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J J Calvete

Publications and source records attributed to J J Calvete.

At least 55 records · Page 3Linked to original sources
PubMed1997

Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 A resolution: a bovine polypeptide of the spermadhesin family.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa polypeptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 A resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds. On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family.

Amino Acid Sequence↗
PubMed1997

The 2.4 A resolution crystal structure of boar seminal plasma PSP-I/PSP-II: a zona pellucida-binding glycoprotein heterodimer of the spermadhesin family built by a CUB domain architecture.

The crystal structure of porcine seminal plasma spermadhesin PSP-I/PSP-II heterodimer has been determined in two crystal forms by multiple isomorphous replacement in an hexagonal crystal (space group P6(1)22) and molecular replacement in a trigonal crystal of space group P3(2)21. The crystal structure has been refined at 2.4 A resolution to an R-factor of 20.0% (Rfree = 25.9%) for 14,809 independent reflections with intensities greater than 2 sigma (I), with root-mean-square deviations of 0.009 A and 1.657 degrees from ideal bond lengths and bond angles, respectively. The final model includes 1688 non-hydrogen protein atoms of 221 amino acids and 79 water molecules. PSP-I/PSP-II represents the first crystal structure of a mammalian zona pellucida-binding protein. PSP-II displays a putative carbohydrate-recognition site located around its Asn50. This region shares structural features with sugar binding sites of known lectin structures of the leguminous and galectin families. PSP-I and PSP-II are N-glycosylated at asparagine residues 50 and 98, respectively, and show site heterogeneity. Only the innermost N-acetylglucosamine of PSP-I is defined in the crystal structure. Both subunits of the PSP-I/PSP-II heterodimer are built by a single CUB domain architecture. The CUB domain displays a novel fold, which consists of a compact ellipsoidal beta-sandwich structure (42 A x 27 A x 23 A) organized into two 5-stranded beta-sheets. Each sheet contains parallel and antiparallel beta-strands. Two disulphide bridges, which are conserved in all spermadhesin molecules and many CUB domains, crosslink loop LA and strand beta 4 and loops LE and LG, respectively, at opposite edges of the same face of the domain. The four highly conserved aromatic residues and 15 out of 17 invariant hydrophobic residues, which define the CUB domain signature, display an interior location, suggesting that this hydrophobic core may be essential for maintaining the overall folding of the domain. Most of the hydrophobic core residue characteristics are conserved in the jellyroll topology of certain icosahedral virus capsid proteins, indicating that the CUB domain and the viral proteins share a minimal structural core.

Amino Acid Sequence↗
PubMed1997

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans.

The disulphide bond pattern of the long disintegrin bitistatin (83 amino acids, 14 cysteines) was established using structural information gathered by amino acid analysis, N-terminal sequencing, and molecular mass determination of fragments isolated by reversed-phase HPLC after polypeptide degradation with trypsin and oxalic acid. A computer program was used to calculate all possible combinations of disulphide-bonded peptides matching the mass spectrometric data, and the output was filtered using compositional and sequence data. Disulphide bonds between cysteines 16-34, 18-29, 28-51, 42-48, 47-72, and 60-79 are conserved in medium-long disintegrins flavoridin and kistrin (70 amino acids, 12 cysteines), and the two cysteine residues at positions 5 and 24 found in bitistatin but not in other disintegrin molecules are disulphide-bridged. This linkage creates an extra, large loop, which, depending on whether the NMR structure of flavoridin or kistrin is used for modelling the structure of bitistatin, lies opposite or nearly parallel, respectively, to the biologically active RGD-containing loop.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and characterization of ConBr, the lectin of Canavalia brasiliensis seeds.

ConBr, a lectin isolated from Canavalia brasiliensis seeds, shares with other legume plant lectins from the genus Canavalia (Diocleinae subtribe) primary carbohydrate recognition specificity for D-mannose and D-glucose. However, ConBr exerts different biological effects than concanavalin A, the lectin of Canavalia ensiformis seeds, regarding induction of rat paw edema, peritoneal macrophage spreading in mouse, and in vitro human lymphocyte stimulation. The primary structure of ConBr was established by cDNA cloning, amino acid sequencing, and mass spectrometry. The 237-amino-acid sequence of ConBr displays Ser/Thr heterogeneity at position 96, indicating the existence of two isoforms. The mature Canavalia brasiliensis lectin monomer consists of a mixture of predominantly full-length polypeptide (alpha-chain) and a small proportion of fragments 1-118 (beta-chain) and 119-237 (gamma-chain). Although ConBr isolectins and concanavalin A differ only in residues at positions 58, 70, and 96, ConBr monomers associate into dimers and tetramers in a different pH-dependent manner than those of concanavalin A. The occurrence of glycine at position 58 does not allow formation of the hydrogen bond that in the concanavalin A tetramer exists between Asp58 of subunit A and Ser62 of subunit C. The consequence is that the alpha carbons of the corresponding residues in ConBr are 1.5 A closer that in concanavalin A, and ConBr adopts a more open quaternary structure than concanavalin A. Our data support the hypothesis that substitution of amino acids located at the subunit interface of structurally related lectins of the same protein family can lead to different quaternary conformations that may account for their different biological activities.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1.

