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1.
Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1(c) but not other rat MHC class Ia or Ib molecules. RT1-A1(c) preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1(c) could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1(c) and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1(c) primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1(c)-bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1(c) with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1(c) bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1(c) bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1(c) is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2. 相似文献
2.
Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo. 相似文献
3.
Rudolph MG Stevens J Speir JA Trowsdale J Butcher GW Joly E Wilson IA 《Journal of molecular biology》2002,324(5):975-990
Antigenic peptides are loaded onto class I MHC molecules in the endoplasmic reticulum (ER) by a complex consisting of the MHC class I heavy chain, beta(2)-microglobulin, calreticulin, tapasin, Erp57 (ER60) and the transporter associated with antigen processing (TAP). While most mammalian species transport these peptides into the ER via a single allele of TAP, rats have evolved different TAPs, TAP-A and TAP-B, that are present in different inbred strains. Each TAP delivers a different spectrum of peptides and is associated genetically with distinct subsets of MHC class Ia alleles, but the molecular basis for the conservation (or co-evolution) of the two transporter alleles is unknown. We have determined the crystal structures of a representative of each MHC subset, viz RT1-A(a) and RT1-A1(c), in association with high-affinity nonamer peptides. The structures reveal how the chemical properties of the two different rat MHC F-pockets match those of the corresponding C termini of the peptides, corroborating biochemical data on the rates of peptide-MHC complex assembly. An unusual sequence in RT1-A1(c) leads to a major deviation from the highly conserved beta(3)/alpha(1) loop (residues 40-59) conformation in mouse and human MHC class I structures. This loop change contributes to profound changes in the shape of the A-pocket in the peptide-binding groove and may explain the function of RT1-A1(c) as an inhibitory natural killer cell ligand. 相似文献
4.
Duyar H Dengjel J de Graaf KL Wiesmüller KH Stevanović S Weissert R 《Immunogenetics》2005,57(1-2):69-76
Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW.1AV1 rats share the RT1av1 haplotype. So far, no MHC class II peptide motif of RT1.Da molecules has been described. Sequence alignment of the chain of the rat MHC class II molecule RT1.Da with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.Da and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.Da, after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.Da. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.Da molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.Da molecules. Based on these studies we could define a peptide-binding motif for RT1.Da characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.Da. This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases. 相似文献
5.
6.
A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype 总被引:1,自引:0,他引:1
González-Muñoz AL Le Rolle AF Brun H Hedrich HJ Wedekind D Powis SJ Joly E Butcher GW 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(1):274-284
MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct. 相似文献
7.
Molecular determinants of peptide binding to two common rhesus macaque major histocompatibility complex class II molecules
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Dzuris JL Sidney J Horton H Correa R Carter D Chesnut RW Watkins DI Sette A 《Journal of virology》2001,75(22):10958-10968
Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307-319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4(+) T-lymphocyte epitope encoded within the Rev protein of SIV. 相似文献
8.
Zhang N Qi J Feng S Gao F Liu J Pan X Chen R Li Q Chen Z Li X Xia C Gao GF 《Journal of virology》2011,85(22):11709-11724
The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation. 相似文献
9.
McMahan RH Watson L Meza-Romero R Burrows GG Bourdette DN Buenafe AC 《The Journal of biological chemistry》2003,278(33):30961-30970
The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69-89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the Valpha2 domain connected to the Vbeta8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69-89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness. 相似文献
10.
The aim of these studies was to determine whether auto- and alloreactivity can arise from T cell recognition of MHC-peptides in context of syngeneic MHC. Four synthetic peptides derived from the first domain of the HLA-DR beta 1 * 0101 chain were used in limiting dilution analysis to prime T cells from HLA-DR1- and HLA-DR1+ responders. The frequency of T cells responding to these four peptides was similar in individuals with or without HLA-DR1. In both cases, the peptide corresponding to the nonpolymorphic sequence 43-62, was less immunogenic than peptides corresponding to the three hypervariable regions 1-20, 21-42, and 66-90, eliciting a lower number of reactive T cells. Experiments using a T cell line with specific reactivity to peptide 21-42 showed, however, that this response can be efficiently blocked by adding to the culture a nonpolymorphic sequence peptide. This suggests that alloreactivity can be blocked by use of monomorphic (self) peptides. The binding of both "monomorphic" and "polymorphic" synthetic DR1 peptides to affinity purified HLA-DR 1 and DR 11 molecules was measured using radiolabeled peptides and high performance size exclusion chromatography. The data showed that the polymorphic as well as monomorphic synthetic DR1 peptides bound to both DR1 and DR11 molecules. Competitive inhibition studies indicated that the monomorphic 43-62 peptide can block the binding of the polymorphic peptides, consistent with the results obtained in T cell cultures. Taken together these data suggest that anti-MHC autoreactive T cells are present in the periphery and that both auto and alloreactivity can be elicited by MHC peptides binding to MHC class II molecules. 相似文献
11.
Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. 总被引:17,自引:0,他引:17
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M Valés-Gómez H T Reyburn R A Erskine M López-Botet J L Strominger 《The EMBO journal》1999,18(15):4250-4260
The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells. 相似文献
12.
Samino Y López D Guil S Saveanu L van Endert PM Del Val M 《The Journal of biological chemistry》2006,281(10):6358-6365
Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells. 相似文献
13.
GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the Kb -SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I. 相似文献
14.
Production,crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E.
C. A. O'Callaghan J. Tormo B. E. Willcox C. D. Blundell B. K. Jakobsen D. I. Stuart A. J. McMichael J. I. Bell E. Y. Jones 《Protein science : a publication of the Protein Society》1998,7(5):1264-1266
HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A. 相似文献
15.
Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding 总被引:13,自引:0,他引:13
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T S Jardetzky J C Gorga R Busch J Rothbard J L Strominger D C Wiley 《The EMBO journal》1990,9(6):1797-1803
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. 相似文献
16.
Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus 总被引:7,自引:0,他引:7
Mommaas B Kamp J Drijfhout JW Beekman N Ossendorp F Van Veelen P Den Haan J Goulmy E Mutis T 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(6):3131-3136
The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1(H) and HA-1(R), with a single amino acid difference. Whereas the immunogenicity of the HA-1(R) allele has not yet been shown, the nonameric HA-1(H) peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1(H) or HA-1(R) associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1(H/R) peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1(H/R) peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL(H)/(R)DDL and decameric KECVL(H)/(R)DDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1(H) and HA-1(R) peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1(R) peptide did not induce CTLs that recognize the natural HLA-B60/HA-1(R) ligand. In contrast, dendritic cells pulsed with the nonameric HA-1(H) peptide induced IFN-gamma-secreting T cells specific for the natural HLA-B60/HA-1(H) ligand in three HLA-B60(+) HA-1(RR) individuals, demonstrating the immunogenicity of the HLA-B60/HA-1(H) ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus. 相似文献
17.
Weissert R de Graaf KL Storch MK Barth S Linington C Lassmann H Olsson T 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(12):7588-7599
We dissected the requirements for disease induction of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in MHC (RT1 in rat) congenic rats with overlapping MOG peptides. Immunodominance with regard to peptide-specific T cell responses was purely MHC class II dependent, varied between different MHC haplotypes, and was linked to encephalitogenicity only in RT1.B(a)/D(a) rats. Peptides derived from the MOG sequence 91-114 were able to induce overt clinical signs of disease accompanied by demyelinated CNS lesions in the RT1.B(a)/D(a) and RT1(n) haplotypes. Notably, there was no detectable T cell response against this encephalitogenic MOG sequence in the RT1(n) haplotype in peripheral lymphoid tissue. However, CNS-infiltrating lymphoid cells displayed high IFN-gamma, TNF-alpha, and IL-4 mRNA expression suggesting a localization of peptide-specific reactivated T cells in this compartment. Despite the presence of MOG-specific T and B cell responses, no disease could be induced in resistant RT1(l) and RT1(u) haplotypes. Comparison of the number of different MOG peptides binding to MHC class II molecules from the different RT1 haplotypes suggested that susceptibility to MOG-experimental autoimmune encephalomyelitis correlated with promiscuous peptide binding to RT1.B and RT1.D molecules. This may suggest possibilities for a broader repertoire of peptide-specific T cells to participate in disease induction. We demonstrate a powerful MHC class II regulation of autoaggression in which MHC class II peptide binding and peripheral T cell immunodominance fail to predict autoantigenic peptides relevant for an autoaggressive response. Instead, target organ responses may be decisive and should be further explored. 相似文献
18.
David H. Margulies Maripat Corr Lisa F. Boyd Sergei N. Khilko 《Journal of molecular recognition : JMR》1993,6(2):59-69
Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25°C are in the range of ca 10?4–10?6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions. 相似文献
19.
M Matsumura Y Saito M R Jackson E S Song P A Peterson 《The Journal of biological chemistry》1992,267(33):23589-23595
A soluble form of a mouse class I major histocompatibility antigen (H-2Kb) has been expressed in transfected Drosophila melanogaster cells. These molecules were efficiently secreted (up to 4 mg/liter) as noncovalent heterodimers and purified to homogeneity from cell supernatants. The isolated soluble Kb molecules were devoid of endogenous peptides. Using these molecules, we have characterized the Kb heavy chain-beta 2-microglobulin (beta 2m) assembly as well as peptide binding in vitro. In detergent-free solution the heavy chains readily re-assembled with beta 2m even in the absence of peptides. Kinetic analyses showed that the peptide binding is rapid and reversible and dependent on the heavy chains being assembled with beta 2m. Likewise, peptide dissociated from Kb molecules without the displacement of beta 2m. Equilibrium binding experiments using various peptides confirmed that octapeptides bind to Kb molecules with the highest affinity and form the most stable complexes. However, in contrast to earlier studies, the amino-terminal positioning of peptide to Kb molecules was more crucial than the carboxyl-terminal positioning and amidation of the peptide carboxylate did not affect the binding. Soluble Kb molecules could selectively bind allele-specific peptides among a mixture of randomly synthesized octapeptides in vitro; however, no dominant residue was observed at the carboxyl terminus of bound peptides. This suggests that the previously observed hydrophobic residues at the carboxyl terminus of peptides may reflect the specificity of enzyme(s) or protein(s) involved in peptide processing in vivo. 相似文献
20.
An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries 总被引:10,自引:6,他引:4
Udaka K Wiesmüller KH Kienle S Jung G Tamamura H Yamagishi H Okumura K Walden P Suto T Kawasaki T 《Immunogenetics》2000,51(10):816-828
Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules. 相似文献