Laboratório de Biologia Molecular e Doenças Endêmicas - Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.
Laboratório de Pesquisa em Leishmaniose - Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Pav. 26, Sala 509, Rio de Janeiro, Brazil.
Parasit Vectors. 2015 May 29;8:296. doi: 10.1186/s13071-015-0908-0.
Anopheles aquasalis is a dipteran of the family Culicidae that is widely distributed in the coastal regions of South and Central America. This species acts as a vector of Plasmodium vivax, an important etiological agent of malaria, which represents a serious public health problem. In mosquitoes, trypsin-like serine proteases are important in blood meal digestion, immune responses and reproductive functions. The study of peptidases expressed in the mosquito midgut is essential to understanding the mechanisms of parasite-host interaction and the physiological process of nutrient digestion.
Our study aimed to identify and characterize the proteolytic activities in the midgut of sugar-fed An. aquasalis females using zymographic analyses (substrate-SDS-PAGE), in-solution assays and mass spectrometry.
Here, we used a zymographic analysis to further biochemically characterize the proteolytic profile of the midgut of sugar-feeding An. aquasalis females. The trypsin peptidases migrated between ~17 and ~76 kDa and displayed higher proteolytic activities between pH 7.5 and 10 and at temperatures between 37 °C and 50 °C. Four putative trypsin-like serine peptidases were identified using mass spectrometry and data mining. The molecular masses of these peptidases were similar to those observed using zymography, which suggested that these peptidases could be responsible for some of the observed proteolytic bands.
Taken together, our results contribute to the gene annotation of the unknown genome of this species, to the tissue location of these peptidases, and to the functional prediction of these crucial enzymes, which all impact further studies of this species.
致倦库蚊是双翅目蚊科的一种，广泛分布于南美洲和中美洲的沿海地区。这种物种是间日疟原虫的传播媒介，间日疟原虫是疟疾的一个重要病原体，这是一个严重的公共卫生问题。在蚊子中，胰蛋白酶样丝氨酸蛋白酶在血液消化、免疫反应和生殖功能中起着重要作用。研究蚊子中肠表达的肽酶对于理解寄生虫-宿主相互作用的机制和营养消化的生理过程至关重要。
我们的研究旨在使用凝胶电泳分析（底物-SDS-PAGE）、溶液内测定和质谱法鉴定和表征吸食糖的致倦库蚊雌性中肠的蛋白水解活性。
在这里，我们使用凝胶电泳分析进一步对吸食糖的致倦库蚊雌性中肠的蛋白水解图谱进行了生化特征分析。胰蛋白酶肽酶在17 到76 kDa 之间迁移，并且在 pH 7.5 到 10 之间以及在 37°C 到 50°C 之间显示出更高的蛋白水解活性。使用质谱法和数据挖掘鉴定了四种假定的胰蛋白酶样丝氨酸肽酶。这些肽酶的分子量与凝胶电泳观察到的相似，这表明这些肽酶可能负责观察到的一些蛋白水解带。
总之，我们的结果有助于该物种未知基因组的基因注释、这些肽酶在组织中的定位以及这些关键酶的功能预测，所有这些都对该物种的进一步研究产生了影响。
