Metabolism of lipid-linked oligosaccharide intermediates in rat spleen lymphocytes. Evidence for ectoglycosyltransferase activities.

Double-labelling experiments show that intact lymphocytes as well as lymphocyte homogenates can utilize GDP-[14C]mannose and UDP-N-[3H]acetylglucosamine to synthesize lipid-linked oligosaccharide intermediates. However, the intermediates formed are quantitatively and qualitatively different in the two systems. The amount of dolichyl diphosphate oligosaccharides synthesized in both cases was calculated by using external labelling by sodium boro[3H]hydride reduction of the glycan moiety obtained after mild acid treatment of [14C]mannose-labelled dolichyl diphosphate oligosaccharides. This showed that, due to the liberation of intracellular enzymes, a larger amount of dolichyl diphosphate oligosaccharides was synthesized by homogenate. However, this higher glycosyltransferase activity was not detected by the direct measurement of incorporation of labelled GDP-[14C]mannose and UDP-N-[3H]acetylglucosamine, due to isotopic dilution caused by both endogenous soluble UDP-N-acetylglucosamine and membrane-bound dolichyl phosphate mannose accumulated during the homogenization process. In addition, endogenous UDP-glucose allowed the formation, by homogenate, of glucosylated dolichyl diphosphate oligosaccharides which were not observed with intact cells unless exogenous UDP-glucose was added. These striking differences between the lipid intermediates synthesized by homogenate or by intact cells exclude the possibility that intracellular glycosyltransferases could account for the glycosyltransferase activities observed with whole lymphocyte suspensions. This allows us to conclude that ectoglycosyltransferases involved in the dolichol cycle are present at the outer surface of lymphocytes.