Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Mar 2;124(6):935–947. doi: 10.1083/jcb.124.6.935

A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

PMCID: PMC2119965  PMID: 8132715

Abstract

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein- GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI- PLC, cell-associated gp63 could not be detected in immunoblots. Pulse- chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Abeijon C., Hirschberg C. B. Topography of glycosylation reactions in the endoplasmic reticulum. Trends Biochem Sci. 1992 Jan;17(1):32–36. doi: 10.1016/0968-0004(92)90424-8. [DOI] [PubMed] [Google Scholar]
  2. Armstrong C., Schubert J., Ueda E., Knez J. J., Gelperin D., Hirose S., Silber R., Hollan S., Schmidt R. E., Medof M. E. Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants. J Biol Chem. 1992 Dec 15;267(35):25347–25351. [PubMed] [Google Scholar]
  3. Bangs J. D., Hereld D., Krakow J. L., Hart G. W., Englund P. T. Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc Natl Acad Sci U S A. 1985 May;82(10):3207–3211. doi: 10.1073/pnas.82.10.3207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bangs J. D., Uyetake L., Brickman M. J., Balber A. E., Boothroyd J. C. Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J Cell Sci. 1993 Aug;105(Pt 4):1101–1113. doi: 10.1242/jcs.105.4.1101. [DOI] [PubMed] [Google Scholar]
  5. Bordier C., Etges R. J., Ward J., Turner M. J., Cardoso de Almeida M. L. Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5988–5991. doi: 10.1073/pnas.83.16.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Broomfield S. J., Hooper N. M. Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase. Biochim Biophys Acta. 1993 Feb 9;1145(2):212–218. doi: 10.1016/0005-2736(93)90291-7. [DOI] [PubMed] [Google Scholar]
  7. Bölow R., Griffiths G., Webster P., Stierhof Y. D., Opperdoes F. R., Overath P. Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei. J Cell Sci. 1989 Jun;93(Pt 2):233–240. doi: 10.1242/jcs.93.2.233. [DOI] [PubMed] [Google Scholar]
  8. Bülow R., Nonnengässer C., Overath P. Release of the variant surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei. Mol Biochem Parasitol. 1989 Jan 1;32(1):85–92. doi: 10.1016/0166-6851(89)90132-1. [DOI] [PubMed] [Google Scholar]
  9. Bülow R., Overath P. Purification and characterization of the membrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei. J Biol Chem. 1986 Sep 5;261(25):11918–11923. [PubMed] [Google Scholar]
  10. Caras I. W., Weddell G. N., Davitz M. A., Nussenzweig V., Martin D. W., Jr Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science. 1987 Nov 27;238(4831):1280–1283. doi: 10.1126/science.2446389. [DOI] [PubMed] [Google Scholar]
  11. Cardoso de Almeida M. L., Turner M. J. The membrane form of variant surface glycoproteins of Trypanosoma brucei. Nature. 1983 Mar 24;302(5906):349–352. doi: 10.1038/302349a0. [DOI] [PubMed] [Google Scholar]
  12. Chapman A., Fujimoto K., Kornfeld S. The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose. J Biol Chem. 1980 May 25;255(10):4441–4446. [PubMed] [Google Scholar]
  13. Chaudhuri G., Chaudhuri M., Pan A., Chang K. P. Surface acid proteinase (gp63) of Leishmania mexicana. A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages. J Biol Chem. 1989 May 5;264(13):7483–7489. [PubMed] [Google Scholar]
  14. Davitz M. A., Low M. G., Nussenzweig V. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J Exp Med. 1986 May 1;163(5):1150–1161. doi: 10.1084/jem.163.5.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Doering T. L., Masterson W. J., Englund P. T., Hart G. W. Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J Biol Chem. 1989 Jul 5;264(19):11168–11173. [PubMed] [Google Scholar]
  16. Elhay M. J., McConville M. J., Curtis J. M., Bacic A., Handman E. Identification of truncated forms of lipophosphoglycan in mutant cloned lines of Leishmania major that are deficient in mature lipophosphoglycan. Parasitol Res. 1993;79(5):435–438. doi: 10.1007/BF00931836. [DOI] [PubMed] [Google Scholar]
  17. Englund P. T. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu Rev Biochem. 1993;62:121–138. doi: 10.1146/annurev.bi.62.070193.001005. [DOI] [PubMed] [Google Scholar]
  18. Etges R., Bouvier J., Bordier C. The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid. EMBO J. 1986 Mar;5(3):597–601. doi: 10.1002/j.1460-2075.1986.tb04252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ferguson M. A., Homans S. W., Dwek R. A., Rademacher T. W. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988 Feb 12;239(4841 Pt 1):753–759. doi: 10.1126/science.3340856. [DOI] [PubMed] [Google Scholar]
  20. Field M. C., Medina-Acosta E., Cross G. A. Characterization of a glycosylphosphatidylinositol membrane protein anchor precursor in Leishmania mexicana. Mol Biochem Parasitol. 1991 Oct;48(2):227–229. doi: 10.1016/0166-6851(91)90118-p. [DOI] [PubMed] [Google Scholar]
  21. Field M. C., Medina-Acosta E., Cross G. A. Inhibition of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana by mannosamine. J Biol Chem. 1993 May 5;268(13):9570–9577. [PubMed] [Google Scholar]
  22. Fox J. A., Duszenko M., Ferguson M. A., Low M. G., Cross G. A. Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei. J Biol Chem. 1986 Nov 25;261(33):15767–15771. [PubMed] [Google Scholar]
  23. Grab D. J., Webster P., Ito S., Fish W. R., Verjee Y., Lonsdale-Eccles J. D. Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes. J Cell Biol. 1987 Aug;105(2):737–746. doi: 10.1083/jcb.105.2.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gupta D., Tartakoff A., Tisdale E. Metabolic correction of defects in the lipid anchoring of Thy-1 in lymphoma mutants. Science. 1988 Dec 9;242(4884):1446–1448. doi: 10.1126/science.2904699. [DOI] [PubMed] [Google Scholar]
  25. Hereld D., Hart G. W., Englund P. T. cDNA encoding the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8914–8918. doi: 10.1073/pnas.85.23.8914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hereld D., Krakow J. L., Bangs J. D., Hart G. W., Englund P. T. A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. J Biol Chem. 1986 Oct 15;261(29):13813–13819. [PubMed] [Google Scholar]
  27. Hillmen P., Bessler M., Mason P. J., Watkins W. M., Luzzatto L. Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5272–5276. doi: 10.1073/pnas.90.11.5272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Inoue N., Kinoshita T., Orii T., Takeda J. Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a novel expression cloning strategy. J Biol Chem. 1993 Apr 5;268(10):6882–6885. [PubMed] [Google Scholar]
  29. Kamitani T., Chang H. M., Rollins C., Waneck G. L., Yeh E. T. Correction of the class H defect in glycosylphosphatidylinositol anchor biosynthesis in Ltk- cells by a human cDNA clone. J Biol Chem. 1993 Oct 5;268(28):20733–20736. [PubMed] [Google Scholar]
  30. Kamitani T., Menon A. K., Hallaq Y., Warren C. D., Yeh E. T. Complexity of ethanolamine phosphate addition in the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells. J Biol Chem. 1992 Dec 5;267(34):24611–24619. [PubMed] [Google Scholar]
  31. Kapler G. M., Coburn C. M., Beverley S. M. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. doi: 10.1128/mcb.10.3.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kardami E., Pearson T. W., Beecroft R. P., Fandrich R. R. Identification of basic fibroblast growth factor-like proteins in African trypanosomes and Leishmania. Mol Biochem Parasitol. 1992 Apr;51(2):171–181. doi: 10.1016/0166-6851(92)90067-t. [DOI] [PubMed] [Google Scholar]
  33. Kelleher M., Bacic A., Handman E. Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):6–10. doi: 10.1073/pnas.89.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krakow J. L., Hereld D., Bangs J. D., Hart G. W., Englund P. T. Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein. J Biol Chem. 1986 Sep 15;261(26):12147–12153. [PubMed] [Google Scholar]
  35. LeBowitz J. H., Smith H. Q., Rusche L., Beverley S. M. Coupling of poly(A) site selection and trans-splicing in Leishmania. Genes Dev. 1993 Jun;7(6):996–1007. doi: 10.1101/gad.7.6.996. [DOI] [PubMed] [Google Scholar]
  36. Lee H. C., Shoda R., Krall J. A., Foster J. D., Selhub J., Rosenberry T. L. Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor. Biochemistry. 1992 Mar 31;31(12):3236–3243. doi: 10.1021/bi00127a027. [DOI] [PubMed] [Google Scholar]
  37. Masterson W. J., Doering T. L., Hart G. W., Englund P. T. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. McConville M. J., Homans S. W., Thomas-Oates J. E., Dell A., Bacic A. Structures of the glycoinositolphospholipids from Leishmania major. A family of novel galactofuranose-containing glycolipids. J Biol Chem. 1990 May 5;265(13):7385–7394. [PubMed] [Google Scholar]
  40. McConville M. J., Thomas-Oates J. E., Ferguson M. A., Homans S. W. Structure of the lipophosphoglycan from Leishmania major. J Biol Chem. 1990 Nov 15;265(32):19611–19623. [PubMed] [Google Scholar]
  41. Medof M. E., Walter E. I., Roberts W. L., Haas R., Rosenberry T. L. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry. 1986 Nov 4;25(22):6740–6747. doi: 10.1021/bi00370a003. [DOI] [PubMed] [Google Scholar]
