The structure of the O-linked carbohydrate chain of bovine seminal plasma protein PDC-109 revised by H-NMR spectroscopy A correction.
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Animals↗
Biomedical subjects
J J Calvete
Publications and source records attributed to J J Calvete.
Crystallization and preliminary X-ray diffraction analysis of boar seminal plasma spermadhesin PSP-I/PSP-II, a heterodimer of two CUB domains.
Boar spermadhesin PSP-I/PSP-II (M(r) 29 000-30 000), a non-covalent heterodimer of two CUB domains, was crystallized in two crystal forms. Complete diffraction data sets for hexagonal (space group P6(1,5)22) and trigonal (space group P3(1,2)21) crystals have been collected up to 2.9 and 2.5 angstrom resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are a=b=87.2 angstrom, c=152.4 angstrom, and a=b=96.2 angstrom, c=70.8 angstrom, respectively. The calculated packing parameters (Vm) are 2.8 and 3.2 angstrom(3)/DA for the hexagonal and trigonal crystal forms, respectively, indicating that, in both cases, the asymmetric unit is constituted by one PSP-I/PSP-II heterodimer. This paper reports the first crystals of a protein built up by a CUB domain architecture.
Amino Acid Sequence↗
Mapping the heparin-binding domain of boar spermadhesins.
Boar spermadhesins are a group of seminal plasma, heparin-binding proteins which appear to be involved in sperm capacitation and gamete interaction. Using a proteolytic protection assay we have identified regions of AQN-1, AQN-3, PSP-I and AWN which remain attached to a heparin-Sepharose column following in-column digestion of bound spermadhesins with chymotrypsin and elastase. In addition, the complete amino acid sequence of spermadhesin AWN was synthesized as overlapping peptides, and their ability to bind to a heparin-Sepharose column and to inhibit the interaction of soluble heparin with purified ELISA plate-coated AWN was tested. Both approaches gave similar results and as a whole showed that different regions of AWN may converge in its tertiary structure to form a composite heparin-binding site. The conformational heparin-binding surface resides on the GFCC'C'' face of the proposed structural model for AWN and is in an opposite location to the carbohydrate-binding region of the spermadhesin.
Amino Acid Sequence↗
A procedure for the large-scale isolation of major bovine seminal plasma proteins.
A method for the stepwise purification of bovine seminal plasma proteins aSFP, PDC-109, TIMP-2, RNAse, and BSP-30K/PDC-109 complex is presented. It is based on the use of nondenaturing, classical chromatographic procedures. The isolation protocol is reproducible, inexpensive, easy to carry out in a common laboratory, and yields large amounts of pure proteins for biochemical and structural studies.
Amino Acid Sequence↗
Sperm-associated protein candidates for primary zona pellucida-binding molecules: structure-function correlations of boar spermadhesins.
The initial binding of mammalian spermatozoa and the zona pellucida of their homologous oocytes appears to be mediated by a protein-carbohydrate recognition mechanism. Subsequent protein-protein interaction triggers the acrosome reaction leading to sperm penetration. Spermatozoa possess a complement of surface-associated proteins that mediate interactions with the zona pellucida of the oocyte at fertilization. Both integral membrane and peripherally associated zona pellucida-interacting molecules have been described in a number of species. Some of these proteins have been shown to possess carbohydrate-binding activity and are therefore classified as candidates for primary gamete recognition molecules. The participation of any of these proteins in the in vivo situation is, however, a matter of debate. Here, we summarize current knowledge of the most thoroughly studied sperm-associated zona pellucida- and carbohydrate-binding molecules, paying special attention to recent evidence from our laboratories for considering sperm-adhesin AWN, and probably other members of its protein family, as a bona fide candidate for the primary gamete adhesion lectin.
Animals↗
Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.
The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.
Animals↗
Effect of glycosylation on the heparin-binding capability of boar and stallion seminal plasma proteins.
