Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada.
Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Sci Rep. 2023 Aug 29;13(1):14123. doi: 10.1038/s41598-023-41389-7.
Angiotensin-converting enzyme II (ACE2) is a monocarboxypeptidase expressed throughout multiple tissues and its catalysis of bioactive peptides regulates the renin-angiotensin system mediating blood pressure homeostasis. ACE2 is implicated in a variety of diseases, including obesity, diabetes, and cardiovascular diseases, and is the obligate entry receptor for SARS-CoV-2 infection. Disease-associated genetic variants of ACE2 are increasingly being identified but are poorly characterized. To aid this problem, we introduce a fluorometric cell-based assay for evaluating surface-expressed ACE2 catalytic activity that preserves the native glycosylation of the host environment and is amenable to high-throughput analysis of ACE2 variants in multi-well plates. We demonstrate sensitivity to detecting catalysis of the key ACE2 substrates, Angiotensin II, Apelin-13, and des-Arg-bradykinin, and impact of a catalytically-deficient ACE2 variant. Normalizing catalytic measures to surface ACE2 expression accounts for variability in ACE2 variant transfection, surface delivery or stability. This assay provides a convenient and powerful approach for investigating the catalytic characteristics of ACE2 variants involved in cardiovascular peptide cascades and homeostasis of multiple organs.
血管紧张素转化酶 2（ACE2）是一种单羧肽酶，在多种组织中表达，其对生物活性肽的催化作用调节肾素-血管紧张素系统，介导血压稳态。ACE2 与多种疾病有关，包括肥胖、糖尿病和心血管疾病，并且是 SARS-CoV-2 感染的必需进入受体。越来越多的与疾病相关的 ACE2 遗传变异被识别出来，但特征描述很差。为了解决这个问题，我们引入了一种用于评估表面表达的 ACE2 催化活性的荧光细胞测定法，该方法保留了宿主环境的天然糖基化，并且适用于多孔板中 ACE2 变体的高通量分析。我们证明了该测定法对检测关键 ACE2 底物血管紧张素 II、Apelin-13 和 des-Arg-缓激肽的催化作用的敏感性，以及对催化缺陷型 ACE2 变体的影响。将催化测量值与表面 ACE2 表达归一化可解释 ACE2 变体转染、表面递呈或稳定性的变异性。该测定法为研究涉及心血管肽级联和多个器官稳态的 ACE2 变体的催化特性提供了一种方便而强大的方法。
