title: Dolichol is not a necessary moiety for lipid-linked oligosaccharide substrates of the mannosyltransferases involved in in vitro N-linked-oligosaccharide assembly.
abstract: Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of glycosyltransferases. The role of dolichol in enzyme substrate recognition is investigated. Thus the biosynthetic intermediate NN'-diacetylchitobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as substrates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[3H]Mannosylated lipids from this reaction were then used as substrates for the subsequent mannosyltransferases from yeast or rat liver microsomes. It was found that both the dolichyl- and phytanyl-linked substrates were easily mannosylated to form Man5GlcNAc2, with some further mannosylation to Man7GlcNAc2 and Man9GlcNAc2 at low concentrations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransferases in the biosynthetic pathway for N-glycosylation.
authors.affiliation: Dyson Perrins Laboratory, University of Oxford, U.K.
authors.forename: I B
authors.initials: IB
authors.lastname: Wilson
authors.forename: M C
authors.initials: MC
authors.lastname: Webberley
authors.forename: L
authors.initials: L
authors.lastname: Revers
authors.forename: S L
authors.initials: SL
authors.lastname: Flitsch
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004286:Dolichols / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014316:Tritium
chemical_list: D004286:Dolichols; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014316:Tritium; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D008358:Mannose