Dolichol‐Dependent Synthesis of Chitobiosyl Proteins and Their Further Mannosylation

Incubation of whole lymphocytes with UDP‐N‐acetyl [ 3 H]glucosamine used as the only preeursor leads to the formation of dolichyl diphosphate [ 3 H]chitobiose, DolPP‐(GlcNAc) 2 , and dolichyl diphosphate N ‐acetyl‐ [ 3 H]glucosamine, DolPP‐GlcNAc. Although very few dolichyl diphosphate oligosaccharidcs are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [ 3 H]chitobiosyl and N ‐acetyl[ 3 H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DoIPP‐(GIcNAc) 1−2 and the incorporation of the N ‐acetyl [ 3 H] glucobaminyl 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 matcrial. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DoIPP‐(GIcNAc) 1−2 is not recovered on proteins. This fact allows to show that part of the [ 3 H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP‐mannose. This direct niannosylation of chitobiosyl‐proteins may represent a second route for the N‐glycosylation of proteins.