Biomedical subjects

Libia Sanz

Publications and source records attributed to Libia Sanz.

At least 19 recordsLinked to original sources

PubMed2007

Snake venomics of Bitis gabonica gabonica. Protein family composition, subunit organization of venom toxins, and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2.

The protein composition of the venom of the East African Gaboon viper (Bitis gabonica gabonica) was analyzed using RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting, and CID-MS/MS. In total, 35 proteins of molecular masses in the range of 7-160 kDa and belonging to 12 toxin families were identified. The most abundant proteins were serine proteinases (26.4%), Zn2+-metalloproteinases (22.9%), C-type lectin-like proteins (14.3%), PLA2s (11.4%), and bitiscystatin (9.8%). Other protein classes, that is, bradykinin-potentiating peptides, dimeric disintegrins, Kunitz-type inhibitor, DC-fragments, sv-VEGF, CRISP, and L-amino acid oxidase, comprised between 1.3 and 3.4% of the total venom proteome. Only 11 venom-secreted proteins matched any of the previously reported 22 partial or full-length venom gland transcripts. In addition, venome and transcriptome depart in their relative abundances of different toxin families. The proteomic characterization of purified B. gabonica gabonica proteins run under nonreducing and reducing SDS-PAGE conditions revealed their aggregation state and subunit composition. Multimeric proteins included heterodimeric disintegrins, homodimeric sv-VEGF-A, heterodimeric (alphabeta) and tetrameric (alphabeta)4 C-type lectins, and multimeric PIII Zn2+-metalloproteinases. Determination of the complete primary structure and subunit composition of the two major dimeric disintegrins, bitisgabonin-1 and bitisgabonin-2, showed that each comprised a distinct RGD- and MLD-bearing subunit and a common, N-terminal-blocked, RGD-containing subunit identical to the disintegrin domain of the PII Zn2+-metalloproteinase 4. Cell adhesion inhibition assays showed that bitisgabonin-1 (RGD-RGD) is a potent inhibitor of integrin alpha5beta1, whereas bitisgabonin-2 (MLD-RGD) is a better antagonist of integrins alpha4beta1 and alpha9beta1.

Amino Acid Sequence↗

PubMed2006

Loss of introns along the evolutionary diversification pathway of snake venom disintegrins evidenced by sequence analysis of genomic DNA from Macrovipera lebetina transmediterranea and Echis ocellatus.

Analysis of cDNAs from Macrovipera lebetina transmediterranea (Mlt) and Echis ocellatus (Eo) venom gland libraries encoding disintegrins argued strongly for a common ancestry of the messengers of short disintegrins and those for precursors of dimeric disintegrin chains. We now report the sequence analysis of disintegrin-coding genes from these two vipers. Genomic DNAs for dimeric disintegrin subunits Ml_G1 and Ml_G2 (Mlt) and Eo_D3 (Eo) contain single 1-kb introns exhibiting the 5'-GTAAG (donor)/3'-AG (acceptor) consensus intron splicing signature. On the other hand, the short RTS-disintegrins Ml_G3 (Mlt) and Eo_RTS (Eo) and the short RGD-disintegrin ocellatusin (Eo) are transcribed from intronless genomic DNA sequences, indicating that the evolutionary pathway leading to the emergence of short disintegrins involved the removal of all intronic sequences. The insertion position of the intron within Ml_G1, Ml_G2, and Eo_D3 is conserved in the genes for vertebrate ADAM (A disintegrin and metalloproteinase) protein disintegrin-like domains and within the gene for the medium-size snake disintegrins halystatins 2 and 3. However, a comparative analysis of currently available disintegrin(-like) genes outlines the view that a minimization of both the gene organization and the protein structure underlies the evolution of the snake venom disintegrin family.

Amino Acid Sequence↗

PubMed2006

YPI1 and SDS22 proteins regulate the nuclear localization and function of yeast type 1 phosphatase Glc7.

We have recently characterized Ypi1 as an inhibitory subunit of yeast Glc7 PP1 protein phosphatase. In this work we demonstrate that Ypi1 forms a complex with Glc7 and Sds22, another Glc7 regulatory subunit that targets the phosphatase to substrates involved in cell cycle control. Interestingly, the combination of equimolar amounts of Ypi1 and Sds22 leads to an almost full inhibition of Glc7 activity. Because YPI1 is an essential gene, we have constructed conditional mutants that demonstrate that depletion of Ypi1 leads to alteration of nuclear localization of Glc7 and cell growth arrest in mid-mitosis with aberrant mitotic spindle. These phenotypes mimic those produced upon inactivation of Sds22. The fact that progressive depletion of either Ypi1 or Sds22 resulted in similar physiological phenotypes and that both proteins inhibit the phosphatase activity of Glc7 strongly suggest a common role of these two proteins in regulating Glc7 nuclear localization and function.

Cell Nucleus↗

PubMed2006

Zinc ions induce the unfolding and self-association of boar spermadhesin PSP-I, a protein with a single CUB domain architecture, and promote its binding to heparin.

Spermadhesins are a family of seminal plasma proteins composed of a single CUB domain, which appear to be involved in various aspects of the fertilization process in pigs. PSP-I and PSP-II, the most abundant porcine spermadhesins, occur in seminal plasma as noncovalent heterodimers devoid of heparin-binding capability. Of note is the stability of this dimer, which is significantly affected by physiologically relevant conditions such as Zn2+ ions. Here, we show that PSP-I and PSP-II when separated appear to conserve the overall fold of the CUB domain observed in the crystal structure of the PSP-I/PSP-II heterodimer, as concluded from gel filtration, analytical ultracentrifugation, differential scanning calorimetry, and circular dichroism analyses. However, Zn2+ concentrations in the range of those found in boar seminal plasma induce the unfolding and self-association of PSP-I, apparently as a consequence of the exposure of hydrophobic core residues, whereas they have no effect on PSP-II. Remarkably, Zn2+-denatured and self-associated (but not structured monomeric) PSP-I is retained on a heparin column, resembling the behavior of free PSP-I and homologous spermadhesins of the heparin-binding fraction of boar seminal plasma, which also exhibit different aggregation states. Thus, the modulation of the structural organization and heparin-binding ability of PSP-I by Zn2+ might be a physiological phenomenon in seminal plasma.

Animals↗

PubMed2006

Molecular cloning of Echis ocellatus disintegrins reveals non-venom-secreted proteins and a pathway for the evolution of ocellatusin.

We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations.

Amino Acid Sequence↗

PubMed2006

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa.

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.

Animals↗

PubMed2006

Molecular cloning of disintegrin-like transcript BA-5A from a Bitis arietans venom gland cDNA library: a putative intermediate in the evolution of the long-chain disintegrin bitistatin.

We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis arietans, that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn(2+)-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA(2) (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis arietans. The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.

Amino Acid Sequence↗

PubMed2006

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system.

We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.

Amino Acid Sequence↗

PubMed2006

Conformational diversity of the Goodpasture antigen, the noncollagenous-1 domain of the alpha3 chain of collagen IV.

The noncollagenous-1 domain of the alpha3 chain of collagen IV networks of basement membranes is the target of an antibody-mediated inflammatory response in Goodpasture autoimmune disease. This domain when excised from basement membranes by bacterial collagenase digestion exists in two molecular forms, M(H) and M(L), that differ in cleavage site and mobility in SDS-PAGE. In the present study, M(H) and M(L) were shown to also differ with respect to epitope exposure, susceptibility to endoprotease digestion, and redox states of specific cystene residues, as determined by MS. Moreover, M(H) and M(L) assemble to form different quaternary structures, critically influencing pathogenic epitope(s) exposure and autoantibody binding. Collectively, our findings reveal that M(H) and M(L) are conformational isomers stabilized by a distinct disulfide bond connectivity, and coexist in basement membranes. The hitherto unrecognized conformational diversification of the Goodpasture autoantigen may be of relevance in pathogenesis.

Animals↗

PubMed2006

Venom proteomes of closely related Sistrurus rattlesnakes with divergent diets.

The protein composition of the venoms of the three subspecies of Sistrurus catenatus (S. c. catenatus, tergeminus, and edwardsii) and a basal species, Sistrurus miliarius barbouri, were analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting, and CID-MS/MS. The venoms of the four Sistrurus taxa contain proteins from 11 families. The protein family profile and the relative abundance of each protein group in the different venoms are not conserved. Myotoxins and 2-chain PLA2s were detected only in S.c. catenatus and S.c. tergeminus, whereas C-type BPP and Kunitz-type inhibitors were exclusively found in S.c. edwardsii and Sistrurus miliarius barbouri. Among major protein families, taxa were most similar in their metalloproteases (protein similarity coefficient value: 34%) and most divergent in PLA2s (12%), with values for disintegrins and serine proteases lying between these extremes (25 and 20%, respectively). The patterns of venom diversity points to either a gain in complexity in S. catenatus taxa or a loss of venom diversity occurring early on in the evolution of the group involving the lineage connecting S. milarius to the other taxa. The high degree of differentiation in the venom proteome among recently evolved congeneric taxa emphasizes the uniqueness of the venom composition of even closely related species that have different diets. Comparative proteomic analysis of Sistrurus venoms provides a comprehensive catalog of secreted proteins, which may contribute to a deeper understanding of the biology and ecology of these North American snakes and may also serve as a starting point for studying structure-function correlations of individual toxins.

Amino Acid Sequence↗

PubMed2006

cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds.

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Acetylglucosamine↗

PubMed2005

Dissecting the protective effect of the seminal plasma spermadhesin PSP-I/PSP-II on boar sperm functionality.

To dissect the protective activity of PSP-I/PSP-II, the effect of the isolated subunits PSP-I and PSP-II and their affinity-purified tryptic peptide and glycan fractions on the viability, mitochondrial activity, and motility of highly diluted boar spermatozoa was investigated. High dilution exerted a negative effect on control spermatozoa. Incubation of spermatozoa with PSP-I/PSP-II or with its PSP-II subunit had a protective effect on sperm functionality, high mitochondrial membrane potential, and sperm motility. These effects were less pronounced when spermatozoa were incubated with the PSP-I subunit. It was noteworthy that motility was abolished by incubation of spermatozoa with isolated PSP-I. Trypsin-degraded PSP-I/PSP-II, PSP-I, and PSP-II reproduced the effects of the native proteins. Incubating spermatozoa with the glycan-depleted tryptic-peptide fraction of PSP-I/PSP-II for 5 hours preserved a higher percentage of viable spermatozoa than when sperm was incubated for the same time with the native heterodimer, trypsin-digested PSP-I/PSP-II, the glycan fraction or without added proteins. However, sperm motility decreased as the concentration of added peptide fraction increased. On the other hand, spermatozoa incubated with the glycan fraction showed lower values than spermatozoa incubated with the peptide fraction. We concluded that the subunits of the PSP-I/PSP-II heterodimeric spermadhesin exert different activities on sperm functions. The finding that the beneficial effect of the native PSP-I/PSP-II on the functionality of highly diluted boar spermatozoa is largely preserved in its isolated PSP-II subunit and does not appear to require the glycan moiety points to a peptide moiety as a potential sperm function-preserving additive of highly diluted boar spermatozoa.

Animals↗

PubMed2005

cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha1beta1 integrin.

Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional (1)H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.

Amino Acid Sequence↗

PubMed2005

Snake venom disintegrins: evolution of structure and function.

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Amino Acid Motifs↗

PubMed2005

Boar spermatozoa in the oviduct.

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boar's large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 2-3 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa--usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus--are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization.

Animals↗

PubMed2005

Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina.

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.

Amino Acid Sequence↗

PubMed2005

Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction.

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.

Animals↗

PubMed2005

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH.

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.

Acids↗