Ricardo J.S. Torquato

A double headed serine proteinase inhibitor — human plasma kallikrein and elastase inhibitor — from Boophilus microplus larvae

Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae (BmTI) were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris-HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins (BmTI-B and BmTI-A, respectively). Both purified proteins inhibited trypsin with Ki of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase (Ki 1.4 nM) and plasma kallikrein (Ki 120 nM). BmTI-A did not change prothrombin time (PT) and thrombin time (TT), but it increased activated partial thromboplastin time (APTT) was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor (BPTI)-Kunitz type inhibitor family. These inhibitors (by their properties) play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable.

Structural Study of Binding of Flagellin by Toll-Like Receptor 5

In order to predict the binding regions within the complex formed by Toll-like receptor 5 (TLR-5) and flagellin, a complementary hydropathy between the two proteins was sought. A region common to the flagellins of Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Listeria monocytogenes was shown to be hydropathically complementary to the 552-to-561 fragment of TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profile of this region is shared with flagellins of 377 bacterial species out of a total of 723 publicly available sequences. A conformational analysis of the predicted binding site of TLR-5, whose structure is still unknown, was carried out with a methodology already applied to similar problems. To sample the conformations available to the peptide chain, a plot of the number of conformations per unit energy interval (density of states) versus energy was built. Following a theoretical argument, conformations belonging to maxima in this plot were selected. The most stable structure obtained in this search, an alpha-helical conformation, was shown to form the electrostatic interactions Glu552-Gln89, Asp555-Arg92, and Arg558-Glu93 with the predicted binding site of the flagellin of S. enterica serovar Typhimurium, formed by the 88-to-97 chain fragment (LQRVRELAVQ), which is likewise alpha helical.

Baccharis dracunculifolia, the main source of green propolis, exhibits potent antioxidant activity and prevents oxidative mitochondrial damage

Baccharis dracunculifolia DC (Asteraceae) is the main botanical source used by honeybees to produce Brazilian green propolis whose hepatoprotective properties have been already described. In this work we investigated the protective effects of the glycolic extract of B. dracunculifolia (GEBd) against oxidative stress in isolated rat liver mitochondria (RLM). The GEBd was prepared by fractionated percolation using propylene glycol as solvent. The total phenols and flavonoids, which are substances with recognized antioxidant action, were quantified in GEBd and the phytochemical analysis was carried out by HPLC. GEBd exhibited significant scavenger activity towards DPPH radicals and superoxide anions in a concentration-dependent manner, and also a Fe2+ chelating activity. GEBd decreased the basal H2O2 generation and the Fe2+- or t-BuOOH-induced ROS production in isolated mitochondria. Lipid oxidation of mitochondrial membranes, protein thiol groups and GSH oxidation were also prevented by GEBd. This shows that B. dracunculifolia exhibit potent antioxidant activity protecting liver mitochondria against oxidative damage and such action probably contribute to the antioxidant and hepatoprotective effects of green propolis.

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech‐derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2×104 mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1–P4′ positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI‐2T (K8R, I9V, S10, K11W, P12A), LDTI‐5T (K8R, I9V, S10, K11S, P12L) and LDTI‐10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI‐2T and ‐5T, prolong the blood clotting time, inhibit thrombin (K i 302 nM and 28 nM) and trypsin (K i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI‐10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1′ (I9V). The data indicate further that LDTI‐5T might be a model candidate for generation of active‐site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme‐inhibitor interactions.

Rhipicephalus sanguineus trypsin inhibitors present in the tick larvae: isolation, characterization, and partial primary structure determination

Blood sucking animals are a rich source of proteinase inhibitors mainly those that interfere in their host hemostatic systems. The tick Rhipicephalus sanguineus is an ectoparasite of dogs and other animals. The aims of this work were the purification and characterization of serine proteinase inhibitors present in R. sanguineus larvae (RsTI). The inhibitors (RsTI) were isolated by affinity chromatography on trypsin-Sepharose and ion exchange chromatographies in Resource Q and Mono S columns. These RsTIs were separated in around 12 different protein peaks, when they showed molecular masses between 8 and 18 kDa, by SDS-PAGE. Purified RsTIs presented differences in the specificity for different serine proteinases. RsTIQ2 was, better inhibitor than RsTIQ7 and RsTIS5 for neutrophil elastase, plasmin, and HuPK with dissociation constants (K(i)) of 1.3, 3.2, and 22 nM, respectively. Other inhibitors such as RsTIQ7, RsTIS3, and RsTIS5 also affected neutrophil elastase and plasmin with K(i) in the nM range. The RsTIQ2, RsTIQ7, and RsTIS5 amino acid sequence data allowed classifying them as members of the Kunitz-type serine proteinase inhibitor family, even though the RsTI role is still unknown. Our present results showed that serine proteinase inhibitors from R. sanguineus are similar to inhibitors from Boophilus microplus other hard tick species, suggesting a similar role of these inhibitors in hard tick species and also as a potential tool to generate or improve vaccine against different ectoparasites with an unique antigen.

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut.

Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquitos development.

Serine proteinase inhibitors from eggs and larvae of tick Boophilus microplus: purification and biochemical characterization.

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as E1, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCl buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant Ki determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTIs) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The Ki for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.

Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes.

A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). The putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. In this work, we reported the biochemical characterization of BmCI. The recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of 1mgL(-1) of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K(i) (6.2pM). The BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. The cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC(50)=8microM). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. In conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity.

The Kazal-type inhibitors infestins 1 and 4 differ in specificity but are similar in three-dimensional structure.

Blood coagulation is an important process in haemostasis, and disorders of blood coagulation can lead to an increased risk of haemorrhage and thrombosis. Coagulation is highly conserved in mammals and has been comprehensively studied in humans in the investigation of bleeding or thrombotic diseases. Some substances can act as inhibitors of blood coagulation and may affect one or multiple enzymes throughout the process. A specific thrombin inhibitor called infestin has been isolated from the midgut of the haematophagous insect Triatoma infestans. Infestin is a member of the nonclassical Kazal-type serine protease inhibitors and is composed of four domains, all of which have a short central α-helix and a small antiparallel β-sheet. Domains 1 and 4 of infestin (infestins 1 and 4) possess specific inhibitory activities. Infestin 1 inhibits thrombin, while infestin 4 is an inhibitor of factor XIIa, plasmin and factor Xa. Here, the structure determination and structural analysis of infestin 1 complexed with trypsin and of infestin 4 alone are reported. Through molecular modelling and docking, it is suggested that the protein-protein binding site is conserved in the infestin 1-thrombin complex compared with other Kazal-type inhibitors. Infestin 4 is able to bind factor XIIa, and the F9N and N11R mutants selected by phage display were shown to be more selective for factor XIIa in comparison to the wild type.