MANNOSYLPHOSPHORYLDOLICHOL-MEDIATED REACTIONS IN OLIGOSACCHARIDE-P-P-DOLICHOL BIOSYNTHESIS - RECOGNITION OF THE SATURATED ALPHA-ISOPRENE UNIT OF THE MANNOSYL DONOR BY PIG BRAIN MANNOSYLTRANSFERASES

The specificity of Man-P-Dol:Man5-8GlcNAC2-P-P-Dol (Oligo-P-P-Dol) man nosyltransferase activity in pig brain was investigated by comparing a variety of mannosylphosphorylisoprenols as mannosyl donors. For this comparison the beta-Man-P-isoprenols were synthesized using a partiall y purified preparation of mannosylphosphorylundecaprenol (Man-P-Undec) synthase from Micrococcus luteus. The bacterial mannosyltransferase e fficiently catalyzed the transfer of mannose from GDP-[H-3]Man to a se ries of defined isoprenyl monophosphate substrates. Two alpha-Man-P-do lichols were synthesized chemically and also examined as substrates. W hen exogenous beta-[H-3]Man-P-Dol95, was tested as a substrate for Man -P-Dol:Oligo-P-P-Dol mannosyltransferase activity in pig brain microso mes, [H-3]mannose was actively transferred to endogenous Oligo-P-P-Dol acceptors. The major enzymatically labeled product was Man9GlcNAC2-P- P-Dol. Under identical conditions beta-[H-3]mannosylphosphorylpolypren ol (Man-P-Poly95) was an extremely poor substrate, indicating that the saturated alpha-isoprene unit of the dolichyl moiety is critical for recognition of the lipophilic mannosyl donor by the endoplasmic reticu lum-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 hydrophobici ty of the polyisoprenol chain. The anomeric configuration of the manno syl moiety is apparently essential because the brain mannosyltransfera ses exhibited a strong preference for beta-Man-P-dolichols over the co rresponding chemically synthesized alpha-stereoisomers. These results: 1) describe a simple two-step procedure for obtaining a partially pur ified preparation of Man-P-Undec synthase that efficiently synthesizes a variety of beta-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 bet a-Man 1-P group attached to the saturated alpha-isoprene unit of dolic hol; and 3) emphasize the importance of the reduction of the alpha-iso prene unit in the biosynthesis and function of Dol-P in mammalian cell s.