Abstract
Biosynthetic intermediates for the mammalian glycosylphosphatidylinositol (GPI) anchor have been described. The earliest GPI anchor precursor is N-acetylglucosaminylphosphatidylinositol, which is deacetylated to give glucosaminylphosphatidylinositol. This is followed by fatty acylation of the inositol ring, sequential addition of mannose residues donated by dolichyl mannosyl phosphate, and finally addition of ethanolamine phosphate. Here, we show that the final steps of GPI anchor biosynthesis are more complex than we have previously reported. Six distinct GPI anchor precursors were found to contain at least 1 ethanolamine phosphate residue. The headgroups of these glycolipids were purified and analyzed by a combination of Bio-Gel P4 chromatography and high resolution thin-layer chromatography. The sizes of neutral glycans were determined following HF dephosphorylation. The position of the ethanolamine phosphate residue was inferred from results of alpha-mannosidase treatment. Finally, the number of negative charges on the headgroups were determined by Mono Q chromatography. Our results show that the addition of ethanolamine phosphate is controlled by at least two different genes. Thus, the class F mutant, though unable to add ethanolamine phosphate to the third mannose residue, does incorporate ethanolamine phosphate into the first and second mannose residues. Only the wild type cells are capable of incorporating ethanolamine phosphate into the third mannose residue. Furthermore, the GPI core contains up to 3 ethanolamine phosphate residues. These results should facilitate the elucidation of the biochemical defects in paroxysmal nocturnal hemoglobinuria.
Highlights
The costs of publication of this article were defrayed in part by the phosphorylated by HF treatment
Our results show that ethanolamine phosphate cabne added to allof the mannose residues in the GPcIore
The disproportionately The headgroup ofM1 (ELIIF) was completely resistant high radioactivity inthe free mannose peak (Fig. 9D) is t o a-mannosidase treatmentbecause ethanolamine phosphate expected since the M3 (EL4/F) sample was prepared from must be attached to the one and only mannose residue (Fig. cells after only short term metabolic labeling. 9F).It should be emphasized that we routinely added a second The a-mannosidaseresults for derived from the EL4/ aliquot of a-mannosidase to the reaction mixture after the F mutant clearly show that ethanolamine phosphate can be initial incubation to ensure complete treatment
Summary
GPIs-We have previously described a GPI precursor called “core”which contains inositol, mannose, glucosamine, and ethanolamine (11, 12, 16). Core could contain additional polarmodifications in the glycan structure Inordertodistinguish between these possibilities, we treated [3H]mannose o[r 3H]ethanolamine-labeledglycolipids withnitrous acid and analyzed the releasedradiolabeled glycans(termed“headgroup”hereafter) by gel filtration chromatographyon Bio-Gel P4. YH 16.33 chromatographed the products on a Bio-Gel P4 column.A portion of the purified coreglycolipid was rechromatographed on a TLC plate to confirm its identit(yFig. 2A, lane 3 ) .The headgroup derived from thecore region gave rise to two peaks (I and; Fig. 2B). These resultssuggested that theheadgroup of core contained additional modificationswhen compared with P2 and P3.
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