Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis.
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.
Du
Xiaohan
X
Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A.
Cai
Jiewei
J
Zhou
Jian-zhong
JZ
Stevens
Victoria L
VL
Low
Martin G
MG
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D009154:Mutation; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D010738:Type C Phospholipases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase