Opperman Roza C M, Bosch Sofie, Nazmi Kamran, Bikker Floris J, Brand Henk S, Jimenez Connie R, de Meij Tim G J, Dekker Evelien, de Boer Nanne K H, Kaman Wendy E
Department of Gastroenterology and Hepatology, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
Amsterdam Gastroenterology Endocrinology Metabolism (AGEM) Research Institute, 1081 HV Amsterdam, The Netherlands.
Anal Chem. 2024 Dec 24;96(51):20239-20246. doi: 10.1021/acs.analchem.4c04586. Epub 2024 Dec 12.
identification and removal of advanced adenomas (AA) reduce colorectal cancer (CRC) incidence and potentially mortality. CRC screening often uses fecal immunochemical testing to select high-risk individuals for colonoscopy, despite its low sensitivity for AA and relatively high false-positivity rate. Previous studies have linked proteases to CRC development through their ability to facilitate angiogenesis and immunoregulation. This study aims to identify colorectal neoplasia-associated proteases and their substrates as a potential noninvasive screening test, introducing an innovative application of fecal protease profiling, which has previously been limited to tissue samples.
eighteen fluorogenic substrates were designed based on literature. Proteolytic degradation of these substrates was measured in fecal samples of patients with CRC ( = 12), AA ( = 9), nonadvanced adenomas ( = 10), and controls ( = 14). Substrate degradation was correlated to a matched human proteome data set, and underlying proteases were identified based on their recognition patterns. Experiments with protease inhibitors and ZnCl were performed to further characterize the involved proteases.
in total, 7 of the 18 substrates tested showed a significantly decreased proteolytic degradation in feces from patients with any colorectal neoplasia compared to the control group. The l-aspartic acid-l-glutamic acid substrate (ED) showed significantly decreased degradation in AA and CRC patients. ED degradation significantly decreased with the addition of ZnCl and the cysteine protease inhibitor NEM.
we successfully developed colorectal neoplasia-specific fluorogenic substrates, highlighting the ED substrate as a potential substrate for the detection of AA and CRC. Although the responsible proteases require further identification, our results suggest an association with calcium-dependent cysteine proteases.
识别并切除进展期腺瘤（AA）可降低结直肠癌（CRC）的发病率，并有可能降低死亡率。CRC筛查通常使用粪便免疫化学检测来选择结肠镜检查的高危个体，尽管其对AA的敏感性较低且假阳性率相对较高。先前的研究已将蛋白酶与CRC的发展联系起来，因为它们具有促进血管生成和免疫调节的能力。本研究旨在识别与结直肠肿瘤相关的蛋白酶及其底物，作为一种潜在的非侵入性筛查测试，引入粪便蛋白酶谱分析的创新应用，该方法此前仅限于组织样本。
根据文献设计了18种荧光底物。在CRC患者（n = 12）、AA患者（n = 9）、非进展期腺瘤患者（n = 10）和对照组（n = 14）的粪便样本中测量这些底物的蛋白水解降解。底物降解与匹配的人类蛋白质组数据集相关，并根据其识别模式鉴定潜在的蛋白酶。进行了蛋白酶抑制剂和ZnCl₂的实验，以进一步表征所涉及的蛋白酶。
总共，与对照组相比，在任何结直肠肿瘤患者的粪便中，测试的18种底物中有7种显示出蛋白水解降解显著降低。L-天冬氨酸-L-谷氨酸底物（ED）在AA和CRC患者中显示出降解显著降低。添加ZnCl₂和半胱氨酸蛋白酶抑制剂NEM后，ED降解显著降低。
我们成功开发了结直肠肿瘤特异性荧光底物，突出了ED底物作为检测AA和CRC的潜在底物。尽管负责的蛋白酶需要进一步鉴定，但我们的结果表明与钙依赖性半胱氨酸蛋白酶有关。
