ABSTRACT
The decapacitating fraction of human seminal plasma, which strongly interacts with concanavalin A, is constituted by high mannose-type N-linked glycoproteins, most of them of less than 44 kDa. Each component with apparent molecular mass of 30, 18, and 17 kDa respectively, as judged by SDS-PAGE, was submitted to "in gel" digestion with trypsin followed by HPLC separation of the peptides and sequencing. They were characterized at microscale as gp17, an aspartyl protease that possibly contributes to liquefaction of the seminal plasma coagulum, two fragments of human acid phosphatase (17 and 30 kDa, respectively), and a 17-kDa fragment of carboxypeptidase E. Neither the fragments of prostatic acid phosphatase nor that of carboxypeptidase E had been described before in the human seminal fluid. Very weak bands, of apparent molecular masses 44 and 52 kDa, are consistent with presence of small amounts of parent compounds, prostatic acid phosphatase and carboxypeptidase E.
Subject(s)
Carboxypeptidase H/isolation & purification , Concanavalin A/metabolism , Glycoproteins/isolation & purification , Peptide Fragments/isolation & purification , Protein Tyrosine Phosphatases/isolation & purification , Semen/chemistry , Acid Phosphatase , Amino Acid Sequence , Carboxypeptidase H/metabolism , Chromatography, Affinity , Chromatography, High Pressure Liquid , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Peptide Fragments/metabolism , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Semen/enzymologyABSTRACT
These studies showed that the fractionation of bovine seminal plasma based on lectin agarose affinity chromatography, employing lectins specific to asparagine linked oligosaccharides, and a lectin specific for fucosylated glycans, lead to products with an inhibitory effect on the acrosine-like protease activity. This effect decreases when glycocompounds containing fucosylated Lewis(x) structures are removed, suggesting that these compounds might have some role in the modulation of this activity in the bull. In the fraction devoid of high mannose, hybrid and non-bisecting lactosaminic oligosaccharide-containing glycocompounds, PDC-109 and aSFP proteins were detected and characterized at microscale.
Subject(s)
Chromatography, Affinity/methods , Endopeptidases/metabolism , Protease Inhibitors/pharmacology , Proteins/analysis , Proteins/chemistry , Semen/chemistry , Amino Acid Sequence , Animals , Cattle , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Endopeptidases/chemistry , Lectins/chemistry , Male , Protease Inhibitors/chemistry , Seminal Plasma ProteinsABSTRACT
A semiquantitative immunohistochemical technique for the detection of N-acetyl alpha-D-neuraminic acid, N-acetyl beta-D-glucosamine and its beta-(1 leads to 4)-linked internal chains, alpha-D-glucopyranosyl and alpha-D-mannopyranosyl and its alpha-(1 leads to 2)-linked internal chains and sterically related, nonreducing, end-chain residues of oligosaccharide chains of glycoproteins or glycolipids on the surface of membranes was developed using Con A and wheat germ lectins. When this method was applied to the localization of carbohydrate receptors on the membrane of the normal human spermatozoa, it was found that the Con A and wheat germ lectin receptors were mainly located in the equatorial and post nuclear cap with few receptors located in the acrosome and neck. None of them were found in the intermediate segment plus tail. Con A receptors were alpha-D-mannopyranosyl end-chain residues and wheat germ lectin receptors were N-acetyl beta-D-glucosamine (1 leads to 4)-linked internal chains. These groups occur together in the oligomannosidic type of N-glycosidic-linked oligosaccharide chains of glycoproteins and so the use of both lectins on desialycated membranes or on those which contain nonclustered N-acetyl neuraminic acid residues may be of help to localize this type of glycoprotein oligosaccharide chains. Con A receptors were not removed after proteases digestion, suggesting the possibility that they are part of intrinsic spermatozoal antigens.
