Biomedical subjects

L Sanz

Publications and source records attributed to L Sanz.

At least 73 records · Page 4Linked to original sources

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

Molecular characterization of a novel transcription factor that controls stromelysin expression.

Stromelysins, which are the metalloproteinases with the widest substrate specificities, play a critical role in tumor invasion and metastasis. We have previously reported an element (SPRE) of the stromelysin promoter located between nucleotides -1221 and -1203 that is necessary and sufficient for the control of stromelysin gene expression by mitogenic activation, which induces a nuclear activity that binds to this sequence. Using a concatenated probe with several copies of this element to screen a lambda gt11 cDNA expression library from mouse Swiss 3T3 fibroblasts, we report here the molecular cloning of a cDNA coding for a novel protein (SPBP) of 937 amino acids that binds to this element and has several features of a transcription factor, such as a putative leucine zipper region, a nuclear localization signal, and a basic domain with homology to the DNA-binding domains of Fos and Jun. Evidence that SPBP is at least a critical component of the mitogen-induced SPRE nuclear binding activity is presented here. Furthermore, the transfection of an expression plasmid for SPBP transactivates reporter chloramphenicol acetyltransferase plasmids containing either the full-length stromelysin promoter or a single copy of the SPRE cloned upstream of the herpes simplex virus thymidine kinase minimal promoter. Therefore, the results presented here identify a novel transcription factor critically involved in the control of stromelysin expression.

3T3 Cells↗

PubMed1995

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↗

PubMed1994

Evidence for the in vitro and in vivo interaction of Ras with protein kinase C zeta.

The zeta isoform of protein kinase C (zeta PKC) has been shown to be involved in the maturation of Xenopus oocytes and mitogenic signaling in fibroblasts. zeta PKC also regulates the important transcription factor nuclear factor kappa B, most probably by phosphorylation of the inhibitory molecule I kappa B. The mechanisms that control zeta PKC activity are still poorly characterized. This kinase is not activated by diacylglycerol but is potently stimulated in vitro by the products of phosphatidylinositol 3-kinase (PI 3-kinase), which suggests that zeta PKC is at least one of the critical targets of PI 3-kinase-triggered signals, and strengthens its role in cell proliferation. PI 3-kinase has been shown, like Raf, to be a direct effector of Ras. zeta PKC is a required step for Ras mitogenic signaling. Therefore, it is possible that zeta PKC directly interacts with Ras during mitogenic activation. We demonstrate here that Ras interacts in vitro with the regulatory domain of zeta PKC as well as that the association of zeta PKC with Ras in vivo is triggered by platelet-derived growth factor. It is also shown here that the expression of a dominant negative mutant of Ras (Asn-17) severely impairs the activation of zeta PKC in mouse fibroblasts.

Base Sequence↗

PubMed1994

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↗

PubMed1994

Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase.

Recent evidence demonstrates that the protein kinase C zeta (zeta PKC) isoform is required for the activation of nuclear factor kappa B (NF-kappa B) and mitogenic signaling in Xenopus oocytes and mammalian cells. The mechanism whereby zeta PKC regulates NF-kappa B most probably involves the activation of a putative I kappa B kinase of molecular mass approximately 50 kDa, which phosphorylates and inactivates I kappa B. Tumor necrosis factor alpha (TNF alpha) and interleukin-1, besides activating the phospholipase C-mediated breakdown of phosphatidylcholine, also generate ceramide, which is produced by stimulation of sphingomyelin hydrolysis. We show here that exogenous addition of sphingomyelinase (SMase) to NIH-3T3 fibroblasts transactivates a kappa B-dependent chloramphenicol acetyltransferase reporter plasmid, to an extent similar to that produced by TNF alpha or phosphatidylcholine/phospholipase C. More importantly, the ability of SMase to stimulate this parameter is severely impaired by transfection of a zeta PKC kinase-defective dominant negative mutant, which suggests a critical role of zeta PKC in SMase signaling. In keeping with this notion, we also demonstrate here that zeta PKC is activated in vitro by ceramide and in vivo by treatment of NIH-3T3 fibroblasts with SMase.

