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. 1991 Aug;10(8):2041–2045. doi: 10.1002/j.1460-2075.1991.tb07734.x

Phenylmethanesulphonyl fluoride inhibits GPI anchor biosynthesis in the African trypanosome.

W J Masterson 1, M A Ferguson 1
PMCID: PMC452885  PMID: 1829674

Abstract

A wide variety of eukaryotic membrane proteins are anchored to the outside of cells by covalent linkage to glycosyl phosphatidylinositol (GPI). One of the best characterized examples is the variant surface glycoprotein (VSG) of the protozoan parasite, Trypanosoma brucei. The structure of the GPI precursor is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2-PI; the phosphoethanolamine moiety forms an amide linkage to the VSG polypeptide alpha-COOH group during its attachment to protein. Here we report that the serine esterase inhibitor, phenylmethanesulphonyl fluoride (PMSF), inhibits phosphoethanolamine incorporation into the GPI precursor resulting in the accumulation of a Man3GlcNH2-PI intermediate. PMSF exerts this effect both in living trypanosomes and in a trypanosome-derived cell-free system. This is the first report of an inhibitor which affects GPI biosynthesis but not N-glycosylation. A model of the mechanism of phosphoethanolamine incorporation into the GPI precursor, based on the known properties of PMSF, is presented.

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Selected References

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  1. 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]
  2. 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]
  3. 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]
  4. Dotti C. G., Parton R. G., Simons K. Polarized sorting of glypiated proteins in hippocampal neurons. Nature. 1991 Jan 10;349(6305):158–161. doi: 10.1038/349158a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. GOLD A. M. SULFONYL FLUORIDES AS INHIBITORS OF ESTERASES. 3. IDENTIFICATION OF SERINE AS THE SITE OF SULFONYLATION IN PHENYLMETHANESULFONYL ALPHA-CHYMOTRYPSIN. Biochemistry. 1965 May;4:897–901. doi: 10.1021/bi00881a016. [DOI] [PubMed] [Google Scholar]
  8. Homans S. W., Ferguson M. A., Dwek R. A., Rademacher T. W., Anand R., Williams A. F. Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein. Nature. 1988 May 19;333(6170):269–272. doi: 10.1038/333269a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Robinson P. J., Millrain M., Antoniou J., Simpson E., Mellor A. L. A glycophospholipid anchor is required for Qa-2-mediated T cell activation. Nature. 1989 Nov 2;342(6245):85–87. doi: 10.1038/342085a0. [DOI] [PubMed] [Google Scholar]
  21. Rothberg K. G., Ying Y. S., Kolhouse J. F., Kamen B. A., Anderson R. G. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. doi: 10.1083/jcb.110.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Zamze S. E., Wooten E. W., Ashford D. A., Ferguson M. A., Dwek R. A., Rademacher T. W. Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins. Eur J Biochem. 1990 Feb 14;187(3):657–663. doi: 10.1111/j.1432-1033.1990.tb15350.x. [DOI] [PubMed] [Google Scholar]

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