Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Oct;10(10):2731–2739. doi: 10.1002/j.1460-2075.1991.tb07821.x

A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: lipid structure and biosynthesis.

M C Field 1, A K Menon 1, G A Cross 1
PMCID: PMC452981  PMID: 1655402

Abstract

Cells of the insect (procyclic) stage of the life cycle of the African trypanosome, Trypanosoma brucei, express an abundant stage-specific glycosylated phosphatidylinositol (GPI) anchored glycoprotein, the procyclic acidic repetitive protein (PARP). The anchor is insensitive to the action of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it contains an acyl-inositol. We have recently described the structure of a PI-PLC resistant glycosylphosphatidylinositol, PP1, which is specific to the procyclic stage, and have presented preliminary evidence that the phosphatidylinositol portion of the protein-linked GPI on PARP has a similar structure. In this paper we show, by metabolic labelling with [3H]fatty acids, that the PARP anchor contains palmitate esterified to inositol, and stearate at sn-1, in a monoacylglycerol moiety, a structure identical to PP1. Using pulse-chase labelling, we show that both fatty acids are incorporated into the GPI anchor from a large pool of metabolic precursors, rather than directly from acyl-CoA. We also demonstrate that the addition of the GPI anchor moiety to PARP is dependent on de novo protein synthesis, excluding the possibility that incorporation of fatty acids into PARP can occur by a remodelling of pre-existing GPI anchors. Finally we show that the phosphatidylinositol (PI) species that are utilized for GPI biosynthesis are a subpopulation of the cellular PI molecular species. We propose that these observations may be of general validity since several other eukaryotic membrane proteins (e.g. human erythrocyte acetylcholine esterase and decay accelerating factor) have been reported to contain palmitoylated inositol residues.