3T3 Cells↗

PubMed1994

zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro.

The zeta isotype of protein kinase C (zeta PKC), a distinct PKC unable to bind phorbol esters, is required during NF-kappa B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by zeta PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es) with zeta PKC. The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained. MBP-zeta PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of approximately 50 kDa molecular weight able to regulate I kappa B-alpha function. Activation of the I kappa B kinase was dependent on zeta PKC enzymatic activity and ATP, suggesting that zeta PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase. Importantly, zeta PKC immunoprecipitates from TNF-alpha-stimulated NIH-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings. We also show here that zeta PKC associates with and activates MKK-MAPK in vitro, suggesting that one of the mechanisms whereby overexpression of zeta PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex. However, neither MKK nor MAPK is responsible for the putative I kappa B phosphorylating activity. These data provide a decisive step towards understanding the functions of zeta PKC.

Animals↗

PubMed1994

Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa.

Spermadhesins are multifunctional proteins involved in boar sperm capacitation and gamete recognition. Using anti-AWN antibodies, we have followed the fate of spermadhesin AWN along the maturation and capacitation stages of boar spermatozoa. In addition, the amount of spermadhesins AQN-1, AQN-2, and AQN-3 relative to that of AWN was determined by amino acid analysis after reverse-phase HPLC isolation. Our data show that AWN-1 is the only spermadhesin on the surface of epididymal sperm and that a large amount of AQN-1, AQN-2, AQN-3, AWN-1 and AWN-2 become coated on ejaculated spermatozoa. The number of spermadhesin molecules on ejaculated sperm (12-60 x 10(6)/spermatozoa) is sufficient to form a many-molecules-thick coat over the sperm head. However, 50-75% of the AQN-1, AQN-2, and AQN-3 population, and around 90% of coated AWN (1 + 2) are released from ejaculated sperm during capacitation. This indicates that a large subpopulation of each boar spermadhesin is loosely associated to the sperm surface and may function as decapacitation factors. The remaining spermadhesin molecules, which are tightly bound to the sperm head's surface may play a role as either positive capacitation factors and/or in gamete recognition and binding.

Animals↗

PubMed1994

Zeta PKC plays a critical role during stromelysin promoter activation by platelet-derived growth factor through a novel palindromic element.

Stromelysin is a metalloproteinase with the widest substrate specificity that plays a critical role in the induction of the metastatic phenotype in cancer cells. The mechanisms whereby growth factors and oncogenes control stromelysin expression are beginning to be characterized. We have recently demonstrated that protein kinase C isotypes down-regulatable by chronic exposure to phorbol esters are not involved in stromelysin gene expression in response to platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C. We also identified a region in the stromelysin promoter, distinct from the 12-O-tetradecanoylphorbol-13-acetate-responsive element, responsible for the promoter activity in response to these stimulants. In this paper, we further characterize that promoter fragment and demonstrate that the region encompassing nucleotides -1218 to -1202, including the palindromic sequence ACTAGT, is necessary and sufficient for the control of stromelysin gene expression. The involvement of zeta-protein kinase C but not of c-raf in the stimulation of stromelysin promoter activity in response to platelet-derived growth factor is also demonstrated here. All these data suggest the existence of a bifurcation downstream of ras in the signaling mechanisms leading to stromelysin expression and DNA synthesis.

3T3 Cells↗

PubMed1994

Different influence of superoxide anions and hydrogen peroxide on endothelial function of isolated cat cerebral and pulmonary arteries.

