Abstract
We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.
Highlights
We have developed model systems inwhich the bind- major histocompatibility complex (MHC)’ class I or class I1 ing of purified, genetically engineered, soluble ana- molecule on the antigen presentingcell surface [1].The MHC logues of majorhistocompatibility complex (MHC) molecules of both class I and class I1 are polymorphic nonclass I molecules to immobilized antigenic peptides can covalent heterodimers containing one or two membrane-anchored glycoprotein subunits, which present nance (SPR)
In the past several years our in the sequence of an H-2Ld-specific viral peptide, understanding of the structure of the class I molecule and its pMCMV (YPHFMPTNL), was used for coupling, and relation to function has advanced in large part as a result of the resulting surfaces were tested for binding of the the determination of a number of x-ray crystallographic strucsoluble analogue of H-2Ld, H-2Ld
H-2Ld/pMCMV three-dimen- residues) and sequences of peptides that canbind or that sional structuraml odel, only those residuesof the pep- copurify with these molecules [14,15,16,17,18,19]
Summary
(Received for publication, February 11, 1993, and in revised form, April 9, 1993). We have developed model systems inwhich the bind- major histocompatibility complex (MHC)’ class I or class I1 ing of purified, genetically engineered, soluble ana- molecule on the antigen presentingcell surface [1].The MHC logues of majorhistocompatibility complex (MHC) molecules of both class I and class I1 are polymorphic nonclass I molecules to immobilized antigenic peptides can covalent heterodimers containing one (class I) or two (class be monitored in real timeusing surface plasmon reso- 11) membrane-anchored glycoprotein subunits, which present nance (SPR). MATERIALS ANDMETHODS tide subunits,using incorporation of Pz-mas anindication of MHC Class I Molecules the formation of the complex [31,32,33,34] Such assays suffer from the inherent lack of a direct correspondence between peptide binding and the parameter measured. The biosensor was continuously washed with 10 mM HEPES,pH 7.5,150 mM NaCI, 3.4 mM EDTA, 0.005% BIAcore as resonance units (RU) [44] Such SPR measurements have been successfullyapplied to kinetic and equilibrium studies of biomolecular models including antigen-antibody and hapten-antibody systems (4850), receptor-ligand binding [51], and protein-DNA interacsurfactant P20 (Pharmacia) (HBS). Method C-streptavidin was immobilized exactly as described in coupled to a solid-phase
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