Semisynthesis of homogeneous spike RBD glycoforms from SARS-CoV-2 for profiling the correlations between glycan composition and function

Abstract

Vaccines have been the primary remedy in the global fight against coronavirus disease 2019 (COVID-19). The receptor-binding domain (RBD) of the spike protein, a critical viral immunogen, is affected by the heterogeneity of its glycan structures and relatively low immunogenicity. Here, we describe a scalable synthetic platform that enables the precise synthesis of homogeneously glycosylated RBD, facilitating the elucidation of carbohydrate structure–function relationships. Five homogeneously glycosylated RBDs bearing biantennary glycans were prepared, three of which were conjugated to T-helper epitope (Tpep) from tetanus toxoid to improve their weak immune response. Relative to natural HEK293-derived RBD, synthetic RBDs with biantennary N-glycan elicited a higher level of neutralising antibodies against SARS-CoV-2 in mice. Furthermore, RBDs containing Tpep elicited significant immune responses in transgenic mice expressing human angiotensin-converting enzyme 2. Our collective data suggest that trimming the N-glycans and Tpep conjugation of RBD could potentially serve as an effective strategy for developing subunit vaccines providing efficient protection.

Investigation of N-Glycan Functions in Receptor for Advanced Glycation End Products V Domain through Chemical Glycoprotein Synthesis

“…N-Glycosylation is a widely distributed PTM in eukaryotic proteins that plays pivotal roles in protein biosynthesis, , structural stabilization, enzymatic activity, and protein–protein interactions. , Two N-glycosylation sites, Asn3 and Asn59, located at the VD of RAGE, have been identified and suggested to influence ligand binding and the activation of inflammation-related pathways. ,, However, similar to many other examples of protein glycosylations, the potential modulatory roles of N-glycans and their structure–function relationships remain to be elucidated, facing daunting challenges posed by the heterogeneous nature of biosynthetically generated glycoprotein samples. Recent advances in probing glycan functions have utilized chemically synthesized N-glycoproteins, such as EPO, , IL-6, IFN β, IFN γ, IL-17A, SARS-CoV RBD, , and the ectodomain of herpes simplex virus-1 glycoprotein D . We hypothesized that the homogeneous N-glycosylated VD of RAGE may serve as a tool for studying the potential roles of N-glycosylation in ligand–receptor interactions.…”

“…23,30,31 However, similar to many other examples of protein glycosylations, the potential modulatory roles of N-glycans and their structure−function relationships remain to be elucidated, facing daunting challenges posed by the heterogeneous nature of biosynthetically generated glycoprotein samples. Recent advances in probing glycan functions have utilized chemically synthesized N-glycoproteins, such as EPO, 32,33 IL-6, 34 IFN β, 35 IFN γ, 36 IL-17A, 37 SARS-CoV RBD, 38,39 and the ectodomain of herpes simplex virus-1 glycoprotein D. 40 We hypothesized that the homogeneous N-glycosylated VD of RAGE may serve as a tool for studying the potential roles of N-glycosylation in ligand− receptor interactions. In this study, we describe the chemical synthesis of RAGE-VD in a series of homogeneous Nglycosylated forms and explore its structure−activity relationships with HMGB1 and S100B.…”