Legumain 是一种与炎症广泛相关的半胱氨酸蛋白酶。据报道，它可以裂解并激活蛋白酶激活受体 2，从而引发与口腔癌相关的疼痛。除了胃癌和结肠癌之外，关于legumain在胃肠道内的作用的报道很少。使用基于legumain选择性活性的探针LE28，我们报告legumain在小鼠结肠的结肠细胞和巨噬细胞内被激活，并且在急性实验性结肠炎模型中上调。我们证明，通过药理学抑制或基因删除，结肠细胞中legumain活性的丧失对体外上皮通透性没有影响。此外，legumain抑制或缺失对与硫酸葡聚糖钠诱导的结肠炎相关的症状或组织学特征没有明显影响，这表明其蛋白水解活性对于结肠炎的引发是可有可无的。为了深入了解legumain在结肠内的潜在功能，我们对野生型和legumain缺陷小鼠的幼稚和发炎结肠组织进行了场不对称波形离子淌度光谱法辅助的定量蛋白质组学和N末端组学分析。我们鉴定了 16 个带有天冬酰胺酰内肽酶特征的改变的切割位点，其可能是legumain 的直接底物，另外 16 个切割位点可能是由legumain 间接介导的。我们还分析了与肠道结肠炎广泛相关的蛋白质丰度和蛋白水解事件的变化，这可以与最近对炎症性肠病患者粘膜活检的分析进行比较。总的来说，这些结果揭示了legumain的潜在功能，并强调了它在从炎症到结直肠癌转变中的潜在作用。© 2024 作者。 《细胞生理学杂志》由 Wiley periodicals LLC 出版。

Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.