Antigen presentation. One size fits all.
The structure of a complex between an MHC class II molecule and a specific peptide reveals both similarities and differences in peptide binding by MHC class I and class II molecules.
Amino Acid Sequence↗
Biomedical subjects
J B Rothbard
Publications and source records attributed to J B Rothbard.
Role of the polymorphic residues in HLA-DR molecules in allele-specific binding of peptide ligands.
Analysis of peptide binding to a set of HLA-DR alleles has allowed the proteins to be segregated into functional subsets, depending on the amino acids at positions 57 and 86 of the beta-chain. DR proteins with glycine at 86 beta and aspartic acid at 57 beta bound a simplified peptide with significantly lower IC50 values than alleles that did not have this combination of amino acids. The size of the amino acid at 86 beta seemed to modify the steric requirements for the single most important side chain of the peptide. Within each of the four subgroups, other polymorphic amino acids define allele-specific binding requirements. These were explored by analyzing the ability of eight different DR alleles to bind 13 known T cell determinants. The side chains in the peptides that seemed to be responsible for allele specificity were determined by correlating their common structural features with complementary polymorphic residues in the binding site. The importance of these residues was tested by incorporating them into a polyalanine backbone, and was confirmed by the ability of these residues to transfer allele specificity to these simplified analogues. Even though polymorphic contacts affected peptide affinity, the majority of the free energy of binding in all cases arose from interactions with the peptide backbone and the single hydrophobic amino acid at the third position. These constraints seem to orient all peptides in a similar location, forcing them to adopt a closely related conformation in the binding site. The corresponding side chain in each peptide contacts the same pocket in the binding site, regardless of the allele. This apparent similarity should allow any DR allele to be analyzed by extrapolation from the DR1 crystal structure.
Alleles↗
Exploration of requirements for peptide binding to HLA DRB1*0101 and DRB1*0401.
The individual amino acid contacts responsible for peptide binding to DRB1*0101 and/or DRB1*0401 were defined using a quantitative binding assay. The differential contribution of each amino acid in two well studied T cell determinants, HA307-319 and RMBP 90-102, was delineated by comparing the IC50 values of analogues of varying length. This analysis confirmed the importance of a hydrophobic amino acid located near the amino-terminus for binding to both alleles and revealed that the contribution of the carboxyl-terminal amino acids differed between DRB1*0101 and DRB1*0401. Taking advantage of previous experiments demonstrating that all of the residues could be replaced with alanine, with the exception of the key hydrophobic amino acid, simplified analogues composed of polyalanines were used to prove 1) optimal binding depended on the position of the hydrophobic side chain relative to the amino- and carboxyl-termini; 2) aromatic amino acids were superior to aliphatic side chains at this position; and 3) a significant amount of free energy of binding arises from hydrogen bonding between the class II binding site and the amide bonds of the ligand. The role of each carbonyl and amide nitrogen was measured by assaying analogues containing reduced peptide bonds or N-methyl amino acids. Serine, but not glycine, could be used as a framework amino acid for peptide ligands, indicating that the beneficial aspects of these simplified structures was the combination of retaining the correct orientation of the peptide bonds, the restriction of the conformational freedom by limiting the possible phi/psi angles of the peptide, and avoidance of deleterious side-chain contacts. Collectively, these data were consistent with the peptide binding in a nonrepeating conformation with the vast majority of the free energy of binding arising from hydrogen bonds with the peptide backbone and a single, key hydrophobic side chain interacting in a conserved pocket in both DRB1*0101 and DRB1*0401.
Amino Acid Sequence↗
Detergent-enhanced dissociation of endogenous peptides from PI-DRB1*0401.
A variety of detergents have been shown to catalyze the dissociation of bound peptides from a soluble from of DRB1*0401. By using a class II molecule lacking the hydrophobic transmembrane region, the need for solubilizing the transmembrane protein was removed and enabled the specific interaction between the class II protein and the amphiphile to be identified. The presence of detergent increased the rate of association of added peptide and the percent occupancy of the receptor, presumably because the dissociation of endogenous peptide was the rate-limiting step in binding. The data help explain the differences reported between peptide binding to class II proteins on the surface of cells and binding to class II proteins solubilized in detergent. The interaction did not correlate with the critical micellar concentration of the detergent nor were all amphiphilic structures equally effective, consistent with a specific interaction between the amphiphile and the MHC class II protein. Of the eight detergents examined, octyl glucoside was the most efficient. These experiments did not distinguish between an allosteric mechanism or direct competition with the peptide for binding.
Amino Acid Sequence↗
Responses of NOD congenic mice to a glutamic acid decarboxylase-derived peptide.
Type 1 diabetes in man and the NOD (H-2g7) mouse is frequently associated with an autoimmune response to two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. GAD-specific autoantibodies produced by B cells and GAD-specific T cells have been observed in both species. In the current study, the response to a GAD65-derived peptide, GAD65 524-543, previously reported to be an epitope recognized by spleen cells obtained from 3-week-old NOD mice, was assessed in NOD MHC and non-MHC congenic strains. Although spontaneous reactivity to GAD65 524-543 was not observed in NOD mice, the peptide was immunogenic in NOD mice as well as in two NOD congenic strains which are both H-2g7, B10.H-2g7 and NOD.B6Il2-Tshb. This was surprising since the B10.H-2g7 strain does not develop diabetes or insulitis and fewer than 3% of NOD.B6Il2-Tshb mice develop diabetes. The response to GAD65 524-543 was shown to be controlled by the MHC since neither the B10 nor the NOD.H-2b strain, both of which are H-2b, responded to the peptide. This study demonstrates that T cell responsiveness to GAD-derived peptides can be elicited in strains of mice that are resistant to the development of spontaneous diabetes, suggesting that peripheral tolerance to GAD is not associated with protection from diabetes.
Amino Acid Sequence↗
Prediction of peptide affinity to HLA DRB1*0401.
Hydrogen bonding between conserved amino acids in the HLA DR and the peptide backbone of the ligand both provide the majority of free energy of binding and force the peptide ligands to adopt a similar extended conformation. Consequently the corresponding side chains of all peptides interact with similar pockets in the binding site. For peptides of a common length the contribution of the peptide backbone can be treated as a constant and the differential affinity can be viewed as a simple sum of the side chain interactions. These can be quantified by measuring the effects of each of the naturally occurring amino acids in the context of a simplified polyalanine backbone containing an aromatic amino acid to orient the peptide unequivocally in the binding site. The dataset of the relative contributions can be used to predict quantitatively the affinity of any peptide sequence.
Amino Acid Sequence↗
Comparison of three different forms of HLA-DR4Dw4 proteins.
The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI-DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected chinese hamster ovary cells. Recombinant and B cell-derived DR4Dw4 molecules bound monoclonal anti-DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10-20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI-DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.
Amino Acid Sequence↗
A method for rapid screening of recombinant proteins for recognition by T lymphocytes.
A simple, cost-effective method is described that allows rapid screening of recombinant protein sequences for their ability to stimulate T cells. Individual microcultures of E. coli each expressing a gene product or peptide sequence fused to protein A are grown in 96-well plates. Following lysis of the bacteria, the fusion peptide is readily captured with immobilized immunoglobulin in tissue culture wells. No further purification is required. T lymphocytes plus appropriate antigen-presenting cells are added directly to the wells and assayed for proliferation. The DNA in bacteria from wells stimulating T cell proliferation is then sequenced. The technique allows rapid mapping of T cell epitopes by facilitating screening of truncation mutants without extensive purification. Described here is a further application of the technique to study monosubstituted analogues of a known T cell epitope.
Animals↗
Synthetic peptides as vaccines.
The economics of vaccines has been a major limitation in the commercial research and development of new approaches. This coupled with the natural scientific desire to simplify and define the composition of effective vaccines argues that the future of vaccines lies in novel approaches that will discover effective and less expensive components. Peptides, whether they are chemically synthesized or produced in bacteria, are an attractive possibility. To substitute linear peptides for complex mixtures of proteins would be a major technical advance and would stimulate tremendous commercial interest. However, at the present time I view this approach still unlikely to be of major practical importance. I conclude this because of the complexity of immunological responses to microorganisms. Even though, in some instances, a cytotoxic T-cell response or even the majority of the antibody response to a pathogen can be defined by a short linear peptide, most people believe that multiple effector functions of the immune system should be stimulated in optimal vaccines. For a small cocktail of peptides to reproduce the diversity of responses elicited by a virus, parasite, or bacterium is unlikely. However, I fully realize that remarkable progress has occurred towards understanding the structural requirements necessary to stimulate cellular and humoral immune responses, and peptides have been integral in the development of this field. Also, the success of several research groups in developing effective antiviral vaccines using short linear peptides argues that I might be painting too dark of a picture. As someone who has used this strategy to explore peptide-MHC and peptide-antibody interactions, I am a strong scientific supporter of the approach. In this forum I am purposely cautious in my optimism. As the details of the complex molecular and cellular interactions that control the immune system are elucidated, both the number of strategies and the possible applications of modulating the immune response will increase as well. In addition to protective immunity to pathogens, cancer therapy could be revolutionized if tumor-specific cytotoxic T-cells could be generated routinely. Novel therapeutic approaches to allergy, autoimmunity, and transplantation can be envisioned if the T-lymphocytes responsible for these syndromes could be modulated without total immune suppression. Consequently, I am confident that the experiments described in this chapter will be central to developing exciting new therapeutic and prophylactic compounds, but I am not sure that they will resemble naturally occurring peptides. The one aspect I am confident of is that the capacity of the immune response to protect the organism will continually surprise us.
Animals↗
Positioning of a peptide in the cleft of HLA-A2 by complementing amino acid changes.
Several mutant HLA-A2 molecules have been constructed and expressed in the mutant human B-cell line C1R, which lacks HLA-A and HLA-B antigens, and examined for presentation of a previously defined peptide epitope derived from the influenza matrix protein to appropriate human cytotoxic T-lymphocyte lines. When leucine residue 66 in this matrix peptide containing residues 57-68 (matrix peptide 57-68) was replaced by arginine, the resulting matrix peptide 57-68 R66 was not presented to HLA-A2, but the mutation Y116D (tyrosine to aspartic acid at residue 116) in the floor of the peptide binding cleft near its right end dramatically restored peptide presentation. A similar result was obtained by substitution of ornithine for leucine at residue 66. These data provide strong support for a model in which the peptide is orientated with its amino terminus at the left end of the cleft of HLA-A2 and its carboxyl terminus at the right.
Amino Acid Sequence↗
Effect of natural polymorphism at residue 86 of the HLA-DR beta chain on peptide binding.
Class I and class II MHC glycoproteins are highly polymorphic molecules that bind antigenic peptides and present them on cell surfaces for recognition by T lymphocytes. Even though MHC polymorphism has long been known to affect both peptide binding and recognition by the TCR, the role of individual amino acids of MHC proteins in these interactions is poorly understood. To examine the effect of a small number of amino acid residues on T cell stimulation, B lymphoblastoid cell lines homozygous for the closely related DR1 subtypes, Dw1 and Dw20, and the DR4 subtypes, Dw4 and Dw14, were compared for their ability to present an immunogenic influenza hemagglutinin peptide (HA307-319) to an Ag-specific, DR1,4-restricted T cell clone. B cell lines expressing DR1 Dw20 and DR4 Dw14 presented HA307-319 much less efficiently than DR1 Dw1 and DR4 Dw4 and bound a biotinylated analogue of the same peptide less well. Analysis of DRB1 gene sequences suggested that polymorphism at residue 86 had a major effect on peptide binding. Differences in binding of a set of HA307-319 analogues biotinylated at each residue to cells expressing DR1 Dw1 and DR1 Dw20 suggested that the polymorphism affected the interactions of many peptide residues with the class II molecule. In inhibition assays, DR1 Dw1 and DR4 Dw4 were shown to differ from DR1 Dw20 and DR4 Dw14 in their length requirements for peptide binding. Using a larger panel of homozygous B cell lines expressing many class II haplotypes, a Ser-309 substituted HA307-319 analogue was shown to bind to most B cell lines expressing Val-86 containing alleles (including DR1 Dw20 and DR4 Dw14) but failed to bind most B cell lines expressing Gly-86 alleles (including DR1 Dw1 and DR4 Dw4). The results indicated that polymorphism at residue 86 influenced the specificity and affinity of peptide binding and affected the conformation of peptide-DR protein complexes without completely eliminating T cell recognition.
Alleles↗
Conformational and structural characteristics of peptides binding to HLA-DR molecules.
A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.
Amino Acid Sequence↗
A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA.
The sequences of the peptide binding domains of 33 70 kd heat shock proteins (hsp70) have been aligned and a consensus secondary structure has been deduced. Individual members showed no significant deviation from the consensus, which showed a beta 4 alpha motif repeated twice, followed by two further helices and a terminus rich in Pro and Gly. The repeated motif could be aligned with the secondary structure of the functionally equivalent peptide binding domain of human leucocyte antigen (HLA) class I maintaining equivalent residues in structurally important positions in the two families and a model was built based on this alignment. The interaction of this domain with the ATP domain is considered. The overall model is shown to be consistent with the properties of products of chymotryptic cleavage.
Adenosine Triphosphate↗
An in vitro model of peptide-mediated immunomodulation of the human T cell response to Dermatophagoides spp (house dust mite).
Allergic sensitivity of Dermatophagoides spp (house dust mites) is mediated by specific IgE antibody, the production of which requires the presence of CD4+ helper T cells. Attempts to hyposensitize this response in allergic individuals have depended on the administration of extracts of specific allergen. However, the ability of peptides derived from unrelated antigens to inhibit specific immune responses offers an alternative approach to therapy. We have addressed this question by examining the ability of a nonstimulatory peptide analogue derived from influenza virus hemagglutinin to modulate T cell recognition of house dust mite. The peptide inhibited the response of mite-specific CD4+ T cell clones restricted by either the HLA-DRAB1 or DRAB3 gene products. Furthermore, mite-induced polyclonal T cell responses were negatively modulated by the peptide, whereas recognition of common recall antigens remained intact. The inhibitory effects were mediated at the level of the antigen-presenting cell, since no inhibition of mitogen or anti-CD3 antibody-driven T cell proliferation was observed. In direct binding assays, the peptide analogue bound to selected HLA-DR molecules expressed on the membrane of antigen-presenting cells, with specificity predominantly for those class II proteins capable of restricting house dust mite-allergen T cell recognition.
Adult↗
Interactions between immunogenic peptides and MHC proteins.
The MHC class-I and class-II molecules are highly polymorphic membrane proteins, which bind and transport to the surface of cells peptide fragments of intact proteins. The peptide-MHC complexes are recognized by the antigen-specific receptor of T lymphocytes and are the basis by which the cellular immune system distinguishes self from nonself. In order to perform this function, MHC proteins simultaneously display a large spectrum of structurally divergent peptides for a sufficiently long period of time for the T cell repertoire to scan the cell effectively. Consistent with the protein's biological role, the rates of association and dissociation at physiological pH are very slow relative to other known receptor-ligand interactions. The mechanism by which the proteins do this is still poorly understood, but recent experimental results indicate that the rate determining step may be a conformational change that results in the entrapment of the peptide. A variety of binding assays have been developed that allow study of the detailed kinetics and specificity of the interaction. The optimal peptide length for binding is between 8 and 12 amino acids with the central 5-7 residues contributing the majority of the specific contacts. Determining the conformation of bound peptides has been hampered by the inherent ability of the receptor to bind manifold sequences. Consequently, strategies employing monosubstituted analogs have had only limited success. Approaches using biotinylated amino acids and other bulky substituents or multiple substitutions have generated more information. Recent experiments demonstrating that peptides with polyalanine, polyproline, or polyglycine bind well to MHC proteins have proven that the structural requirements for binding are quite minimal. In fact, a significant factor of the selectivity for binding appears to be the avoidance of deleterious contacts, rather than the need for a large number of critical interactions. Binding experiments also have shown that several peptides can bind a large number of MHC class-I and class-II alleles. The degenerate binding indicated that the binding site of MHC proteins must have a significant number of conserved features. Solution of the crystal structures of the MHC class-I alleles A2 and Aw68 has identified a putative antigen-combining site whose overall dimensions were quite similar between the two structures. The detailed surface topology of the site varied between the two alleles due to the size and chemical properties of the side chains of the polymorphic amino acids composing the cleft.(ABSTRACT TRUNCATED AT 400 WORDS)
Alleles↗
Detection of peptide-MHC class II complexes on the surface of intact cells.
The interaction of peptides with major histocompatibility complex proteins on the surface of cells is required for their recognition by T lymphocytes. Many studies characterizing the formation of peptide-MHC class II complexes have used either assays for T cell responses or for peptide binding to purified class II molecules. Recently, specific peptide-class II interactions have been demonstrated convincingly on the surface of intact cells. The effects of varying peptide and class II structure have been examined in order to identify structural requirements for binding to cell surface class II molecules and to examine the conformation adopted by immunogenic peptides when bound.
Amino Acid Sequence↗
Prediction and identification of bacterial and parasitic T-cell antigens and determinants.
Explore the source record for details and available documents.
Animals↗
Interactions between immunogenic peptides and HLA-DR molecules.
Explore the source record for details and available documents.
HLA-DR Antigens↗