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J J Calvete

Publications and source records attributed to J J Calvete.

114 records · Page 7Linked to original sources
PubMed1986

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane.

We describe a new procedure for isolation of glycoproteins IIb (GPIIb) and IIIa (GPIIIa) from human platelet plasma membrane with high yields (2.7 mg of GPIIb and 3.3 mg of GPIIIa per 100 mg of starting platelet membrane proteins), equivalent to a recovery of 35% and 55% respectively of the total GPIIb and GPIIIa of the membrane. The procedure involves Triton X-100 differential extraction of platelet membranes, SDS solubilization of the 4%-Triton X-100 supernatant, zonal centrifugation in a sucrose density gradient, and preparative high-performance size-exclusion chromatography. The weight percentage of sugar is 15.7% for GPIIb and 12.5% for GPIIIa. Neuraminic acid is present in both glycoproteins, representing 30% and 15% respectively of the total sugar weight of GPIIb and GPIIIa. Mannose, galactose and glucosamine account for 45%, 13% and 28% respectively of the sugars of GPIIIa, whereas galactosamine was not detected. Mannose, galactose, glucosamine and galactosamine represent 17%, 21%, 24% and 10% respectively of the sugar content of GPIIb. The molar percentages of half-cystine and methionine are 4-fold and 2-fold higher respectively in GPIIIa than in GPIIb. From the amino acid and sugar compositions we confirmed the acidic nature of both glycoproteins. The Mr values obtained, 136,500 for GPIIb and 91,500 for GPIIIa, are in very good agreement with those obtained by physical methods. The apparent lack of free thiol groups in both glycoproteins indicates that the tertiary structure of GPIIIa is maintained by 21 intrachain disulphide bonds, and that there are eight intrachain and interchain disulphide groups in GPIIb.

Amino Acids↗
PubMed1986

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

Amino Acids↗
PubMedDate not supplied

Immunohistochemical localization in the stallion genital tract, and topography on spermatozoa of seminal plasma protein SSP-7, a member of the spermadhesin protein family.

SSP-7 is a protein originally isolated from stallion seminal plasma. It has extensive amino acid sequence homology with boar spermadhesin AWN, and, like its porcine counterpart, SSP-7 displays zona pellucida-binding activity. Strikingly, however, immunohistochemical studies presented here show that the stallion and the boar spermadhesin homologues are secreted at different places of the male genital tract. Furthermore, indirect immunofluorescence shows that the topography of SSP-7 on the surface of stallion spermatozoa is restricted to the equatorial segment, whereas boar AWN epitopes cover the entire acrosomal cap membrane. The different cellular origin and compartimentalization of spermadhesin molecules in different species suggest that structurally related proteins could be involved in species-specific aspects of mammalian fertilization.

Acrosome↗
PubMedDate not supplied

Spermadhesins: a new protein family. Facts, hypotheses and perspectives.

Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN-1, AQN-2, PSP-I and PSP-II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand-binding abilities, e.g. carbohydrates, sulfated glycosaminoglycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation-dependent manner with a Kd in a low micromolar range, and AWN, AQN-1 and AQN-3 display similar binding affinity for glycoproteins containing Gal beta(1-3)-GalNAc and Gal beta(1-4)-GlcNAc sequences in O-linked and N-linked oligosaccharides, respectively. During sperm passage through the epididymis AQN-3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid-bound spermadhesins. AWN and AQN-3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg. The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). However, they belong to the superfamily of proteins with a CUB domain with a predicted all-beta structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP-I/PSP-II has been solved, showing that the overall structure of both spermadhesins consists of a beta-sandwich with five (parallel and antiparallel) beta-strands. It is the first three-dimensional structure of a zona pellucida-binding protein and reveals the architecture of the CUB domain. The spermadhesins represent a novel class of lectins that may be involved in sequential steps of fertilization, at least in the pig.

Animals↗
PubMedDate not supplied

Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida binding characteristics.

AQN-1 and AQN-3 form part of the complement of surface-associated lectins which coat the plasma membrane overlying the acrosomal cap of in vitro capacitated boar spermatozoa. They belong to the spermadhesin protein family and have binding affinity for glycoconjugates of the zona pellucida, the extracellular investment surrounding mammalian eggs. The oligosaccharide and zona pellucida binding characteristics of spermadhesins AQN-1 and AQN-3 were investigated using a solid-phase assay and biotinylated glycoprotein ligands. Both sperm proteins bind glycoproteins containing Gal beta (1-4)-GlcNAc and Gal beta-(1-3)-GalNAc oligosaccharide sequences with dissociation constants (Kd) of 0.08 to 0.8 microM, and to zona pellucida glycoproteins with Kd = 0.15-0.25 microM. However, 5-N-acetyl neuraminic acid alpha (2-3/6)-linked to the galactose residue decreases the affinity of glycosylated ligands to AQN-1 three-fold, although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glycoproteins with either a linear or tri- and tetraantennary carbohydrates than to those containing diantennary N-acetyllactosamine structures. The similar but distinct oligosaccharide recognition capabilities of spermadhesins AQN-1 and AQN-3 (this work) and AWN-1 (Dostálová, Z, Calvete, J.J., Sanz, L., and Töpfer-Petersen, E. (1995) Eur. J. Biochem. 230, 329-336) suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying similar although distinct carbohydrate-recognition abilities.

Animals↗