Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.
A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.
Thomas
L J
LJ
Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
Urakaze
M
M
DeGasperi
R
R
Kamitani
T
T
Sugiyama
E
E
Chang
H M
HM
Warren
C D
CD
Yeh
E T
ET
D000818:Animals; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D006457:Hemoglobinuria, Paroxysmal / Q000378:metabolism*; D006825:Hybridomas / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols