Detection of Glycophospholipid Anchors on Proteins

Tamara L. Doering1, Paul T. Englund2, Gerald W. Hart2

1 University of California, Berkeley, California, 2 Johns Hopkins Medical School, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.8
DOI:  10.1002/0471142727.mb1708s22
Online Posting Date:  May, 2001
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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 protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.

     
 
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Table of Contents

  • 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
     
 
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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), ice‐cold
  • recipePrecondensed Triton X‐114 stock solution in TBS ( protocol 3support protocol), 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)
  • 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; GIBCO/BRL), 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
  • Acetone, −20°C
  • Appropriate enzyme buffer: recipeGPI‐PLD buffer, recipeGPI‐PLC buffer, and recipePI‐PLC buffer
  • Phospholipase enzyme: 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)
  • recipePrecondensed Triton X‐114 solution (first protocol 3support protocol)
  • Centrifuge and rotor (e.g., SS‐34)

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

  Materials
  • Protein(s)
  • recipe0.1 M acetate buffer, pH 3.5
  • 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 (second protocol 5alternate protocol and third protocol 7basic protocols)
  • recipeWater‐saturated n‐butanol

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

  Materials
  • Protein radiolabeled with fatty acid
  • 0.2 M KOH in methanol
  • Methanol
  • recipe1 M hydroxylamine·HCl, pH 7.5, made fresh
  • 1 M Tris·Cl, pH 7.5 ( appendix 22)
  • Additional reagents and equipment for one‐dimensional gel electrophoresis (unit 10.2), staining of gels (unit 10.6), and autoradiography ( appendix 3A)
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Figures

  •   FigureFigure 17.8.1 Schematic representation of the glycan core structure common to all GPI anchors. The sites of cleavage of phospholipase C enzymes (PI‐PLC, GPI‐PLC), phospholipase D (GPI‐PLD), and nitrous acid (HONO) are indicated, as are lipid products resulting from these cleavages: PI (phosphatidylinositol), PA (phosphatidic acid), DAG (diacylglycerol). Other abbreviations: Man, mannose; GlcN, glucosamine; EthN, ethanolamine; P, phosphate group. Phospholipase C treatment generates inositol cyclic phosphate, the major epitope contributing to the cross‐reacting determinant (CRD). Lipid products may vary from those depicted, depending on features of the anchor (see background information).
  •   FigureFigure 17.8.2 Flowchart for the detection of GPI‐anchored proteins.

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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.
   Cross, G.A.M. 1990. Glycolipid anchoring of plasma membrane proteins. Annu. Rev. Cell Biol. 6:1‐39.
   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.
   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., 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.
   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. 1991. Lipid anchors on membrane proteins. Current Opinion in Structural Biology. 1:522‐529.
   Ferguson, M.A.J. 1992. The chemical and enzymic 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.
   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.
   Low, M.G. 1989. The glycosyl‐phosphatidylinositol anchor of membrane proteins. Biochim. Biophys. Acta 988:427‐454.
   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.
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.
   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 methods 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); Mayor and Menon (pp. 297‐305)—as well as related topics.
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