NMR study on the interaction between MHC class I protein and its antigen peptide

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.     

CML characterization of a product of a second class I locus in the rat MHC

In the rat, genes that control the expression of target antigens detected by cell-mediated lympholysis (CML) are present in the major histocompatibility complex (MHC). The relationship of these loci, CT and Ag-L, to each other and to other loci within the MHC is unknown. In this report, we demonstrate the existence of a CML target antigen in the (DA × BN)F₁ anti-DA.11(BI) strain combination. The gene coding for this antigen is linked to the RT1 complex as indicated by the CML reactivity of targets from backcross and congenic animals. Inhibition studies demonstrated that this antigen has the widespread tissue distribution characteristic of class I antigens, and the gene coding for this CML antigen maps coincident with the RT1.E class I locus as indicated by the lysis of targets from the recombinant strains r10 and r11. The CML can be blocked by antisera directed against a product of the RT1.E locus. The locus controlling this CML reactivity, like CT and Ag-L, has been separated from RT1.A by recombination; unlike CT and Ag-L, the product of this CML locus appears to be identical with an RT1.E allelic product that has been serologically identified and biochemically characterized.Abbreviations used in this paper MHC major histocompatibility complex - CML cell-mediated lympholysis - Con A concanavalin A - SD standard deviation - HEPES N-2-hydroxy-piperazine-N-2-ethanesulfonic acid - CPM counts per minute - grc growth and reproduction complex     

Serological and molecular diversity in the cattle MHC class I region

Information on major histocompatibility complex (MHC) diversity in cattle is important to aid our understanding of immune responses and may contribute to maintenance of healthy cattle populations. Equally, understanding the mechanisms involved in generating this diversity may shed light on the complex nature of mammalian MHC evolution. The aim of this study was to assess molecular and serological variation within cattle MHC class I molecules and to study the mechanisms generating diversity. To address this aim, sequence variation was examined in 12 serologically assigned alleles from three putative loci and correlated with monoclonal antibody (mAb) binding data. The results demonstrate that both alloantisera and mAbs often fail to distinguish gene products that differ by a significant number of amino acids. Conversely, some mAbs could distinguish alleles differing by only one or two amino acids. Examination of the sequences demonstrates sharing of motifs between alleles, some encoded at distinct loci, supporting the occurrence of interlocus recombination within the cattle MHC class I region. The implications of this for MHC sequence diversity, and functional capability, are discussed.     

An analysis of insulin receptor expression and binding on MHC class I positive and negative human lymphoblastoid cells

Recent studies have demonstrated a specific membrane association between the polymorphic class I antigens of the MHC and the insulin receptor (IR). In some reports, variation in IR binding is attributed to the class I phenotype. To extend these observations, we have examined whether MHC class I products influence IR function, in particular, high affinity ligand-specific binding. Using two independent methods, we have compared IR affinity and density on a human B-lymphoblastoid cell line expressing HLA-A, -B products vs a null mutant and a set of transfectants where HLA-A3, -B7, or -Bw58 expression has been restored through gene transfer. The results from radioreceptor assay and RIA measuring IR binding and expression indicate that although there is a decrease in high affinity insulin binding sites in the HLA-A, -B null mutant, ligand binding cannot be restored in the transfectants expressing HLA-A or -B alleles. We conclude that the MHC class I products do not determine the insulin-binding phenotype of the cells examined in this study. Alternatively, we propose that insulin receptor heterogeneity in affinity and density may be influenced by other undetermined factors inherent to clonally expanded cells, thereby complicating dissection of IR/MHC interactions.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

Comparative molecular dynamics analysis of tapasin-dependent and -independent MHC class I alleles

MHC class I molecules load antigenic peptides in the endoplasmic reticulum and present them at the cell surface. Efficiency of peptide loading depends on the class I allele and can involve interaction with tapasin and other proteins of the loading complex. Allele HLA-B*4402 (Asp at position 116) depends on tapasin for efficient peptide loading, whereas HLA-B*4405 (identical to B*4402 except for Tyr116) can efficiently load peptides in the absence of tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Comparative unrestrained molecular dynamics simulations on the alpha(1)/alpha(2) peptide binding domains performed in the presence of bound peptides resulted in structures in close agreement with experiments for both alleles. In the absence of peptides, allele-specific conformational changes occurred in the first segment of the alpha(2)-helix that flanks the peptide C-terminal binding region (F-pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B*4402, a shift toward an altered F-pocket structure deviating significantly from the bound form was observed. Subsequent free energy simulations on induced F-pocket opening in B*4402 confirmed a conformation that deviated significantly from the bound structure. For B*4405, a free energy minimum close to the bound structure was found. The simulations suggest that B*4405 has a greater tendency to adopt a peptide receptive conformation in the absence of peptide, allowing tapasin-independent peptide loading. A possible role of tapasin could be the stabilization of a peptide-receptive class I conformation for HLA-B*4402 and other tapasin-dependent alleles.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

 Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and β₂-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP. Received: 20 March 1997 / Revised: 2 June 1997     

Reoxidation of the class I disulfides of the rat adipocyte insulin receptor is dependent upon the presence of insulin: the class I disulfide of the insulin receptor is extracellular

Elements of the quaternary structure of the native and dithiothreitol- (DTT) reduced rat adipocyte insulin receptor have been elucidated by vectorial probing and subunit cross-linking. The charged reducing agents glutathione and beta-mercaptoethylamine were used to reduce the class I disulfides of the receptor in intact adipocytes, demonstrating the extracellular location of the disulfide directly. This interpretation was confirmed by use of DTT as a reducing agent and the nonpermeant sulfhydryl blocking reagent Thiolyte MQ to prevent the reoxidation of the class I sulfhydryl groups which occurred when they were not blocked. It was found that the above reoxidation of the receptor is dependent on the concentration of insulin in the nanomolar range, not occurring measurably at 4 degrees C in its absence. Cross-linking studies with ethylene glycol bis(succinimidyl succinate) demonstrated that the alpha subunits could not be cross-linked to each other after reduction of the class I disulfides, suggesting that the interaction between the receptor heterodimers may be due primarily to the disulfide bonds.     

Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils

As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.     

Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer

The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit beta(2)-microglobulin (beta(2)m), and a peptide. Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2. Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/beta(2)m heterodimer. However, we identify two pathways for peptide dissociation from the heterotrimer: (1) initial peptide dissociation leaving a heavy chain/beta(2)m heterodimer and (2) initial dissociation of beta(2)m, followed by peptide dissociation from the heavy chain. Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding. We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues. Thermodynamic and kinetic analyses of peptide-MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity. Finally, our data suggest a role for beta(2)m in dampening conformational dynamics in the heavy chain. Remaining conformational variability in the heavy chain once beta(2)m has bound may be a mechanism to promote promiscuity in peptide binding.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

Effect of glycosylation on structure and dynamics of MHC class I glycoprotein: a molecular dynamics study

Complex carbohydrates linked to glycoproteins are recently being implicated to play a variety of biological roles. The lack of well-resolved crystallographic coordinates of the carbohydrates makes it difficult to assess the contributions of the glycan chain on protein structure and dynamics. We have modeled two different oligosaccharides NeuNAc2Gal3Man3GlcNAc5Fuc and Man3GlcNAc4 to generate two glycosylation variants of major histocompatibility complex (MHC) class I glycoprotein. Molecular dynamics simulations of the isolated fourteen- and seven-residue oligosaccharides have been done in vacuo and in solution. The dynamics of the two glycoforms of MHC class I protein have been simulated in solution in the free as well as in the peptide-bound form. Good agreement between the calculated solution conformations of the oligosaccharides in isolated and conjugated forms and the average conformations obtained from x-ray or NMR data was observed for most of the glycosidic linkages. These molecular dynamics simulations of the isolated glycan chains and the glycoconjugates reveal the details of the conformational flexibility of the glycan chains; they also provide atomic level details of protein-carbohydrate interactions and the effect of the ligand binding on the carbohydrate structure and dynamics. It was found that though there is some flexibility in some of the glycosidic linkages in the isolated oligosaccharides, in the protein-conjugated form the linkages adopt more restricted conformations. The glycan chains protrude out into the solvent and might hinder the lateral association of the proteins. The presence of the bulky glycan chains does not affect the average backbone fold of the protein but induces local changes in protein structure and dynamics. It has been noted that the extent of the changes depends upon the nature of the attached glycan chain. The glycan chains do not appear to influence the peptide binding property of the protein directly, but may stabilize the protein residues that are involved in ligand binding.     

Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.     

Conserved MHC class I peptide binding motif between humans and rhesus macaques

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.