Measurement of Peptide Dissociation from MHC Class II Molecules

Francisco A. Chaves1, Andrea J. Sant1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.14
DOI:  10.1002/0471142735.im1814s77
Online Posting Date:  May, 2007
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This unit describes a method of measuring the kinetic stability of complexes formed between purified MHC class II molecules and antigenic N‐terminal fluorescein‐labeled peptides. An HPLC‐SEC allows for the separation of fluoresceinated MHC class II:peptide complexes from excess free dissociating peptides, while the fluorescence detector allows for the quantification of the remaining complexes throughout the dissociation time‐course.

Keywords: Peptide; MHC class II molecules; epitope; immunodominance; vaccine design; dissociation assay

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Table of Contents

  • Basic Protocol 1: Measurement of Dissociation Kinetics of MHC Class II:Peptide Complexes
  • Support Protocol 1: Purification of MHC Class II Molecules
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Measurement of Dissociation Kinetics of MHC Class II:Peptide Complexes

  • 100 nM purified MHC class II molecules (see protocol 2)
  • 250 µM fluorescein isothiocyanate (FITC)–labeled peptide of interest
  • 10× McIlvaine's buffer, pH 5.3 (see recipe)
  • 10 mg/ml BSA in binding buffer (see recipe), pH 5.3
  • Binding buffer (see recipe), pH 5.3
  • 250 µM unlabeled competitor peptide
  • PBS ( appendix 2A), pH 7.4
  • 1.5‐ml amber conical tubes
  • 37°C water bath with temperature control to ±0.1°C
  • Microspin columns (Bio‐Rad, no. 732‐6223)
  • High performance liquid chromatograph (HPLC) with isocratic delivery system of flow rate range 0.001 to 9.999 ml/min and high flow rate accuracy (±2% or ±2 µl/min) and analytical size exclusion column (separation range from 103 to 105 Da)
  • Spectrofluorometric detector coupled to the chromatograph
  • Additional reagents and equipment for viable cell counting ( appendix 3B) and flow cytometry (see Chapter 5)

Support Protocol 1: Purification of MHC Class II Molecules

  • 1010 cells expressing MHC class II molecules, such as insect cells (e.g., Sf9, SD2, H5), transfected fibroblasts, CHO cells, B cell lymphoma cells (e.g., A20 (H‐2d), or CH27 (H‐2k), or human lymphoblastoid cell lines.
  • Lysis buffer, pH 8.0 (see recipe)
  • PMSF
  • Protein G– or protein A–Sepharose (unit 2.7)
  • Immunoaffinity resin (unit 8.2) prepared with monoclonal anti‐MHC class II antibody (see recipe; collected from culture supernatant of antibody producing hybridomas or in purified form)
  • Protease inhibitor cocktail (see recipe)
  • 50 mM Tris⋅Cl ( appendix 2A), pH 7.4, containing 0.025% (w/v) NaN 3
  • Enzyme buffer (see recipe)
  • Phosphatidylinositol phospholipase C for GPI‐linked MHC class II molecules
  • 50 mM Tris⋅Cl, pH 7.4
  • 2 M Tris⋅Cl ( appendix 2A), pH 6.8
  • Elution buffer, pH 11.0 (see recipe)
  • PBS, pH 7.4, with 0.025% (w/v) NaN 3
  • Rocking platform
  • Refrigerated centrifuge
  • Empty 10‐ml capacity Econo‐columns (Bio‐Rad)
  • Fraction collector
  • Dialysis membranes with MWCO of 12,000 to 14,000
  • 10,000 MWCO centrifugal protein concentrators (15‐ml capacity)
  • Additional reagents and equipment for Bradford protein assay (unit 2.11), SDS‐PAGE (unit 8.1), and immunoblotting (unit 8.10)
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Literature Cited

Literature Cited
   Berezhkovskiy, L.M. 1999. The analysis of peptide affinity and its binding kinetics to DR1DW1 major histocompatibility complex protein. Biophys. Chem. 77:183‐194.
   Busch, R., Rinderknecht, C.H., Roh, S., Lee, A.W., Harding, J.J., Burster, T., Hornell, T.M., and Mellins, E.D. 2005. Achieving stability through editing and chaperoning: Regulation of MHC class II peptide binding and expression. Immunol. Rev. 207:242‐260.
   Chaves, F.A., Hou, P., Wu, S., and Sant, A.J. 2005. Replacement of the membrane proximal region of I‐Ad MHC class II molecule with I‐E‐derived sequences promotes production of an active and stable soluble heterodimer without altering peptide‐binding specificity. J. Immunol. Methods 300:74‐92.
   Hausmann, D.H., Yu, B., Hausmann, S., and Wucherpfennig, K.W. 1999. pH‐dependent peptide binding properties of the type I diabetes‐associated I‐Ag7 molecule: Rapid release of CLIP at an endosomal pH. J. Exp. Med. 189:1723‐1734.
   Lazarski, C.A., Chaves, F.A., Jenks, S.A., Wu, S., Richards, K.A., Weaver, J.M., and Sant, A.J. 2005. The kinetic stability of MHC class II:peptide complexes is a key parameter that dictates immunodominance. Immunity. 23:29‐40.
   Liang, M.N., Beeson, C., Mason, K., and McConnell, H.M. 1995. Kinetics of the reactions between the invariant chain (85‐99) peptide and proteins of the murine class II MHC. Int. Immunol. 7:1397‐1404.
   Marin‐Esteban, V., Falk, K., and Rotzschke, O. 2004. “Chemical analogues” of HLA‐DM can induce a peptide‐receptive state in HLA‐DR molecules. J. Biol. Chem. 279:50684‐50690.
   Rabinowitz, J.D., Vrljic, M., Kasson, P.M., Liang, M.N., Busch, R., Boniface, J.J., Davis, M.M., and McConnell, H.M. 1998. Formation of a highly peptide‐receptive state of class II MHC. Immunity 9:699‐709.
   Sette, A., Buus, S., Colon, S., Miles, C., and Grey, H.M. 1988. I‐Ad‐binding peptides derived from unrelated protein antigens share a common structural motif. J. Immunol. 141:45‐48.
   Stratmann, T., Apostolopoulos, V., Mallet‐Designe, V., Corper, A.L., Scott, C.A., Wilson, I.A., Kang, A.S., and Teyton, L. 2000. The I‐Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder. J. Immunol. 165:3214‐3225.
   Wettstein, D.A., Boniface, J.J., Reay, P.A., Schild, H., and Davis, M.M. 1991. Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid‐linked form with enhanced peptide/soluble MHC complex formation at low pH. J. Exp. Med. 174:219‐228.
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