Recognition by natural killer (NK) cells of major histocompatibility complex (MHC) class I molecules on target cells inhibits NK-mediated lysis. Here, inhibition of NK clones by HLA-B*2705 molecules mutated at single amino acids in the peptide binding site varied among HLA-B*2705-specific NK clones. In addition, a subset of such NK clones was inhibited by only one of several self peptides loaded onto HLA-B*2705 molecules expressed in peptide transporter-deficient cells, showing that recognition was peptide-specific. These data demonstrate that specific self peptides, complexed with MHC class I, provide protection from NK-mediated lysis.
Identification of the targets of autoreactive T cells is important for understanding the pathogenesis of many autoimmune diseases. In multiple sclerosis, myelin proteins are thought to be the targets of autoreactive T-cell responses. To date only major histocompatibility complex class 11-restricted CD4+ T-cell responses to myelin proteins have been investigated. In the present study, the ability of self peptides derived from human myelin proteins to induce autoreactive CD8+ T-cell responses has been assessed. Peptide sequences from human myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein have been identified that bind to and form stable complexes with HLA-A2. MBP 110-118, PLP 80-88, MAG 287-295, MAG 509-517, and MAG 556-564 were all able to induce peptide-specific HLA-A2-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro in HLA-A2+ individuals. CTLs specific for MBP 110-118 and MAG 556-564 could recognize endogenously processed antigens presented by HLA-A2. CTL clones reactive to MBP 110-118 and MAG 556-564 produced tumor necrosis factor a and a subset ofthese clones also produced interferon y. These results demonstrate that (i) self peptides derived from human myelin proteins can induce autoreactive CD8+ CTLs and (ii) these CD8+ T cells produce cytokines thought to be important in mediating demyelinating disease. These studies provide an experimental approach for the assessment of CD8+ T-cell responses in such autoimmune diseases.sively by MHC class I molecules (17, 18), and adult oligodendrocytes constitutively express MHC class I molecules but do not express class II MHC molecules, even when stimulated by interferon y (IFN-y) (19). Thus, if adult oligodendrocytes are targets for T-cell reactivity, they will most likely be recognized by MHC class I-restricted CD8+ T cells but not by class II-restricted CD41 T cells. (iii) In an animal model of demyelinating disease, experimental allergic encephalomyelitis (EAE), CD8+ T cells have been shown to have an immunoregulatory effect on the course of the disease (20, 21). Despite these implications that autoreactive CD8+ T-cell responses may play an important role in MS, a successful experimental approach to this question has not yet been reported.To investigate the possible role(s) of CD8+ T-cell responses to myelin proteins in the pathogenesis of demyelinating disorders, we have taken advantage of recent advances in the ability to predict peptide epitopes presented by the HLA class I molecule, . A computer-based algorithm has been devised that predicts the stability of HLA-A2-peptide complexes by quantitating positive and negative effects on binding by each amino acid within a nonamer peptide (22). This algorithm has been used to predict HLA-A2 binding nonamer peptides from the myelin proteins MBP, PLP, MAG, and MOG. In this report we have identified HLA-A2-restricted epitopes derived from these proteins that are capable of inducing CD8+ cytotoxic T-lymphocyte (CTL)...
Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA-B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9.
With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.
Cytotoxic T lymphocytes kill virally infected cells when they detect antigenic fragments presented by class I major histocompatibility complex (MHC) antigens (HLA in humans). The crystal structures of HLA-A2 and HLA-Aw68 reveal that peptide-antigen forms an integral part of the HLA structure, being retained in a prominent groove even after purification and crystallization. Here we report that the heavy chain and beta 2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, beta 2-microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. Reconstitution is not stimulated by either of two peptides not restricted to HLA-A2. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, Np (85-94), crystallizes under conditions previously used to crystallize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence.
As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system (Gygi et al. (1999) Nat. Biotechnol. 17, 994 -999) was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed. Molecular & Cellular Proteomics 3:625-659, 2004.One of the goals of proteomic research is to identify (correctly) and quantify as many proteins as possible in the biological system of choice (see Refs. 1 and 2 for general reviews). We chose a yeast system for this study because it is possible to get large quantities of biological material from yeast cells using fermentation, and the yeast genome encodes Ͻ6600 rather well-characterized proteins whose abundance can be estimated based on the codon adaptation index (3). At the biological level, we chose to study two yeast strains that were related by the knockout of Upf1p (the protein encoded by the UPF1 gene), a protein that is crucial to the process of nonsense-mediated mRNA decay (4, 5).1 We used the isotope-coded affinity tag (ICAT) 2 reagent approach (6) to
The kinetics of dissociation of iodinated beta 2-microglobulin (beta 2m) from the papain-solubilized class I histocompatibility antigen HLA-B7 have been investigated. In the presence of unlabeled beta 2m, most of the HLA dissociates according to a single rate constant, whereas in the absence of unlabeled beta 2m, the system approaches an equilibrium dependent upon the initial HLA concentration. When iodinated beta 2m is incubated with unlabeled HLA-B7, the rate of incorporation of beta 2m into the complex is much less dependent on the concentration than is expected for a simple association/dissociation system; instead, the system behaves as if the "activity" (in a thermodynamic sense) of the HLA heavy-chain intermediate cannot surpass a critical concentration. The dissociation rate for each class I specificity is a function of temperature, ionic strength, pH, and the status of the heavy chain (papain solubilized vs. detergent solubilized). High temperature, high ionic strength, and extremes of pH promote dissociation. The intact molecule dissociates about 10 times more slowly than the papain-solubilized molecule. In contrast, the rate of dissociation of all papain-solubilized class I antigens tested falls within the range of about a factor of 2. The presence of the carbohydrate has no effect on the rate of dissociation. The possibility that HLA class I antigen dissociation may occur in vivo within acidic internal vesicles is discussed.
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