Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Insect Biochem Mol Biol. 2009 Nov;39(11):782-91. doi: 10.1016/j.ibmb.2009.09.003. Epub 2009 Oct 6.
Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.
家蝇幼虫的前肠和中肠内容物中存在一种蛋白水解酶，其最适 pH 值为 3.0-3.5，且具有与组织蛋白酶 D 相似的动力学特性。本研究从家蝇中肠 cDNA 文库中克隆了 3 个编码前组织蛋白酶 D 样蛋白酶（ppCAD1、ppCAD2、ppCAD3）的 cDNA。编码的蛋白质序列包括信号肽、前肽和成熟酶，其中包含了在牛溶酶体组织蛋白酶 D 中发现的所有保守的催化和底物结合残基。然而，ppCAD2 和 ppCAD3 缺乏特征性的脯氨酸环和糖基化位点。对来自某些脊椎动物和家蝇以及埃及伊蚊、黑腹果蝇、赤拟谷盗和家蚕基因组中的组织蛋白酶 D 样酶序列进行比较发现，只有蝇类具有缺乏脯氨酸环的酶（根据 motif：DxPxPx(G/A)P 定义），因此类似于脊椎动物的胃蛋白酶。ppCAD3 应该对应于酶测定中发现的消化组织蛋白酶 D 样蛋白酶（CAD），原因如下：（1）根据 EST 的出现频率，它似乎是表达最丰富的 CAD。（2）CAD3 的 mRNA 仅在前肠和近端中肠中表达。（3）重组原组织蛋白酶 D 样蛋白酶（pCAD3）在自动激活后最适 pH 值为 2.5-3.0，接近家蝇中肠的腔液 pH 值。（4）用抗 pCAD3 血清进行免疫印迹的蛋白质分析显示，仅在前肠和中肠组织及内容物的样本中呈阳性。（5）CAD3 在内分泌泡内和前肠和中肠细胞的微绒毛周围用免疫胶体金进行定位。这些数据支持这样一种观点，即家蝇的消化 CAD 通过与细胞内组织蛋白酶 D 相同的原始基因适应在富含细菌的酸性中肠区域中发挥作用，与脊椎动物的情况类似。缺乏脯氨酸环可能与胃蛋白酶和消化 CAD3 的细胞外作用有关。