1. Exogenous superoxide dismutase (SOD) or catalase did not modify isolated cat middle cerebral arterial basal tone. Catalase but not SOD reduced ACh relaxation. 2. H2O2 induced endothelium-independent relaxation which was abolished by catalase. 3. 3-Amino-1,2,4-triazole (AT) evoked endothelium-dependent contractions and diminished ACh relaxation. 4. Diethyldithio carbamic acid (DETC) induced endothelium-independent relaxation and did not modify ACh vasodilatation. 5. ACh relaxation of cat isolated pulmonary arteries was unaffected by SOD, catalase or AT, and diminished by DETC. 6. Endothelial catalase but neither SOD nor superoxide anions is involved in EDRF cerebral vasodilatation and H2O2 participates in ACh relaxation. In pulmonary arteries, only endothelial SOD activity plays a role.

Acetylcholine↗

PubMed1994

Glycosylated boar spermadhesin AWN-1 isoforms. Biological origin, structural characterization by lectin mapping, localization of O-glycosylation sites, and effect of glycosylation on ligand binding.

Spermadhesin AWN-1 is a 133-residues boar sperm surface lectin with capability to bind different ligands, e.g. glycoproteins from zona pellucida (ZP), soybean trypsin inhibitor and heparin, and is involved in capacitation and binding of spermatozoa to the homologous zona pellucida. Here, we report the characterization of N- and O-glycosylated isoforms of AWN-1. Non-glycosylated AWN-1 is present in seminal plasma and on epididymal and ejaculated spermatozoa whereas its N- and O-glycosylated isoforms are only secretory products of the seminal vesicles. Lectin mapping indicated the presence of the glycosylated AWN-1 isoform mixture of both fucosylated and non-fucosylated N-glycans, and of two different classes of O-linked carbohydrate chains. These N- and O-linked oligosaccharide chains are neither sialylated nor contain terminal Gal beta (1-4)-GlcNAc sequences. Noteworthy, N- and O-glycosylation (either class) are mutually exclusive on the same protein molecule, indicating that each glycosylated AWN-1 molecule contains a single oligosaccharide chain. Peptide mapping was used to locate the N- and the O-glycosylation sites. Glycosylation of AWN-1 with either of the carbohydrate chain types greatly impaired the ability of the spermadhesin to bind biotinylated zona pellucida glycoproteins and soybean trypsin inhibitor, suggesting that the blocking effect may be due to steric hindrance of the ligand-binding pocket.

Acrosin↗

PubMed1994

Immunolocalization and quantitation of acidic seminal fluid protein (aSFP) in ejaculated, swim-up, and capacitated bull spermatozoa.

Acidic seminal fluid protein (aSFP, 12.9 kDa), a major protein of bull seminal plasma, belongs to the spermadhesin protein family. Boar spermadhesins become bound to the sperm head's surface at ejaculation and are thought to play a role as capacitation factors and/or in gamete recognition and binding. Here, we have investigated the topographical distribution and fate of bovine spermadhesin aSFP during sperm capacitation in order to assess whether aSFP could be involved in similar aspects of the fertilization process as its boar homologous proteins. 5.7 +/- 2.1 x 10(6) molecules/spermatozoa were quantitated on the surface of fresh ejaculated and washed sperm. The binding site of aSFP was restricted to a thin coat at the apical part of the acrosomal cap. The amount of aSFP in swim-up sperm was 1.8 +/- 1.0 x 10(6) molecules/spermatozoa, but decreased dramatically to 22 +/- 10 x 10(3) and to undetectable levels after incubation of sperm for 1.5 h and 18 h, respectively, in capacitation medium. This indicates that the bull spermatozoa surface may be completely depleted of spermadhesin aSFP before spermatozoa reach the surroundings of the investing egg. Therefore, our results suggest that aSFP may act as a decapacitation factor on bull spermatozoa rather than as a zona pellucida binding molecule.

Animals↗

PubMed1993

Characterization of two glycosylated boar spermadhesins.

