Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The N-linked oligosaccharides that were released by hydrazinolysis from glycoproteins of zonae pellucidae of bovine ovarian eggs, were composed of neutral (23%) and acidic (77%) carbohydrate chains; almost all the acidic chains were neutralized by sialidase digestion. Sugar mapping analysis of pyridylaminated N-linked chains by reverse-phase and normal-phase HPLC and 500-MHz 'H-NMR spectroscopy revealed that the major neutral chain is a high-mannose-type oligosaccharide and the acidic chains are di-, tri-, and tetra-antennary, fucosylated complex-type chains that have N-acetyllactosamine repeats in the non-reducing regions. The structures of the neutral chain and the core regions of the acidic chains of N-linked oligosaccharides from the zona proteins of fertilized eggs, which were obtained by the in vitro fertilization method, were essentially the same as those of the ovarian egg zonae. The amount, however, of the acidic chains decreased to 32 mol/l00 mol in the fertilized egg zonae, which suggests that a sialidase released from the oocyte during fertilization operates on the zona glycoproteins.Keywords: zona pellucida; carbohydrate structure ; glycoprotein ; mammalian fertilization ; egg.The mammalian oocyte is surrounded by an extracellular matrix called the zona pellucida. The zona plays important roles during fertilization including species-specific sperm binding, blocking of polyspermy, and protection of the embryo. The zona of most mammals consists of three glycoprotein components, which have N-linked and 0-linked carbohydrate chains. It has been proposed that oligosaccharides of the zona protein are involved in sperm binding [I, 21. In the mouse, the a-galactosyl residue [3] or N-acetylglucosaminyl residue [4] at the non-reducing end of the 0-linked chain of the ZP3 glycoprotein possesses sperm receptor activity. In the pig, neutral N-linked chains of pZP3a glycoprotein have been shown to have receptor activity (5, 61.We have elucidated the structure1 features of the N-linked chains of the porcine PZP3 family and mouse ZP2 and ZP3 components [S, 7, 81. N-linked chains of PZP3 consist of di-, tri-, and tetra-antennary, complex-type neutral (28 %) and acidic (72%) chains. The acidic chains have sialic acids at the nonreducing ends of N-acetyllactosamine repeats and sulfates at the C6 position of GlcNAc residues of the repeats. core regions. The sulfate contents in the mouse ZP2 and ZP3 are much smaller than those in the porcine PZP3 family, and , 8-GalNAc and a-Gal residues, which are absent from the porcine chains, are found at the non-reducing ends of mouse N-linked chains.We characterized the protein moieties of zona glycoproteins from the unfertilized and fertilized bovine eggs [9]. Furthermore, we have observed that N-linked chains of bovine zona proteins possess the sperm receptor activity; the number of sperm binding to the bovine zona is reduced after fertilization (Mitsui, s., Katsumata, T., Yonezawa, N. and Nakano, M., unpublished results). In this study, we analyzed the structures of N-linked ...

Section: 'supporting

confidence: 65%

Section: Monosaccharide Composition the Monosaccharide Compositions supporting

confidence: 65%

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Structural Characterization of the N‐Linked Carbohydrate Chains of the Zona Pellucida Glycoproteins from Bovine Ovarian and Fertilized Eggs

Katsumata

Noguchi

Yonezawa

et al. 1996

“…Values in parentheses were measured at 65°C with internal acetone (6 = 2.220 pprn in D,O). Data of reference compounds were from [19] (200.4 and 210.4) and [20] …”

Section: 'H-nmr Spectroscopymentioning

confidence: 99%

Structures of the N‐linked oligosaccharides on porcine plasma vitronectin

Yoneda

Ogawa

Matsumoto

et al. 1993

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...

“…Triantennary and tetraantennary oligosaccharides without fucose (300.8 and 400.16) were prepared from 3,-acid glycoprotein [21]. Triantennary oligosaccharides with one or two terminal GalP residues (300.3 and 300.5) were prepared by fucosidase digestion of the corresponding oligosaccharides from porcine pancreatic kallikrein [22]. Triantennary oligosaccharides with a terminal Galpl3GlcNAc sequence (300.22) were prepared from fetuin [23].…”

Section: Standard Oligosaccharidesmentioning

confidence: 99%

Detailed structural analysis of asparagine‐linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica

Shoji

Takahashi

Nishimura

et al. 1992

Self Cite

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo califovnica Biochem. 157, 233-2421 to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor.The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by highresolution proton NMR.Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal Nacetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal a-galactose residue(s) linked to C3 of p-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Galp1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.The nicotinic acetylcholine receptor (AcChoR) is a ligandgated ion channel that mediates synaptic transmission at the neuro-muscular junction [l, 21. AcChoR is a transmembrane Correspondence to K. Hayashi,