Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Jul 1;114(1):61–71. doi: 10.1083/jcb.114.1.61

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system

PMCID: PMC2289052  PMID: 1828808

Abstract

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI- anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Bailey C. A., Gerber L., Howard A. D., Udenfriend S. Processing at the carboxyl terminus of nascent placental alkaline phosphatase in a cell-free system: evidence for specific cleavage of a signal peptide. Proc Natl Acad Sci U S A. 1989 Jan;86(1):22–26. doi: 10.1073/pnas.86.1.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Berne P. F., Schmitter J. M., Blanquet S. Peptide and protein carboxyl-terminal labeling through carboxypeptidase Y-catalyzed transpeptidation. J Biol Chem. 1990 Nov 15;265(32):19551–19559. [PubMed] [Google Scholar]
  4. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  5. Bowles D. J., Marcus S. E., Pappin D. J., Findlay J. B., Eliopoulos E., Maycox P. R., Burgess J. Posttranslational processing of concanavalin A precursors in jackbean cotyledons. J Cell Biol. 1986 Apr;102(4):1284–1297. doi: 10.1083/jcb.102.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Chalifour R. J., Spiro R. G. Effect of phospholipids on thyroid oligosaccharyltransferase activity and orientation. Evaluation of structural determinants for stimulation of N-glycosylation. J Biol Chem. 1988 Oct 25;263(30):15673–15680. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Conzelmann A., Riezman H., Desponds C., Bron C. A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 1988 Jul;7(7):2233–2240. doi: 10.1002/j.1460-2075.1988.tb03063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conzelmann A., Spiazzi A., Bron C. Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. Biochem J. 1987 Sep 15;246(3):605–610. doi: 10.1042/bj2460605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cross G. A. Crossreacting determinants in the C-terminal region of trypanosome variant surface antigens. Nature. 1979 Jan 25;277(5694):310–312. doi: 10.1038/277310a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Cross G. A. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology. 1975 Dec;71(3):393–417. doi: 10.1017/s003118200004717x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Dorner A. J., Wasley L. C., Kaufman R. J. Protein dissociation from GRP78 and secretion are blocked by depletion of cellular ATP levels. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7429–7432. doi: 10.1073/pnas.87.19.7429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fasel N., Rousseaux M., Schaerer E., Medof M. E., Tykocinski M. L., Bron C. In vitro attachment of glycosyl-inositolphospholipid anchor structures to mouse Thy-1 antigen and human decay-accelerating factor. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6858–6862. doi: 10.1073/pnas.86.18.6858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Ferguson M. A., Haldar K., Cross G. A. Trypanosoma brucei variant surface glycoprotein has a sn-1,2-dimyristyl glycerol membrane anchor at its COOH terminus. J Biol Chem. 1985 Apr 25;260(8):4963–4968. [PubMed] [Google Scholar]
  21. 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]
  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. Hart G. W., Brew K., Grant G. A., Bradshaw R. A., Lennarz W. J. Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins. Studies with natural and synthetic peptides. J Biol Chem. 1979 Oct 10;254(19):9747–9753. [PubMed] [Google Scholar]
  24. He H. T., Finne J., Goridis C. Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule. J Cell Biol. 1987 Dec;105(6 Pt 1):2489–2500. doi: 10.1083/jcb.105.6.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  27. Izaki K., Matsuhashi M., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. 8. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains of Escherichia coli. J Biol Chem. 1968 Jun 10;243(11):3180–3192. [PubMed] [Google Scholar]
  28. Kaetzel D. M., Singh N., Kennedy G. C., Virgin J. B., Farr G., Kitagawa Y., Nilson J. H., Tartakoff A. M. Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1. J Biol Chem. 1990 Sep 15;265(26):15932–15937. [PubMed] [Google Scholar]
  29. Kane P. M., Yamashiro C. T., Wolczyk D. F., Neff N., Goebl M., Stevens T. H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 1990 Nov 2;250(4981):651–657. doi: 10.1126/science.2146742. [DOI] [PubMed] [Google Scholar]
  30. Kaplan H. A., Welply J. K., Lennarz W. J. Oligosaccharyl transferase: the central enzyme in the pathway of glycoprotein assembly. Biochim Biophys Acta. 1987 Jun 24;906(2):161–173. doi: 10.1016/0304-4157(87)90010-4. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. Low M. G. The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):427–454. doi: 10.1016/0304-4157(89)90014-2. [DOI] [PubMed] [Google Scholar]
  35. Lutek M. K., Hofmann T., Deber C. M. Transpeptidation reactions of porcine pepsin. Formation of tetrapeptides from dipeptide substrates. J Biol Chem. 1988 Jun 15;263(17):8011–8016. [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Micanovic R., Kodukula K., Gerber L. D., Udenfriend S. Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7939–7943. doi: 10.1073/pnas.87.20.7939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Moreno S. N., Ip H. S., Cross G. A. An mRNA-dependent in vitro translation system from Trypanosoma brucei. Mol Biochem Parasitol. 1991 Jun;46(2):265–274. doi: 10.1016/0166-6851(91)90050-g. [DOI] [PubMed] [Google Scholar]
  45. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  46. Parodi A. J., Behrens N. H., Leloir L. F., Carminatti H. The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3268–3272. doi: 10.1073/pnas.69.11.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. 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]
  50. Schwarz R. T., Datema R. Inhibition of the dolichol pathway of protein glycosylation. Methods Enzymol. 1982;83:432–443. doi: 10.1016/0076-6879(82)83041-3. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Takami N., Ogata S., Oda K., Misumi Y., Ikehara Y. Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring. J Biol Chem. 1988 Feb 25;263(6):3016–3021. [PubMed] [Google Scholar]
  53. 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]
  54. Tipper D. J., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. XII. Inhibition of cross-linking by penicillins and cephalosporins: studies in Staphylococcus aureus in vivo. J Biol Chem. 1968 Jun 10;243(11):3169–3179. [PubMed] [Google Scholar]
  55. Turco S. J., Stetson B., Robbins P. W. Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4411–4414. doi: 10.1073/pnas.74.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES