ABSTRACT
Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective inâ vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pHâ 7.5 in 1â h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.
Subject(s)
Azides/chemistry , COVID-19 Vaccines/chemistry , Gluconates/chemistry , Glycine/chemistry , Histidine/chemistry , Lactones/chemistry , Vaccines, Virus-Like Particle/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Azides/immunology , COVID-19 Vaccines/immunology , Gluconates/immunology , Glycine/immunology , Histidine/immunology , Humans , Lactones/immunology , Models, Molecular , Molecular Structure , Vaccines, Virus-Like Particle/immunologyABSTRACT
The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.