Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl‐activated subtilisin‐like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin‐like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55–60°C, 25–30% NaCl and 70–100 mmol l^?1 CaCl₂. Enzymes showed stability at 0–30% NaCl and <20 mmol l^?1 CaCl₂ and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19‐, 34‐ and 44‐kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI‐TOF and LC–MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F‐like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F‐like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.     

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.     

Human and bovine brain cathepsin L and cathepsin H: Purification,physico-chemical properties,and specificity

Cathespin L (EC 3.4.22.15) and cathepsin H (EC 3.4.22.16) have been purified from brain cortex to apparent homogeneity by a simultaneous procedure involving acid extraction of homogenate at pH 4.2, ammonium sulfate fractionation (30–80%), chromatography on pepstatin-Sepharose, CM-Sephadex C-50, DEAE-Sephadex A-50, phenyl- and concanavalin A-Sepharose and isoelectric focusing. Cathepsin L and cathepsin H were assayed in the presence of dithiothreitol and Na₂EDTA (2 mM each) with Z-Phe-Arg-NHMec (pH 5.5) and Lys-NNa (pH 6.5) respectively. Cathepsin L consists of 2 polypeptide chains with M_r 25 000 and 5 000, M_r of cathepsin H is 28 000. Cathepsin L exists in brain tissue in two multiple forms with pI values 5.7 and 5.9, pI of cathepsin H is 6.8. Substrate specificity of these thiol proteinases was tested with proteins (pyridoxyl-hemoglobin, azocasein) and low M_r naphthylamide and methylcoumarylamide substrates: Lys-NNa, Arg-NNa, Dz-Arg-NNa, Z-Arg-Arg-NNaOMe, Z-Phe-Arg-NHMec, Z-Phe, Val-Arg-NHMec, Z-Gly-Gly-Arg-NHMec. Z-Phe-Arg-NHMec is the best substrate for cathepsin L (K_M=5 M, K_cat=21 s^–1), Arg-NNa—for cathepsin H (K_M=0.1 mM, K_cat=1.93 s^–1), being endoaminopeptidase cathepsin H also hydrolyses Bz-Arg-NNa (K_M=0.7 mM, K_cat=1.3 s^–1). Both proteinases are inhibited by traditional inhibitors of cysteine proteinases and E-64, but leupeptin turned to be more effective inhibitor of cathepsin L (K_i=2.4 nM) than of cathepsin H (K_i=9.2 M), the latter enzyme being sensitive to puromycin and benzethonium chloride as well. Z-Phe-Phe-CHN₂ and Z-Phe-Ala-CHN₂ are potent irreversible inhibitors of brain cathepsin L with K_2nd 150 000 and 137 000 M^–1 s^–1 respectively. Properties of the enzymes from human and bovine brain are similar.Special Issue Dedicated to Dr. Abel Lajtha.     

Midgut proteinases in three divergent species of Coleoptera

Cysteine proteinases have been found in some families of Coleoptera and, based on this, these enzymes were supposed to be characteristic of Coleoptera. To test this hypothesis, we studied midgut homogenates of three phylogenetically distant Coleoptera species: Tenebrio molitor (Tenebrionidae) larvae, Pyrearinus termitilluminans (Elateridae) larvae, and Pheropsophus aequinoctialis (Carabidae) adults. T. molitor display two cysteine proteinases (pHo 6.8) resolved in Superose (FPLC) with Mr 31,000 and 51,000. These enzymes are inhibited by E-64 and pHMB, are activated by EDTA + cysteine and hydrolyze benzoyl-DL-arginine-β-naphthylamide. T. molitor enzymes differ from a cysteine proteinase (Mr 64,000 using Superose) present in the wheat meal ingested by the insect. The cysteine proteinases predominate in the anterior two thirds of T. molitor midgut, probably because they are unstable in the higher luminal pH observed in the posterior third of the midgut. P. termitilluminans and P. aequinoctialis do not display cysteine proteinases, although they have trypsins (Mr 15,000, 25,000 and 41,000 for P. termitilluminans; Mr 26,000, 33,000 and 52,000 for P. aequinoctialis) and chymotrypsins (Mr 38,000 and 25,000 for P. aequinoctialis and Mr 15,000 for P. termitilluminans). Our results, together with literature data, suggest that cysteine proteinases occur in the Cucujiformia ancestor, which corresponds to the ancestor of most Coleoptera which ingest seeds rich in serine proteinase inhibitors.     

