RESUMO
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
Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. In this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. In contrast, AaTIΔ (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. In the competition assay, rAaTI, AaTIΔ or C-terminal AaTI acidic tail-thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. In conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTIΔ form also prolongs thrombin time.
Assuntos
Aedes , Antitrombinas/farmacologia , Proteínas de Insetos/farmacologia , Trombina/antagonistas & inibidores , Inibidor da Tripsina Pancreática de Kazal/química , Sequência de Aminoácidos , Animais , Antitrombinas/química , Antitrombinas/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Masculino , Dados de Sequência MolecularRESUMO
Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatoryand immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilatorsand platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegyptishowed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands,carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor).Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. In this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able toprolong prothrombin time, activated partial thromboplastin time and thrombin time. In contrast, AaTID(rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. In the competition assay, rAaTI, AaTID or C-terminal AaTI acidic tailethrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibitionassay of rAaTI showed an uncompetitive inhibition mechanism. In conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTID form also prolongs thrombin time.
Assuntos
Aedes/classificação , Trombina/análogos & derivados , Trombina/antagonistas & inibidores , Glândulas Salivares , Inibidor da Tripsina Pancreática de Kazal , Mucosa IntestinalRESUMO
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.