Ziegler Alexander R, Anderson Bethany M, Latorre Rocco, McQuade Rachel M, Dufour Antoine, Schmidt Brian L, Bunnett Nigel W, Scott Nichollas E, Edgington-Mitchell Laura E
Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.
Department of Molecular Pathobiology, New York University College of Dentistry, New York, New York, USA.
J Cell Physiol. 2025 Jan;240(1):e31466. doi: 10.1002/jcp.31466. Epub 2024 Oct 11.
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.
天冬酰胺内肽酶是一种与炎症广泛相关的半胱氨酸蛋白酶。据报道，它可切割并激活蛋白酶激活受体2，引发与口腔癌相关的疼痛。除胃癌和结肠癌外，关于天冬酰胺内肽酶在胃肠道中的作用鲜有报道。我们使用基于天冬酰胺内肽酶选择性活性的探针LE28，发现天冬酰胺内肽酶在小鼠结肠的结肠细胞和巨噬细胞中被激活，并且在急性实验性结肠炎模型中上调。我们证明，通过药物抑制或基因缺失使结肠细胞中天冬酰胺内肽酶活性丧失，对体外上皮通透性没有影响。此外，抑制或缺失天冬酰胺内肽酶对硫酸葡聚糖钠诱导的结肠炎相关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起始并非必需。为了深入了解天冬酰胺内肽酶在结肠中的潜在功能，我们对野生型和天冬酰胺内肽酶缺陷型小鼠的未发炎和发炎结肠组织进行了场不对称波形离子迁移谱辅助的定量蛋白质组学和N端蛋白质组学分析。我们鉴定出16个具有天冬酰胺内肽酶特征的改变的切割位点，它们可能是天冬酰胺内肽酶的直接底物，另外还有16个切割位点可能由天冬酰胺内肽酶间接介导。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，这使得我们能够与最近对炎症性肠病患者黏膜活检的分析进行比较。总的来说，这些结果揭示了天冬酰胺内肽酶的潜在功能，并突出了其在从炎症向结直肠癌转变中的潜在作用。