Boar and stallion seminal plasmas were fractionated using affinity chromatography on heparin-Sepharose. In both species, among other proteins, the heparin-binding (H+) and non-heparin-binding (H-) fractions each contained glycoforms of either porcine PSP-I or equine HSP-1 and HSP-2. However, porcine H+/PSP-I eluted as a monomeric protein, whereas H-/PSP-I formed a heterodimer with PSP-II, another major seminal plasma protein. On the other hand, the stallion proteins H+/HSP-1 and H+/HSP-2 eluted together as an aggregate of relative molecular mass (M(r)) 90,000, whereas H-/HSP-1 and H-/HSP-2 eluted as monomers (15,000). Remarkably, when PSP-I and PSP-II from the H- fraction were separated, both proteins bound to heparin. Altogether these data show that glycosylation has an indirect effect on the heparin-binding ability of PSP-I, HSP-1 and HSP-2 through modulation of their aggregation state.
Amino Acid Sequence↗
Amino acid sequence of HSP-1, a major protein of stallion seminal plasma: effect of glycosylation on its heparin- and gelatin-binding capabilities.
We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.
Amino Acid Sequence↗
Boar spermadhesin PSP-II: location of posttranslational modifications, heterodimer formation with PSP-I glycoforms and effect of dimerization on the ligand-binding capabilities of the subunits.
Spermadhesin PSP-II was isolated from the non-heparin-binding fraction of boar seminal plasma; its disulphide bridge pattern, and the location of a single N-glycosylation site were established. PSP-II forms a heterodimer with specific N-glycoforms of PSP-I. Although both subunits possess heparin-binding capability, the PSP-I/PSP-II complex does not. The heterodimer contains binding sites for zona pellucida glycoproteins and soybean trypsin inhibitor located in the PSP-II subunit. However, the PSP-I/PSP-II heterodimer binds only loosely to the sperm surface and is easily removed during in vitro capacitation, suggesting that the zona pellucida binding activity may not be relevant for gamete interaction. Our results show that dimerization of spermadhesins PSP-I and PSP-II markedly affects their binding capabilities.
Amino Acid Sequence↗
Boar spermadhesin AWN-1. Oligosaccharide and zona pellucida binding characteristics.
We have analyzed the oligosaccharide recognition ability of boar spermadhesin AWN-1 using biotinylated glycoproteins with defined carbohydrate chains as probes. Our results show that AWN-1 bound to proteins containing O-linked NeuAc alpha(2-3/6)-Gal beta(1-3)-GalNAc (PDC-109 and fetuin) with a Kd = 0.7 microM. AWN-1 also bound to NeuAc alpha(2-3/6)-Gal beta(1-4)-GlcNAc sequences in N-linked triantennary structures of fetuin, but not to the same oligosaccharide in the diantennary structures of IgG or fibrinogen. The absence of terminal sialic acid decreased fivefold the binding affinity. By competitive ELISA, peptides containing the N-linked oligosaccharide sequence inhibited the binding of the parent glycoprotein to immobilized AWN-1 5-45 times less effectively than those carrying O-linked NeuAc alpha(2-3/6)-Gal beta(1-3)-GalNAc structures. In addition, AWN-1 bound with a Kd = 0.3 microM to solubilized, biotinylated porcine zona pellucida glycoproteins. PDC-109 competed effectively with zona pellucida glycoproteins for AWN binding, whereas fetuin was a poor competitor. On the other hand, AWN epitopes were demonstrated on in vitro capacitated boar spermatozoa which were able to bind to, and penetrate, zona-encased oocytes. These data indicate that spermadhesin AWN-1 may play a role in pig fertilization as a sperm-associated lectin of broad specificity though preferential affinity for certain O-linked oligosaccharide structures of the oocyte's zona pellucida.
Amino Acid Sequence↗
Immunohistochemical localization of spermadhesin AWN in the porcine male genital tract.
Boar spermadhesin (AWN) is a 14-kDa multifunctional protein, attached to the surface of the spermatozoa and involved in sperm capacitation and zona pellucida binding. The cellular origin of AWN was previously unknown. Moreover, the region of the male genital tract in which AWN becomes attached to the surface of spermatozoa was also uncertain. By using monospecific polyclonal antibodies against AWN, the immunohistochemical distribution pattern of AWN epitopes has been investigated in tissue sections of the porcine male genital tract. Our study has revealed that AWN is synthesized in the rete testis and in the epithelium of the seminal vesicles. The latter are also the major contributors of seminal plasma AWN. In addition, immunoblotting analysis has shown that AWN is present on epididymal spermatozoa. Our results indicate that the cellular origin of spermadhesins is species-specific. The attachment of AWN to epididymal spermatozoa is probably important in developing the capacity for fertilization.
