O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure

Abstract

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus’s entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

1. Introduction

Protein O-linked glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. More than 80% of secreted and cell membrane proteins undergo O-glycosylation [1]. This type of glycosylation plays crucial roles in modulating various functions, including ligand-receptor interactions, proprotein processing, and subcellular localization sorting [2,3]. The initiation of protein O-glycosylation is facilitated by a glycosyltransferase family known as polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which transfer GalNAc from UDP-GalNAc to the Thr/Ser residues of substrate proteins or peptides [4]. In human cells, the ppGalNAc-T family contains up to 20 isoenzymes, exhibiting distinct spatial and temporal expression patterns in tissues and cells. Furthermore, these ppGalNAc-T isoenzymes share both redundant and partially specific preferences for protein substrates [5,6]. Therefore, protein O-glycosylation is a kind of systematic modification in human cells.

Viruses, as simple life forms, exploit the host’s transcription, translation, and modification systems to synthesize and package their required proteins and nucleic acids [7]. Consequently, viral proteins carry post-translational modifications that are homologous to those of the host cells, such as glycosylation. Many envelope proteins of pathogenic viruses, including HIV, MERS, and SARS, are heavily glycosylated [8]. Viruses utilize host glycosylations to evade host immunity, bind to host receptor cells, and regulate hydrolysis by host proteases [9]. Therefore, glycosylation plays a crucial role in interactions between viruses and their hosts.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is responsible for the novel coronavirus disease 2019 (COVID-19), posed a pandemic threat to global public health during 2019–2023 [10]. Currently, both long COVID-19 and sporadic COVID-19 cases continue to burden global health systems [11]. The spike protein (S protein) of SARS-CoV-2 facilitates membrane fusion between the virus and host cells [12]. This type I membrane protein is a homotrimer [13]. It has been reported that two trimeric S proteins can simultaneously bind to one angiotensin converting enzyme 2 (ACE2) homodimer from host cells, and then mediate the membrane fusion process [14]. Each monomer of the S protein is coated with 22 N-glycan sites and numerous O-glycan sites in diverse expression systems [15]. N-glycosylation of the S protein has been extensively studied and found to directly affect interactions.