In the bovine, seminal plasma heparin-binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin-like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin- and phosphorylcholine-binding proteins of stallion and boar seminal plasma. Horse seminal plasma proteins HSP-1 and HSP-2, and boar protein pB1, belong to the same family as the bull heparin- and phosphorylcholine-binding proteins BSP-A1/2, BSP-A3, and BSP-30K. We have determined the amino acid sequence and posttranslational modifications of boar glycoprotein pB1. It contains 105 amino acids arranged into a mosaic structure consisting of a N-terminal 18-residue O-glycosylated polypeptide followed by two tandemly organized 40-45-residue fibronectin type II domains. pB1 displays 60-65% amino acid sequence similarity with its equine and bovine homologues. However, in their respective seminal plasmas, the BSP and the HSP proteins associate into 90-150-kDa oligomeric complexes, whereas pB1 forms a 35-40-kDa complex with spermadhesin AQN-1. In addition, pB1 appears to be identical to the recently described leukocyte adhesion regulator of porcine seminal fluid pAIF-1. Our results tie in with the hypothesis that homologous proteins from different mammalian species may display distinct biological activities, which may be related to species-specific aspects of sperm physiology.

Amino Acid Sequence↗
PubMed1997

The crystal structure of Canavalia brasiliensis lectin suggests a correlation between its quaternary conformation and its distinct biological properties from Concanavalin A.

Canavalia brasiliensis lectin was isolated from the seeds of a Brazilian autochthonous Leguminosae plant. Despite extensive amino acid sequence similarity with Concanavalin A, C. brasiliensis lectin exerts in vitro and in vivo cellular effects that are markedly different from those displayed by Concanavalin A. We have solved the crystal structure of the C. brasiliensis lectin at 3.0 A resolution. The three-dimensional structure of the lectin monomer can be superimposed onto that of Concanavalin A with a root-mean-square deviation for all C alpha atoms of 0.65 A. However, this parameter is 0.84 and 1.62 A when the C. brasiliensis lectin dimer and tetramer, respectively, are compared with the same structures of Concanavalin A. We suggest that these differences in quaternary structure may account for the different biological properties of these two highly related Leguminosae lectins.

Concanavalin A↗
PubMed1997

Crystallization and preliminary X-ray diffraction analysis of bovine seminal plasma PDC-109, a protein composed of two fibronectin type II domains.

PDC-109 is a 13 kDa glycoprotein and the major phosphorylcholine- and heparin-binding protein of bull seminal plasma. It is built by an acidic 23-residue N-terminal sequence followed by a tandem of fibronectin type II domains. Full-length PDC-109 was crystallized in complex with o-phosphorylcholine by vapor diffusion in sitting drops. Crystals grew to maximal size of 0.5 x 0.3 x 0.2 mm3, diffract x-rays beyond 2.6 A resolution, and belong to space group P321 with unit cell dimensions a = b = 93.6 A, c = 52.7 A.

Animals↗
PubMed1997

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.

Animals↗
PubMed1997

Fractionation and characterization of boar seminal plasma spermadhesion PSP-II glycoforms reveal the presence of uncommon N-acetylgalactosamine-containing N-linked oligosaccharides.

Lectin mapping, carbohydrate analysis and electrospray mass spectrometry of boar seminal plasma PSP-II glycoforms show that its single N-glycosylation site displays a repertoire of carbohydrate structures consisting of the basic pentasaccharide core Man alpha 1-6[Man alpha 1-3]Man beta1-4GlcNAc beta1-4GlcNAc with a fucosyl residue alpha1-6-linked to the innermost N-acetylglucosamine residue. Other glycoforms display fucosylated hybrid-type or monoantennary complex-type chains, some of which contain alpha2-6-linked sialic acid. N-acetylgalactosamine, possibly in Gal beta1-3GalNAc sequence, is present in most of the PSP-II glycoforms.

Acetylglucosamine↗
PubMed1997

The crystal structures of two spermadhesins reveal the CUB domain fold.

Spermadhesins, 12,000-14,000 M(r) mammalian proteins, include lectins involved in sperm-egg binding and display a single CUB domain architecture. We report the crystal structures of porcine seminal plasma PSP-I/PSP-II, a heterodimer of two glycosylated spermadhesins, and bovine aSFP at 2.4 A and 1.9 A resolution respectively.

Amino Acid Sequence↗
PubMed1997

Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows.

