Metabolism of Lipid‐Linked Oligosaccharide Intermediates in Rat Spleen Lymphocytes

Abstract

Double-labeling experiments show that intact lymphocytes and lymphocyte homogenates can utilize GDP-[14C]mannose and UDP-N-[3H]acetylglucosamine to synthesize lipid-linked oligosaccharide intermediates. The intermediates formed are quantitatively and qualitatively different in the 2 systems. The amount of dolichyl diphosphate oligosaccharides synthesized in both cases was calculated by using external labeling by sodium boro[3H]hydride reduction of the glycan moiety obtained after mild acid treatment of [14C]mannose-labeled dolichyl diphosphate oligosaccharides. Due to the liberation of intracellular enzymes, a larger amount of dolichyl diphosphate oligosaccharides was synthesized by homogenate. This higher glycosyltransferase activity was not detected by the direct measurement of incorporation of labeled 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. 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. Ectoglycosyltransferases involved in the dolichyl cycle are present at the outer surface of lymphocytes.