Detection of Glycophospholipid Anchors on Proteins

Tamara L. Doering1, Paul T. Englund2, Gerald W. Hart3

1 University of California, Berkeley, 2 Johns Hopkins Medical School, Baltimore, 3 University of Alabama, Birmingham
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.5
DOI:  10.1002/0471140864.ps1205s02
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI‐anchored proteins. First, the detergent‐partitioning behavior of a protein of interest is examined for characteristics of GPI‐linked species. The partitioning of total cellular and isolated proteins with Triton X‐114 is described in this unit, and precondensation of Triton X‐114, which is necessary to remove hydrophilic contaminants before partitioning, is outlined in a . The protein may also be subjected to specific enzymatic or chemical cleavages to release it from its GPI anchor. Phospholipase cleavage (starting with intact cells or membranes, or with isolated protein) is detailed, and chemical cleavage with nitrous acid is also described. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. A protocol for separation of lipid moieties released from proteins is provided and base hydrolysis of proteins is also presented.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Extraction and Partitioning of Total Proteins from Cells or Membranes with Triton X‐114
  • Alternate Protocol 1: Partitioning of Isolated Proteins with Triton X‐114
  • Support Protocol 1: Precondensation of Triton X‐114 Detergent
  • Basic Protocol 2: Identification of GPI‐Anchored Proteins by PI‐PLC Digestion of Intact Cells
  • Alternate Protocol 2: Identification of GPI Anchorage by Phospholipase Treatment of Isolated Proteins
  • Support Protocol 2: Detection of Products After Phospholipase Treatment by Reactivity with Anti‐CRD Antibody
  • Basic Protocol 3: Nitrous Acid Cleavage of GPI‐Anchored Proteins
  • Support Protocol 3: Separation of Lipid Moiety to Detect Cleavage of GPI‐Anchored Proteins
  • Basic Protocol 4: Base Hydrolysis of Radiolabeled Proteins
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Extraction and Partitioning of Total Proteins from Cells or Membranes with Triton X‐114

  Materials
  • Cells, membrane fraction, or other source of protein
  • recipeTris‐buffered saline (TBS; see recipe), ice‐cold
  • Precondensed Triton X‐114 stock solution in recipeTBS (see protocol 3), ice‐cold
  • 15‐ml polypropylene centrifuge tubes
  • Centrifuges: low‐speed (tabletop) and equipped with appropriate rotor (e.g., SS‐34), at 4°C and room temperature

Alternate Protocol 1: Partitioning of Isolated Proteins with Triton X‐114

  Materials
  • Triton X‐114 detergent
  • recipeTris‐buffered saline (TBS; see recipe)
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge

Support Protocol 1: Precondensation of Triton X‐114 Detergent

  Materials
  • Cells (or membrane preparation)
  • Bacterial phosphatidylinositol‐specific phospholipase C (PI‐PLC; e.g., from Bacillus thuringiensis, Oxford GlycoSystems)
  • Hanks balanced salt solution (HBSS; e.g., Life Technologies), buffered saline, or culture medium
  • Tabletop centrifuge and appropriate centrifuge tubes

Basic Protocol 2: Identification of GPI‐Anchored Proteins by PI‐PLC Digestion of Intact Cells

  Materials
  • Isolated protein (see protocol 1)
  • Acetone, −20°C
  • Appropriate enzyme buffer: recipeGPI‐PLD buffer, recipeGPI‐PLC buffer, and recipePI‐PLC buffer (see reciperecipes)
  • Phospholipase enzymes: GPI‐PLD from rat, rabbit, or human whole serum, GPI‐PLC from Trypanosoma brucei (Oxford GlycoSystems), and PI‐PLC from Bacillus thuringiensis (Oxford GlycoSystems) or B. cereus (Boehringer Mannheim or Sigma)
  • recipeTris‐buffered saline (TBS; see recipe)
  • Precondensed Triton X‐114 solution (see protocol 3)
  • Centrifuge and rotor (e.g., SS‐34)

Alternate Protocol 2: Identification of GPI Anchorage by Phospholipase Treatment of Isolated Proteins

  Materials
  • Protein of interest
  • recipe0.1 M acetate buffer, pH 3.5 (see recipe)
  • 0.5 M NaNO 2, made fresh
  • 0.5 M NaCl

Support Protocol 2: Detection of Products After Phospholipase Treatment by Reactivity with Anti‐CRD Antibody

  Materials
  • Radiolabeled protein, cleaved by phospholipase or nitrous acid treatment (see protocol 5 and protocol 7)
  • recipeWater‐saturated n‐butanol (see recipe)

