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Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines.

ABSTRACT: Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma. It could potentially regulate the surface expression of GPI-anchored proteins, but it remains to be established which tissue(s) or cell type(s) are the principal sources of the circulating enzyme. Here we report that all the myeloid cell lines tested, including K562 (multipotential blast), KG-1 (human myeloblast), HL-60, NB4, PLB-985 (human promyelocyte), U937 (human promonocyte), THP-1 (human monocyte) and J774, RAW264.7 (mouse monocyte/macrophage), contained GPI-degrading activity. T.l.c. analysis of reaction products confirmed the activity as a phospholipase D. These cells also exhibited positive immunofluorescent staining with an anti-GPI-PLD monoclonal antibody. The expression of GPI-PLD activity was not substantially reduced when the cells were cultured in either serum-free medium or GPI-PLD-depleted regular medium. Both granulocytic and monocytic differentiation of myelomonoblastic lines (e.g. HL-60) induced by dimethyl sulphoxide or phorbol diester respectively was accompanied by a 2-3-fold increase in GPI-PLD activity. J774 and HL-60 cells secreted GPI-PLD into the medium constitutively. Taken together, these data suggest that myeloid cells are a potential contributor to the circulating GPI-PLD pool. As leucocytes express many important GPI-anchored surface antigens, these cells may prove to be a valuable model system for studying the physiological functions of GPI-PLD.

SUBMITTER: Xie M

PROVIDER: S-EPMC1137868 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis.

Project description:Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214-3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.

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Project description:Mammalian serum and plasma contain high levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). Previous studies with crude serum or partially purified GPI-PLD have shown that this enzyme is capable of degrading the GPI anchor of several purified detergent-solubilized cell surface proteins yet is unable to act on GPI-anchored proteins located in intact cells. Treatment of intact ROS17/2.8, WISH or HeLa cells (or membrane fractions prepared from them) with GPI-PLD purified from bovine serum by immunoaffinity chromatography gave no detectable release of alkaline phosphatase into the medium. However, when membranes were treated with GPI-PLD in the presence of 0.1% Nonidet P-40 substantial GPI anchor degradation (as measured by Triton X-114 phase separation) was observed. The mechanism of this stimulatory effect of detergent was further investigated using [3H]myristate-labelled variant surface glycoprotein and human placental alkaline phosphatase reconstituted into phospholipid vesicles. As with the cell membranes the reconstituted substrates exhibited marked resistance to the action of purified GPI-PLD which could be overcome by the inclusion of Nonidet P-40. Similar results were obtained when crude bovine serum was used as the source of GPI-PLD. These data indicate that the resistance of cell membranes to the action of GPI-PLD is not entirely due to the action of serum or membrane-associated inhibitory factors. A more likely explanation is that, in common with many other eukaryotic phospholipases, the action of GPI-PLD is restricted by the physical state of the phospholipid bilayer in which the substrates are embedded. These data may account for the ability of endothelial and blood cells to retain GPI-anchored proteins on their surfaces in spite of the high levels of GPI-PLD present in plasma.

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