Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins.
ZP3, a preparation of the 55K families of porcine oocyte zona pellucida, possesses carbohydrate-dependent ligand activity for boar sperm. The aim of the present study was to analyze ZP3 N- and O-linked oligosaccharides with respect to size distribution, composition, and role in sperm-zona recognition events. Digestion of denatured ZP3 with peptide N-glycosidase F (PNGaseF) released the majority of N-glycans which fractionated on Sephadex G-75 resin as a polydisperse population with apparent molecular masses ranging from 1,900-8,200 Da. The higher molecular weight N-glycans were characterized by the presence of strongly anionic sulfated/sialylated polylactosamine structures. Alkaline-borohydride treatment of the PNGaseF-digested core proteins liberated O-glycans as a heterogeneous population of oligosaccharide alcohols, which were fractionated on a Sephadex G-50 column. Compositional analyses indicated sulfated polylactosamine units associated with the higher molecular weight O-glycans. Preincubation of boar sperm with ZP3 or purified O-glycans, but not N-glycans, inhibited subsequent attachment to zona-encased oocytes. Purified O-glycans were, however, 2 to 3 orders of magnitude less effective than ZP3 as competitive ligands. The results document the extreme heterogeneity of the ZP3 carbohydrate moiety, in large part attributable to a broad spectrum of variably sized N- and O-linked sulfated polylactosamines. Ligand competition bioassays suggest that O-glycans mediate, at least in part, the sperm adhesive properties of ZP3 and strongly imply that high-affinity interaction of ZP3 sugar chains with complementary sperm receptors is dependent upon their covalent association with core proteins.
Yurewicz
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Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan 48201.
Pack
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Sacco
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D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004527:Egg Proteins; D005260:Female; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism; D013552:Swine; D015044:Zona Pellucida / Q000737:chemistry* / Q000378:metabolism; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins