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.     

Molecular Cloning of Testican-2

We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.     

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.     

A [Ca2+ + Mg2+]-ATPase and active Ca2+ transport in the plasma membranes isolated from ram sperm flagella

Plasma membranes isolated from the flagella of ram ejaculated sperm were found to contain a [Ca²⁺ + Mg²⁺]-ATPase. Freeze-fracture electron microscopy showed the membranes occur as vesicles. The membrane vesicles actively accumulate Ca²⁺, uptake was reversed by the ionophore A23187 and inhibited by either ruthenium red or La³⁺. The plasma membranes contain two major proteins, designated proteins A and B, with molecular weights of 109,000 and 18,300 daltons, respectively. Protein B is not detected in plasma membranes isolated from ram epididymal sperm. The plasma membrane Ca²⁺ pump may be modulated by protein factors present in seminal plasma.     

A [Ca + Mg]-ATPase and active Ca transport in the plasma membranes isolated from ram sperm flagella

Plasma membranes isolated from the flagella of ram ejaculated sperm were found to contain a [Ca²⁺ + Mg²⁺]-ATPase. Freeze-fracture electron microscopy showed the membranes occur as vesicles. The membrane vesicles actively accumulate Ca²⁺, uptake was reversed by the ionophore A23187 and inhibited by either ruthenium red or La³⁺. The plasma membranes contain two major proteins, designated proteins A and B, with molecular weights of 109,000 and 18,300 daltons, respectively. Protein B is not detected in plasma membranes isolated from ram epididymal sperm. The plasma membrane Ca²⁺ pump may be modulated by protein factors present in seminal plasma.     

Characterization of acid and neutral alpha-mannosidases in bull semen and reproductive organs

Acid and neutral alpha-mannosidase activities were studied in the bull reproductive tissues, isolated spermatozoa, epididymal and seminal vesicle secretion and seminal plasma. The acid enzyme in the seminal plasma mainly derived from the epididymal secretion, while the neutral one was enriched in the sperm cells. The latter activity in the seminal plasma appears to be due to an enzyme released from the cytoplasmic droplets in the epididymis. The acid enzyme had a molecular weight of 220,000-320,000, pI 7.3-6.0 and an optimum at pH 4.0. It was sensitive to swainsonine but was stimulated by Zn2+. The neutral enzyme had a molecular weight of 360,000-460,000, pI 5.4-4.7 and showed double optima at pH 5.5 and 6.0-7.0. It was resistant to swainsonine but was markedly activated by Co2+ or Fe2+. The neutral enzyme was also more sensitive to thermal inactivation than the acid one.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems

Centrifugal counter-current distribution (CCCD) in a dextran, Ficoll, poly(ethylene glycol) two-phase system was used to study the effect of seminal plasma proteins on the partition behaviour of ram spermatozoa exposed to thermal shock. Ram spermatozoa freed from seminal plasma by a ‘swim-up’ procedure were submitted to thermal shock and fractionated by CCCD. Cell viability decreased from 68% to 18% after the treatment, showing a slight displacement of the cells from the right (where a higher enrichment of live cells is found) to the centre of the profile. A change of the distribution profile was shown in the presence of either ram or bull seminal plasma. Bull seminal plasma was able to move the profile to the right, whereas ram seminal plasma increased the proportion of cells with enhanced affinity for the lower dextran-rich phase. Plasma proteins isolated from both seminal plasmas moved the profile to the right. In addition, cell viability rose to 48% after the CCCD run in the presence of ram plasma proteins. This restoring effect was lost when ram plasma proteins were thermally denatured. Bovine serum albumin was not only unable to move the profile to the right but even promoted displacement of the profile to the left. This negative effect was also observed when proteins from bull seminal plasma were in the presence of protein-free ram seminal plasma. However, proteins isolated from ram seminal plasma still restored the profile in the presence of bull seminal plasma freed from proteins. The results presented here strongly suggest that seminal plasma proteins are absorbed by a spermatozoal surface previously exposed to thermic shock. These proteins would exert a highly specific protective effect on ram spermatozoa. In addition, in the ram seminal plasma there must be some factor which avoids this adsorption.     

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.     

金属蛋白质组学研究锌离子匮乏对肺炎链球菌的影响

【目的】研究锌离子缺乏对肺炎链球菌的影响,找到其适应性生长机制。【方法】以肺炎链球菌为模型,利用加锌和不加锌的培养基对细菌进行培养,收集细胞蛋白,采用双向凝胶电泳,结合金属亲和层析和质谱技术鉴定差异表达蛋白,进而通过生物信息学分析蛋白质相互关系,从中找到细菌适应锌离子匮乏条件的关键代谢通路和蛋白。【结果】测定了在限制培养条件下肺炎链球菌的最适生长浓度,建立了锌离子调控蛋白双向凝胶电泳图谱,鉴定到了96个差异表达蛋白斑点,共67个差异蛋白,其中32个表达下调,35个表达上调,锌离子调控蛋白的作用可能主要体现在糖代谢、核酸代谢、氧化还原作用、辅助蛋白质翻译、合成及折叠等方面。建立了锌结合蛋白的差异表达图谱,鉴定到了10个差异表达蛋白斑点,共7个差异蛋白,其中1个表达下调,6个表达上调。锌离子结合蛋白的作用可能主要体现在应对压力、蛋白质折叠和转运、氨基酸代谢等方面。【结论】肺炎链球菌主要通过调控碳水化合物代谢和核酸代谢等多个代谢通路来应对宿主锌金属离子匮乏的环境,从而使自身能够存活并对宿主形成感染。本研究为揭示细菌在宿主环境,特别是金属离子匮乏条件下的适应性生长机制提供理论基础。     

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically

Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.     

Regulation of calcium content in bovine spermatozoa

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.     

Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.     

Mechanism of liquefaction of the human ejaculate. II. Role of collagenase-like peptidase and seminal proteinase.

Collagenase-like peptidase and seminal proteinase were isolated from human testis and human seminal plasma. The effects of both enzymes upon proteins isolated from the human ejaculate were studied. Both enzymes degraded ejaculate proteins. The data suggest that collagenase-like peptidase is responsible for the first, and seminal proteinase for the second, phase of human ejaculate liquefaction in vitro.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.