Boar spermadhesins AQN-1, AQN-3 and AWN form a recently described protein family, synthesized by the sexual accessory glands, and become associated with the sperm head upon ejaculation. They contain 109-133 amino acid residues, two conserved disulphide bridges, are not glycosylated, and have 40-60% primary structure identity. These boar polypeptides are multifunctional proteins, which possess heparin-, serine-protease-inhibitor- and/or zona-pellucida-glycoprotein-binding capability and have, therefore, been implicated in sperm capacitation and sperm-oocyte attachment. AQN-2 (18-20 kDa), however, is unique among boar spermadhesins in that it is the only member of the family which is known to be glycosylated and which possesses weak zona-pellucida-binding but not seminal-plasma-inhibitor-binding ability. In this study we report the structural and functional characterization of the two glycoproteins contained in the AQN-2 fraction. One component is identical with PSP-I, a major porcine seminal plasma protein whose function has not yet been identified, while the second protein is a glycosylated isoform of AQN-3. Here we show that the inability of the glycosylated boar spermadhesins to bind seminal-plasma protease inhibitors as well as the weak binding of glycosylated AQN-3 to zona pellucida glycoproteins is due to the presence of the oligosacharide chain on a conserved asparagine residue. This indicates that modification of a spermadhesin polypeptide framework may serve to modulate its ligand-binding capabilities.

Amino Acid Sequence↗

PubMed1993

Characterization of AWN-1 glycosylated isoforms helps define the zona pellucida and serine proteinase inhibitor-binding region on boar spermadhesins.

Spermadhesin AWN-1 (14 kDa) belongs to a recently described family of boar sperm surface-associated proteins. AWN-1 is a multifunctional protein which possesses heparin-, serine proteinase inhibitor-, and zona pellucida glycoprotein-binding capability. Therefore it has been implicated in sperm capacitation and sperm-oocyte attachment. Here, we report the characterization of 22-25 kDa isoforms of AWN-1 isolated by heparin-affinity chromatography, which fail to bind to zona pellucida glycoproteins or serine proteinase inhibitors. Our results show that the structure of the high and low molecular mass AWN-1 forms differ in that the former is N-glycosylated at Asp50 and truncated at the C-terminus. The inability of the glycosylated AWN-1 molecules to bind ligands is due solely to the presence of the oligosaccharide moieties, however. This indicates that glycosylation of AWN-1 may modulate its ligand-binding capabilities. On the other hand, the effect of glycosylation on ligand-binding suggests that both the zona pellucida- and the serine proteinase inhibitor binding domain(s) may be located around the glycosylation point.

Animals↗

PubMed1993

Protein kinase C zeta isoform is critical for mitogenic signal transduction.

The requirement of protein kinase C zeta (zeta PKC) for maturation of X. laevis oocytes in response to insulin, p21ras, and phosphatidylcholine-hydrolyzing phospholipase C has recently been shown. Here we present experimental evidence demonstrating that activation of zeta PKC is not only necessary but also sufficient by itself to activate maturation in oocytes and to produce deregulation of growth control in mouse fibroblasts. Furthermore, by using a dominant kinase-defective mutant of zeta PKC, we confirm that this kinase is required for mitogenic activation in oocytes and fibroblasts. These results permit us to propose zeta PKC as a critical step downstream of p21ras in mitogenic signal transduction.

3T3 Cells↗

PubMed1993

Isolation and biochemical characterization of heparin-binding proteins from boar seminal plasma: a dual role for spermadhesins in fertilization.

Sperm surface-coated heparin-binding proteins originating from secretions of the male sexual accessory glands, are known to play a pivotal role as extrinsic regulatory factors during sperm capacitation in many mammalian species. They interact with glycosaminoglycans present in the female genital tract and enhance the subsequent zona pellucida-induced acrosome reaction. We have isolated heparin-binding proteins from boar seminal plasma by affinity chromatography on heparin-Sepharose and reverse-phase HPLC. N-Terminal sequence analysis of these proteins identified a boar counterpart of the bovine capacitation factors BSP-A1/2 (also called PDC-109) and BSP-A3. Several carbohydrate- and zona pellucida-binding proteins, which belong to the newly described spermadhesin family, were also identified as heparin-binding proteins. Our results imply that, besides other capacitation factors, members of the spermadhesin family may play a dual role in sperm capacitation and fertilization in the pig.

Amino Acid Sequence↗