Abstract
In the endoplasmic reticulum (ER), MHC class I molecules associate with several specialized proteins, forming a large macromolecular complex referred to as the “peptide-loading complex” (PLC). In the PLC, antigenic peptides undergo a stringent selection process for binding onto MHC class I molecules. This ensures that the immune system elicits robust CD8+ T-cell responses to viruses and solid tumors. The ability to reconstitute in vitro MHC class I molecules in association with key proteins of the PLC provides a mean for studying at the molecular level how antigenic peptides are selected for presentation to CD8+ T-cells. Here, we describe practical procedures for generating a cell-free system involving MHC class I molecules and tapasin, a critical protein of the PLC, that can be used as a versatile tool for biochemical and mechanistic studies of peptide loading and exchange.
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References
Burgess RR (2009) Refolding solubilized inclusion body proteins. Methods Enzymol 463:259–282
Garboczi DN, Hung DT, Wiley DC (1992) HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A 89:3429–3433
Busch R, Pashine A, Garcia KC, Mellins ED (2002) Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers. J Immunol Methods 263:111–121
Scott CA, Garcia KC, Carbone FR, Wilson IA, Teyton L (1996) Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules. J Exp Med 183:2087–2095
Kalandadze A, Gallenno M, Foncerrada L, Strominger JL, Wucherpfennig KW (1996) Expression of recombinant HLA-DR2 molecules. Replacement of the hydrophobic transmembrane motif allows the assembly and secretion of soluble DR alpha beta heterodimers. J Biol Chem 271:20156–20162
Chen M, Bouvier M (2007) Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection. EMBO J 26:1681–1690
Bouvier M, Wiley DC (1998) Structural characterization of a soluble and partially folded class I major histocompatibility/β2m heterodimer. Nat Struct Biol 5:377–382
Chen M, Stafford WF, Diedrich G, Khan A, Bouvier M (2002) A characterization of the lumenal region of human tapasin reveals the presence of two structural domains. Biochemistry 41:14539–14545
Gakamsky DM, Davis DM, Strominger JL, Pecht I (2000) Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by real-time fluorescence resonance energy transfer. Biochemistry 39:11163–11169
Binz AK, Rodriguez RC, Biddison WE, Baker BM (2003) Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer. Biochemistry 42:4954–4961
Springer S, Doring K, Skipper JCA, Townsend ARM, Cerundolo V (1998) Fast association rates suggest a conformational change to the MHC class I molecules H-2Db upon peptide binding. Biochemistry 37:3001–3012
Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954
Wearsch PA, Cresswell P (2007) Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer. Nat Immunol 8:873–881
Acknowledgement
This work was supported by the National Institutes of Health Grant AI045070 from NIAID.
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Bouvier, M. (2013). Studying MHC Class I Peptide Loading and Exchange In vitro. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology™, vol 960. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-218-6_7
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DOI: https://doi.org/10.1007/978-1-62703-218-6_7
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Publisher Name: Humana Press, Totowa, NJ
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