Animals↗
Identification by affinity chromatography of boar sperm membrane-associated proteins bound to immobilized porcine zona pellucida. Mapping of the phosphorylethanolamine-binding region of spermadhesin AWN.
We have identified boar sperm membrane components recovered by affinity chromatography on a porcine zona pellucida affinity column. The major zona pellucida-bound proteins were spermadhesins AWN and AQN-3, the heparin-binding protein pAIF, and a homolog of the mouse milk fat globule membrane protein. All these proteins are phospholipid-binding proteins peripherally associated with the plasma membrane. Our data suggest that coating proteins tightly bound to the external lipid bilayer may act as major zona pellucida-binding molecules. Using a synthetic peptide approach we show that the regions of spermadhesin AWN comprising residues 6-12 and 104-108 possess affinity for phosphorylethanolamine. These two amino acid sequences are in close proximity in the predicted structural model for AWN, and in opposite location to its carbohydrate-recognition domain. Taken together, our data provide further evidence for the possible involvement of members of the porcine spermadhesin protein family in gamete interaction and suggest a model for the ultrastructural disposition of functional domains of spermadhesin AWN bound to the sperm surface.
Acrosin↗
Interaction of non-aggregated boar AWN-1 and AQN-3 with phospholipid matrices. A model for coating of spermadhesins to the sperm surface.
Boar spermadhesins are 12-14 kDa lectins which coat the entire acrosomal cap sperm head surface. The large amount of spermadhesins AQN-1, AQN-2, AQN-3, and AWN-1 present on in vitro capacitated spermatozoa (approximately 7 x 10(6) molecules of each spermadhesin per cell) suggested that they may bind to major component(s) of the sperm surface. We have investigated both the aggregation state of spermadhesins in seminal plasma using gel filtration chromatography, and their ability to bind to the major phospholipids of the boar sperm plasma membrane, i.e. phosphorylcholine and phosphorylethanolamine. The bulk (90%) of spermadhesins AQN-3 and AWN-1 were eluted as aggregated proteins (Mr > 50,000) with the void volume of a Sephadex G-50 column; the remaining 10% of the total amount of seminal plasma AWN-1 and AQN-3 were recovered, together with the whole amount of AQN-1 and AQN-2, in a fraction containing low-molecular-mass proteins (Mr 16,000-30,000). None spermadhesin of either gel-filtration fraction bound to a phosphorylcholine affinity matrix. On the other hand, low-molecular-mass (monomeric or dimeric) AQN-3 and AWN-1 were the only spermadhesins retained in a phosphorylethanolamine affinity column. Both AQN-3 and AWN-1 purified from seminal plasma by reverse-phase HPLC retained their lipid-binding capability. In addition, immobilization of AQN-3 and AWN-1 onto a phosphorylethanolamine matrix did not interfere with the ability of the proteins to bind bovine glycoprotein PDC-109, indicating that the structural determinants for the binding lipid and carbohydrates lay on different structural domains.(ABSTRACT TRUNCATED AT 250 WORDS)
Acrosin↗
Advanced ovulation in gilts by the intrauterine application of a low molecular mass pronase-sensitive fraction of boar seminal plasma.
The shortening of the time interval between the onset of oestrus and ovulation in sows by the transcervical administration of seminal plasma was investigated in 23 German Landrace gilts, using the technique of single horn infusions (Mariensee model) in combination with the transcutaneous sonographic monitoring of ovaries. Preparative surgery comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. The left ovary was loosely tied to the ventral abdominal wall for better sonographic distinction. The animals were used in two to four consecutive cycles. After detection of oestrus by the teaser boar, the patent (right) horns were filled by transcervical infusion of 100 ml of a variety of test solutions. Ovulation was probed by transcutaneous sonography at intervals of 4 h thereafter. Native seminal plasma provoked ovulation in the ipsilateral ovary of the treated horn 10.7 h earlier than in the contralateral ovary. This effect was reduced to 7.3 h after charcoal treatment of seminal plasma; addition of 10 micrograms oestradiol restored the effect in full, while 10 micrograms of oestradiol in PBS shortened the time interval to only 3.3 h versus the control ovary. Little effect was seen with oestrone sulfate, none with prostaglandins in PBS or with PBS alone. The preliminary characterization of the nonsteroidal component of seminal plasma advancing ipsilateral ovulation after transcervical infusion suggests a proteinaceous nature. The activity resides in the 1-10 kDa fraction separated by ultrafiltration and is lost after treatment with pronase.
