Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibited peptidoglycan synthesis in Escherichia coli in a concentration-dependent manner. The inhibition of peptidoglycan synthesis appears to be a cause rather than a consequence of growth inhibition as it was observed soon after the addition of the antibiotic even in E. coli cells whose growth was totally inhibited by chloramphenicol.     

Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles.

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)+ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)+ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Calcium-binding protein in bull seminal vesicle secretion and seminal plasma.

A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.     

Molecular cloning and expression of a synthetic DNA coding for the antimicrobial protein of bull seminal plasma

A DNA carrying the coding sequence for the antimicrobial protein from bull seminal plasma (SAP) was obtained by enzymic ligation of six synthetic oligonucleotides. The 162 bp synthetic DNA fragment was cloned into the C-terminal part of the lacZ-gene employing the vector pUR289. Expression in E. coli in the presence of the inducer isopropylthiogalactoside (IPTG) led to the formation of a fusion protein, which was shown by immuno-blotting to contain immuno-reactive antimicrobial protein. Approximately 90 min after induction, the cells stopped growing and the culture was found to contain no viable cells 3 h after induction. We conclude from this observation that the beta-galactosidase-antimicrobial protein fusion product was toxic for the E. coli cell and that the SAP-residue attached to beta-galactosidase was responsible for the cytotoxicity.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Purification and spectral characterization of seminalplasmin, an antimicrobial protein from bull semen

A new method for the purification of seminalplasmin, an antimicrobial protein from bull semen, was developed. The last step of the procedure involved preparative high performance liquid chromatography on a reversed phase column. Highly purified seminalplasmin was characterized by CD, absorption, fluorescence spectroscopy, double immunodiffusion and biological activity. Analytical ultracentrifugation revealed a molecular mass of 6300 Da. Amino-acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Induction of autolysis in starved Escherichia coli cells by seminalplasmin, an antimicrobial protein from bovine seminal plasma

Abstract A pair of relA ⁺ and relA E. coli strains, otherwise isogenic, were studied with regard to the susceptibility of starved cells to lysis induced by the natural peptide seminalplasmin. Starved relA cells were more sensitive to seminalplasmin-induced lysis when compared to starved relA ⁺ cells. Nevertheless, pronounced lysis of starved relA ⁺ cells was observed with increase in the concentration of seminalplasmin. In conctrast, ampicillin could not lyse starved relA ⁺ cells even at very high concentrations. Further, seminalplasmin could cause loss of viability and degradation of peptidoglycan in starved relA ⁺ cells. These observations suggest that, unlike many other antibiotics, seminalplasmin can induce autolysis under the conditions of a stringent response.     

Mass spectrometry study of ecto-5'-nucleotidase from bull seminal plasma.

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.     

The major protein of bull seminal plasma: Biosynthesis and biological function

We isolated the major protein of apparent M_r of 15,000–16,000 from seminal plasma as well as from seminal veiscle 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 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 from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent M_r of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Eschet al., Biochem. Biophys. Res. Comm. 113:861–867, 1983).The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP.Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a phyisological process presumably related to the onset of sperm motility.     

Testicular proteins, nucleic acids and their synthesis following gamma irradiation

The effects of two doses (250 and 1000 rads) of local gamma irradiation on testes of adult rats are reported after 1, 2, 4 and 16 weeks. There was a significant increase in DNA content per gm testes at 1 week; a gradual decrease at 2 and 4 week intervals was followed by a trend towards recovery at 16 weeks post-irradiation. The rate of synthesis of both DNA and RNA as studied by the incorporation of (3H)-thymidine and (3H)-uridine, showed similar results. Total protein content per gm testis declined with both doses and at all post-irradiation intervals. Histological observation showed loss of spermatogenic cells suggestive of DNA loss.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

Effects of dithiothreitol, dithioerythritol and chelating agents on 5'-nucleotidase from bull seminal plasma

5'-Nucleotidase from bull seminal plasma is inhibited by dithiothreitol and dithioerythritol. These reactives proved to dissociate the dimeric glycoprotein 5'-nucleotidase of Mr 160 000 into two subunits of apparent Mr 80 000, indicating that the subunits are held together by interchain disulfide bridges. HPLC determinations of cysteic acid and carboxymethylcysteine protein derivatives resulted in 50 +/- 3 half-cystine plus cysteine residues, while 1.9 +/- 0.4 free cysteine residues were estimated by HPLC analysis. The enzyme is inhibited by EDTA and EGTA, and the inhibition appears to be of the non-competitive type for both the chelating agents. Experiments for the enzyme activity recovery by MgCl2 and CaCl2 additions, after the EDTA and EGTA treatments in the presence of 8 M urea, are reported.     

Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-Kilodaltons), and their quantification in seminal plasma and sperm

Three proteins, BSP-A1/-A2, BSP-A3, and BSP-30 kilodaltons (collectively called BSP proteins), represent the major proteins of bovine seminal plasma (BSP). At ejaculation, these proteins bind to the sperm surface and induce molecular changes in the plasma membrane that are deemed to be essential for sperm capacitation. The present study was carried out to develop specific radioimmunoassays (RIAs) for the quantification of each of the BSP proteins in BSP and sperm. RIAs were developed using polyclonal antibodies raised in rabbits against each BSP protein. The purified and iodinated BSP proteins were used as standard and tracer, respectively. The RIAs that were developed were shown to be specific for each protein and the cross-reactivity toward various antigens was negligible (<2%). The average sensitivity limit was 5 ng/ml of sample for BSP-A1/-A2 and BSP-A3, and 40 ng/ml of sample for BSP-30-kDa. The concentration of BSP proteins was determined by analyzing the RIA data with spline function. BSP proteins represented 40% to 57% of seminal plasma total protein (25% to 47% of BSP-A1/-A2, 3% to 5% of BSP-A3, and 3% to 7% of BSP-30 kDa) and 4% to 6% of sperm total protein (2.5% to 4% of BSP-A1/-A2, 0.4% to 0.9% of BSP-A3, and 0.5% to 1% of BSP-30-kDa). We also determined the concentration of BSP proteins that were sperm-bound after semen cryopreservation in Tris-egg yolk-glycerol extender. A significant decrease (70%-80%) in sperm-bound BSP proteins was noted after cryopreservation. The availability of reliable RIA procedures should aid in the further understanding of the role of BSP proteins in sperm function as well as their effect on sperm cryopreservation.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.