Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

A novel receptor on allograft (H-2d)-induced macrophage (H-2b) toward an allogeneic major histocompatibility complex class I molecule, H-2Dd, in mice

The generation of knockout mice demonstrated that noncytotoxic CD4(+), but not cytotoxic CD8(+), T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograftinduced macrophages (AIM) in mice lysed allografts with H-2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H-2D(b)) for an allogeneic major histocompatibility complex (MHC) class I molecule, H-2D(d), by using H-2D(d) tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181-bp) encoded a 342-amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H-2D(d), but not H-2L(d), H-2K(d), H-2D(b), H-2K(b), H-2D(k), or H-2K(k), molecules; and the H-2D(d) binding was suppressed by the addition of R15 or anti-H-2D(d) mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H-2D(d), but not H-2D(b) or H-2D(k), molecules. The dissociation constant of AIM toward H-2D(d) tetramers was 1.9 x 10(-9) M ; and the binding was completely inhibited by the addition of R15 or anti-H-2D(d) mAb. These results reveal that a novel receptor for an allogeneic H-2D(d) molecule was induced on effector macrophages responsible for allograft (H-2(d)) rejection in H-2(b) mice.     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.     

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.     

Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule

Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0 A resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 beta-chain are mediated by a zinc ion, and 22% of the buried surface of peptide.MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I.peptide.DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic beta-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.     

Physical association of the K3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class I molecules with impaired peptide and beta(2)-microglobulin assembly

To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.     

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide‐empty intermediate

The cell biology and biochemistry of peptide exchange on major histocompatibility complex class I (MHC‐I) proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross‐presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC‐I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction. We find that low pH stabilizes the peptide‐empty forms of MHC‐I that occur as intermediates of the exchange reaction, and that is synergizes with dipeptides and with disulfide‐mediated stabilization of MHC‐I.     

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d)

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.     

Secondary structure and 3D homology modeling of grass carp (Ctenopharyngodon idellus) major histocompatibility complex class I molecules

No information to date is available on the structure of fish major histocompatibility complex (MHC) class I and beta2-microglobulin (beta2m) proteins. In the present study, grass carp (Ctenopharyngodon idellus) MHC class I (Ctid-MHC I) and beta(2)-microglobulin (Ctid-beta2m) genes were expressed as soluble maltose binding protein (MBP)-proteins and purified in a pMAL-p2X/Escherichia coli TB1 system. The expressed proteins were purified on amylase affinity columns followed by DEAE-Sepharose. The purified products were identified by Western blotting with anti-MBP polyclonal antibodies. The MBP-Ctid-MHC I and MBP-Ctid-beta2m were cleaved separately with Factor Xa, mixed together and purified on DEAE-Sepharose. The secondary structures were analyzed by circular dichroism (CD) spectrophotometry. The three-dimensional (3D) structure of their peptide-binding domain (PBD) was modeled based sequence homology. The sequence lengths of the alpha-helix, beta-sheet, turn, and random coil in the Ctid-MHC I protein were 79aa, 75aa, 20aa, and 99aa, respectively. In the 97aa of Ctid-beta2m, the contents of the alpha-helix, beta-sheet, turn, and random coil were 0aa, 41aa, 12aa, and 44aa, respectively. The Ctid-beta2m protein displayed a typical beta-sheet. Homology modeling of the Ctid-MHC I and Ctid-beta2m proteins demonstrated similarities with the structure of human MHC class I proteins.     

A bovine class I major histocompatibility complex molecule plus a Theileria parva antigen resembles mouse H-2Kd

Two BoT2+, BoT8+ cytotoxic T cell clones were generated from peripheral blood of a steer immunized with the intracellular protozoan parasite Theileria parva. Both cytotoxic T cell clones appeared to be restricted by the same major histocompatibility complex (MHC) molecule and were specific for the immunizing parasite clone. However, one of the two clones also recognized uninfected mouse cell lines carrying the H-2d haplotype. Inhibition of cytotoxicity with monoclonal antibodies specific for polymorphic determinants on H-2 molecules confirmed that this CTL clone recognized the H-2Kd MHC molecule. These results extend to the bovine system observations in other species that foreign MHC mimics self MHC plus antigen.     

Beta-amino acid scan of a class I major histocompatibility complex-restricted alloreactive T-cell epitope

An HLA-B27-restricted self-octapeptide known to react with an alloreactive T-cell receptor has been modified by systematic substitution of a beta-amino acid for the natural alpha-amino acid residue, over the whole length of the parent epitope. All modified peptides were shown to bind to recombinant HLA-B*2705 and induce stable major histocompatibility complex-peptide complexes, but with some variation depending on the position of the beta-amino acid on the peptide sequence. Alteration of the natural peptide sequence at the two N-terminal positions (positions 1 and 2) decreases binding affinity and thermodynamic stability of the refolded complex, but all other positions (from position 3 to the C-terminal residue) were insensitive to the beta-amino acid substitution. All modified peptides were recognized by an alloreactive T-cell clone specific for the parent epitope with decreased efficiency, to an extent dependent of the position that was modified. Furthermore, the introduction of a single beta-amino acid at the first two positions of the modified peptide was shown to be sufficient to protect them against enzymatic cleavage. Thus, beta-amino acids represent new interesting templates for alteration of T-cell epitopes to design either synthetic vaccines of T-cell receptor antagonists.     

Regulation of insulin receptor functions by a peptide derived from a major histocompatibility complex class I antigen

A 25 residue peptide, Dk-(61-85), derived from the alpha 1 domain of a murine MHC class I molecule (H-2Dk), enhances cellular glucose uptake, prolongs the effect of insulin, and inhibits insulin receptor internalization without affecting insulin binding or dissociation. Full effect of the peptide is obtained at 10-100 microM. The magnitude of the peptide-mediated enhancement of glucose uptake is insulin dependent and is at maximum approximately 50% above that of full insulin stimulation, excluding a merely insulinomimetic action of the peptide. Dk-(61-85) does not interact directly with the glucose transporter molecule. Furthermore, the peptide-mediated inhibition of insulin receptor internalization results in 2-3 times more receptors in the plasma membrane. The peptide also causes hypoglycemia in rats. The biological activity of Dk-(61-85) suggests that an important nonimmunological role of MHC class I molecules is to affect some of the key functions of ligand-activated receptors.     

The energetic basis underpinning T-cell receptor recognition of a super-bulged peptide bound to a major histocompatibility complex class I molecule

Although the major histocompatibility complex class I (MHC-I) molecules typically bind short peptide (p) fragments (8-10 amino acids in length), longer, "bulged" peptides are often be presented by MHC-I. Such bulged pMHC-I complexes represent challenges for T-cell receptor (TCR) ligation, although the general principles underscoring the interaction between TCRs and bulged pMHC-I complexes are unclear. To address this, we have explored the energetic basis of how an immunodominant TCR (termed SB27) binds to a 13-amino acid viral peptide (LPEPLPQGQLTAY) complexed to human leukocyte antigen (HLA) B*3508. Using the crystal structure of the SB27 TCR-HLA B*3508(LPEP) complex as a guide, we undertook a comprehensive alanine-scanning mutagenesis approach at the TCR-pMHC-I interface and examined the effect of the mutations by biophysical (affinity measurements) and cellular approaches (tetramer staining). Although the structural footprint on HLA B*3508 was small, the energetic footprint was even smaller in that only two HLA B*3508 residues were critical for the TCR interaction. Instead, the energetic basis of this TCR-pMHC-I interaction was attributed to peptide-mediated interactions in which the complementarity determining region 3α and germline-encoded complementarity determining region 1β loops of the SB27 TCR played the principal role. Our findings highlight the peptide-centricity of TCR ligation toward a bulged pMHC-I complex.     

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.     

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.