The mannosylation of dolichol-diphosphate oligosaccharides in relation to the formation of oligosaccharides and glycoproteins in pig-liver endoplasmic reticulum.

Abstract

In the chain-lengthening of the oligosaccharide chains of endogenous dolichol-diphosphate oligosaccharides (DOL-P-P-oligosaccharides) by a pig liver microsomal preparation dolichol-monophosphate mannose (Dol-P-Man) was a more efficient donor of mammose (a maximum of 35% transferred by 5 min) than was GDP-Man (reaching 16% by 45 min). The effects of an excess of GDP, an excess of GDP-Man, a lack of Mn2+ and an excess of EDTA showed that the transfer from GDP-Man was via Dol-P-Man. The evidence also indicated the presence of two pools of Dol-P-Man one of which was difficult to extract and which was possibly closely associated with the Dol-P-P-oligosaccharides and the appropriate transferase. After 1 h of incubation transfer of 14C to 'insoluble polymer' from GDP-[14C]Man, Dol-P-[14C]Man and Dol-P-P-[14C]oligosaccharides reached approximately 3%, 3% and 13% respectively, of that available. The result of adding excess unlabelled GDP-Man to an incubation with GDP-[14C]Man in progressconfirmed the sequence GDP-Man leads to Dol-P-Man leads to Dol-P-P-oligosaccharide leads to insoluble polymer. Solubilisation with sodium dodecyl sulphate of the radioactive 'insoluble polymer' followed by gel chromatography showed the presence of radioactive glycoprotein and oligosaccharide when either GDP-[14C]Man or Dol-P-P-[14C]oligosaccharide was used as donor. The proportion of oligosaccharide formed rose sharply when excess EDTA was present and GDP-[14C]Man was the donor. Under these conditions the oligosaccharide contained 5--6 units and all of the radioactivity could be released by alpha-mannosidase. The glycoprotein was susceptible to proteolysis.