The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose.

Thy-1mutant mouse lymphoma cells of the class E complementation group are unable to synthesize the normal GlclManBGlcNAcz lipid-linked oligosaccharide, but instead accumulate a smaller lipid-linked species with the structure Manal -+ 2Manal 3 tMana1 -+ 3(Manal+ G)Ma*l-+ 4GlcNAc/?l+ 4GlcNAc (Trowbridge, I., and Hyman, R. (1979) Cell 17, 503-508 and Chapman, A., Trowbridge, I., Hyman, R., and Kornfeld, S. (1979) Cell 17, 509-515). The present study demonstrates that the primary defect in the Thy-1cells is an inability to synthesize dolichol-P-mannose. Thus, intact Thy-1cells incubated with [2-3H]mannose failed to incorporate any detectable radioactivity into dolicholP-mannose and crude membrane preparations of Thy1cells were unable to transfer mannose from GDP[3H]mannose to dolichol-P. However, these membrane preparations did incorporate [3H]mannose into lipidlinked oligosaccharides up to the size of Man6GlcNAcz, suggesting that these species are formed from mannosyl donors other than dolichol-P-mannose. When class E Thy-1membrane preparations were incubated with exogenous dolichol-P-f3H]mannose, lipid-linked oligosaccharides ranging in size from MansGlcNAcz to MansGlcNAcz were formed. This result demonstrates that the mutant cells have the a1,3-mannosyltransferase necessary for the conversion of the Man5GlcNAcz species to Man,GlcNAcz, but lack the appropriate mannosy1 donor (dolichol-P-mannose) necessary for the formation of the larger lipid-linked oligosaccharides. We conclude that at least two mannosyl donors are involved in the synthesis of lipid-linked oligosaccharides. GDP-mannose is the probable donor for the formation of the Manl&1cNAc2 species, while dolicho1-Pmannose donates the 6th mannosyl residue and probably mannose residues 7 through 9.