Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides

Tussey, Lynda; Frelinger, Jeffrey A.

doi:10.1016/0198-8859(91)90055-e

Cited by 5 publications

(3 citation statements)

References 31 publications

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“…The ability of the peptide to bind HLA‐B*44:02 in these in vitro conditions despite the absence of tapasin might be related to the presence of detergent in the assay. Indeed, a low detergent concentration was previously suggested to generate empty HLA molecules or stabilize an HLA structure that is more prone to bind peptide []. In any case, our results demonstrate that the inability of the MAGE‐A1 peptide to associate with surface HLA‐B*44:02 is not caused by a particularly low affinity of this peptide for the HLA molecule.…”

Section: Resultssupporting

confidence: 51%

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

et al. 2012

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Anti-tumor CTLs recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by “cancer-germline” genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

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Section: Resultssupporting

confidence: 51%

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

et al. 2012

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“…Finally, for some of the in vitro assays, class I molecules were isolated from cells via detergent lysis and immunoprecipitation. However, the presence of detergent has been shown to influence the binding behavior of class I ( , ) and the closely related MHC class II molecules (). Also, the binary complexes of class I heavy chains and β 2 m are unstable overnight in the presence of detergent (), which makes peptide binding and denaturation competing reactions in these experiments.…”

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confidence: 99%

Fast Association Rates Suggest a Conformational Change in the MHC Class I Molecule H-2D^bupon Peptide Binding

et al. 1998

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Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

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“…MHC/peptide complexes were iodinated by previously described methods [24]. Protein integrity was analyzed by reactivity to the anti-MHC mAb W6/32 and gel filtration chromatography.…”

Section: Direct Binding Assaysmentioning

confidence: 99%

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

et al. 1997

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T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.

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Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides

Cited by 5 publications

References 31 publications

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

Fast Association Rates Suggest a Conformational Change in the MHC Class I Molecule H-2D^bupon Peptide Binding

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

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Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides

Cited by 5 publications

References 31 publications

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

Inefficient exogenous loading of a tapasin‐dependent peptide onto HLA‐B*44:02 can be improved by acid treatment or fixation of target cells

Fast Association Rates Suggest a Conformational Change in the MHC Class I Molecule H-2Dbupon Peptide Binding

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

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Fast Association Rates Suggest a Conformational Change in the MHC Class I Molecule H-2D^bupon Peptide Binding