Mannobiose

Abstract: 1 In a crude particulate fraction from yeast mannose is transferred from GDP-mannose to an endogenous “lipid” fraction, the monophosphates of dolichol-14 to dolichol-18 The same membrane fraction also catalyzes the mannosylation of glycoproteins More than 80% of the total radioactivity in the glycoprotein fraction obtained from GDP-[14C]mannose is released by β-elimination The small-sized radioactive products of β-elimination are mannose, mannobiose and mannotriose 2 Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP-[14C]mannose to dolichol monophosphate To the same extent the amount of [14C]mannose obtained after β-elimination of the glycoproteins decreases The amount of radioactive mannobiose and mannotriose is, however, much less affected 3 With decreasing GDP-mannose concentrations the amount of [14C]mannose obtained after β-elimination increases as compared to the amount of radioactive mannobiose and mannotriose The Km-value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km-value for the transfer of subsequent mannosyl residues 4 The mannosylation of dolichol monophosphate from GDP-[14C]mannose proceeds in the presence of Mg2+ and Mn2+ ions as well When solely Mg2+ is present in the incubation mixture, β-elimination of the glycoprotein produced yields almost exclusively mannose, and it is only in the presence of Mn2+ that the normal β-elimination pattern is observed 5 Incubation of the particulate fraction with dolichol-monophosphate-[14C]mannose results in the formation of radioactive glycoprotein; again most of the radioactivity is released by β-elimination but only [14C]mannose is obtained as product When dolichol-monophosphate [14C]mannose is incubated together with non-radioactive GDP-mannose, β-elimination yields radioactive mannose, mannobiose and mannotriose Mannobiose is found to be labelled in the reducing end only 6 The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, ie in the formation of mannosyl linkages to serine and/or threonine The subsequent mannosylation proceeds directly from GDP-mannose in a reaction obligatorily requiring Mn2+ 7 After β-elimination of the methanol-insoluble material mannosylated in the presence of dolichol-monophosphate [14C] mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase Mannose, therefore, is transferred from dolichol-monophosphate mannose also to glycoprotein positions not involving OH-groups of serine or threonine