  42. Menon A. K., Mayor S., Ferguson M. A., Duszenko M., Cross G. A. Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins. J Biol Chem. 1988 Feb 5;263(4):1970–1977. [PubMed] [Google Scholar]
  43. Mensa-Wilmot K., Englund P. T. Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli. Mol Biochem Parasitol. 1992 Dec;56(2):311–321. doi: 10.1016/0166-6851(92)90180-r. [DOI] [PubMed] [Google Scholar]
  44. Mensa-Wilmot K., Hereld D., Englund P. T. Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei. Mol Cell Biol. 1990 Feb;10(2):720–726. doi: 10.1128/mcb.10.2.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Miyata T., Takeda J., Iida Y., Yamada N., Inoue N., Takahashi M., Maeda K., Kitani T., Kinoshita T. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science. 1993 Feb 26;259(5099):1318–1320. doi: 10.1126/science.7680492. [DOI] [PubMed] [Google Scholar]
  46. Murray P. J., Spithill T. W., Handman E. The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen. J Immunol. 1989 Dec 15;143(12):4221–4226. [PubMed] [Google Scholar]
  47. Müller G., Schubert K., Fiedler F., Bandlow W. The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol. J Biol Chem. 1992 Dec 15;267(35):25337–25346. [PubMed] [Google Scholar]
  48. Nakakuma H., Nagakura S., Kawaguchi T., Horikawa K., Kagimoto T., Kawakita M., Tomita M., Takatsuki K. Increased plasma decay-accelerating factor levels in paroxysmal nocturnal hemoglobinuria. Int J Hematol. 1992 Apr;55(2):121–125. [PubMed] [Google Scholar]
  49. Ramamoorthy R., Donelson J. E., Paetz K. E., Maybodi M., Roberts S. C., Wilson M. E. Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form. J Biol Chem. 1992 Jan 25;267(3):1888–1895. [PubMed] [Google Scholar]
  50. Rivas L., Kahl L., Manson K., McMahon-Pratt D. Biochemical characterization of the protective membrane glycoprotein GP46/M-2 of Leishmania amazonensis. Mol Biochem Parasitol. 1991 Aug;47(2):235–243. doi: 10.1016/0166-6851(91)90183-7. [DOI] [PubMed] [Google Scholar]
  51. Rosse W. F. Paroxysmal nocturnal hemoglobinuria. Curr Top Microbiol Immunol. 1992;178:163–173. doi: 10.1007/978-3-642-77014-2_10. [DOI] [PubMed] [Google Scholar]
  52. Schneider P., Ferguson M. A., McConville M. J., Mehlert A., Homans S. W., Bordier C. Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease. J Biol Chem. 1990 Oct 5;265(28):16955–16964. [PubMed] [Google Scholar]
  53. Schneider P., Rosat J. P., Bouvier J., Louis J., Bordier C. Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages. Exp Parasitol. 1992 Sep;75(2):196–206. doi: 10.1016/0014-4894(92)90179-e. [DOI] [PubMed] [Google Scholar]
  54. Stevens V. L., Raetz C. R. Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. J Biol Chem. 1991 Jun 5;266(16):10039–10042. [PubMed] [Google Scholar]
  55. Takahashi M., Takeda J., Hirose S., Hyman R., Inoue N., Miyata T., Ueda E., Kitani T., Medof M. E., Kinoshita T. Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria. J Exp Med. 1993 Feb 1;177(2):517–521. doi: 10.1084/jem.177.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Takami N., Oda K., Ikehara Y. Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol. J Biol Chem. 1992 Jan 15;267(2):1042–1047. [PubMed] [Google Scholar]
  57. 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]
  58. Tartakoff A. M., Singh N. How to make a glycoinositol phospholipid anchor. Trends Biochem Sci. 1992 Nov;17(11):470–473. doi: 10.1016/0968-0004(92)90491-q. [DOI] [PubMed] [Google Scholar]
  59. Tisdale E. J., Schimenti J. C., Tartakoff A. M. Sodium butyrate causes reexpression of three membrane proteins on glycolipid-anchoring mutants. Somat Cell Mol Genet. 1991 Jul;17(4):349–357. doi: 10.1007/BF01233060. [DOI] [PubMed] [Google Scholar]
  60. Turco S. J., Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol. 1992;46:65–94. doi: 10.1146/annurev.mi.46.100192.000433. [DOI] [PubMed] [Google Scholar]
  61. Vidugiriene J., Menon A. K. Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. J Cell Biol. 1993 Jun;121(5):987–996. doi: 10.1083/jcb.121.5.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Webb J. R., Button L. L., McMaster W. R. Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani. Mol Biochem Parasitol. 1991 Oct;48(2):173–184. doi: 10.1016/0166-6851(91)90113-k. [DOI] [PubMed] [Google Scholar]
  63. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  64. Zamze S. E., Ferguson M. A., Collins R., Dwek R. A., Rademacher T. W. Characterization of the cross-reacting determinant (CRD) of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein. Eur J Biochem. 1988 Oct 1;176(3):527–534. doi: 10.1111/j.1432-1033.1988.tb14310.x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES