results agreed well with the reactivities of the vitronectins toward lentil lectin, that is pVN was stained strongly with lentil-lectin-horseradish-peroxidase, while hVN was only very faintly [4].…”

“…The monoclonal antibody, anti-sialyl Lewis" : 1H4 (lot 92201), was purchased from Seikagaku Kogyo (Japan) and anti- Preparation and analysis of pyridylamino-oligosaccharides from hVN. Oligosaccharides were prepared by the same procedure as that used for pVN [13]. Briefly, hVN (5 mg) was digested with a-chymotrypsin (1 %, by mass) at 37°C for 6 h. After stopping the digestion by adding EDTA and phenylmethanesulfonyl fluoride (each 1 mM), oligosaccharides were released from the glycopeptides by exhaustive digestion with Nglycosidase F (3 units) at pH 8.6 at 37°C for 24 h. The supernatant was lyophilized and the reducing ends of the oligosaccharides were pyridylaminated, with which sialyl linkages are not released.…”

“…The major pyridylamino-asialooligosaccharide fraction, E, was desalted and deuterium-exchanged [13]. 'H-NMR spectroscopic analysis was performed with a JEOL GX-400 spectrometer operating at 400 MHz with a probe temperature of 30°C or 65°C.…”

“…In our previous study, we determined the structures of N-linked oligosaccharides on porcine vitronectin (pVN) from plasma [13]. In that study, we examined the utility of Salmonella sialidase, which exhibits a kinetic preference for sialyl a2-3 linkages [ 141, in the structural analysis of pyridylamino-oligosaccharides.…”