Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)

Charles J. Parker1

1 University of Utah School of Medicine and The Veterans Affairs Medical Center, Salt Lake City, Utah
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.5
DOI:  10.1002/0471142735.im1305s11
Online Posting Date:  May, 2001
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Abstract

The complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement‐mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay‐accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b‐9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement‐mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of CD55 and CD59
  • Support Protocol 1: Functional Assay for CD59
  • Support Protocol 2: Functional Assay for CD55
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of CD55 and CD59

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A2; for suppliers, see appendix 55.
  • Phosphate‐buffered saline (PBS; appendix 2A2)
  • 4 units (∼1 liter) packed human red blood cells
  • recipeGhosting buffer (see recipe), ice‐cold and room temperature
  • BCA protein assay kit (Pierce)
  • 3 M NaCl
  • n‐butanol
  • 20 mM sodium phosphate buffer (pH 7.5)/20 mM NaCl
  • Mono Q starting buffer I: 20 mM sodium phosphate buffer (pH 7.5)/20 mM NaCl/0.1% (v/v) NP‐40
  • 20 mM sodium phosphate buffer (pH 7.5)/500 mM NaCl/0.1% (v/v) NP‐40
  • Hydroxylapatite (Bio‐Gel HT, Bio‐Rad)
  • 20 mM Tris⋅Cl, pH 8.5
  • Hydroxylapatite starting buffer: 20 mM Tris⋅Cl (pH 8.5)/0.1% (v/v) NP‐40
  • 40 mM sodium phosphate (pH 7.5)/0.1% (v/v) NP‐40
  • 40 mM sodium phosphate buffer (pH 7.5)/300 mM NaCl
  • Phenyl Superose starting buffer: 40 mM sodium phosphate buffer (pH 7.5)/300 mM NaCl/0.1% (v/v) NP‐40
  • 40 mM sodium phosphate buffer (pH 9.5)/50 mM NaCl/1% (v/v) NP‐40
  • 10 mM Tris⋅Cl (pH 8.0)/10 mM NaCl
  • Mono Q starting buffer II: 10 mM Tris⋅Cl (pH 8.0)/10 mM NaCl/0.1% (w/v) NP‐40
  • 10 mM Tris⋅Cl (pH 8.0)/500 mM NaCl/0.1% (v/v) NP‐40
  • PBS containing 0.02% (w/v) sodium azide
  • 250‐ml centrifuge bottles
  • Beckman J2‐HS centrifuge and JA‐14 rotor (or equivalent)
  • 20‐liter carboys
  • Pellicon ultrafiltration device and PTHK filters (Millipore)
  • Peristaltic pump
  • FPLC system (Pharmacia Biotech)
  • Mono Q HR 16/10 and HR 5/5 FPLC columns (Pharmacia Biotech)
  • Phenyl Superose HR 5/5 column (Pharmacia Biotech)
  • Additional reagents and equipment for dialysis ( appendix 3A3), denaturing SDS‐PAGE (unit 8.4), and silver staining (unit 8.9)

Support Protocol 1: Functional Assay for CD59

  Additional Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A2; for suppliers, see appendix 55.
  • Crude Naja naja naja cobra venom factor (CoF; Miami Serpentarium) or pure Naja naja kaouthia CoF (Sigma)
  • Normal human serum (fresh or frozen at −70°C)
  • 0.1 M EDTA, pH 7.4
  • Veronal‐buffered saline (VBS; unit 13.2)
  • GVBE (unit 13.1), room temperature and ice‐cold
  • Guinea pig whole blood diluted 1/9 in recipeacid‐citrate‐dextrose (ACD; see recipe)
  • Low‐speed centrifuge (e.g., Sero‐Fuge II, Becton Dickinson)

Support Protocol 2: Functional Assay for CD55

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A2; for suppliers, see appendix 55.
  • Sheep erythrocytes stored in recipeACD (acid‐citrate‐dextrose; see recipe), or equivalent
  • GVB (unit 13.1)
  • IgM rabbit anti–sheep red blood cell antibody (hemolysin; Organon Teknika Cappel)
  • GVBE (unit 13.1), room temperature and ice‐cold
  • Normal human serum
  • VBS++: VBS (GBV without gelatin; unit 13.2) containing 0.15 mM CaCl 2 and 0.5 mM MgCl 2
  • Low‐speed centrifuge (e.g., Sero‐Fuge II, Becton Dickinson)
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Figures

