Gstöttner Christoph, Zhang Tao, Resemann Anja, Ruben Sophia, Pengelley Stuart, Suckau Detlev, Welsink Tim, Wuhrer Manfred, Domínguez-Vega Elena
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany.
Anal Chem. 2021 May 4;93(17):6839-6847. doi: 10.1021/acs.analchem.1c00893. Epub 2021 Apr 19.
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising - and -glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type -glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.
由于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）大流行仍在持续并对我们的生活产生巨大影响，对用于诊断、疫苗开发和研究的重组病毒蛋白的需求非常高。刺突（S）蛋白，特别是其受体结合域（RBD），介导与宿主细胞上血管紧张素转换酶2（ACE2）受体的相互作用，并且可能受其结构特征的调节。因此，充分表征的重组RBD至关重要。我们对在中国仓鼠卵巢（CHO）细胞和人胚肾293（HEK293）细胞中表达的RBD进行了深入的结构和功能表征。为了从结构上表征天然RBD（包含N-聚糖和O-聚糖以及其他翻译后修饰），采用了多级质谱方法。释放的聚糖和糖肽分析与完整质量分析、聚糖酶切分析和自上而下测序相结合，以全面注释RBD蛋白变体。数据显示CHO-RBD和HEK293-RBD具有不同的糖基化，后者表现出触角岩藻糖基化、更高水平的唾液酸化以及核心1型和核心2型O-聚糖的组合。此外，使用基于O-糖基化N端切割的另一种方法，将先前未知的O-糖基化位点定位在T323。对于这两种RBD，研究了它们与阳性患者的SARS-CoV-2抗体的结合以及与ACE2受体的亲和力，结果显示具有可比性。这项工作不仅提供了对RBD结构和功能特征的见解，还提供了一种用于表征新RBD和批次间比较的分析工作流程。
