Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington DC, 20007, USA.
Department of Pathological Anatomy, Bogomolets National Medical University, Kyiv, 01601, Ukraine.
Arch Biochem Biophys. 2023 Oct 15;748:109769. doi: 10.1016/j.abb.2023.109769. Epub 2023 Sep 27.
The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.
2019 年冠状病毒病（COVID-19）大流行是由严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）引起的，该病毒通过其刺突蛋白与血管紧张素转换酶 2（ACE2）的相互作用进入宿主细胞。ACE2 是一种肽酶，可切割血管紧张素 II，这是一种关键的病理介质。本研究探讨了刺突蛋白与 ACE2 的结合是否会影响其肽酶活性。刺突/ACE2 结合测定表明，SARS-CoV-2、SARS-CoV 和 MERS-CoV 的刺突蛋白，但不是 HKU1，与 ACE2 结合。S1 和受体结合域（RBD），而不是 S2、细胞外域（ECD）或 CendR 域，与 ACE2 结合。虽然在 HEK293 细胞中制备的糖基化刺突蛋白与 ACE2 结合，但在大肠杆菌中制备的非糖基化蛋白则不结合。在 HEK293 细胞中表达的刺突蛋白中的半胱氨酸残基完全氧化，而在大肠杆菌中表达的蛋白中的半胱氨酸残基则被还原。HEK 细胞产生的蛋白的去糖基化减弱了 ACE2 的结合，而大肠杆菌蛋白的氧化则不会促进结合。SARS-CoV-2 的 S1 蛋白增强了 ACE2 的肽酶活性，而 SARS-CoV、MERS-CoV 或 HKU1 则没有。RBD 增强了 ACE2 的活性，但 ECD 或 CendR 则没有。与在 HEK293 细胞和大肠杆菌中表达的蛋白的不同 ACE2 结合能力不同，在这两种系统中表达的刺突蛋白均增强了 ACE2 的活性。因此，SARS-CoV-2 的刺突蛋白而非其他冠状病毒通过其 RBD 以非糖基化依赖的方式增强 ACE2 的肽酶活性。
