The mannosylation of dolichol diphosphate oligosaccharides and the formation of oligosaccharides and glycoproteins in pig liver microsomal preparations.

After incubation of GDP-[14C]mannose with pig liver microsomal preparations [14C]mannose can be recovered in dolichol monophosphate mannose (dol-P-man), dolichol diphosphate oligosaccharides (dol-P-P-oligosac), oligosaccharide and glycoprotein. The identification and formation of dolichol phosphate mannose has been described in detail (Richards & Hemming, 1972; Evans & Hemming, 1973). Identification of dolichol diphosphate oligosaccharides was based on hydrolytic and chromatographic data. Mobility on t.1.c. and on DEAE-cellulose was typical of dolichol diphosphate sugars. Stability to mild treatment with alkali, but lability to mild treatment with acid confirmed this view. In addition, strong treatment with alkali released 14Clabelled oligosaccharide phosphate which was susceptible to calf intestinal alkaline phosphatase. The nature of the 14C-labelled oligosaccharide portion was investigated after its release by mild treatment with acid. T.1.c. showed this product to be a mixture of seven 14C-labelled oligosaccharides containing seven to 13 sugar residues each. The effects of strong treatment with alkali of the oligosaccharide mixture, followed by acetylation, were monitored by electrophoresis. This indicated that the essentially neutral oligosaccharide contained at least two N-acetylhexosamine residues per oligosaccharide chain. Strong treatment with acid yielded [14C]mannose. Periodateoxidation and NaBH, reduction showed that 60 % of the [14C]mannose was at the non-reducing terminus of the oligosaccharide chains, the other 40% being internal. It was argued that the microsomal preparation had catalysed the transfer of one [14C]mannose residue (sometimes two) to the non-reducing end of the sugar chains of endogenous dolichol diphosphate oligosaccharides. Reduction of the reducing termini of the acid-released mixture of [“C]oligosaccharides with NaB3H4 followed by preparative chromatography and 3H assay enabled assessment of the molar proportions of the oligosaccharides. Determination of the 14C/3H ratios of each oligosaccharide showed that the quantitatively minor components of the mixture had incorporated on average one, or slightly more than one, new mannose residue per chain, but that the major components contained on average only one newly incorporated mannose residue in every four chains.