RESUMO
Rhipicephalus microplus is an ectoparasite responsible for transmissions of babesiosis and anaplasmosis causing large losses to livestock production. To survive R. microplus tick produces several active molecules, such as protease inhibitors. This ectoparasite has been described as a rich source of serine protease inhibitors most of them are Kunitz-BPTI members named BmTIs which have no clear function yet. In the present work, we described the expression and functional characterization of rBmTI-A which showed to be similar to the native BmTI-A, a double-headed Kunitz-BPTI inhibitor, capable to inhibit trypsin, human neutrophil elastase (HNE), human plasma kalikrein (HuPK) and human plasmin. rBmTI-A was able to cause a decrease of HUVEC cell viability. Besides, the rBmTI-A showed to be a potent inhibitor of "in vitro" vessel formation. Our results suggested that BmTI-A may participate in the blood acquisition process interfering in the vessel formation during the tick parasite life stage, around 20 days. In conclusion, BmTI-A is a promising molecule to be used in the drug design and development of new method of R. microplus control.
Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Rhipicephalus/genética , Rhipicephalus/metabolismo , Inibidores de Serina Proteinase , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Enzimas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologia , TranscriptomaRESUMO
The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's 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.
Assuntos
Inibidores de Cisteína Proteinase/imunologia , Resistência à Doença/imunologia , Hemócitos/imunologia , Rhipicephalus/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Resistência à Doença/genética , Escherichia coli/imunologia , Escherichia coli/fisiologia , Corpo Adiposo/imunologia , Corpo Adiposo/metabolismo , Expressão Gênica/imunologia , Hemócitos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Dados de Sequência Molecular , Interferência de RNA/imunologia , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhipicephalus/genética , Rhipicephalus/microbiologia , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Óvulo/metabolismo , Rhipicephalus/classificação , Rhipicephalus/metabolismo , Calicreínas Teciduais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ativação Enzimática , Feminino , Dados de Sequência Molecular , Ligação Proteica , Especificidade da Espécie , Distribuição TecidualRESUMO
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.
Assuntos
Aedes/enzimologia , Aedes/genética , Tripsina/genética , Aedes/metabolismo , Sequência de Aminoácidos , Animais , Digestão/fisiologia , Sistema Digestório/enzimologia , Sistema Digestório/metabolismo , Expressão Gênica , Larva/enzimologia , Larva/genética , Larva/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de Proteína , Tripsina/metabolismoRESUMO
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.
Assuntos
Cisteína Proteases/metabolismo , Rhipicephalus/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisteína Proteases/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Rhipicephalus/genética , Especificidade por SubstratoRESUMO
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 mosquito's development.
Assuntos
Anticoagulantes/farmacologia , Trombina/farmacologia , Inibidores da Tripsina/farmacologia , Aedes , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator andantimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. Inthis 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 withinsect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptaseinhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized therecombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (Ki) 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.