INVOLVEMENT OF MANNOSYL-PHOSPHORYL-DOLICHOLS IN MANNOSE TRANSFER TO BRAIN GLYCOPROTEINS

Abstract— Mannose was transferred from GDP-¹⁴C]mannose by homogenates of embryonic chick and adult rat brain to mannolipids with properties identical to manriosyl-phosphoryl-dihydropolyisoprenols. Embryonic chick brain formed six-fold larger quantities of mannolipid than adult rat brain. The reaction was stimulated by Mn²⁺ ions and Triton X-100 but inhibited by EDTA. Phosphoenolpyruvic acid had no effect on the reaction. A crude mitochondrial fraction was two to three times more active than the microsomal fraction. All radioactivity in the mannolipid could be displaced by the addition of non-radioactive GDP-mannose. The endogenous lipid acceptor in brain was readily labelled in vivo by injection of ³H]mevalonate into the amniotic sac of 7-day-old embryos. The mannolipid formed had the properties of an acidic phospholipid on column and TLC, was stable to dilute alkali but readily cleaved by dilute acid. Synthesis was markedly stimulated by the addition of pig liver or calf brain dolichol phosphate in the presence of Triton X-100 and Mn²⁺. The mannolipid so formed displayed chemical characteristics identical to the endogenous lipid acceptor. Incubation of the purified radioactive mannolipid with the 'post-nuclear' fraction from 14-day-old embryonic chick brain in the presence of EDTA and Triton X-100 resulted in the transfer of 40-50 per cent of the radioactive mannose to protein and 40-45 per cent to water soluble compounds. The efficiency of transfer of radioactivity from endogenously formed mannolipid with or without the addition of dolichol phosphate was similar to exogenously added highly purified mannolipid. These results are compatible with the hypothesis that synthesis of the mannose core of brain glycoproteins involves the synthesis first of mannosyl-phosphoryl-dolichols followed by transfer of the mannose to glycoprotein.