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

Abstract

Glucosaminyl(acyl)phosphatidylinositol [GlcN(acyl)PI], the third intermediate in the mammalian glycosylphosphatidylinositol (GPI) anchor pathway, is undetectable in most cells. This intermediate was previously shown to accumulate, however, in murine lymphoma mutant E and in yeast mutant dpm1, both of which lack dolicholphosphomannose synthase activity. Here we report that a mammalian HeLa S3 subline, denoted D, produces large amounts of GlcN(acyl)PI. The level of GlcN(acyl)PI in this subline is twice that in the murine lymphoma mutant E and 4 times that in the parental S3 line. This HeLa D subline differs from the previously reported mutants that accumulate GlcN(acyl)PI because no defects in the synthesis or utilization of dolicholphosphomannose were found. Kinetic analysis indicated that in this HeLa subline there is an increased rate of synthesis of GlcN(acyl)PI, whereas the rate of metabolism for this GPI is comparable to that in wild-type cells. Furthermore, HeLa D cells accumulate GlcN(acyl)PI without a block in the synthesis of the downstream mannosylated GPI anchor precursors and GPI-anchored proteins. These findings might be relevant for understanding the regulation of the GPI pathway.

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

“…To establish that these species are in fact derived from GPI-PLD hydrolysis, we repeated the above experiment, but with 3 H-inositol-labelled HeLa D cells. HeLa D is a subline of HeLa S3 cells that accumulates GlcN-(acyl)PI [27], the third intermediate of the GPI biosynthetic pathway. The advantage of using this approach is that GlcN-(acyl)Inos, the GPI-PLD cleavage product of GlcN-(acyl)PI, can be easily identified, because it moves further away from the origin of the TLC plate [29], in contrast with the GPI-PLD products of mannosylated GPI anchor intermediates that stay at or very close to the origin.…”

“…3-fold higher activity in the medium and in cell lysates (using 3 H-mannose-labelled GPI anchor intermediates as substrates) compared with that of untransfected HeLa cells, was used for the studies. HeLa D cells, isolated in our laboratory [27], were labelled with 10 µCi of [ 3 H]inositol for 3 days in two 100 mm plates in inositol-free DMEM, supplemented similar to HeLa medium but with 10 % (v/v) dialysed newborn calf serum instead of 10 % foetal calf serum. After the incubation period, the cells were washed and one-fifth of the cells were extracted in CMW (chloroform/methanol/water, 10:10:3).…”

Glycosylphosphatidylinositol-anchor intermediates associate with Triton-insoluble membranes in subcellular compartments that include the endoplasmic reticulum

“…The HeLa D cell line isolated in our laboratory from HeLa S3, as described in Sevlever et al [32], was maintained in DMEM supplemented with 10 % newborn calf serum, 100 i.u.\ml penicillin, 100 µg\ml streptomycin and 2 mM glutamine in an atmosphere of 5 % CO # . Erythroleukaemia K562 cells and the derived mutant K cells [33] were cultured in RPMI 1640 supplemented with DMEM.…”

Metabolism of exogenous sn-1-alkyl-sn-2-lyso-glucosaminyl-phosphatidylinositol in HeLa D cells: accumulation of glucosaminyl(acyl)phosphatidylinositol in a metabolically inert compartment

“…[22]. While these cells accumulated unusual amounts of GlcN(acyl)PI (10( molecules\cell determined by metabolic labelling with [$H]inositol [15]), mannosylated GPIs were observed in amounts typical of other cell lines.…”

“…[14], even though GlcN(acyl)PI was effectively labelled and accumulated in a relatively large amount. To investigate the difficulty in labelling mannosylated GPIs in these cells, we asked whether the large amount of GlcN(acyl) [ [22].…”