Dolichol‐Dependent Synthesis of Chitobiosyl Proteins and Their Further Mannosylation

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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 was characterized, using a 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 protein acceptors from their dolichol derivatives. Chase experiments indicated that the DolPP-(GlcNAc)1-2 were utilized in 2 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. 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 2nd route for the N-glycosylation of proteins.