Membrane Glycoproteins: I. ENZYMATIC SYNTHESIS OF MANNOSYL PHOSPHORYL POLYISOPRENOL AND ITS ROLE AS A MANNOSYL DONOR IN GLYCOPROTEIN SYNTHESIS

Abstract

Membranous particulate preparations of hen oviduct and bovine thyroid catalyze the transfer of mannose from GDP-mannose to endogenous acceptors. One of the mannosylated acceptors is a lipid, which has been partially purified and characterized. Mild acid hydrolysis of labeled mannosyl lipid yields [14C]mannose, whereas alkaline hydrolysis produced [14C]mannose phosphate. The mannosyl lipids formed in both enzyme preparations are chromatographically identical to synthetic mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannosyl lipid synthesis in oviduct preparations 7-fold. Synthesis of mannosyl lipid is freely reversible; in the presence of GDP the transfer of mannosyl groups from endogenously labeled mannosyl lipid to GDP-mannose is observed. These results indicate that the mannosyl lipid has the structure of mannosyl phosphoryl polyisoprenol; the precise chain length of the polyisoprenyl moiety remains to be established. A kinetic study of the transfer of mannose from GDP-mannose to endogenous acceptor in oviduct membranes revealed that, in addition to mannosyl phosphoryl polyisoprenol, two other labeled components are formed. One of these, termed soluble mannosylated endogenous acceptor (mannosyl s-acceptor), has the unique property of being insoluble in chloroform-methanol (2:1) and in water, but is soluble in chloroform-methanol-water (1.0:1.0:0.3). The structure of mannosyl s-acceptor is unknown. The other labeled component, termed residual mannosylated endogenous acceptor (mannosyl r-acceptor), is insoluble in all of the above named solvents. Based on gel filtration and electrophoresis studies performed on mannosyl r-acceptor before and after treatment with proteolytic enzymes it is concluded that this component consists of one or more mannosylated proteins. Several lines of evidence indicate that in oviduct membranes mannosyl phosphoryl polyisoprenol is an obligatory intermediate in the synthesis of mannosyl s-acceptor and the mannosylated protein(s) from GDP-mannose. (a) The formation and turnover of mannosyl phosphoryl polyisoprenol is rapid. Mannosyl s-acceptor is formed more slowly and turns over more slowly. The mannosylated protein(s) is formed even more slowly, and does not turn over. (b) The effect of a chase with unlabeled GDP-mannose on synthesis of labeled mannosyl phosphoryl polyisoprenol, mannosyl s-acceptor and the mannosylated protein(s) was immediate cessation of synthesis of labeled mannosyl phosphoryl polyisoprenol; in contrast, a marked delay was observed in cessation of synthesis of labeled mannosyl s-acceptor and of the mannosylated protein(s). (c) Exogenous, partially purified mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of mannosyl s-acceptor and the mannosylated protein(s). Because both the enzymes involved in these reactions and the mannosylated protein(s) produced are associated with membranous components of the cell-free preparations, it is possible that this enzyme system plays an important role in assembly of membrane glycoproteins.