title: The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.
abstract: Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Petrescu
authors.forename: T D
authors.initials: TD
authors.lastname: Butters
authors.forename: G
authors.initials: G
authors.lastname: Reinkensmeier
authors.forename: S
authors.initials: S
authors.lastname: Petrescu
authors.forename: F M
authors.initials: FM
authors.lastname: Platt
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: M R
authors.initials: MR
authors.lastname: Wormald
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D000520:alpha-Glucosidases / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D006023:Glycoproteins; D008351:Mannans; D009844:Oligosaccharides; D011134:Polysaccharides; D017485:1-Deoxynojirimycin; C059896:miglustat; D000520:alpha-Glucosidases