Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4.

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Immunoglobulin A supplementation abrogates bacterial translocation and preserves the architecture of the intestinal epithelium.

BACKGROUND: Breast milk has been shown to prevent gut-origin infections in neonates through undefined mechanisms. Putative protective factors in breast milk include immunoglobulin (Ig)A, IgG, and lactoferrin. We examined their role in bacterial translocation in neonatal rabbits. METHODS: IgA, IgG, and lactoferrin were isolated from rabbit breast milk through gel filtration and ion-exchange chromatography. Neonates were randomized to receive breast milk, formula alone, or formula supplemented with IgA, IgG, or lactoferrin. Quantitative cultures were performed on day 7 for bacterial translocation. Hematoxylin-eosin-stained sections of distal ileum were examined by light microscopy. Transmucosal bacterial passage was determined in vitro, and the ileal mucosal membranes were examined by confocal microscopy. RESULTS: IgA supplementation abrogated bacterial translocation. IgG and lactoferrin had no significant effect. Neonates that received IgA or breast milk gained more weight than those in the other groups. IgA reduced transmucosal bacterial passage in vitro. In contrast to the normal-appearing distal ileum of neonates fed breast milk, intestinal epithelium from neonates that received formula or formula with IgG or IgA demonstrated prominent vacuoles by light microscopy. Those fed formula alone or formula with lactoferrin had slightly shortened villi. CONCLUSIONS: IgA supplementation prevents bacterial translocation by enhancing gut mucosal barrier function.

HLA-DP2: self peptide sequences and binding properties.

Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.

Human class II MHC molecule HLA-DR1: X-ray structure determined from three crystal forms.

The three-dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA-DR1, was determined to 3.3 A by X-ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 A using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen, Staphylococcus aureus enterotoxin B (SEB), bound to each DR1 molecule. Single-isomorphous replacement phasing followed by iterative two- and fourfold non-crystallographic real-space averaging between the two crystal forms resulted in 4.2 A resolution electron-density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 A for the two lymphocyte-derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexed in vitro with a specific antigenic peptide. Iterative sixfold non-crystallographic real-space averaging resulted in an electron- density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 alphabeta heterodimer in the three crystal forms are discussed.

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8.

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.

Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen.

The structure of a bacterial superantigen, Staphylococcus aureus enterotoxin B, bound to a human class II histocompatibility complex molecule (HLA-DR1) has been determined by X-ray crystallography. The superantigen binds as an intact protein outside the conventional peptide antigen-binding site of the class II major histocompatibility complex (MHC) molecule. No large conformational changes occur upon complex formation in either the DR1 or the enterotoxin B molecules. The structure of the complex helps explain how different class II molecules and superantigens associate and suggests a model for ternary complex formation with the T-cell antigen receptor (TCR), in which unconventional TCR-MHC contacts are possible.

Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide.

An influenza virus peptide binds to HLA-DR1 in an extended conformation with a pronounced twist. Thirty-five per cent of the peptide surface is accessible to solvent and potentially available for interaction with the antigen receptor on T cells. Pockets in the peptide-binding site accommodate five of the thirteen side chains of the bound peptide, and explain the peptide specificity of HLA-DR1. Twelve hydrogen bonds between conserved HLA-DR1 residues and the main chain of the peptide provide a universal mode of peptide binding, distinct from the strategy used by class I histocompatibility proteins.

Comparison of the P2 specificity pocket in three human histocompatibility antigens: HLA-A*6801, HLA-A*0201, and HLA-B*2705.

Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.

The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.

The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC.

Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.

Structural analysis of class II major histocompatibility complex proteins.

Numerous studies have contributed much data that, taken together, provide considerable insight into the structural features of class II molecules. The putative peptide-binding region has been modeled from the crystal structure of the class I molecule HLA-A2. Mutational analyses and peptide-binding studies support this model. Direct structural studies have established features of the noncovalent association of the alpha and beta chains of class II molecules, and detergents and phospholipids have been shown to affect class II protein structure and/or peptide-binding capacity. Spectroscopic studies have revealed dynamic features of class II molecules in solution, and suggest differences in the overall structures of class I and II molecules that may reflect differences in function.

Crystallization and preliminary X-ray diffraction studies of the human major histocompatibility antigen HLA-B27.

The class I major histocompatibility (MHC) antigen HLA-B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG-2. Detergent-soluble HLA-B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water-soluble extracellular fragments were obtained in hanging drops by the vapor-diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 A, b = 71.0 A, c = 83.7 A, alpha = 79.4 degrees, beta = 88.5 degrees, gamma = 89.9 degrees, and diffract beyond 2.5 A resolution.

The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation.

X-ray crystallography reveals electron density in the antigen-binding site of HLA-B27 that is an interpretable image of nonameric peptides in a largely extended conformation. Clear density exists for the main chain and several side chains and is consistent with the sequence of 11 nonameric self-peptides eluted from HLA-B27. Pockets in the antigen-binding cleft bind four side chains and the amino and carboxyl termini of the peptide.

Crystallization of HLA-DR antigens.

The class II major histocompatibility antigens HLA-DR1, DR2, DR3, DR4, DR7 and DR8 were purified by immunoaffinity chromatography from homozygous human B lymphoblastoid cell lines. The purified, detergent-soluble molecules were cleaved with the protease papain to remove the hydrophobic transmembrane regions and cytoplasmic tails. Crystals were obtained for each of the papain-solubilized fragments. DR1 crystallized under a variety of different conditions, resulting in two different orthorhombic crystal forms, one of which diffracts as far as 3.5A. Crystals of DR2, DR3, DR4 and DR8 have the same unit cell dimensions as the DR1 crystals, and crystals of DR3 and DR4 have the same diffracting power as the DR1 crystals. The best DR7 crystals obtained thus far are hexagonal and diffract to only about 8A. Crystals of similar hexagonal form have also been observed for most of the other DR subsets.

Analysis of the permissive association of a malaria T cell epitope with DR molecules.

In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding.

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.

Comparison of the secondary structures of human class I and class II major histocompatibility complex antigens by Fourier transform infrared and circular dichroism spectroscopy.

We have examined the secondary structures of human class I and class II histocompatibility antigens in solution by Fourier transform infrared spectroscopy and circular dichroism in order to compare the relative amounts of alpha-helix, beta-sheet, and other structures, which are crucial elements in the comparison of the protein structures. Quantitation of infrared spectra of papain-solubilized HLA-A2, HLA-B7, and DR1 in phosphate buffer gave alpha-helix contents of 17%, 8%, and 10% and beta-sheet contents of 41%, 48%, and 53%, respectively. By circular dichroism, papain-solubilized HLA-A2, HLA-B7, and DR1 were also found to have comparable alpha-helix contents (e.g., 8%, 20%, and 17%, respectively). Circular dichroism analysis for beta-sheet gave 29% for papain-solubilized HLA-B7 and 42% for papain-solubilized DR1. The value for papain-solubilized HLA-A2 (74%) was anomalous. It is proposed that Trp-107 of HLA-A2, missing in both HLA-B7 and DR1, may be responsible for much of the anomaly. Due to the uncertainties inherent in quantitation of the amounts of secondary structures by both spectral methods, the differences in the contents of alpha-helix and beta-sheet in the three proteins are not considered significant. However, differences in the nature of the beta-sheet structures are suggested by infrared spectroscopy. These results provide physical evidence for an overall structure of class II antigens modeled on that of class I antigens.