Full text

PDF
2733

Images in this article

2732Image
on p.2732
2734Image
on p.2734
2736Image
on p.2736

Selected References

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

  1. 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]
  2. Brennan P., Ballou C. E. Biosynthesis of mannophosphoinositides by Mycobacterium phlei. Enzymatic acylation of the dimannophosphoinositides. J Biol Chem. 1968 Jun 10;243(11):2975–2984. [PubMed] [Google Scholar]
  3. Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
  4. Chattopadhyay P. K., Wu H. C. Biosynthesis of the covalently linked diglyceride in murein lipoprotein of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5318–5322. doi: 10.1073/pnas.74.12.5318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cho K. S., Hong S. D., Cho J. M., Chang C. S., Lee K. S. Studies on the biosynthesis of acylphosphatidylglycerol in Escherichia coli B and B/r. Biochim Biophys Acta. 1976 Jan 18;486(1):47–54. doi: 10.1016/0005-2760(77)90068-6. [DOI] [PubMed] [Google Scholar]
  6. Clayton C. E. Most proteins, including fructose bisphosphate aldolase, are stable in the procyclic trypomastigote form of Trypanosoma brucei. Mol Biochem Parasitol. 1988 Feb;28(1):43–46. doi: 10.1016/0166-6851(88)90178-8. [DOI] [PubMed] [Google Scholar]
  7. Clayton C. E., Mowatt M. R. The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification. J Biol Chem. 1989 Sep 5;264(25):15088–15093. [PubMed] [Google Scholar]
  8. Cross G. A. Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell Biol. 1990;6:1–39. doi: 10.1146/annurev.cb.06.110190.000245. [DOI] [PubMed] [Google Scholar]
  9. Dahl C. E., Dahl J. S. Phospholipids as acyl donors to membrane proteins of Mycoplasma capricolum. J Biol Chem. 1984 Sep 10;259(17):10771–10776. [PubMed] [Google Scholar]
  10. DeGasperi R., Thomas L. J., Sugiyama E., Chang H. M., Beck P. J., Orlean P., Albright C., Waneck G., Sambrook J. F., Warren C. D. Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene. Science. 1990 Nov 16;250(4983):988–991. doi: 10.1126/science.1978413. [DOI] [PubMed] [Google Scholar]
  11. Dixon H., Ginger C. D., Williamson J. The lipid metabolism of blood and culture forms of Trypanosoma lewisi and Trypanosoma rhodesiense. Comp Biochem Physiol B. 1971 Jun 15;39(2):247–266. doi: 10.1016/0305-0491(71)90168-4. [DOI] [PubMed] [Google Scholar]
  12. Doering T. L., Masterson W. J., Hart G. W., Englund P. T. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J Biol Chem. 1990 Jan 15;265(2):611–614. [PubMed] [Google Scholar]
  13. Ferguson M. A., Duszenko M., Lamont G. S., Overath P., Cross G. A. Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor. J Biol Chem. 1986 Jan 5;261(1):356–362. [PubMed] [Google Scholar]
  14. 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]
  15. Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
  16. Field M. C., Menon A. K., Cross G. A. Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein. J Biol Chem. 1991 May 5;266(13):8392–8400. [PubMed] [Google Scholar]
  17. Henderson L. E., Oroszlan S., Konigsberg W. A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction. Anal Biochem. 1979 Feb;93(1):153–157. [PubMed] [Google Scholar]
  18. Jackowski S., Rock C. O. Transfer of fatty acids from the 1-position of phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli. J Biol Chem. 1986 Aug 25;261(24):11328–11333. [PubMed] [Google Scholar]
  19. Krakow J. L., Doering T. L., Masterson W. J., Hart G. W., Englund P. T. A glycolipid from Trypanosoma brucei related to the variant surface glycoprotein membrane anchor. Mol Biochem Parasitol. 1989 Oct;36(3):263–270. doi: 10.1016/0166-6851(89)90174-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lisanti M. P., Field M. C., Caras I. W., Menon A. K., Rodriguez-Boulan E. Mannosamine, a novel inhibitor of glycosylphosphatidylinositol incorporation into proteins. EMBO J. 1991 Aug;10(8):1969–1977. doi: 10.1002/j.1460-2075.1991.tb07726.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luhrs C. A., Slomiany B. L. A human membrane-associated folate binding protein is anchored by a glycosyl-phosphatidylinositol tail. J Biol Chem. 1989 Dec 25;264(36):21446–21449. [PubMed] [Google Scholar]
  24. 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]
  25. Masterson W. J., Raper J., Doering T. L., Hart G. W., Englund P. T. Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors. Cell. 1990 Jul 13;62(1):73–80. doi: 10.1016/0092-8674(90)90241-6. [DOI] [PubMed] [Google Scholar]
  26. Mayor S., Menon A. K., Cross G. A., Ferguson M. A., Dwek R. A., Rademacher T. W. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6164–6173. [PubMed] [Google Scholar]
  27. Mayor S., Menon A. K., Cross G. A. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6174–6181. [PubMed] [Google Scholar]
  28. Mayor S., Menon A. K., Cross G. A. Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system. J Cell Biol. 1991 Jul;114(1):61–71. doi: 10.1083/jcb.114.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Menon A. K., Mayor S., Schwarz R. T. Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosoma brucei: involvement of mannosyl-phosphoryldolichol as the mannose donor. EMBO J. 1990 Dec;9(13):4249–4258. doi: 10.1002/j.1460-2075.1990.tb07873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Menon A. K., Schwarz R. T., Mayor S., Cross G. A. Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates. J Biol Chem. 1990 Jun 5;265(16):9033–9042. [PubMed] [Google Scholar]
  32. Ohga Y., Nishijima M., Akamatsu Y. Chinese hamster ovary cell mutants defective in myo-inositol transport. J Biol Chem. 1990 Oct 25;265(30):18083–18086. [PubMed] [Google Scholar]
  33. Omura S., Tomoda H., Xu Q. M., Takahashi Y., Iwai Y. Triacsins, new inhibitors of acyl-CoA synthetase produced by Streptomyces sp. J Antibiot (Tokyo) 1986 Sep;39(9):1211–1218. doi: 10.7164/antibiotics.39.1211. [DOI] [PubMed] [Google Scholar]
  34. Orlean P. Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Nov;10(11):5796–5805. doi: 10.1128/mcb.10.11.5796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Raetz C. R., Dowhan W. Biosynthesis and function of phospholipids in Escherichia coli. J Biol Chem. 1990 Jan 25;265(3):1235–1238. [PubMed] [Google Scholar]
  36. Roberts W. L., Santikarn S., Reinhold V. N., Rosenberry T. L. Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry. J Biol Chem. 1988 Dec 15;263(35):18776–18784. [PubMed] [Google Scholar]
  37. Roditi I., Schwarz H., Pearson T. W., Beecroft R. P., Liu M. K., Richardson J. P., Bühring H. J., Pleiss J., Bülow R., Williams R. O. Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei. J Cell Biol. 1989 Feb;108(2):737–746. doi: 10.1083/jcb.108.2.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sadeghi H., da Silva A. M., Klein C. Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5512–5515. doi: 10.1073/pnas.85.15.5512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schneider A., Eichenberger W., Seebeck T. A microtubule-binding protein of Trypanosoma brucei which contains covalently bound fatty acid. J Biol Chem. 1988 May 15;263(14):6472–6475. [PubMed] [Google Scholar]
  40. Schultz A. M., Henderson L. E., Oroszlan S. Fatty acylation of proteins. Annu Rev Cell Biol. 1988;4:611–647. doi: 10.1146/annurev.cb.04.110188.003143. [DOI] [PubMed] [Google Scholar]
  41. Stadler J., Keenan T. W., Bauer G., Gerisch G. The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type. EMBO J. 1989 Feb;8(2):371–377. doi: 10.1002/j.1460-2075.1989.tb03387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Takahashi N., Breitman T. R. Retinoylation of HL-60 proteins. Comparison to labeling by palmitic and myristic acids. J Biol Chem. 1990 Nov 5;265(31):19158–19162. [PubMed] [Google Scholar]
  43. Thomas J. R., Dwek R. A., Rademacher T. W. Structure, biosynthesis, and function of glycosylphosphatidylinositols. Biochemistry. 1990 Jun 12;29(23):5413–5422. doi: 10.1021/bi00475a001. [DOI] [PubMed] [Google Scholar]
  44. Towler D. A., Gordon J. I., Adams S. P., Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99. doi: 10.1146/annurev.bi.57.070188.000441. [DOI] [PubMed] [Google Scholar]
  45. Vance D., Goldberg I., Mitsuhashi O., Bloch K. Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem Biophys Res Commun. 1972 Aug 7;48(3):649–656. doi: 10.1016/0006-291x(72)90397-x. [DOI] [PubMed] [Google Scholar]
  46. Walter E. I., Roberts W. L., Rosenberry T. L., Ratnoff W. D., Medof M. E. Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage. J Immunol. 1990 Feb 1;144(3):1030–1036. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES