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. 2002 Jan 1;361(Pt 1):113–118. doi: 10.1042/0264-6021:3610113

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

Xiaohan Du 1, Jiewei Cai 1, Jian-zhong Zhou 1, Victoria L Stevens 1, Martin G Low 1
PMCID: PMC1222285  PMID: 11742535

Abstract

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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Selected References

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  1. Almeida I. C., Camargo M. M., Procópio D. O., Silva L. S., Mehlert A., Travassos L. R., Gazzinelli R. T., Ferguson M. A. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J. 2000 Apr 3;19(7):1476–1485. doi: 10.1093/emboj/19.7.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Azzouz N., Kedees M. H., Gerold P., Becker S., Dubremetz J. F., Klenk H. D., Eckert V., Schwarz R. T. An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection. Glycobiology. 2000 Feb;10(2):177–183. doi: 10.1093/glycob/10.2.177. [DOI] [PubMed] [Google Scholar]
  3. Baumann N. A., Vidugiriene J., Machamer C. E., Menon A. K. Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells. J Biol Chem. 2000 Mar 10;275(10):7378–7389. doi: 10.1074/jbc.275.10.7378. [DOI] [PubMed] [Google Scholar]
  4. Davitz M. A., Hom J., Schenkman S. Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma. J Biol Chem. 1989 Aug 15;264(23):13760–13764. [PubMed] [Google Scholar]
  5. De Angelis D. A., Miesenböck G., Zemelman B. V., Rothman J. E. PRIM: proximity imaging of green fluorescent protein-tagged polypeptides. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12312–12316. doi: 10.1073/pnas.95.21.12312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Du X., Low M. G. Down-regulation of glycosylphosphatidylinositol-specific phospholipase D induced by lipopolysaccharide and oxidative stress in the murine monocyte- macrophage cell line RAW 264.7. Infect Immun. 2001 May;69(5):3214–3223. doi: 10.1128/IAI.69.5.3214-3223.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferguson M. A. Site of palmitoylation of a phospholipase C-resistant glycosylphosphatidylinositol membrane anchor. Biochem J. 1992 Jun 1;284(Pt 2):297–300. doi: 10.1042/bj2840297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Güther M. L., Leal S., Morrice N. A., Cross G. A., Ferguson M. A. Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei. EMBO J. 2001 Sep 3;20(17):4923–4934. doi: 10.1093/emboj/20.17.4923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang K. S., Li S., Fung W. J., Hulmes J. D., Reik L., Pan Y. C., Low M. G. Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D. J Biol Chem. 1990 Oct 15;265(29):17738–17745. [PubMed] [Google Scholar]
  10. Iyer V. R., Eisen M. B., Ross D. T., Schuler G., Moore T., Lee J. C., Trent J. M., Staudt L. M., Hudson J., Jr, Boguski M. S. The transcriptional program in the response of human fibroblasts to serum. Science. 1999 Jan 1;283(5398):83–87. doi: 10.1126/science.283.5398.83. [DOI] [PubMed] [Google Scholar]
  11. Jones D. R., Avila M. A., Sanz C., Varela-Nieto I. Glycosyl-phosphatidylinositol-phospholipase type D: a possible candidate for the generation of second messengers. Biochem Biophys Res Commun. 1997 Apr 17;233(2):432–437. doi: 10.1006/bbrc.1997.6475. [DOI] [PubMed] [Google Scholar]
  12. Küng M., Bütikofer P., Brodbeck U., Stadelmann B. Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells. Biochim Biophys Acta. 1997 Jul 24;1357(3):329–338. doi: 10.1016/s0167-4889(97)00044-x. [DOI] [PubMed] [Google Scholar]
  13. LeBoeuf R. C., Caldwell M., Guo Y., Metz C., Davitz M. A., Olson L. K., Deeg M. A. Mouse glycosylphosphatidylinositol-specific phospholipase D (Gpld1) characterization. Mamm Genome. 1998 Sep;9(9):710–714. doi: 10.1007/s003359900851. [DOI] [PubMed] [Google Scholar]
  14. Leidich S. D., Drapp D. A., Orlean P. A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis. J Biol Chem. 1994 Apr 8;269(14):10193–10196. [PubMed] [Google Scholar]
  15. Li J. Y., Low M. G. Studies of the role of the integrin EF-hand, Ca2+-binding sites in glycosylphosphatidylinositol-specific phospholipase D: reduced expression following mutagenesis of residues predicted to bind Ca2+. Arch Biochem Biophys. 1999 Jan 1;361(1):142–148. doi: 10.1006/abbi.1998.0969. [DOI] [PubMed] [Google Scholar]
  16. Low M. G., Huang K. S. Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the membrane anchors of cell surface proteins. Biochem J. 1991 Oct 15;279(Pt 2):483–493. doi: 10.1042/bj2790483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Low M. G., Stiernberg J., Waneck G. L., Flavell R. A., Kincade P. W. Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C. J Immunol Methods. 1988 Oct 4;113(1):101–111. doi: 10.1016/0022-1759(88)90386-9. [DOI] [PubMed] [Google Scholar]
  18. Lu C. F., Montijn R. C., Brown J. L., Klis F., Kurjan J., Bussey H., Lipke P. N. Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol. 1995 Feb;128(3):333–340. doi: 10.1083/jcb.128.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maguire G. A., Gossner A. Glycosyl phosphatidyl inositol phospholipase D activity in human serum. Ann Clin Biochem. 1995 Jan;32(Pt 1):74–78. doi: 10.1177/000456329503200107. [DOI] [PubMed] [Google Scholar]
