Characterization of SARS-CoV-2 Receptor-Binding Domains using SmartEnzymes™

Scientists at Leiden University Medical Center (LUMC) present a multilevel mass spectrometry approach using SmartEnzymes for in-depth characterization of mammalian SARS-CoV-2 receptor-binding (RBD) domains.

The COVID-19 disease caused by the SARS-CoV-2 virus has affected more that 100 million individuals in the ongoing pandemic. The enveloped RNA corona virus contains three structural proteins in the membrane, including the heavily glycosylated spike protein carrying 22 N-glycosylation sites. The spike protein in turn consists of two subunits, S1 and S2, where the receptor-binding domain (RBD) of S1 directly interacts with the ACE2 receptor in the human respiratory tract and facilitates host cell entry. Considering the relevance of RBD glycosylation on ACE2 binding and recognition by neutralizing antibodies, the use of well-characterized S proteins is essential for continued research and development of diagnostic tools and vaccines.

In this work, scientists at LUMC performed an in-depth functional and structural analysis of RBDs expressed in mammalian cells, Chinese hamster (CHO) and human (HEK293). Using a multilevel mass spectrometry approach in combination with several SmartEnzymes, the scientists were able to perform a comprehensive characterization of the RBD proteoforms and glycoforms on N- and O-glycosylation sites. The use of sequentially different enzymes proved to be a powerful tool for the annotation of the complex intact RBD spectra. Intact protein analysis using the SialEXO, GalactEXO, FucosEXO, OglyZOR and OpeRATOR enzymes not only revealed a complex pattern of different N- and O-glycans, but also enabled the scientists to map previously undetermined O-glycosylation sites.

In short, this work highlights recombinantly produced S proteins as essential tools in the fight against SARS-CoV-2, and the versatility of SmartEnzymes in analytical workflows contributing to further research for improved diagnostics and vaccines.

Gstöttner et al., 2021. Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells. Anal. Chem. doi: 10.1021/acs.analchem.1c00893

EXOglycosidases – SmartEnzymes for specific hydrolysis of Galactose, Sialic acids, Fucose or GalNac residues.

Enzymes for O-glycans – SmartEnzymes for the analysis and mapping of O-glycans on native glycoproteins.