Animals↗
On the structure and function of platelet integrin alpha IIb beta 3, the fibrinogen receptor.
Platelet membrane glycoprotein (GP) IIb/IIIa (alpha IIb beta 3), a Ca(2+)-dependent heterodimer, serves as an inducible receptor for fibrinogen and other adhesive plasma proteins, and is the most thoroughly studied integrin receptor. Intensive research during the past several years has elucidated the major features of its biosynthetic pathway, covalent structure, domain organization, and topography, and we are beginning to get an insight into the cellular mechanisms controlling integrin function. The emerging picture indicates that platelet-specific elements initiate at the cytoplasmic domains of GPIIb/IIIa a signal that leads to conformational changes within the integrin's extracellular domains and expression of the fibrinogen receptor. The simultaneous occupancy on adjacent platelets of receptors with dimeric fibrinogen molecules leads to platelet aggregation. Further structural alterations promote clustering of occupied GPIIb/IIIa complexes and their attachment to the remodelling cytoskeletal network. This interaction provides the physical link for clot retraction to occur and appears to regulate the compartmentalization, and local activation, of a multienzymatic complex which translates the ligand-binding information into time-dependent irreversibility of the fibrinogen-GPIIb/IIIa interaction. Platelet GPIIb/IIIa plays, thus, a central role in thrombus formation both in health and disease: abnormalities in the platelet adhesive mechanisms responsible for the formation of the hemostatic plug, lead to major pathophysiologic disorders, ranging from severe bleeding to thrombosis. It is, therefore, not surprising that GPIIb/IIIa has been the subject of intensive research during the last decades, since a detailed knowledge of the molecular biology and the mechanism underlying the platelet activation and aggregation processes may aid in the rational design of both an effective gene replacement therapy, and of potent and specific anti-thrombotic drugs. The aim of this minireview is to summarize many functional and structural data from different laboratories in the perspective of an emerging model that may help us to understand structure-function relationships of GPIIb/IIIa and of other members of the integrin family.
Amino Acid Sequence↗
Molecular mechanisms of the interaction between sperm and the zona pellucida in mammals: studies on the pig.
Research carried out on mammalian fertilization is summarized here. The main focus is on current knowledge of porcine gamete interaction. Attention has been given to the structural characterization of zona pellucida glycoproteins with sperm receptor activity and the primary zona pellucida-binding proteins of the spermadhesin family on spermatozoa, and to the role of acrosin as a secondary ligand for sulphated glycoproteins of the zona pellucida. Established similarities and differences between mammalian species are described to highlight the existence of both common and species-specific mechanisms.
Animals↗
Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109. Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa.
PDC-109 (13 kDa) is the most abundant component, and the major heparin-binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC-109 contains a single O-linked oligosaccharide (NeuNAc alpha(2-6)-Gal beta(1-3)-GalNAc-) attached to Thr11. Immunoquantitation of PDC-109 indicates that its concentration in seminal plasma is 15-20 mg/ml. Though PDC-109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 +/- 0.3) x 10(6) molecules of PDC-109/cell. This value remained constant in swim-up sperm and decreased to (7.7 +/- 0.4) x 10(6)/spermatozoon after incubation for 24 h in capacitation medium at 39 degrees C. These data substantiate the hypothesis that PDC-109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin-like glycosaminoglycans present in the female genital tract.
Amino Acid Sequence↗
Characterization of the cross-linking site of disintegrins albolabrin, bitistatin, echistatin, and eristostatin on isolated human platelet integrin GPIIb/IIIa.
Disintegrins, a family of low molecular weight, RGD-containing peptides found in snake venoms prevent the binding of adhesive ligands to a number of integrin receptors. Albolabrin, bitistatin, echistatin, and eristostatin bind to the platelet fibrinogen receptor (GPIIb/IIIa) acting thus as potent inhibitors of platelet aggregation. Here, we have determined the cross-linking of these disintegrins on isolated GPIIb/IIIa. The cross-linking site of all of them was within GPIIIa 217-302, a domain that has been implicated in a number of receptor functions including heterodimer association, activation-dependent conformational changes, and fibrinogen binding.
Amino Acid Sequence↗