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

ABSTRACT

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 mosquito’s development.