SARS-CoV-2 Spike Protein-Priming Cysteine Cathepsin L Acting at Microvilli of Intestine Epithelial Cells

导致COVID-19的SARS-CoV-2通过包括小肠肠细胞在内的上皮细胞进入。虽然蛋白酶ACE2和TMPRSS2被认为是SARS-CoV-2靶细胞的进入共受体，但半胱氨酸肽酶cathepsin L的参与机制尚不清楚，原则上它可以作为病毒刺突蛋白激活酶。因此，本研究将测试一种新的、非常规的SARS-CoV-2刺突蛋白激活机制，即组织蛋白酶L作用于小肠上皮细胞的微绒毛。我们基于我们之前的工作证明组织蛋白酶L从肠上皮细胞和杯状细胞分泌到肠腔，在那里它与粘膜细胞表面重新结合。因此，组织蛋白酶L存在于小肠上皮的暴露良好的微绒毛中，它可能参与冠状病毒刺突蛋白的启动，从而促进病毒的进入和传播，尽管ACE2和TMPRSS2在这方面也发挥着重要作用。因此，本项目将利用我们建立的肠细胞和杯状细胞组成的类器官-典型共培养模型，研究组织蛋白酶L、ACE2和TMPRSS2在小肠原位表达和亚细胞共定位，以及组织蛋白酶L在肠上皮细胞微绒毛处的酶活性。此外，我们将通过模拟肠道腔的预期条件来研究组织蛋白酶l介导的刺突蛋白在体外切割的可能性。最后，我们将分析组织蛋白酶L活性对小肠细胞微绒毛维持的意义。我们希望阐明组织蛋白酶L是否在SARS-CoV-2小肠感染中发挥重要作用。这一基础科学项目的成果将有助于更全面地研究冠状病毒进入靶细胞的治疗性预防。

SARS-CoV-2, responsible for causing COVID-19, enters via epithelial cells including enterocytes of the small intestine. While the proteases ACE2 and TMPRSS2 are recognized as entry co-receptor/s of SARS-CoV-2 target cells, it is not sufficiently understood how the cysteine peptidase cathepsin L involves, which can act as a viral spike protein-activating enzyme in principle. Thus, the proposed research shall test a novel and non-conventional mechanism of SARS-CoV-2 spike protein activation, namely, by cathepsin L acting at microvilli of small intestine epithelial cells. We base this proposal on our previous work demonstrating that cathepsin L is secreted from intestine epithelial and goblet cells into the gut lumen from where is re-associates with the surface of the mucosal cells. Hence, cathepsin L is present at well-exposed microvilli of the small intestine epithelium, where it might be involved in priming of the coronaviral spike protein, thereby promoting viral entry and spreading, despite and in addition to the prominent tasks that ACE2 and TMPRSS2 play in this regard. Therefore, this project will address expression and sub-cellular co-localization of cathepsin L, ACE2 and TMPRSS2 in the small intestine in situ as well as the enzymatic activity of cathepsin L at microvilli of intestine epithelial cells using our established organoid-typical co-culture model consisting of enterocytes and goblet cells. In addition, we will investigate the possibility of cathepsin L-mediated spike protein cleavage in vitro by simulating conditions expected for the gut lumen. Eventually, the significance of cathepsin L activity for maintenance of microvilli at cells of the small intestine will be analyzed. We expect to clarify whether cathepsin L can play an important role in SARS-CoV-2 infection of the small intestine. The results of this basic science project will help to more comprehensively approach therapeutic prevention of coronavirus entry into target cells.