Effect of chronic ingestion of the cysteine proteinase inhibitor, E-64, on Colorado potato beetle gut proteinases

Chronic ingestion of the highly active, specific cysteine proteinase inhibitor, E-64, has a profound effect on Colorado potato beetle (CPB) larval growth, development and survival, as well as on adult fecundity. However, the number of insects surviving to the adult stage did not decrease below 26% with increasing E-64 concentration above 1.5 g E-64 cm^–2 leaf surface. The development time to the pupal stage was increased from 13 days, when larvae were reared on control leaves, to 21 days at a concentration of 1.5 g E-64 cm^–2 . The most significant effect of dietary E-64 was on adult fecundity, with mated females reared on untreated leaves laying an average 62 ± 5.7 eggs daily in the first 10 days, and those maintained on 0.5 g E-64 cm^–2, laying only 16 ± 2.4 eggs day^–1. Females given 1 g E-64 cm^–2 laid few if any eggs, but started producing egg masses as large as control insects about 5 days after being switched to control leaves. These effects on the insect life cycle were directly related to the degree of inhibition of cysteine proteinase activity in gut extracts. The general proteinase activity in control extracts was 6.5 ± 0.16 units min^–1 mg gut^–1, which decreased to 1.9 ± 0.16 in guts of insects reared on 1 g E-64 cm^–2. The proportion of proteinase activity inhibitable by E-64 decreased from 66% in control guts to 10-15% in guts from larvae reared on 1 g E-64 cm^–2. The aspartate proteinase inhibitor, pepstatin, decreased proteinase activity by 35% in control guts. There was no induction of pepstatin-inhibitable proteinases in response to inhibition by E-64, and no inhibition of gut enzyme activity by soybean trypsin inhibitor from larvae fed any of the E-64 concentrations. This study demonstrates that proteinase levels must be significantly reduced to have a pronounced effect on larval growth and survival, while fecundity of mated females is affected by lower concentrations of inhibitor. It also suggests that the CPB may be a difficult pest to control using a more specific, plant-derived cysteine proteinase inhibitor, such as oryzacystatin.     

Purification and Substrate Specificity of Two Cysteine Proteinases of Giardia lamblia

The proteinase activity present in homogenates of trophozoites of Giardia lamblia , active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of M_r= 95,000 and 35,000 ± 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (M_r= 95,000) and proteinase II (M_r= 35,000) were active against the β-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1–6, 8–18, and 20–30 of the insulin β-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Effects of Proteinase Inhibitor Ingestion on Survival,Learning Abilities and Digestive Proteinases of the Honeybee

The impact on beneficial insects of proteinase inhibitors expressed in pest-resistant transgenic crops needs to be assessed before the release of these plants into the environment. Three proteinase inhibitors, suitable for incorporation into oilseed rape, were tested on worker bees: the chicken egg white cystatin, oryzacystatin I (OCI) and Bowman-Birk soyabean inhibitor (BBI). Ingestion of low doses of the inhibitors did not cause short-term mortality, and a conditioned proboscis extension assay showed that olfactory learning performances were unchanged when the inhibitors were added to the reward. Long-term ingestion of BBI or OCI did not disrupt total digestive proteolytic activity, but ingestion of BBI induced a new proteinase form, suggesting the existence of a mechanism of control of proteinase synthesis in the honeybee.     

Cysteine proteinases of microorganisms and viruses

This review considers properties of secreted cysteine proteinases of protozoa, bacteria, and viruses and presents information on the contemporary taxonomy of cysteine proteinases. Literature data on the structure and physicochemical and enzymatic properties of these enzymes are reviewed. High interest in cysteine proteinases is explained by the discovery of these enzymes mostly in pathogenic organisms. The role of the proteinases in pathogenesis of several severe diseases of human and animals is discussed.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Evaluation of cardosin A as a proteolytic probe in the presence of organic solvents

This investigation showed that cardosin A not only is active in media with organic solvents, cleaving the β-chain of oxidised insulin at three susceptible peptide bonds, but also maintains its specificity in all media tested. Additionally, the presence of organic solvents in the reaction media led to modifications of enzyme selectivity, which enabled the detection of intermediate products. While solvents like ethyl acetate induced a decrease in enzymatic activity, both by reducing the amount of active enzyme and presumably due to an inhibiting effect of ethyl acetate (which might compete with the substrate for the active site of the enzyme), n-hexane caused an increase in the hydrolysis velocity of one peptide bond. In view of the activity and specificity of cardosin A (which shows high preference for hydrophobic residues), it is proposed as a reliable probe for limited proteolysis in the presence of organic solvents. This may become particularly useful for structural characterisation of membrane proteins.