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Well characterized recombinant SARS-CoV-2 proteins are essential instruments in the fight against COVID-19. They are currently employed for diagnostic purposes, vaccine development and many other research activities. Our project aimed to perform an in-depth structural and functional characterization of commercial receptor-binding domains (RBDs) expressed in different mammalian systems (CHO and HEK293 cells). To achieve our goal, we applied several SmartEnzymes from the Genovis portfolio including the FucosEXO, GalactEXO, SialEXO, OglyZOR and OpeRATOR enzymes.
How did SmartEnzymes enhance your project?
We analyzed the RBDs by MS after complete removal of the N and O-glycans, permitting us to detect various modifications in the protein backbone (e.g. cysteinylation, pyroglutamic acid). By sequentially trimming of the N- and O-glycans using a combination of different exoglycosidases, we could assign the N- and O-glycans at the intact level, overcoming potential glycoform bias as observed by glycopeptide analysis. Overall, we observed tremendous differences in the glycosylation between CHO- and HEK293 produced RBDs with the latter exhibiting a combination of core 1 and core 2 type O-glycans. RBDs present two potential O-glycosylation sites in close proximity, difficulting the localization of the O-glycosylation site. With the help of the OpeRATOR enzyme, we demonstrated the presence of a single, fully occupied O-glycosylation site to the threonine 323 in both mammalian production systems. Furthermore, we investigated the functional differences between the glycosylated and deglycosylated RBDs with the help of SmartEnzymes. Our data suggested that RBD glycosylation plays a role in conformational stabilization of the spike protein rather than a direct involvement in ACE2 binding.
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Glycan profiling
