Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Abstract

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

Inhibition of Protein N- Glycosylation Blocks SARS-CoV-2 Infection

“…Differences in baseline N- glycosylation profiles between Vero and HEK cells ( Fig. S3 A, File S1 ) as well as the differential expression of glycosylation enzymes may account for variations between cell lines, particularly the Golgi endo-α-1,2-mannosidase (MANEA) alternative pathway ( 30 ) that can rescue glycoproteins improperly glycosylated by malfunctioning ER α-glucosidases. Cell viability assays were run in parallel to rule out poor metabolism or toxicity as the main cause of infection decrease ( Fig.…”

Evolution and phylogenetic distribution ofendo-α-mannosidase

“…3B). A tyrosine at position 189 switches the −2 sugar residue preference to Glc by forming a water-mediated hydrogen bond with the −2 sugar hydroxyl group 35,41 (Fig. 3A).…”

“…5C). As residue N197 forms a hydrogen bond with the O4 of the -2 glucose 41 (Fig. 3), the N197H variation might change the substrate binding affinity, possibly increasing it, as the electronegativity difference would be greater in case of the NH-O bond than in case of the OH-O bond.…”

Protein N-glycosylation is essential for SARS-CoV-2 infection

“…1). Differences in baseline N-glycosylation profiles between Vero and HEK cells (Fig S4A; File S1 and S2) and expression of the Golgi's endo-a-1,2-mannosidase (MANEA) (27) independent pathway may account for such variations. Cell viability assays were run in parallel to rule out poor metabolism or toxicity as the main cause of infection decrease (Fig.…”