Mannosylphosphoryldolichol-mediated reactions in oligosaccharide-P-P-dolichol biosynthesis: Recognition of the saturated α-isoprene unit of the mannosyl donor by pig brain mannosyltransferases

The specificity of Man-P-Dol:Man_5-8GlcNAc₂-P-P-Dol (Oligo-P-P-Dol) mannosyltransferase activity in pig brain was investigated by comparing a variety of mannosylphosphorylisoprenols as mannosyl donors. For this comparison the β-Man-P-isoprenols were synthesized using a partially purified preparation of mannosylphosphorylundecaprenol (Man-P-Undec) synthase from Micrococcus luteus. The bacterial mannosyltransferase efficiently catalyzed the transfer of mannose from GDP-[³H]Man to a series of defined isoprenyl monophosphate substrates. Two α-Man-P-dolichols were synthesized chemically and also examined as substrates. When exogenous β-[³H]Man-P-Dol₉₅ was tested as a substrate for Man-P-Dol:Oligo-P-P-Dol mannosyltransferase activity in pig brain microsomes, [³H]mannose was actively transferred to endogenous Oligo-P-P-Dol acceptors. The major enzymatically labeled product was Man₉GlcNAc₂-P-P-Dol. Under identical conditions β-[³H]mannosylphosphorylpolyprenol (Man-P-Poly₉₅) was an extremely poor substrate, indicating that the saturated α-isoprene unit of the dolichyl moiety is critical for recognition of the lipophilic mannosyl donor by the endoplasmic reticulum-associated mannosyltransferase(s). When Man-P-dolichols containing 2, 11, or 19 isoprene units were compared, the initial rates for the mannosyl transfer reactions and the affinity of the enzyme(s) for the mannophospholipid substrate increased with the length and hydrophobicity of the polyisoprenol chain. The anomeric configuration of the mannosyl moiety is apparently essential because the brain mannosyltransferases exhibited a strong preference for β-Man-P-dolichols over the corresponding chemically synthesized α-stereoisomers. These results: 1) describe a simple two-step procedure for obtaining a partially purified preparation of Man-P-Undec synthase that efficiently synthesizes a variety of β-Man-P-isoprenols; 2) indicate that pig brain Man-P-Dol:Oligo-P-P-Dol mannosyltransferase activity is relatively specific for lipophilic mannosyl donors containing 19 isoprene units with a β-Man 1-P group attached to the saturated α-isoprene unit of dolichol; and 3) emphasize the importance of the reduction of the α-isoprene unit in the biosynthesis and function of Dol-P in mammalian cells.