Background: Despite clinical success with anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines has been compromised by rapidly spreading SARS-CoV-2 variants. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host's immune response and attenuate antibody efficiency. However, it remains unclear if targeting glycosylation on viral spike protein can impair infectivity of SARS-CoV-2 and its variants. Methods: We adopted flow cytometry, ELISA, and BioLayer interferometry approaches to assess binding of glycosylated or deglycosylated spike with ACE2. Viral entry was determined by luciferase, immunoblotting, and immunofluorescence assays. Genome-wide association study (GWAS) revealed a significant relationship between STT3A and COVID-19 severity. NF-kB/STT3A-regulated N-glycosylation was investigated by gene knockdown, chromatin immunoprecipitation, and promoter assay. We developed an antibody-drug conjugate (ADC) that couples non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) to specifically target SARS-CoV-2-infected cells. Findings: The receptor binding domain and three distinct SARS-CoV-2 surface N-glycosylation sites among 57,311 spike proteins retrieved from the NCBI-Virus-database are highly evolutionarily conserved (99.67%) and are involved in ACE2 interaction. STT3A is a key glycosyltransferase catalyzing spike glycosylation and is positively correlated with COVID-19 severity. We found that inhibiting STT3A using N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 infectivity and that of its variants [Alpha (B.1.1.7) and Beta (B.1.351)]. Most importantly, 4G10-ADC enters SARS-CoV-2-infected cells and NGI-1 is subsequently released to deglycosylate spike protein, thereby reinforcing the neutralizing abilities of antibodies, vaccines, or convalescent sera and reducing SARS-CoV-2 variant infectivity. Interpretation: Our results indicate that targeting evolutionarily-conserved STT3A-mediated glycosylation via an ADC can exert profound impacts on SARS-CoV-2 variant infectivity. Thus, we have identified a novel deglycosylation method suitable for eradicating SARS-CoV-2 variant infection in vitro. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section

β-1,6-N-acetylglucosaminyltransferase 2 (GCNT2), which encodes a key glycosyltransferase for blood group I antigen synthesis, is induced upon epithelial-mesenchymal transition (EMT). Our results indicate that GCNT2 is upregulated upon EMT induced with epidermal growth factor and basic FGF in cultured human colon cancer cells. GCNT2 knockdown or overexpression decreases or increases, respectively, malignancy-related characteristics of colon cancer cells and I antigen levels. MiR-199a/b-5p is markedly downregulated upon EMT in colon cancer cells. Here, we find that miR-199a/b-5p consistently regulates GCNT2 expression in reporter assays and that it binds directly to the GCNT2 3' untranslated region intracellularly in RNA-induced silencing complex-trap assays. Overexpression of miR-199a/b-5p decreases GCNT2 expression and suppresses I antigen production. Based on these findings, we propose that miR-199a/b-5p regulates GCNT2 and I antigen expression in colon cancer cells undergoing EMT.

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Human Toll-like receptor (TLR) signaling plays a vital role in intestinal inflammation by activating the NF-κB pathway. By querying GENT2 datasets, we identified the gene expression level of TLR2 and TLR4 as being substantially increased in colorectal cancer. Introduction of shRNAs for TLR4 but not TLR2 dramatically recovered disialyl Lewis a and sialyl 6-sulfo Lewis x glycans, which are preferentially expressed in non-malignant colonic epithelial cells and could serve as ligands for the immunosuppressive molecule Siglec-7. We screened several TLR4 ligands and found that among them BGN is highly expressed in cancers and is involved in the epigenetic silencing of Siglec-7 ligands. Suppression of BGN expression substantially downregulated NF-κB activity and the marker H3K27me3 in the promoter regions of the SLC26A2 and ST6GalNAc6 genes, which are involved in the synthesis of those glycans, and restored expression of normal glycans as well as Siglec-7 binding activities. We show that in the presence of TLR4, inflammatory stimuli initiate a positive loop involving NF-κB that activates BGN and further enhances TLR4 activity. Present findings indicate a putative mechanism for the promotion of carcinogenesis by loss of immunosuppressive ligands by the BGN/TLR4/ NF-κB pathway.