Basic Protocol 3: Nitrous Acid Cleavage of GPI‐Anchored Proteins

  Materials
  • Protein radiolabeled with fatty acid (unit 12.2)
  • 0.2 M KOH in methanol
  • Methanol
  • recipe1 M hydroxylamine⋅HCl, pH 7.5, made fresh (see recipe)
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 2E)
  • Additional reagents and equipment for one‐dimensional SDS (unit 10.1) and staining of gels (unit 10.5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X‐114 solution. J. Biol. Chem. 256:1604‐1607.
   Brown, D.A. and Rose, J.K. 1992. Sorting of GPI‐anchored proteins to glycolipid‐enriched membrane subdomains during transport to the apical surface. Cell 68:533‐544.
   Cardoso de Almeida, M.L. and Turner, M.J. 1983. The membrane form of variant surface glycoproteins of Trypanosoma brucei. Nature 302:349‐352.
   Conzelmann, A., Puoti, A., Lester, R.L., and Desponds, C. 1992. Two different types of lipid moieties are present in glycophosphoinositol‐anchored membrane proteins of Saccharomyces cerevisiae. EMBO J. 11:457‐466.
   Doering, T.L., Masterson, W.J., Hart, G.W., and Englund, P.T. 1990a. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J. Biol. Chem. 265:611‐614.
   Doering, T.L., Raper, J., Buxbaum, L.U., Hart, G.W., and Englund, P.T. 1990b. Biosynthesis of glycosyl phosphatidylinositol protein anchors. Methods 1:288‐296.
   Englund, P.T. 1993. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Ann. Rev. Biochem. 62:121‐138.
   Ferguson, M.A.J. 1992. The chemical and enzymatic analysis of GPI fine structure. In Lipid Modifications of Proteins: A Practical Approach (A.J. Turner and N. Hooper, eds.) pp.191‐230. IRL Press, Oxford.
   Ferguson, M.A.J. and Cross, G.A.M. 1984. Myristylation of the membrane form of Trypanosoma brucei variant surface glycoprotein. J. Biol. Chem. 259:3011‐3015.
   Ferguson, M.A.J., Duszenko, M., Lamont, G.S., Overath, P., and Cross, G.A.M. 1986. Biosynthesis of Trypanosoma brucei variant surface glycoprotein: N‐glycosylation and addition of a phosphatidylinositol membrane anchor. J. Biol. Chem. 261:356‐362.
   Ferguson, M.A.J., Haldar, K., and Cross, G.A.M. 1985. Trypanosoma brucei variant surface glycoprotein has a sn‐1,2‐dimyristoyl glycerol membrane anchor at its COOH‐terminus. J. Biol. Chem. 260:4963‐4968.
   Field, M.C. and Menon, A.K. 1991. Biosynthesis of glycolipid anchors in Trypanosoma brucei. Trends Genet. 3:107‐115.
   Hereld, D., Krakow, J.L., Bangs, J.D., Hart, G.W., and Englund, P.T. 1986. A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. J. Biol. Chem. 261:13813‐13819.
   Hooper, N.M. and Bashir, A. 1991. Glycosyl‐phosphatidylinositol‐anchored membrane proteins can be distinguished from transmembrane polypeptide‐anchored proteins by differential solubilization and temperature‐induced phase separation in Triton X‐114. Biochem. J. 280:745‐751.
   Ikezawa, H. 1991. Bacterial PIPLcs—unique properties and usefulness in studies on GPI anchors. Cell Biol. Int. Rep. 15:1115‐1131.
   Kodukula, K., Gerber, L.D., Amthauer, R., Brink, L., and Udenfriend, S. 1992. Biosynthesis of glycosylphosphatidylinositol (GPI)‐anchored membrane proteins in intact cells: Specific amino acid requirements adjacent to the site of cleavage and GPI attachment. J. Cell Biol. 120:657‐664.
   Leidich, S.D., Drapp, D.A., and Orlean, P.A. 1994. A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis. J. Biol. Chem. 269:10193‐10196.
   Lisanti, M.P., Field, M.C., Caras, I.W., Menon, A.K., and Rodriguez‐Boulan, E. 1991. Mannosamine, a novel inhibitor of glycosyl‐phosphatidylinositol incorporation into proteins. EMBO J. 10:1969‐1977.
   Low, M.G. 1989. The glycosyl‐phosphatidylinositol anchor of membrane proteins. Biochim. Biophys. Acta 988:427‐454.
   McConville, M.J. and Ferguson, M.A.J. 1993. The structure, biosynthesis, and function of glycosylated phosphatidylinositols in parasitic protozoa and higher eukaryotes. Biochem. J. 294:305‐324.
   Pan, Y.‐T., Kamitani, T., Bhuvaneswaran, C., Hallaq, Y., Warren, C.D., Yeh, E.T.H., and Elbein, A.D. 1992. Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine. J. Biol. Chem. 267:21250‐21255.
   Ralton, J.E., Milne, K.G., Guther, M.L.S., Field, R.A., and Ferguson, M.A.J. 1993. The mechanism of inhibition of glycosylphosphatidylinositol anchor biosynthesis in Trypanosoma brucei by mannosamine. J. Biol. Chem. 268:24183‐24189.
   Rosenberry, T.L. 1991. A chemical modification that makes glycoinositol phospholipids resistant to phospholipase C cleavage: Fatty acid acylation of inositol. Cell Biol. Int. Rep. 15:1133‐1149.
   Watkins, S. 1989. Immunohistochemistry. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.14.6.1‐14.6.13. John Wiley & Sons, New York.
Key References
   Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. 1988. Glycosyl‐phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science 239:753‐759.
  This landmark paper first described the core glycan of GPI anchors, providing a scheme for their structural analysis.
   McConville and Ferguson, 1993. See above.
  Provides an excellent overview of the GPI field, with an extensive bibliography and useful compilations of structural information.
   Post‐translational Modifications of Proteins by Lipids: A Laboratory Manual. 1988. (U. Brodbeck and C. Bordier, eds.) Springer‐Verlag, N.Y.
  A useful laboratory manual of methods dealing with various post‐translational modifications of proteins by lipids; many relate to GPI‐linked proteins.
   Methods: A Companion to Methods in Enzymology, Vol.1, Number 3. (P. Casey, ed.) Academic Press, San Diego, Calif.
  This issue of Methods, titled “Covalent modification of proteins by lipids,” contains two chapters specifically about GPIs—Doering et al., (see above) and Mayor and Menon (pp. 297‐305)—as well as material on related topics.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library