In insects, cathepsin D is a lysosomal aspartic endopeptidase involved in several functions such as digestion, defense and reproduction. Jack Bean Urease (JBU) is the most abundant urease isoform obtained from the seeds of the plant Canavalia ensiformis. JBU is a multifunctional protein with entomotoxic effects unrelated to its catalytic activity, by mechanisms not yet fully understood. In this work, we employed nymphs of the hematophagous insect Dipetalogaster maxima as an experimental model in order to study the effects of JBU on D. maxima CatD (DmCatD). In insects without treatment, immunofluorescence assays revealed a conspicuous distribution pattern of DmCatD in the anterior and posterior midgut as well as in the fat body and hemocytes. Western blot assays showed that the active form of DmCatD was present in the fat body, the anterior and posterior midgut; whereas the proenzyme was visualized in hemocytes and hemolymph. The transcript of DmCatD and its enzymatic activity was detected in the anterior and posterior midgut as well as in fat body and hemocytes. JBU injections induced a significant increase of DmCatD activity in the posterior midgut (at 3 h post-injection) whereas in the hemolymph, such an effect was observed after 18 h. These changes were not correlated with modifications in DmCatD mRNA and protein levels or changes in the immunofluorescence pattern. In vitro experiments might suggest a direct effect of the toxin in DmCatD activity. Our findings indicated that the tissue-specific increment of cathepsin D activity is a novel effect of JBU in insects.

在昆虫中，组织蛋白酶D是一种溶酶体天冬氨酸内肽酶，具有多种功能，例如消化，防御和繁殖。Jack Bean Urease（JBU）是从植物Canavalia ensiformis的种子中获得的最丰富的脲酶同工型。JBU是一种多功能蛋白，具有尚不完全了解的机制，其毒性与它的催化活性无关。在这项工作中，我们使用食血昆虫极大的Dipetalogaster的若虫作为实验模型，以研究JBU对极大D.的影响CatD（DmCatD）。在未经治疗的昆虫中，免疫荧光分析显示DmCatD在前中肠和后中肠以及脂肪体和血细胞中的分布模式明显。蛋白质印迹分析表明，DmCatD的活性形式存在于脂肪体，前中肠和后中肠。而在血细胞和血淋巴中可以看到原酶。DmCatD的成绩单并在中肠前，后肠以及脂肪体和血细胞中检测到了其酶活性。JBU注射后中肠（注射后3小时）诱导DmCatD活性显着增加，而在血淋巴中，在18小时后观察到这种作用。这些变化与DmCatD mRNA和蛋白质水平的修饰或免疫荧光模式的变化无关。体外实验可能表明该毒素对DmCatD活性具有直接作用。我们的发现表明组织蛋白酶D活性的组织特异性增加是JBU在昆虫中的一种新型作用。