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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Real-time measurement of antigenic peptide binding to empty and preloaded single-chain major histocompatibility
D M Ojcius1, F Godeau, J P Abastado
1Institut Pasteur, INSERM U.277, Paris, France.
Insights
Cytotoxic T lymphocytes (CTL) use major histocompatibility complex (MHC) class I to recognize peptides. This study used fluorescence to measure antigenic peptide binding to a soluble MHC class I molecule, revealing stable complex formation but potential for peptide exchange.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Cytotoxic T lymphocytes (CTL) play a crucial role in adaptive immunity by recognizing peptide antigens presented by major histocompatibility complex (MHC) class I molecules.
- The precise mechanisms governing peptide binding to MHC class I molecules remain incompletely understood, hindering a full grasp of immune surveillance and response.
Purpose of the Study:
- To investigate the kinetics and stability of antigenic peptide binding to a soluble, single-chain MHC class I molecule (SC-Kd).
- To characterize the real-time binding and dissociation dynamics of specific peptides to the SC-Kd complex.
Main Methods:
- Utilized a fluorescence-based assay employing dansyl-labeled peptides to monitor binding to a soluble, single-chain Kd (SC-Kd) molecule.
- Measured association and dissociation rate constants, and determined the apparent activation energy for peptide binding.
- Investigated peptide exchange by observing the effect of excess unlabeled peptide on the SC-Kd-peptide complex.
Main Results:
- Dansylated peptides exhibited enhanced fluorescence upon binding to SC-Kd, allowing for real-time monitoring.
- The binding of a specific dansylated Plasmodium berghei circumsporozoite (PbCS) peptide to SC-Kd demonstrated a significant association rate and slow spontaneous dissociation.
- Addition of excess unlabeled peptide led to increased dissociation, but with a much slower association rate, indicating potential for peptide exchange.
Conclusions:
- The Kd-peptide complex is generally stable on antigen-presenting cells.
- High peptide concentrations in the endoplasmic reticulum lumen may facilitate peptide exchange on Kd molecules prior to cell surface presentation.
- These findings provide insights into the dynamic nature of peptide-MHC interactions crucial for T cell recognition.
Abstract:
Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain Kd (SC-Kd) molecule in which the Kd heavy chain was connected by a 15-residue link to beta 2-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated Kd-restricted peptides to SC-Kd resulted in significant fluorescence enhancement, which could be inhibited by unmodified Kd-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263-260 peptide bound to "empty" SC-Kd with an association rate constant of 1140 M-1s-1, and the subsequent spontaneous dissociation of the SC-Kd-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253-260 peptide, but with a slower association constant for unlabeled peptide, 77 M-1s-1. Thus, the Kd-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on Kd before export to the surface. The apparent activation energy for PbCS 253-260 peptide binding to SC-Kd was 6.78 +/- 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions.
