MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats

Summary The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with -elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

Proteolytic activity among various oral Treponema species and cloning of a prtP-like gene of Treponema socranskii subsp. socranskii

The proteolytic activity of 11 treponemal strains representing different phylogenetic groups was investigated by SDS-polyacrylamide gel electrophoresis with copolymerised casein, gelatin and fibrinogen as substrates. The activity was specified to be trypsin- and chymotrypsin-like by the cleavage of synthetic substrates BAPNA and SAAPFNA, respectively. Nine strains degrade casein and the synthetic substrate BAPNA. Chymotrypsin-like activity specifically inhibited by phenylmethylsulfonyl fluoride was found in four treponemes. Southern blot analysis using a Treponema socranskii subsp. socranskii-specific prtP probe confirmed the presence of prtP homologous genes in these four strains. The internal fragments of the chymotrypsin-like protease genes were cloned and sequenced after PCR amplification. Here we report the cloning of the complete prtP-like gene of T. socranskii subsp. socranskii, an organism shown to possess epidemiologic relevance in periodontitis.     

CRYSTALLINE TRYPSIN : V. KINETICS OF THE DIGESTION OF PROTEINS WITH CRUDE AND CRYSTALLINE TRYPSIN

The rate of digestion, as determined by the increase in non-protein nitrogen or formol titration, of casein, gelatin, and hemoglobin with crystalline trypsin preparations increases nearly in proportion to the concentration of protein, but with crude pancreatic extract the rate of digestion becomes independent of the protein concentration in concentrations of more than 2.5 per cent. With both enzymes the rate of digestion of mixtures of 5 per cent casein and gelatin is greater than would be expected from the point of view of a compound between enzyme and substrate. The rate of digestion of 5 per cent casein in the presence of 5 per cent gelatin is exactly the same as that of 5 per cent casein alone. This result is obtained with both enzymes. The digestion of casein with crude trypsin follows the course of a monomolecular reaction quite closely while with purified trypsin the velocity constant decreases as the reaction proceeds. In the case of hemoglobin the monomolecular velocity constant decreases with both purified and crude enzyme. When the reaction is followed by changes in the viscosity of the solution the abnormal effect of changing substrate concentration disappears and the reaction is in fair agreement with the monomolecular equation. The results as a whole indicate that the abnormalities of the reaction are due to the occurrence of several consecutive reactions rather than to the formation of a substrate enzyme compound.     

The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations.

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.     

Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening

Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.     

A gelatin-specific protease from hamster lung-derived cell cultures

Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited by p-chloromecuribenzoate, N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase.     

Involvement of classical bipartite/karyopherin nuclear import pathway components in acrosomal trafficking and assembly during bovine and murid spermiogenesis

This study arose from our finding that SubH2Bv, a histone H2B variant residing in the subacrosomal compartment of mammalian spermatozoa, contains a bipartite nuclear localization signal (bNLS) but in spite of this did not enter the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, which were targeted to the nuclear surface to form the acrosome. On this basis we proposed that SubH2Bv targets proacrosomic/acrosomic vesicles from the Golgi apparatus to the nuclear envelope by utilizing the classical bipartite/karyopherin alpha (KPNA) nuclear import pathway. To test the protein's nuclear targeting ability, SubH2Bv, with and without targeted mutations of the basic residues of bNLS, as well as bNLS alone, were transfected into mammalian cells as GFP-fusion proteins. Only the intact bNLS conferred nuclear entry. Subsequently, we showed that a KPNA, most likely KPNA6, occupies the same sperm head compartment and follows the same pattern of acrosomal association during spermiogenesis as SubH2Bv. Sperm head fractionation combined with Western blotting located this KPNA to the subacrosomal layer of the perinuclear theca, while immunocytochemistry of testicular sections showed that it associates with the surface of proacrosomic/acrosomic vesicles during acrosomal biogenesis. The identical sperm-localization and testicular-expression patterns between KPNA and SubH2Bv suggested a potential binding interaction between these proteins. This was supported by recombinant SubH2Bv affinity pull-down assays on germ cell extracts. The results of this study provide a compelling argument that these two nuclear homing proteins work in concert to direct the acrosomic vesicle to the nucleus. Their final residence in the subacrosomal layer of the perinuclear theca of spermatozoa indicates a role for SubH2Bv and KPNA in acrosomal-nuclear docking.     

Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.     

The Morphology and Physiology of the Acrosome Reaction in the Sperm of the Decapod, Sicyonia ingentis.

Sperm of the shrimp, Sicyonia ingentis , undergo a biphasic acrosome reaction consisting of acrosomal exocytosis and acrosomal filament formation. These events are temporally separated by 10–20 min, in vivo. Using egg water preparations the complete reaction can be induced, in vitro, albeit the temporal separation of the two phases is lengthened. External Ca⁺⁺ is required for the exocytotic phase, while a cytoplasmic acidification and K⁺ efflux are associated with polymerization of the acrosomal filament.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Proteolytic activity of guinea pig spermatozoa after induction of the acrosomal reaction in vitro

After inducing the acrosomal reaction in guinea pig spermatozoa in vitro, the sperm were tested for proteolytic activity by applying them to membranes of fixed gelatin. One to 5% of them showed slight evidence of proteolytic activity, while the rest were completely negative. Sperm that had retained their acrosomes throughout the incubation period displayed intense proteolytic activity. These results suggest that proteinases may be lost from spermatozoa as a result of the acrosomal reaction.     

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.     

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.     

Development of the bovine acrosome

Summary In the present study the development of the bovine acrosome was investigated using conventional electron-microscopical techniques as well as the phosphotungstic-acid (PTA) technique (Rambourg 1967) including enzymatic digestion experiments. As in other species and in accordance with previous light-microscopical studies (Clermont and Leblond 1955) four phases of acrosomal differentiation can be discerned: the Golgi-phase, cap-phase, acrosome-phase, and maturation-phase.In the bull no internal pattern of the acrosomal content can be observed, either with conventional uranyl acetate-lead citrate staining or with the PTA-techniques. Our results support the observation in other species (Fawcett et al. 1971) that no intrinsic polymerization or crystallization process of the acrosomal content is responsible for acrosomal shaping. Some of our results suggest the influence of external forces on acrosomal development in the bull. During the cap-phase and the acrosome-phase accumulations of smooth endoplasmic reticulum and a layer of fine filaments can be observed in the Sertoli-cell cytoplasm, immediately adjacent to the developing acrosome. A temporary influence of these structures on acrosomal development seems possible. The PTA-positive staining of the developing bovine acrosome is probably due to the presence of acrosomal glycoproteins; however, our results do not exclude the possibility that molecules other than glycoproteins contribute to the positive PTA-staining of the developing acrosome.Supported by a grant from the Deutsche Forschungsgemeinschaft     

The stabilizing effect of insect hemolymph on a granulosis virus held in darkness as dry films of the intact capsules

The stabilizing effects of either insect hemolymph, gelatin, or glucose on a granulosis virus of Pieris brassicae held in darkness, as dry films of the intact capsules, have been investigated. The hemolymph solids exert a significant stabilizing action; gelatin is significantly less effective; and glucose almost ineffective. The protective action of hemolymph, which occurs in films deposited on glass and leaf surfaces, is probably due to the proteins present since viruses are known to be stabilized by certain types of foreign proteins. This explanation would also account for the high level of stability of insect viruses in dried films of crude preparations held in darkness. (In daylight there is also a screening action which excludes some UV radiation.)     

A gelatin-specific protease from hamster lung-derived cell cultures

Summary Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited byp-chloromecuribenzoate,N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase. This research was supported by Program Project Grant HL-19717 from the National Heart, Lung, and Blood Institute, Grant AG 000-38-02 from the National Institute of Aging, and National Institute of Health Grant 5T32HL07035.     

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.     

A spectrophotometric method to quantify linear DNA

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.