Dolichol-dependent synthesis of chitobiosyl proteins and their further mannosylation. A second route for glycosylation of proteins in rat-spleen lymphocytes?

Abstract

Incubation of whole lymphocytes with UDP-N-acetyl [3H]glucosamine used as the only precursor leads to the formation of dolichyl diphosphate [3H]chitobiose, DolPP-(GlcNAc)2, and dolichyl diphosphate N-acetyl-[3H]glucosamine, DolPP-GlcNAc. Although very few dolichyl diphosphate oligosaccharides are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N-acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DolPP-(GlcNAc)1-2 and the incorporation of the N-acetyl[3H] glucosaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP-(GlcNAc)1-2 were utilized in two ways: either their transfer onto proteins or their degradation into water-soluble saccharidic material. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DolPP-(GlcNAc)1-2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP-mannose. This direct mannosylation of chitobiosyl-proteins may represent a second route for the N-glycosylation of proteins.