Characterization of abnormal free glycophosphatidylinositols accumulating in mutant lymphoma cells of classes B, E, F, and H.

Several mutant lymphoma lines are unable to add glycophosphatidylinositol membrane anchors to proteins. Some of them accumulate abnormal glycolipids which can be labeled by tritiated myo-inositol, mannose, or ethanolamine and which are not present in the corresponding parental cell lines. The [3H]myo-inositol-labeled abnormal lipids were isolated and characterized using hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, and exoglycosidase treatments alone or in combination. This partial characterization suggests that the class F mutant EL-4-f contains 3 abnormal glycolipids containing 3, 2, or 1 mannose residues, the headgroups of which are Man alpha 1,2Man alpha 1,6(X-->)Man alpha-GlcN-acylinositol, Man alpha 1,6(X-->)Man alpha-GlcN-inositol, and (X-->)Man alpha-GlcN-acylinositol where X represents a charged, hydrofluoric acid-sensitive substituent. A fourth, minor abnormal lipid with a Man alpha 1,6(X-->)Man alpha-GlcN-inositol headgroup but a different lipid moiety is also found. The substituent X is likely to consist of phosphoethanolamine since hydrofluoric acid releases [3H]ethanolamine from the [3H]ethanolamine-labeled version of these lipids. Pulse-chase experiments indicate that the abnormal glycophosphatidylinositols of class F mutants are very long-lived. The class B mutant S1A-b has previously been shown to contain an abnormal Man alpha 1,6(phosphoethanolamine-->)Man alpha-GlcN-acylinositol-P-lipid intermediate. Here we show that S1A-b also accumulates a more polar but less abundant lipid which has the identical headgroup structure but lacks the acyl group on the inositol residue. The class E mutant BW5147-e accumulates a hydrophobic glycolipid with the headgroup structure GlcN-acylinositol. All the abnormal glycolipids except those of EL-4-f are heterogeneous with regard to their lipid moiety since base-resistant as well as base-sensitive lipids are present. This suggests that the base-resistant alkylglycerols typical of mammalian anchors can get integrated into anchors at early stages of glycophosphatidylinositol formation.