DOLICHOL IS NOT A NECESSARY MOIETY FOR LIPID-LINKED OLIGOSACCHARIDE SUBSTRATES OF THE MANNOSYLTRANSFERASES INVOLVED IN IN-VITRO N-LINKED-OLIGOSACCHARIDE ASSEMBLY

Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of g lycosyltransferases. The role of dolichol in enzyme substrate recognit ion is investigated. Thus the biosynthetic intermediate NN'-diacetylch itobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as sub strates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[ H-3]Mannosylated lipids from this reaction were then used as substrate s for the subsequent mannosyltransferases from yeast or rat liver micr osomes. It was found that both the dolichyl- and phytanyl-linked subst rates were easily mannosylated to form Man(5)GlcNAc(2), with some furt her mannosylation to Man(7)GlcNAc(2) and Man9GlcNAc(2) at low concentr ations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransfera ses in the biosynthetic pathway for N-glycosylation.