Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Mar 1;378(Pt 2):641–648. doi: 10.1042/BJ20031326

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism.

Karl J Mann 1, Matthew R Hepworth 1, Nandita S Raikwar 1, Mark A Deeg 1, Daniel Sevlever 1
PMCID: PMC1223959  PMID: 14611645

Abstract

GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown. To investigate whether GPI-PLD plays a role in this regulation, we isolated stable HeLa cells overexpressing the enzyme. GPI-PLD-HeLa (GPI-PLD-transfected HeLa) cells showed a 3-fold increase in intracellular GPI-PLD activity and drastically decreased the levels of GPI-anchored proteins when compared with untransfected HeLa controls. Intracellular cleavage of GPI-anchored proteins has been suggested to occur early in the secretory pathway and, in agreement with this proposal, GPI-PLD activity in GPI-PLD-HeLa cells was detected not only in the endoplasmic reticulum and Golgi apparatus, but also in the plasma membrane. The enzyme was also active in lipid rafts, membrane microdomains in which GPI-anchored proteins and GPI anchor intermediates are concentrated, indicating that intracellular GPI-PLD cleavage may also occur in this compartment. Pulse-chase paradigms revealed the turnover rate of the last intermediate of the GPI anchor pathway in GPI-PLD-HeLa cells to be accelerated compared with the controls. Furthermore, 1,10-phenanthroline, a GPI-PLD inhibitor, reversed this effect. Our studies demonstrated that GPI-PLD can cleave not only GPI-anchored proteins, but also GPI anchor intermediates intracellularly. This observation opens the possibility that GPI-PLD can influence the steady-state levels of GPI-anchored proteins by hydrolysing the anchor before and after its attachment to proteins.

