Refolding of human class II major histocompatibility complex molecules isolated from Escherichia coli. Assembly of peptide-free heterodimers and increased refolding-yield in the presence of antigenic peptide.

Stöckel, J; Meinl, Edgar; Hahnel, Christian; Malotka, Joachim; Seitz, R; Drexler, Klaus; Wekerle, Hartmut; Dornmair, Klaus

doi:10.1016/s0021-9258(18)43918-x

Cited by 23 publications

(6 citation statements)

References 60 publications

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“…Production of empty, peptide-free class II MHC proteins simplified analysis of the binding reaction (24)(25)(26)(27)(28). Truncated class II MHC Rβ subunits comprising the soluble extracellular domain could be produced in recombinant insect cells, which assemble the Rβ heterodimer but do not load it with peptides (25).…”

mentioning

confidence: 99%

A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

2000

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Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M -1 s -1 ) and dissociation (<10 -5 s -1 ) processes. Various linear and branched pathways have been proposed to account for these data. Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins. Our experiments identified an obligate intermediate in the binding reaction. The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change. The same mechanism is observed for different peptide antigens. In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding. The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed.Proteins encoded by the major histocompatibility complex (MHC) 1 gene locus bind peptide antigens and display them at the cell surface for inspection by the immune system as part of the mechanism by which foreign material in the body is recognized and removed (1). Class II MHC proteins generally are found on specialized immune system cells such as B cells, macrophages, and dendritic cells, but they can be expressed by most cell types in response to inflammation or infection (2). Newly synthesized class II MHC R-and -glycoprotein subunits associate with a chaperonin-like invariant chain protein, which places an extended loop in the class II peptide binding site and directs transport to an endosomal compartment (3). Endosomal proteins cleave the invariant chain, and the bound fragment is exchanged for peptides generated from cell-surface and endocytosed proteins, in a poorly characterized process catalyzed by the peptide-exchange factor HLA-DM (4). MHC-peptide complexes then are transported to the cell surface for inspection by T-cell receptors on CD4 + T lymphocytes. Crystal structures have been determined for several human and murine class II MHC proteins in complex with defined peptides (5-13). In each case, the peptide was bound in a polyproline type II-like conformation, with several side chains projecting into specificity-determining pockets within the overall peptide binding groove, and with many additional contacts between the MHC proteins and the main chain of the bound peptide.Initially, in vitro kinetic measurements of peptide binding to purified class II MHC proteins were interpreted in terms of the simple bimolecular reaction shown in Scheme 1 (14): Physical and chemical characterization of purified class II MHC revealed that they carried complex mixtures of tightly bound endogenous peptides (15-19). The stoichiometry of binding for peptide added to these preparations was quite low. Many of the natural peptide ligands had extremely long half-lives, often on the order of ...

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confidence: 99%

A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

2000

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“…33,34 In HLA-Class II, the HLA-DR mAb, L243, has been reported to react to a confirmational epitope present on DRA. [35][36][37] As several exposed AA patches are conserved among HLA Class II alleles, it is impossible to identify any one eplet as the target of these HLA Class II mAbs using SAB/LSA. 38 The difference in the frequency of inter-locus reactive HLA Class I mAbs and inter-locus reactive Class II mAbs could be related to the greater degree of polymorphism seen in HLA Class I alleles.…”

Section: Discussionmentioning

confidence: 99%

“…In HLA‐Class II, the HLA‐DR mAb, L243, has been reported to react to a confirmational epitope present on DRA 35–37 . As several exposed AA patches are conserved among HLA Class II alleles, it is impossible to identify any one eplet as the target of these HLA Class II mAbs using SAB/LSA 38…”

Section: Discussionmentioning

confidence: 99%

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells

Peton,

Taniguchi,

Mangiola

et al. 2023

HLA

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HLA Class I and II expression are known to differ locus‐to‐locus, however, HLA expression on the cell‐surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post‐transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus‐specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid‐phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra‐locus reactivity without also reacting to inter‐locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter‐locus reactivity. The specificity of the HLA mAbs seen in solid‐phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA‐C, HLA‐DR, HLA‐DQ, and HLA‐DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid‐phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

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“…An example is shown for HLA-DRA1*01:01/HLA-DRB1*04:01 (henceforth DRB1*04:01) associated with a H 6 -tagged peptide, HA 307-318(H6) , where the IMAC chromatography was analyzed by absorbance (OD280, Figure 1A), by SDS-PAGE (Figure 1B), and by L243 staining of selected fractions (Figure 1C). L243 is a conformation-dependent monoclonal anti-HLA-DR antibody specific for a monomorphic epitope on the a chain of a properly folded HLA-DR molecule [29], [30] that is strongly supported by peptide binding [19], [31]. For the molecules found in the early eluting peak 1 (Figure 1A), the presence of a and b chains in a 1:1 stoichiometry (Figure 1B), but lack of L243 staining (Figure 1C), demonstrated that the two chains had associated, but failed to fold properly.…”

Section: Generation Of Peptide-hla Class II Monomersmentioning

confidence: 99%

Correction: MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

Braendstrup¹,

Justesen²,

Ãsterbye³

et al. 2013

PLoS ONE

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Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

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Refolding of human class II major histocompatibility complex molecules isolated from Escherichia coli. Assembly of peptide-free heterodimers and increased refolding-yield in the presence of antigenic peptide.

Cited by 23 publications

References 60 publications

A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells

Correction: MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

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