Boar spermadhesin AWN-1 is a sperm surface-associated 14.7-kDa lectin and a major protein of porcine seminal plasma. AWN-1 binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting that this protein might play a role in the primary binding of spermatozoa to the egg's external glycoprotein matrix. We have produced a collection of murine monoclonal antibodies against purified AWN-1. Five monoclonal antibodies recognized sequential antigenic determinants. All these epitopes were located at the C-terminal region of AWN-1 (residues 109-123) by competitive ELISA using overlapping synthetic peptides that cover the complete 133 amino acid sequence of the lectin. In a structural model of spermadhesin AWN-1, the polypeptide stretch 109-123 is fully solvent-exposed, providing a reasonable explanation for its high immunogenicity. In addition to epitope mapping, we have employed anti-AWN monoclonal antibodies for immunolocalization of the protein in the genital tract of inseminated sows. Clusters of AWN epitopes were occasionally found attached to the epithelium of the uterotubal junction and the adjacent lower isthmus. However, neither AWN-1 nor other seminal plasma proteins were found in the isthmic fluid collected 10-26 h after insemination. These results suggest that the whole amount of seminal plasma proteins are absorbed by the epithelium of the female genital tract, supporting the claim that removal of seminal plasma components from spermatozoa might be a major event in both in vitro and in vivo sperm capacitation.

Animals↗
PubMed1997

Analysis of xysA, a gene from Streptomyces halstedii JM8 that encodes a 45-kilodalton modular xylanase, Xys1.

The gene xysA from Streptomyces halstedii JM8 encodes a protein of 461 amino acids (Xys1) which is secreted into the culture supernatant as a protein of 45 kDa (Xys1L). Later, this form is proteolytically processed after residue D-362 to produce the protein Xys1S, which conserves the same xylanolytic activity. The cleavage removes a domain of 99 amino acids that shows similarity to bacterial cellulose binding domains and that allows the protein Xys1L to bind to crystalline cellulose (Avicel). Expression of this monocistronic gene is affected by the carbon source present in the culture medium, xylan being the best inducer. By using an anti-Xys1L serum, we have been able to detect xylanases similar in size to Xys1L and Xys1S in most of the different Streptomyces species analyzed, suggesting the ubiquity of these types of xylanases and their processing mechanism.

Amino Acid Sequence↗
PubMed1996

Primary structure of stallion seminal plasma protein HSP-7, a zona-pellucida-binding protein of the spermadhesin family.

The primary-structure of HSP-7, a 14-kDa protein isolated from stallion seminal plasma, has been determined, HSP-7 belongs to the spermadhesin protein family, shares 98% sequence identity with the boar seminal plasma protein AWN, and, like its boar homolog, displays zona-pellucida-binding activity. Despite these conserved structural and functional features, the equine and porcine spermadhesins differ in their topography on spermatozoa.

Amino Acid Sequence↗
PubMed1996

Chemical cross-linking detects different conformational arrangements of platelet integrin alpha IIb beta III (gpIIb/IIIa).

Treatment of Triton X-100 solubilized platelet membrane with the homobifunctional cross-linker dithiobis(succinimidyl propionate) resulted in covalent cross-linking of the platelet integrin alpha IIb beta 3 (gpIIb/IIIa), the fibrinogen receptor, into three high-molecular-mass complexes with apparent M of 200, 220 and 240 k. Generation of these cross-linked alpha IIb beta 3 aggregates depended on the presence of a native receptor structure and was not influenced by Ca2+ ions and other experimental conditions. Immunoblotting analysis of purified 200/220/240 kDa aggregates revealed that they were made up exclusively by alpha IIb and beta 3 integrin chains in roughly stoichiometric amounts. We therefore conclude that alpha IIb beta 3 integrin occurs in different conformations in the platelet membrane that can be directly detected by chemical cross-linking.

Cross-Linking Reagents↗
PubMed1996

The primary structure of BSP-30K, a major lipid-, gelatin-, and heparin-binding glycoprotein of bovine seminal plasma.

BSP-30K is a major acidic glycoprotein of bovine seminal plasma. It displays heparin-, gelatin-, and phospholipid-binding activities. BSP-30K binds to spermatozoa upon ejaculation and is thought to play a role in sperm capacitation. We have determined its amino acid sequence, disulfide bonds, and 0-glycosylation sites. BSP-30K consists of 158 amino acids arranged in a mosaic structure. BSP-30K has a unique 48-residue N-terminal extension which includes three 7-8- amino acid repeats and the six O-glycosylated threonine residues. The polypeptide stretch 49-71 is homologous to type 'A' domains found in heparin-binding proteins from other mammalian species. The C-terminal portion of BSP-30K is organized in a tandem of 40-44-residue domains each sharing the consensus pattern of the gelatin-binding fibronectin type II module. The mosaic structure of BSP-30K suggests that this glycoprotein might be a factor contributing to the different sperm-capacitating effects exerted by heparin in different mammalian species.

Amino Acid Sequence↗
PubMed1996

Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins.

Echistatin and eristostatin are structurally homologous distintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 AND 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of S-S bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to alpha IIb beta 3, and decreased potency to block vitronectin binding to alpha v beta 3 as compared to wild-type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of alpha IIb beta 3 and alpha v beta 3.

Amino Acid Sequence↗