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.

Abstract

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-i was 17% of the wild-type level, whereas the surface expression of Ly-6A was 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. (J. Clin. Invest.

Compositional analysis of Glucosaminyl(acyl)phosphatidylinositol Accumulated in HeLa S3 Cells

“…These mutants are defective in Dol-P-Man synthase and make no Dol-P-Man, a cofactor necessary both for mannosylation steps immediately downstream from GlcN(acy1)PtdIns in the glycosyl-PtdIns biosynthetic pathway and for the synthesis of N-glycans. Leaky mutants of CHO cells (Singh and Tartakoff, 1991) and T cell hybridomas (Thomas et al, 1992) also have been reported that make very small amounts of Dol-P-Man. Expression of glycosyl-PtdIns-anchored proteins in these mutants is increased from low levels to near normal by culturing with tunicamycin, an agent that blocks N-glycan synthesis but does not inhibit the glycosyl-PtdIns pathway.…”

Activation-induced Aggregation and Processing of the Human Fas Antigen

“…Flow Cytometry Analysis-Indirect immunofluorescent staining was performed as described (15). 1 ϫ 10 6 cells were incubated with 1.25 g/ml of DX2 for 30 min on ice followed by fluorescein isothiocyanatelabeled goat anti-mouse IgG (Caltag Laboratories, San Francisco, CA).…”

Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity

“…GlcN(acyl)PI is the substrate for the first mannosyltransferase, which uses Dol-P-Man as the mannose donor. Two types of defect affecting Dol-P-Man can disrupt the normal mannosylation of GPIs and result in the potential for GlcN(acyl)PI accumulation : (1) total lack of Dol-P-Man synthase activity (as found in mutant E cells) or partial lack of Dol-P-Man synthase activity (as represented in CHO cell [17] and T-cell hybridoma mutants) [23], and (2) impaired Dol-P-Man utilization (as represented in Lec 35). In view of the above findings that HeLa D cells are the highest producers of GlcN(acyl)PI reported, the following experiments were designed to answer whether the defect in these cells involves Dol-P-Man synthesis or utilization.…”

“…Cell mutants that are partly deficient in Dol-P-Man produce smaller amounts of mannosylated-GPIs and GPI-anchored proteins [17,23]. In these leaky Dol-P-Man synthase mutants, the levels of mannosylated-GPIs and GPI-anchored proteins can be boosted by the addition of tunicamycin [17,23]. Tunicamycin inhibits the synthesis of Glu $ Man * GlcNAc # PPDol, the precursor of N-glycans on proteins, and Dol-P-Man is then used only in the GPI pathway.…”