N-端组学分析揭示裸鼠结肠的蛋白酶裂解特征与legumain的作用

N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain

JOURNAL OF CELLULAR PHYSIOLOGY

影响因子:4

分区:生物学3区 / 细胞生物学3区生理学3区

发表日期:2025 Jan

作者: Alexander R Ziegler, Bethany M Anderson, Rocco Latorre, Rachel M McQuade, Antoine Dufour, Brian L Schmidt, Nigel W Bunnett, Nichollas E Scott, Laura E Edgington-Mitchell

DOI: 10.1002/jcp.31466

摘要

Legumain是一种广泛与炎症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，从而引发口腔癌相关的疼痛。除胃癌和结肠癌外，关于legumain在胃肠道中的作用的报道较少。利用选择性针对legumain的活性基质探针LE28，我们发现legumain在小鼠结肠细胞和巨噬细胞中被激活，并在急性实验性结肠炎模型中表达上调。我们证明，在结肠细胞中，通过药理抑制或基因敲除失去legumain活性，未对体外上皮通透性产生影响。此外，legumain的抑制或缺失对DSS诱导的结肠炎的症状或组织学特征没有明显影响，提示其蛋白水解活性在结肠炎的发生中可能不是必需的。为了深入探讨legumain在结肠中的潜在功能，我们对野生型和legumain缺失小鼠的未经处理和炎症结肠组织进行了场不对称波形离子迁移谱（FAIMS）辅助定量蛋白质组学和N-端组学分析。我们鉴定出16个具有天冬氨酰内肽酶（asparaginyl endopeptidase）特征的裂解位点，可能是legumain的直接底物，以及另外16个可能由legumain间接介导的裂解位点。我们还分析了蛋白质丰度和蛋白水解事件的变化，这些变化广泛与肠道结肠炎相关，并与近期对炎症性肠病患者黏膜活检的分析进行了对比。总体而言，这些结果揭示了legumain的潜在功能，并突出了其在炎症向结直肠癌转变中的可能作用。

Abstract

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.