Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.

蛋白酶在扁形动物中发挥诸多关键生理功能，其中多数功能可能在扁形动物门（Platyhelminthes）中具有保守性。该门类包含多种寄生蠕虫，由于其复杂的生命周期以及难以进行遗传操作，这类生物的功能特性常未被充分解析。地中海真涡虫（Schmidtea mediterranea，简称涡虫）因其生命周期简单且可开展RNA干扰（RNAi）等实验技术，成为研究蛋白质消化复杂机制的理想模式生物。本研究旨在解析涡虫肠道内的消化蛋白酶系统。为此，我们开发了酒精诱导反流技术，以富集地中海真涡虫的肠道酶类。通过一组荧光底物实验，我们证实该处理可显著提升蛋白水解活性。这些酶的底物特异性谱广泛且具有多样性。对肠道内容物的蛋白质组学分析显示，其中存在半胱氨酸蛋白酶和金属蛋白酶。但通过类特异性抑制剂处理实验发现，天冬氨酸蛋白酶与半胱氨酸蛋白酶负责了大部分的蛋白质消化过程。对组织蛋白酶B样半胱氨酸蛋白酶（SmedCB）进行特异性RNA干扰敲低，可在体内降低蛋白质降解水平。免疫组织化学与整体原位杂交（WISH）实验证实，SmedCB的全长形式与活性形式均存在于涡虫肠道腔周围的分泌细胞中。最终我们发现，敲低SmedCB会降低组织再生的速度。明确涡虫中蛋白酶的功能，可为解析寄生扁形动物中保守蛋白酶的功能提供参考，亦有望为寄生虫找到潜在药物靶点。