Tatiane S. Soares

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.

Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes.

Trypsin-like enzymes play an important role in the Aedes aegypti digestive process. The trypsin-like enzymes present in adults were characterized previously, but little is known about trypsins in larvae. In the present work, we identified one of the trypsin enzymes from Ae. aegypti larval midgut using a library of trypsin gene fragments, which was the sequence known as AAEL005607 from the Ae. aegypti genome. Quantitative PCR analysis showed that AAEL005607 was transcribed in all larval instars, but it was not present in adult midgut. In order to confirm transcription data, the trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified and sequenced. Purified trypsin showed identity with the amino-terminal sequence of AAEL005607, AAEL005609 and AAEL005614. These three trypsins have high amino acids identity, and could all be used as a template for the design of inhibitors. In conclusion, for the first time, digestive enzymes of 4th larval instar of Ae. aegypti were purified and characterized. The knowledge of digestive enzymes present in Ae. aegypti larvae may be helpful in the development of a larvicide.

Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: Specificity study using a peptide phage display library

The tick Rhipicephalus (Boophilus) microplus is one of the most important bovine ectoparasites, a disease vector responsible for losses in meat and milk productions. A cysteine protease similar to cathepsin L, named BmCL1, was previously identified in R. microplus gut, suggesting a role of the enzyme in meal digestion. In this work, BmCL1 was successfully expressed in Pichia pastoris system, yielding 54.8 mg/L of culture and its activity was analyzed by synthetic substrates and against a R. microplus cysteine protease inhibitor, Bmcystatin. After rBmCl1 biochemical characterization it was used in a selection of a peptide phage library to determine rBmCL1 substrate preference. Obtained sequenced clones showed that rBmCL1 has preference for Leu or Arg at P(1) position. The preference for Leu at position P(1) and the activation of BmCL1 after a Leu amino acid residue suggest possible self activation.

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response.

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in ticks physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.

RmKK, a tissue kallikrein inhibitor from Rhipicephalus microplus eggs

Rhipicephalus microplus is an important ectoparasite that is responsible for transmission of anaplasmosis and babesiosis to cattle. Tissue kallikrein inhibitors might play an important role in R. microplus eggs. In the present work, we purified and characterized, a tissue kallikrein inhibitor presents in R. microplus eggs (RmKK), a protein which contains two Kunitz domain in tandem. Purified inhibitor was confirmed by amino terminal determination and its dissociation constant (Ki) for bovine trypsin and porcine pancreatic kallikrein were 0.6 nM and 91.5 nM, respectively. Using a cDNA library from R. microplus midgut, we cloned the cDNA fragment encoding mature RmKK and expressed the protein in Pichia pastoris system. Recombinant RmKK was purified by ion exchange chromatography and presented molecular mass of 16.3 kDa by MALDI-TOF analysis. Moreover, RmKK showed a tight binding inhibition for serine proteases as bovine trypsin (Ki=0.2 nM) and porcine pancreatic kallikrein (PPK) (Ki=300 nM). We performed, for the first time, the characterization of a tissue kallikrein inhibitor presents in R. microplus eggs, which the transcript is produced in the adult female gut. BmKK seems to be the strongest PPK inhibitor among all BmTIs present in the eggs and larvae (Andreotti et al., 2001; Sasaki et al., 2004). This data suggests that BmKK may participate in the development of tick egg and larvae phase.

Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae

BackgroundDengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4’ selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1).MethodsThe chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4.ResultsThe chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested.ConclusionsThe strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector.