RESUMO
Affinity chromatography on Concanavalin-A Sepharose, followed by gel filtration and hydrophobic interaction chromatography, permits the isolation of low molecular weight N-glycosidically linked oligomannosidic glycopeptides (MGp) from the autoproteolysis products of human seminal plasma. The monosaccharide composition of MGp showed only mannose, N-acetylglucosamine and a small amount of galactose. Structural studies were carried out by methylation analysis and chromium trioxide oxidation, and results were consistent with the structures accepted for high-mannose N-glycans. MGp was capable of inhibiting the sperm acrosomal exocytosis mediated by sperm-surface receptors. These data suggest that MGp act as a "decapacitation" factor preventing premature sperm exocytosis.
Assuntos
Glicopeptídeos/isolamento & purificação , Sêmen/química , Reação Acrossômica/efeitos dos fármacos , Configuração de Carboidratos , Cromatografia de Afinidade , Cromatografia Gasosa , Cromatografia em Gel , Exocitose , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Humanos , Masculino , Manose/química , Oligossacarídeos/químicaRESUMO
Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.
Assuntos
Glicopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Espermatozoides/metabolismo , Humanos , Masculino , Papaína/metabolismo , Pronase/metabolismo , Tripsina/metabolismoRESUMO
Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatmentmainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.