Videos

Literature Cited

Literature Cited
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   Davies, A., Simmons, D.L., Hale, G., Harrison, H.A., Tighe, H., Lachmann, P.J., and Waldman, H. 1989. CD59, an Ly‐6‐like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Exp. Med. 170:637‐654.
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   Holguin, M.H. and Parker, C.J. 1992. Membrane inhibitor of reactive lysis. In Current Topics in Microbiology and Immunology,Vol. 178: Membrane Defenses Against Attack by Complement and Perforins (C.J. Parker, ed.) pp. 61‐85. Springer‐Verlag, New York and Heidelberg.
   Holguin, M.H., Fredrick, L.R., Bernshaw, N.J., and Parker, C.J. 1989. Isolation and characterization of a protein from normal erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria. J. Clin. Invest. 84:7‐17.
   Hughes, T.R., Piddlesden, S.J., Williams, J.D., Harrison, R.A., and Morgan, B.P. 1992. Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement. Biochem. J. 284:169‐176.
   Kinoshita, T., Medof, M.E., Silber, R., and Nussenzweig, V. 1985. Distribution of decay‐accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 162:75‐92.
   Kinoshita, T., Rosenfeld, S.I., and Nussenzweig, V. 1987. A high‐molecular‐weight form of decay‐accelerating factor (DAF‐2) exhibits size abnormalities in paroxysmal nocturnal hemoglobinuria erythrocytes. J. Immunol. 138:2994‐2998.
   Lublin, D.M. 1992. Glycosyl‐phosphatidylinositol anchoring of membrane proteins. In Current Topics in Microbiology and Immunology,Vol. 178: Membrane Defenses Against Attack by Complement and Perforins (C.J. Parker ed.) pp. 141‐162. Springer‐Verlag, New York and Heidelberg.
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   Meri, S., Morgan, B.P., Davies, A., Daniels, R.H., Olavesen, M.G., Waldmann, H., Lachmann, P.J. 1990. Human protectin (CD59), an 18,000‐20,000 MW complement lysis restricting factor, inhibits C5b‐8 catalysed insertion of C9 into lipid bilayers. Immunology 71:1‐9.
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   Nicholson‐Weller, A., Burge, J., Fearon, D.T., Weller, P.F., and Austin, F. 1982. Isolation of a human erythrocyte glycoprotein with decay accelerating activity for C3 convertases of the complement system. J. Immunol. 129:184‐189.
   Nicholson‐Weller, A., March, J.P., Rosenfeld, S.I., and Austen, K.F. 1983. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in decay accelerating factor. Proc. Natl. Acad. Sci. U.S.A. 80:5066‐5070.
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   Parker, C.J. 1991. Paroxysmal nocturnal hemoglobinuria and glycosyl phosphatidylinositol anchored proteins that regulate complement. Clin. Exp. Immunol. 86 (Suppl. 1):36‐42.
   Rosse, W.F. 1990. Clinical Immunohematology: Basic Concepts and Clinical Applications. Blackwell Scientific, Oxford.
   Rosse, W.F. and Parker, C.J. 1985. Paroxysmal nocturnal hemoglobinuria. Clin. Haematol. 14:105‐125.
   Schönermark, S., Rauterberg, E.W., Shin, M.L., Loke, S., Roelcke, D., and Hänsch, G.M. 1986. Homologous species restriction in lysis of human erythrocytes: A membrane‐derived protein with C8‐binding capacity functions as an inhibitor. J. Immunol. 136:1772‐1776.
   Seya, T., Turner, J.R., and Atkinson, J.P. 1986. Purification and characterization of a membrane protein (gp45‐70) that is a cofactor for cleavage of C3b and C4b. J. Exp. Med. 163:837‐855.
   Stefanova, I., Hilgert, I., Kristofova, H., Brown, R., Low, M.G., and Horejsi, V. 1989. Characterization of a broadly expressed human leucocyte surface antigen MEM‐43 anchored in membrane through phosphatidylinositol. Mol. Immunol. 26:153‐161.
   Sugita, Y., Uzawa, M., and Tomita, M. 1987. Isolation of decay‐accelerating factor (DAF) from rabbit erythrocyte membranes. J. Immunol. Methods 104:123‐130.
   Sugita, Y., Nakano, Y., and Tomita, M. 1988. Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels. J. Biochem. 104:663‐667.
   Tomita, A., Radike, E.L., and Parker, C.J. 1993. Isolation of the erythrocyte membrane inhibitor of reactive lysis type II. Identification as glycophorin A. J. Immunol. 151:3308‐3323.
   van den Berg, C.W., Harrison, R.A., and Morgan, B.P. 1993. The sheep analogue of human CD59: Purification and characterisation of its complement inhibitory activity. Immunology 78:349‐359.
   Zalman, L.S., Wood, L.M., and Müller‐Eberhard, H.J. 1986. Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels. Proc. Natl. Acad. Sci. U.S.A. 83:6975‐6979.
Key References
   Holguin, M.H., Fredrick, L.R., Bernshaw, N.J., and Parker, C.J. 1989. See above.
  Original report of the isolation of CD59.
   Nicholson‐Weller, A., Burge, J., Fearon, D.T., Weller, P.F., and Austin, F. 1982. See above.
  Original report of the isolation of CD55 (DAF); the basic protocol is a modification of the method described therein.
   Parker, C.J. (ed). 1992. Current Topics in Microbiology and Immunology, Vol. 178: Membrane Defenses Against Attack by Complement and Perforins. Springer‐Verlag, New York and Heidelberg.
  Contains reviews of CD55, CD59, CD46 (MCP), CD35 (CR1), and HRF, including detailed analysis of biochemical, molecular, and functional characteristics, along with reviews of the GPI anchor and of PNH.
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