Full Text

The Full Text of this article is available as a PDF (152.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arreaza G., Brown D. A. Sorting and intracellular trafficking of a glycosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin-Darby canine kidney epithelial cells. J Biol Chem. 1995 Oct 6;270(40):23641–23647. doi: 10.1074/jbc.270.40.23641. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Brown D. A., Crise B., Rose J. K. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science. 1989 Sep 29;245(4925):1499–1501. doi: 10.1126/science.2571189. [DOI] [PubMed] [Google Scholar]
  4. Brown D. A. Interactions between GPI-anchored proteins and membrane lipids. Trends Cell Biol. 1992 Nov;2(11):338–343. [PubMed] [Google Scholar]
  5. Brown D. A., London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 1998;14:111–136. doi: 10.1146/annurev.cellbio.14.1.111. [DOI] [PubMed] [Google Scholar]
  6. Caro L. H., Tettelin H., Vossen J. H., Ram A. F., van den Ende H., Klis F. M. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast. 1997 Dec;13(15):1477–1489. doi: 10.1002/(SICI)1097-0061(199712)13:15<1477::AID-YEA184>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  7. Cocuzzi E., Szczotka L. B., Brodbeck W. G., Bardenstein D. S., Wei T., Medof M. E. Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF). Clin Exp Immunol. 2001 Feb;123(2):188–195. doi: 10.1046/j.1365-2249.2001.01408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davitz M. A., Hereld D., Shak S., Krakow J., Englund P. T., Nussenzweig V. A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science. 1987 Oct 2;238(4823):81–84. doi: 10.1126/science.2443973. [DOI] [PubMed] [Google Scholar]
  9. Du Xiaohan, Cai Jiewei, Zhou Jian-zhong, Stevens Victoria L., Low Martin G. Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis. Biochem J. 2002 Jan 1;361(Pt 1):113–118. doi: 10.1042/0264-6021:3610113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoener M. C., Stieger S., Brodbeck U. Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain. Eur J Biochem. 1990 Jul 5;190(3):593–601. doi: 10.1111/j.1432-1033.1990.tb15614.x. [DOI] [PubMed] [Google Scholar]
  11. Kinoshita T., Inoue N. Dissecting and manipulating the pathway for glycosylphos-phatidylinositol-anchor biosynthesis. Curr Opin Chem Biol. 2000 Dec;4(6):632–638. doi: 10.1016/s1367-5931(00)00151-4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lisanti M. P., Caras I. W., Davitz M. A., Rodriguez-Boulan E. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol. 1989 Nov;109(5):2145–2156. doi: 10.1083/jcb.109.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Low M. G., Saltiel A. R. Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science. 1988 Jan 15;239(4837):268–275. doi: 10.1126/science.3276003. [DOI] [PubMed] [Google Scholar]
  15. Mann K. J., Sevlever D. 1,10-Phenanthroline inhibits glycosylphosphatidylinositol anchoring by preventing phosphoethanolamine addition to glycosylphosphatidylinositol anchor precursors. Biochemistry. 2001 Feb 6;40(5):1205–1213. doi: 10.1021/bi0024512. [DOI] [PubMed] [Google Scholar]
  16. Martin-Belmonte F., Alonso M. A., Zhang X., Arvan P. Thyroglobulin is selected as luminal protein cargo for apical transport via detergent-resistant membranes in epithelial cells. J Biol Chem. 2000 Dec 29;275(52):41074–41081. doi: 10.1074/jbc.M005429200. [DOI] [PubMed] [Google Scholar]
  17. McConville M. J., Bacic A. The glycoinositolphospholipid profiles of two Leishmania major strains that differ in lipophosphoglycan expression. Mol Biochem Parasitol. 1990 Jan 1;38(1):57–67. doi: 10.1016/0166-6851(90)90205-z. [DOI] [PubMed] [Google Scholar]
  18. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. O'Brien K. D., Pineda C., Chiu W. S., Bowen R., Deeg M. A. Glycosylphosphatidylinositol-specific phospholipase D is expressed by macrophages in human atherosclerosis and colocalizes with oxidation epitopes. Circulation. 1999 Jun 8;99(22):2876–2882. doi: 10.1161/01.cir.99.22.2876. [DOI] [PubMed] [Google Scholar]
  21. Rosen G., Londner M. V., Sevlever D., Greenblatt C. L. Leishmania major: glycolipid antigens recognized by immune human sera. Mol Biochem Parasitol. 1988 Jan 1;27(1):93–99. doi: 10.1016/0166-6851(88)90028-x. [DOI] [PubMed] [Google Scholar]
  22. Rosenberry T. L., Sevlever D., Medof M. E. Metabolism of GPIs in mammalian cells. Braz J Med Biol Res. 1994 Feb;27(2):151–159. [PubMed] [Google Scholar]
  23. 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]
  24. Sevlever D., Humphrey D. R., Rosenberry T. L. Compositional analysis of glucosaminyl(acyl)phosphatidylinositol accumulated in HeLa S3 cells. Eur J Biochem. 1995 Oct 1;233(1):384–394. doi: 10.1111/j.1432-1033.1995.384_1.x. [DOI] [PubMed] [Google Scholar]
  25. Sevlever D., Pickett S., Mann K. J., Sambamurti K., Medof M. E., Rosenberry T. L. Glycosylphosphatidylinositol-anchor intermediates associate with triton-insoluble membranes in subcellular compartments that include the endoplasmic reticulum. Biochem J. 1999 Nov 1;343(Pt 3):627–635. [PMC free article] [PubMed] [Google Scholar]
  26. Sevlever D., Rosenberry T. L. Mannosamine inhibits the synthesis of putative glycoinositol phospholipid anchor precursors in mammalian cells without incorporating into an accumulated intermediate. J Biol Chem. 1993 May 25;268(15):10938–10945. [PubMed] [Google Scholar]
  27. Sevlever D., Schiemann D., Guidubaldi J., Medof M. E., Rosenberry T. L. Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity. Biochem J. 1997 Feb 1;321(Pt 3):837–844. doi: 10.1042/bj3210837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Singh N., Liang L. N., Tykocinski M. L., Tartakoff A. M. A novel class of cell surface glycolipids of mammalian cells. Free glycosyl phosphatidylinositols. J Biol Chem. 1996 May 31;271(22):12879–12884. doi: 10.1074/jbc.271.22.12879. [DOI] [PubMed] [Google Scholar]
  29. Smith Terry K., Crossman Arthur, Paterson Michael J., Borissow Charles N., Brimacombe John S., Ferguson Michael A. J. Specificities of enzymes of glycosylphosphatidylinositol biosynthesis in Trypanosoma brucei and HeLa cells. J Biol Chem. 2002 Jul 29;277(40):37147–37153. doi: 10.1074/jbc.M203371200. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Udenfriend S., Kodukula K. How glycosylphosphatidylinositol-anchored membrane proteins are made. Annu Rev Biochem. 1995;64:563–591. doi: 10.1146/annurev.bi.64.070195.003023. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Xiaotong H., Hannocks Melanie-Jane, Hampson Ian, Brunner Georg. GPI-specific phospholipase D mRNA expression in tumor cells of different malignancy. Clin Exp Metastasis. 2002;19(4):291–299. doi: 10.1023/a:1015545407700. [DOI] [PubMed] [Google Scholar]
  35. Xie M., Low M. G. Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines. Biochem J. 1994 Feb 1;297(Pt 3):547–554. doi: 10.1042/bj2970547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van't Hof W., Rodriguez-Boulan E., Menon A. K. Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells. J Biol Chem. 1995 Oct 13;270(41):24150–24155. doi: 10.1074/jbc.270.41.24150. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES