The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design.

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

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