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

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-a-1,2-mannosidase (MANEA) is the sole endoacting 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

Pathways for glycoprotein degradation in mammalian cells

In mammalian cells, N-glycoproteins comprise the bulk of secretory cargo. N-glycosylation is tightly linked to the mechanisms of co- and post-translational folding and quality control in the endoplasmic reticulum (ER). Aberrant glycoproteins must be cleared from the secretory pathway. Several mechanisms for glycoprotein degradation exist, of which ER-associated degradation (ERAD) is most well-studied. The latter involves retrotranslocation of misfolded glycoproteins into the cytosol, followed by proteasomal destruction of the polypeptide. N-linked oligosaccharides are removed from glycoproteins during degradation to form free oligosaccharides (FOS). FOS analysis has been employed to gain a global, rather than single protein-centred view of the pathways of ERAD and the mechanisms of its regulation. It was established that convergent ERAD pathways, distinct in the site of deglycosylation, as well as sensitivity to a number of modulators, exist in the cell. It was further demonstrated that the pathway leading to the production of ER-localised FOS is linked to retrograde Golgi-to-ER transport and is masked by the activity of Golgi endomannosidase. Investigating the involvement on the latter in ERAD has uncovered an endomannosidase-mediated pre-degradative glycoprotein processing route and showed that endomannosidase can rescue a fraction of glycoproteins from degradation following inhibition of ER glucosidases. Further investigation of endomannosidase activity and specificity in different cell lines showed that the ability of the enzyme to process truncated monoglucosylated oligosaccharides appears to be necessary and sufficient for evolutionary conservation in mammals. Overall, the data presented have demonstrated on the global level the connections between the pathways for glycoprotein degradation and processing in the Golgi apparatus, underlining the complexity of biosynthetic regulation in the secretory pathway.This thesis is not currently available via ORA

Probing replacement of pyrophosphate via click chemistry; synthesis of UDP-sugar analogues as potential glycosyl transferase inhibitors

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding a-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5 mM in an assay against bovine milk b-1,4-galactosyltransferase