Proteinases and amylases of larval midgut of Zabrotes subfasciatus reared on cowpea (Vigna unguiculata) seeds

Proteinase and amylase activities in larval midguts of the bruchid beetle Zabrotes subfasciatus (Boh.) (Coleoptera: Bruchidae) reared on cowpea (Vigna unguiculata (L.) Walp.) seeds were investigated. We could detect and isolate a proteolytic activity with a pH optimum of 5.5 (on azo-casein as substrate) which was activated by thiol reagents and inhibited by several compounds reactive against-SH groups. None of the plant protein inhibitors of serine proteinases utilized were effective inhibitors of this activity. This activity has characteristics of a cysteine class proteinase. We could also detect and isolate a proteolytic activity with a pH optimum of 3.5 (on hemoglobin as substrate) which was not influenced by activators or inhibitors of cysteine, serine, or metalloproteinases. This activity was totally inhibited by pepstatin, a specific inhibitor of aspartic proteinases. We conclude that this activity is due to an aspartic class proteinase. We found also that the aspartic class proteolytic activity is higher than the cysteine class proteinase activity in the midguts of Z. subfasciatus. This seems to be contrary to what is found in Callosobruchus maculatus (F.) larvae midguts. An amylolytic activity with the charateristics of an -amylase was also detected and isolated.

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Résumé Les activités protéinase et amylase ont été étudiées sur l'intestin moyen de larves de Zabrotes subfasciatus Boh. (Coléo, Bruchidae), élevées sur graines de Vigna unguiculata Walp. Nous avons pu déceler et isoler une activité protéolytique optimale à pH 5,5 (sur substrat d'azo-caséine) activée par des réactifs thiol et inhibée par plusieurs composés réagissant aux groupements SH. Aucun inhibiteur végétal des sérine-protéases utilisé n'a inibé efficacement cette activité qui présente les caractéristiques des protéines de la famille des cystéines. Nous avons pu déceler et isoler aussi une activité protéolytique optimale au pH 3,5 (sur hémoglobine comme substrat) qui n'était pas modifiée par les activateurs ou les inhibiteurs de cystéine, de sérine ou de métalloprotéinases. Cette activité était totalement inhibée par la pepstatine, inhibiteur spécifique des protéinases aspartiques. Nous en concluons que l'activité est due à une protéinase de la famille aspartique. Nous avons trouvé aussi que l'activité protéolytique de la famille aspartique était supérieure à l'activité protéinase de la famille cystéine dans l'intestin moyen de Z. subfasciatus. Ceci semble l'inverse de ce qui a été observé dans l'intestin moyen des larves de Callosobruchus maculatus F. (C.P. Silva & al, in litt.). Une activité amylolytique ayant les caractéristiques d'une -amylase a aussi été décelée.

     

Activity of digestive proteinases and carbohydrases in the alimentary tract of the fig leaf roller,Choreutis nemorana Hübner (Lepidoptera: Choreutidae)

.The fig leaf roller or Fig-tree Skeletoniser, Choreutis nemorana (Lep.: Choreutidae), is a destructive pest of fig trees found in some fig-growing areas of Iran. The larvae feed on the upper level of leaves, near the main vein. In this study, digestive carbohydrases including α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and proteinases including trypsin, chymotrypsin and elastase were investigated. The results showed that the carbohydrases were present in the alimentary tracts of the pest. Optimum pH for α-glucosidase and β-glucosidase activity was at pH 6.0 and 7.0, respectively. Maximum activity of α-galactosidase and β-galactosidase occurred at pH 6.0. Total proteolitic activity against the substrate azocasein was optimally occurred at pH 10.0. The greatest activity of trypsin, chymotrypsin and elastase was determined at pH 10.0, 11.0 and 11.0, respectively. Zymogram analyses using nitrocellulose membrane revealed two trypsin isoforms in which one of them was completely inhibited by Soybean Kunitz inhibitor and the other was notably inhibited.     

An unusual orientation for Tyr75 in the active site of the aspartic proteinase from Saccharomyces cerevisiae

The structures of the native Saccharomyces cerevisiae proteinase A have been solved by molecular replacement in the monoclinic and trigonal crystal forms and refined at 2.6-2.7A resolution. These structures agree overall with those of other uninhibited aspartic proteinases. However, an unusual orientation for the side chain of Tyr75, a conserved residue on the flexible "flap" that covers the active site and is important for the activity of these enzymes, was found in the trigonal crystals. A similar conformation of Tyr75 occupying the S1 substrate-binding pocket was previously reported only for chymosin (where it was interpreted as representing a "self-inhibited" state of the enzyme), but for no other aspartic proteinases. Since this orientation of Tyr75 has now been seen in the structures of two members of the family of aspartic proteinases, it might indicate that the placement of that residue in the S1 substrate-binding pocket might have some functional significance, analogous to what was seen for self-inhibited structures of serine proteinases.     

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

We present an analysis of the electrostatic properties in the catalytic site of papain (EC 3.4.22.2), an archetype enzyme of the C1 cysteine proteinase family, and we investigate their possible role in the formation, stabilization and regulation of the Cys25((-))...His159((+)) catalytic ion pair. The electrostatic properties were computed using a reassociation method based in multicentered multipolar expansions obtained from ab initio quantum calculations of overlapping protein fragments. Solvent effects were introduced by coupling the use of multicentered multipolar expansions to two continuum boundary element methods to solve the Poisson and the linearized Poisson-Boltzmann equations. The electrostatic profile found in the proton transfer region of papain showed that this enzyme has a well-defined electrostatic environment to favor the formation and stabilization of the catalytic ion pair. The papain catalytic site electrostatic profile can be considered as an electrostatic fingerprint of the papain family with the following characteristics: (i) the presence of a net electric field highly aligned in the (Cys25)-SG-->(His159)-ND1 direction; (ii) the electrostatic profile has a saddle-point character; (iii) it is basically a local environmental effect. Furthermore, our analysis describes a possible regulatory mechanism (the E(SG-->ND1) attenuation effect) controlling the ion pair reactivity and permits to infer the Asp57 acidic residue as the most probable candidate to act as the electrostatic modulator.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat)

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.     

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr = 4572) containing the first 42 residues from the Bβ-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 μg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-l-Arg-pNA by leuc was competitively inhibited by benzamidine (K_i = 1.61 ± 0.25 mM).