Laboratório de Biologia de Tripanosomatídeos, FIOCRUZ, Avenida Brasil, 4365 Manguinhos, Rio de Janeiro, Brazil.
J Insect Physiol. 2012 Jan;58(1):178-87. doi: 10.1016/j.jinsphys.2011.11.008. Epub 2011 Nov 13.
Triatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects.
巴西锥蝽被认为是恰加斯病的主要传播媒介之一，常见于巴西东北部的半干旱地区。这些昆虫利用羧肽酶 B、氨肽酶和不同的组织蛋白酶来消化血液。在本研究中，鉴定并表征了巴西锥蝽中肠的两个编码组织蛋白酶 L 的基因。成熟的巴西锥蝽组织蛋白酶 L 样蛋白水解酶（TBCATL-1、TBCATL-2）与其他昆虫的组织蛋白酶 L 样蛋白水解酶具有高度的同源性，与 Rhodnius prolixus 的相似度最高。两种组织蛋白酶 L 转录本在后中肠区域高度丰富，是血液消化的主要区域。测定未进食的巴西锥蝽整个肠道的 pH 值显示，前中肠区域（胃）呈碱性，后中肠区域（小肠）呈酸性。凝胶内酶谱法显示，在小肠中至少有四种不同的蛋白酶具有活性，半胱氨酸蛋白酶抑制剂 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) 和组织蛋白酶 B 抑制剂 N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) 被用来表征酶活性。E-64 完全抑制半胱氨酸蛋白酶活性，而在用 CA-074 处理的样品中仍可检测到残留的蛋白酶活性。因此，蛋白水解活性至少部分归因于组织蛋白酶 L。使用特异性抗组织蛋白酶 L 抗体的 Western blot 分析证实了组织蛋白酶 L 存在于昆虫小肠的腔中。
