Specific Serine Proteinase Inhibitors Research Articles

An atypical proprotein convertase in Giardia lamblia differentiation

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

The culture broth ofBacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as glutamyl endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP α-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS–PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A α-chain of fibrinogen first, followed by the B β-chain, and shows no effects on the γ-chain. On fibrin, the enzyme hydrolyzed only the β-chain, leaving the γ-dimer apparently untouched. It was devoid of phospholipase A 2, hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 °C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP α-I is a metalloproteinase and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP α-I activity. Since the BmooMP α-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH 2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity = 21.6 units/mg and 17.5 units/μg, respectively; crude venom = 8.0 units/mg and 9.5 units/μg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala 14-Leu 15 and Tyr 16-Leu 17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 μg) subcutaneously into mice.

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 × 10 4) restricted to the P1–P4 ′ positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor ( K i=0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K i=1.7 nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2–4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.

Purification and Characterization of Alkaline Proteinase from AlkalophilicBacillussp.

Alkaline proteinase was purified from Bacillussp. isolated from soil. The pH optimum was 11.5 at 37°C. Calcium divalent cation was effective in stabilizing the enzyme, especially at higher temperatures. The proteolytic activity was inhibited by the specific serine proteinase inhibitor PMSF (phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, and Hg. The enzyme was stable in the presence of detergents, such as Triton-X100, Tween-80, SDS (sodium dodecyl sulfate), and EDTA (ethylenediaminetetraacetic acid), at pH 11.5 and 37°C for 30 min. The optimum pH was 11.5 at 37°C, and the optimum temperature was 62°C at pH 11.5.

Characterization of Active Components in Food-Grade Proteinase Inhibitors for Surimi Manufacture

Commonly used food-grade inhibitors, beef plasma protein (BPP), egg white, and potato powder, were characterized for their inhibitory activity toward proteinases. BPP and egg white had the highest rate of cysteine and serine proteinase inhibition, respectively. Egg white, containing specific serine proteinase inhibitors, reduced trypsin activity by as much as 99%. Active inhibitory components in the proteinase inhibitors were detected by inhibitory activity staining on sodium dodecyl sulfate (SDS)-substrate gels. Egg white and potato powder contained more serine proteinase inhibitor bands than cysteine proteinase inhibitor bands. No specific cysteine proteinase inhibitory component was found in BPP. Serum albumin was detected on both papain and trypsin inhibitory activity-stained gels of BPP but was not inhibitory to the proteinases. A high molecular weight protein band (HMP) of BPP was also detected on both inhibitory activity-stained gels, but the protein was postulated to be polymerized plasma components resistant to proteolysis. Keywords: Activity staining; proteinase inhibitor; surimi

Biotinylated aprotinin: a versatile probe for the detection of serine proteinases on western blots

The present study was undertaken to provide a highly sensitive detection system for the identification and characterisation of serine proteinases separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Biotinylated aprotinin of high specific activity (88–92% active) was prepared (i) by reaction of aprotinin directly with N-hydroxy sulfosuccinimidyl-6-(biotinamido) hexanoate, and (ii) by reaction of aprotinin-trypsin complex with N-hydroxy succinimidobiotin. Both biotinylated aprotinin samples were suitable as probes for the detection of the serine proteinases, neutrophil elastase and cathepsin G, pancreatic trypsin and chymotrypsin and plasmin on nitrocellulose blots. Specific irreversible chloromethyl ketone proteinase inhibitors used in combination with this detection system enabled respective proteinases to be selectively inactivated and thus positively identified. The biotinylated aprotinin detection system was highly sensitive and could detect as little as 0.2 ng (8.5 fmol) of active proteinase (trypsin). In summary, a method has been developed for the sensitive detection of serine proteinases separated by SDS-PAGE. The method is more sensitive and convenient to perform than conventional zymography and significantly, when used in conjunction with specific serine proteinase inhibitors or specific antibodies can yield appreciable information on the identity of the respective serine proteinases being examined. Furthermore the molecular mass of the serine proteinase may be reliably obtained by this method. This method should find application in identifying the role that serine proteinases play in the etiopathogenesis of connective tissue disorders.

Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2

The molecular masses of purified extracellular serine proteinase of a number of Lactococcus lactis strains vary significantly, and these molecular mass values do not correspond to the values estimated on the basis of genetic data. The discrepancies can only partially be explained by N-terminal processing during maturation of the precursor enzyme and by C-terminal cleaving during the release from the cell envelope. With a monoclonal antibody that binds in the active site region of the L. lactis proteinase, the processing of the released proteinase was followed. At 30 degrees C the proteinase was degraded with a concomitant loss of beta-casein hydrolytic activity. In the presence of CaCl(2), proteinase degradation was inhibited, and new degradation products were detected. The specific serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate also inhibited proteinase degradation. Two major high-molecular-mass proteinase fragments (165 and 90 kDa) were found to have the same N-terminal amino acid sequence as the mature proteinase, i.e., [Asp-1-Ala-2-Lys-3-Ala-4-Asn-5-Ser-6, indicating that both fragments were formed by cleavage at the C terminus. The N terminus of a proteinase fragment with low molecular mass (58 kDa) started with Gln-215. In this fragment part of the active site region was eliminated, suggesting that it is proteolytically inactive. Unlike larger fragments, this 58-kDa fragment remained intact after prolonged incubations. These results indicate that autoproteolysis of the L. lactis subsp. cremoris Wg2 proteinase ultimately leads to inactivation of the proteinase by deletion of the active site region.

Antithrombin activity of a peptide corresponding to residues 54–75 of heparin cofactor II

Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.

Ingensin, a fatty acid-activated serine proteinase from rat liver cytosol

The enzyme responsible for the succinylleucyllvalytyrosine methylcoumarylamide- (SLLVT-) degrading activity was purified from the postmitochondrial supernatant of rat liver (Yamamoto, T., Nojima, M., Ishiura, S. and Sugita, H. (1986) Biochim. Biophys. Acta 882, 297–304). The enzyme, named ingensin, was activated by saturated fatty acids, especially myristic acid, as well as by unsaturated linoleic acid and arachidonic acid. Although 2-mercaptoethanol activated ingensin 2-fold and p-chloromercuribenzoate and HgCl 2 completely inhibited its peptide-hydrolyzing activity, the enzyme is activated by the addition of a thiol-blocking reagent, monoiodoacetic acid. Ingensin was also inhibited by a specific serine proteinase inhibitor, diisopropyl fluorophosphate, but not by a specific cysteine proteinase inhibitor, E-64-c. These results suggest that the enzyme is a serine proteinase with an active thiol group(s) near the active site. We have found that the addition of glycerol and nordihydroguaiaretic acid lowered the extent of its activation by fatty acids as well as its intrinsic peptide-hydrolyzing activity.

Apparent intrinsic prothrombinase activity of human factor X zymogen: Identification with factor VIII inhibitor by passing activity (FEIBA)

Steady state kinetic studies have provided evidence for intrinsic prothrombinase activity of human factor X zymogen in a chromogenic assay system. Using a prothrombin substrate, kinetic parameters have been obtained for the action of factors X and Xa. The K m for prothrombin is of a different order of magnitude for the zymogen as compared with the active enzyme. Using a kinetic approach, we have obtained evidence for the binding of factor X zymogen to cofactors essential for the coagulant activity of factor Xa. Zymogen enzymatic activity is not inhibited by a specific serine proteinase inhibitor, (p-amidino-phenyl)methanesulfonyl flouride (p-APMSF), a potent inhibitor of factor Xa. The apparent slow rate of zymogen inhibition by antithrombin III (AT III) as compared with the active enzyme suggests a different kind of zymogen-antithrombin interaction. Blood clotting studies paralleled the kinetic data. Factor X zymogen evidences factor VIII inhibitor bypassing activity (FEIBA) in an in vitro direct clotting system employing factor VIII deficient inhibitor plasma as substrate in both activated or nonactivated partial thromboplastin assay. Most significantly, zymogen coagulant is refractory to inhibition by p-APMSF or AT III. We conclude that a system consisting of factor X zymogen-phospholipid-factor Va can physiologically initiate blood clotting in the presence of inhibitors and may have a major role in the bypass mechanism of therapeutic prothrombin complex concentrate (PCC).