The protein sequence modulates terminal GalNAc incorporation during N-glycan biosynthesis.

Abstract

The factors governing protein-specific N-glycosylation remain incompletely understood despite extensive knowledge of the biosynthetic machinery involved in glycan assembly. In particular, the contribution of the protein sequence itself to the final glycosylation pattern has received comparatively little attention. Here, we employed isotope-assisted NMR spectroscopy to investigate the N-glycosylation of the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD-SCoV2) produced in HEK293F cells. Previous analyses revealed the unexpected presence of terminal GalNAc-containing epitopes, including LacdiNAc (LDN), sulfated LacdiNAc (4S-LDN), sialylated LacdiNAc (6Sia-LDN), and fucosylated LacdiNAc (LDNF). Inspired by earlier studies showing that certain β4GalNAc-transferases recognize basic peptide motifs, we generated an RBD variant carrying four substitutions of solvent-exposed basic residues located near the N-glycosylation sites. Comparative NMR analysis demonstrated that the mutant protein exhibits a marked reduction in GalNAc-containing terminal epitopes, accompanied by a corresponding increase in Gal-containing structures. The overall protein fold remained unchanged, indicating that the observed glycosylation differences arise from altered glycan processing rather than major structural perturbations. These findings provide direct evidence that local protein sequence features modulate the incorporation of terminal glycan epitopes during Golgi processing. More broadly, this work highlights the power of quantitative NMR spectroscopy for the structural characterization of glycoproteins and for detecting subtle changes in glycan populations that are directly linked to protein sequence.