  20. McConville M. J., Menon A. K. Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (review). Mol Membr Biol. 2000 Jan-Mar;17(1):1–16. doi: 10.1080/096876800294443. [DOI] [PubMed] [Google Scholar]
  21. Metz C. N., Brunner G., Choi-Muira N. H., Nguyen H., Gabrilove J., Caras I. W., Altszuler N., Rifkin D. B., Wilson E. L., Davitz M. A. Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D. EMBO J. 1994 Apr 1;13(7):1741–1751. doi: 10.1002/j.1460-2075.1994.tb06438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Middelhoven P. J., van Buul J. D., Kleijer M., Roos D., Hordijk P. L. Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils. Biochem Biophys Res Commun. 1999 Feb 24;255(3):568–574. doi: 10.1006/bbrc.1999.0244. [DOI] [PubMed] [Google Scholar]
  23. Nakamura N., Inoue N., Watanabe R., Takahashi M., Takeda J., Stevens V. L., Kinoshita T. Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase. J Biol Chem. 1997 Jun 20;272(25):15834–15840. doi: 10.1074/jbc.272.25.15834. [DOI] [PubMed] [Google Scholar]
  24. Priest J. W., Xie L. T., Arrowood M. J., Lammie P. J. The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored. Mol Biochem Parasitol. 2001 Mar;113(1):117–126. doi: 10.1016/s0166-6851(00)00386-8. [DOI] [PubMed] [Google Scholar]
  25. Raymond F. D., Fortunato G., Moss D. W., Castaldo G., Salvatore F., Impallomeni M. Inositol-specific phospholipase D activity in health and disease. Clin Sci (Lond) 1994 Apr;86(4):447–451. doi: 10.1042/cs0860447. [DOI] [PubMed] [Google Scholar]
  26. Rhode H., Lopatta E., Schulze M., Pascual C., Schulze H. P., Schubert K., Schubert H., Reinhart K., Horn A. Glycosylphosphatidylinositol-specific phospholipase D in blood serum: is the liver the only source of the enzyme? Clin Chim Acta. 1999 Mar;281(1-2):127–145. doi: 10.1016/s0009-8981(98)00218-6. [DOI] [PubMed] [Google Scholar]
  27. Roberts W. L., Myher J. J., Kuksis A., Low M. G., Rosenberry T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1988 Dec 15;263(35):18766–18775. [PubMed] [Google Scholar]
  28. Ross D. T., Scherf U., Eisen M. B., Perou C. M., Rees C., Spellman P., Iyer V., Jeffrey S. S., Van de Rijn M., Waltham M. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet. 2000 Mar;24(3):227–235. doi: 10.1038/73432. [DOI] [PubMed] [Google Scholar]
  29. Scallon B. J., Fung W. J., Tsang T. C., Li S., Kado-Fong H., Huang K. S., Kochan J. P. Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D. Science. 1991 Apr 19;252(5004):446–448. doi: 10.1126/science.2017684. [DOI] [PubMed] [Google Scholar]
  30. Smith G. M., Biggs J., Norris B., Anderson-Stewart P., Ward R. Detection of a soluble form of the leukocyte surface antigen CD48 in plasma and its elevation in patients with lymphoid leukemias and arthritis. J Clin Immunol. 1997 Nov;17(6):502–509. doi: 10.1023/a:1027327912204. [DOI] [PubMed] [Google Scholar]
  31. Stevens V. L. Biosynthesis of glycosylphosphatidylinositol membrane anchors. Biochem J. 1995 Sep 1;310(Pt 2):361–370. doi: 10.1042/bj3100361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stevens V. L., Zhang H., Harreman M. Isolation and characterization of a Chinese hamster ovary (CHO) mutant defective in the second step of glycosylphosphatidylinositol biosynthesis. Biochem J. 1996 Jan 1;313(Pt 1):253–258. doi: 10.1042/bj3130253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tachado S. D., Mazhari-Tabrizi R., Schofield L. Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. Parasite Immunol. 1999 Dec;21(12):609–617. doi: 10.1046/j.1365-3024.1999.00268.x. [DOI] [PubMed] [Google Scholar]
  34. Takeda J., Miyata T., Kawagoe K., Iida Y., Endo Y., Fujita T., Takahashi M., Kitani T., Kinoshita T. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993 May 21;73(4):703–711. doi: 10.1016/0092-8674(93)90250-t. [DOI] [PubMed] [Google Scholar]
  35. Tsujioka H., Misumi Y., Takami N., Ikehara Y., Tujioka H. Posttranslational modification of glycosylphosphatidylinositol (GPI)-specific phospholipase D and its activity in cleavage of GPI anchors. Biochem Biophys Res Commun. 1998 Oct 29;251(3):737–743. doi: 10.1006/bbrc.1998.9542. [DOI] [PubMed] [Google Scholar]
  36. Tsujioka H., Takami N., Misumi Y., Ikehara Y. Intracellular cleavage of glycosylphosphatidylinositol by phospholipase D induces activation of protein kinase Calpha. Biochem J. 1999 Sep 1;342(Pt 2):449–455. [PMC free article] [PubMed] [Google Scholar]
  37. Wilhelm O. G., Wilhelm S., Escott G. M., Lutz V., Magdolen V., Schmitt M., Rifkin D. B., Wilson E. L., Graeff H., Brunner G. Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J Cell Physiol. 1999 Aug;180(2):225–235. doi: 10.1002/(SICI)1097-4652(199908)180:2<225::AID-JCP10>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  38. Wong Y. W., Low M. G. Phospholipase resistance of the glycosyl-phosphatidylinositol membrane anchor on human alkaline phosphatase. Clin Chem. 1992 Dec;38(12):2517–2525. [PubMed] [Google Scholar]

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