Peptide-binding Motif Research Articles

Surface Reengineering of RPA70N Enables Cocrystallization with an Inhibitor of the Replication Protein A Interaction Motif of ATR Interacting Protein

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

HLA-A*68

Human leukocyte allele (HLA) class I polymorphism has the greatest impact of human genetic variation on viral load set point. A substantial part of this effect is due to the action of HLA-B and HLA-C alleles. With few exceptions the role of HLA-A molecules in immune control of HIV is unclear. We here study HLA-A*68:02, one of the most highly prevalent HLA-A alleles in C-clade infected sub-Saharan African populations, and one that plays a prominent role in the HIV-specific CD8 T-cell responses made against the virus. We define eight epitopes restricted by this allele and propose the peptide binding motif for HLA-A*68:02. Although one of these epitopes almost exactly overlaps an HLA-B*57-restricted epitope in Gag linked with immune control of HIV, this HLA-A*68:02-restricted Gag-TA10 response imposed only weak selection pressure on the virus and was not associated with significantly lower viral setpoint. The only HLA-A*68:02-restricted responses imposing strong selection pressure on HIV were in the flanking regions of Pol-EA8 and Pol-EA11 and within the Vpr-EV10 epitope (P = 8 × 10). However, targeting of this latter epitope was associated with significantly higher viral loads (P = 0.003), suggesting lack of efficacy. This study is consistent with previous data showing that HLA-A-restricted Gag-specific responses can impose selection pressure on HIV. In the case of HLA-A*68:02 the Gag response is subdominant, and apparently has little impact in natural infection. However, these data suggest the potential for high frequency vaccine-induced Gag responses restricted by this allele to have significant antiviral efficacy in vaccine recipients.

The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV)

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with β-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 61 and B12 from line C determine protection, although another locus from line 61 was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule.

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

The principal genetic determinants for nasopharyngeal carcinoma in China involve the HLA class I antigen recognition groove.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA –B, and HLA -C class I genes (PHLA-A-aa-site-62 = 7.4×10−29; P HLA-B-aa-site-116 = 6.5×10−19; P HLA-C-aa-site-156 = 6.8×10−8 respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China.

37-OR

Aim Cynomolgus macaque (Macaca fascicularis = Mafa) and Rhesus macaque (Macaca mulatta = Mamu) are widely used as alternative nonhuman primate models for studying HIV, transplantation, and vaccines since they are anatomically and physiologically similar to humans. In this study we characterize naturally loaded peptides from two Mafa (A ∗ 31 & B ∗ 49) and one Mamu (B ∗ 47) class I molecule. Methods An MHC Class I-deficient human B cell line was transfected with Mafa and Mamu soluble MHC (sMHC) DNA constructs and selected with Geneticin. Clones were tested for sMHC class I production by ELISA, and “best producers” were single cell sorted, expanded, and retested. Cell pharms were seeded and 25 mg of class I MHC harvested. Mafa and Mamu class I MHC were purified by affinity chromatography, peptide ligands eluted in acetic acid, and the pooled peptide ligands were subjected to 14 cycles of Edman degradation. Pooled peptides were HPLC fractionated and subjected to nanospray mass spectrometry (MS) in order to characterize individual peptide ligands. Results MS analysis data show that Mafa molecules present peptides similar to those presented by human HLA class I molecules. The peptide lengths and source are consistent with previous reports of class I MHC. The two Mafa molecules in this study have peptide-binding motifs characteristic of HLA-B molecules while Mamu peptide ligands are not consistent with previously reported MHC class I. Conclusions Mafa class I molecules exhibit HLA-B-like cross-species peptide presentation, while Mamu class I molecules do not show similarities with human HLA class I molecules. The characterization of endogenously loaded Mafa-B ∗ 49 MHC class I peptides shows that it is comparable to human HLA-B ∗ 4405, and Mafa-A ∗ 31 to human HLA-B ∗ 1509 and B ∗ 1510. These data suggest that the MHC typing of Non-Human Primates for comparable MHC should proceed the use of these animal models when establishing correlates of immunity.

TectoRNP: self‐assembling RNAs with peptide recognition motifs as templates for chemical peptide ligation

TectoRNA, an artificial RNA with self-assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self-assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA-binding peptides were recognized by two peptide-binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg(2+) -dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA-based templates and the extent of acceleration in peptide ligation.

Alternative peptide repertoire of HLA-E reveals a binding motif that is strikingly similar to HLA-A2

The non-classical HLA-E is a conserved class I molecule that mainly presents monomorphic leader peptides derived from other HLA class I molecules. These leader peptides comprise an optimized sequence for tight and deep binding into the HLA-E groove. In a TAP-deficient environment, as it can be generated during viral infection or in tumor tissue, loading of the classical leader peptide sequences is hampered leading to an alternative HLA-E peptide repertoire. In this study, we characterized this alternative peptide repertoire using cells in which TAP activity is inhibited. We identified more than 500 unique peptide sequences carried by HLA-E and found that their binding motif is different from the dominant leader peptides. Hydrophobic amino acids were only found at positions 2 and 9, in close resemblance to the peptide binding motif of HLA-A*0201. HLA-E-eluted peptides were indeed able to bind this classical HLA class I molecule. Our findings suggest that the dominant leader peptides uniquely conform to HLA-E, but that in their absence a peptide pool is presented like that of HLA-A*0201.

Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken

T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8+ T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8+ T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP89–97 and NP198–206, respectively. The results indicate that peptides NP89–97 (PKKTGGPIY) and NP198–206 (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.

The canine MHC class I allele, DLA-88*50801, presents peptides of varying lengths with three key anchor residues (160.14)

Abstract The binding of a peptide to a major histocompatibility (MHC) class I allele depends upon a few key residues within the peptide sequence, which is present only in ~1-2% of peptides in an average protein. Identification of this motif allows one to select candidate epitopes from a protein that may be recognized by cytotoxic T lymphocytes (CTL). While several studies have determined the binding specificities of numerous class I alleles in both humans and mice, no such study has been performed in the domestic dog. The principal aim of this study was to determine the peptide binding motif of a prevalent MHC class I allele, Dog Leukocyte Antigen (DLA) -88*50801. We hypothesized that a conserved binding motif could be identified through sequencing of endogenously presented peptides eluted from DLA-88*50801. To accomplish this goal, a plasmid encoding the DLA-88*50801 gene with an attached flag tag was stably transfected into a canine cell line (DH82). DLA-88*50801 protein was obtained through affinity purification of lysate from ~8 x 109 cells. Bound peptides were eluted from the MHC binding groove utilizing low pH and heat, isolated by size exclusion and affinity purification, and subjected to tandem mass spectrometry analysis to obtain individual peptide sequences. Sequences were manually aligned to determine the binding motif. This study provides essential information to study CTL responses in dogs.

Structural and Functional Studies of trans-Encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) Protein Molecule

MHC class II molecules are composed of one α-chain and one β-chain whose membrane distal interface forms the peptide binding groove. Most of the existing knowledge on MHC class II molecules comes from the cis-encoded variants where the α- and β-chain are encoded on the same chromosome. However, trans-encoded class II MHC molecules, where the α- and β-chain are encoded on opposite chromosomes, can also be expressed. We have studied the trans-encoded class II HLA molecule DQ2.3 (DQA1*03:01/DQB1*02:01) that has received particular attention as it may explain the increased risk of certain individuals to type 1 diabetes. We report the x-ray crystal structure of this HLA molecule complexed with a gluten epitope at 3.05 Å resolution. The gluten epitope, which is the only known HLA-DQ2.3-restricted epitope, is preferentially recognized in the context of the DQ2.3 molecule by T-cell clones of a DQ8/DQ2.5 heterozygous celiac disease patient. This preferential recognition can be explained by improved HLA binding as the epitope combines the peptide-binding motif of DQ2.5 (negative charge at P4) and DQ8 (negative charge at P1). The analysis of the structure of DQ2.3 together with all other available DQ crystal structures and sequences led us to categorize DQA1 and DQB1 genes into two groups where any α-chain and β-chain belonging to the same group are expected to form a stable heterodimer.

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the Dd mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0598-5) contains supplementary material, which is available to authorized users.

Identification of the peptide-binding motif recognized by the pigtail macaque class I MHC molecule Mane-A1*082:01 (Mane A*0301).

Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane-A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.

HLA-B*57 Micropolymorphism shapes HLA allele-specific epitope immunogenicity, selection pressure, and HIV immune control.

The genetic polymorphism that has the greatest impact on immune control of human immunodeficiency virus (HIV) infection is expression of HLA-B*57. Understanding of the mechanism for this strong effect remains incomplete. HLA-B*57 alleles and the closely related HLA-B*5801 are often grouped together because of their similar peptide-binding motifs and HIV disease outcome associations. However, we show here that the apparently small differences between HLA-B*57 alleles, termed HLA-B*57 micropolymorphisms, have a significant impact on immune control of HIV. In a study cohort of >2,000 HIV C-clade-infected subjects from southern Africa, HLA-B*5703 is associated with a lower viral-load set point than HLA-B*5702 and HLA-B*5801 (medians, 5,980, 15,190, and 19,000 HIV copies/ml plasma; P = 0.24 and P = 0.0005). In order to better understand these observed differences in HLA-B*57/5801-mediated immune control of HIV, we undertook, in a study of >1,000 C-clade-infected subjects, a comprehensive analysis of the epitopes presented by these 3 alleles and of the selection pressure imposed on HIV by each response. In contrast to previous studies, we show that each of these three HLA alleles is characterized both by unique CD8(+) T-cell specificities and by clear-cut differences in selection pressure imposed on the virus by those responses. These studies comprehensively define for the first time the CD8(+) T-cell responses and immune selection pressures for which these protective alleles are responsible. These findings are consistent with HLA class I alleles mediating effective immune control of HIV through the number of p24 Gag-specific CD8(+) T-cell responses generated that can drive significant selection pressure on the virus.

Position 45 influences the peptide binding motif of HLA-B*44:08

Position 45 represents a highly polymorphic residue within HLA class I alleles, which contacts the p2 position of bound peptides in 85% of the peptide-HLA structures analyzed, while the neighboring residues 41 and 46 are not involved in peptide binding. To investigate the influence of residue 45 at the functional level, we sequenced peptides eluted from recombinant HLA-B*44:08(41Ala/45Met/46Ala) molecules and compared their features with known peptides from B*44:02(41Thr/45Lys/46Glu). While HLA-B*44:02 has an anchor motif of E at the p2 anchor position, HLA-B*44:08 exhibits Q and L as anchor motif. The 45(Met/Lys) polymorphism contributes to the alteration in the peptide-binding motif and provides further evidence that mismatches at position 45 should be considered as nonpermissive in a transplantation setting.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

SIV-infected Chinese-origin rhesus macaques express specific MHC class I alleles in either elite controllers or normal progressors

SIV-infected Chinese-origin rhesus macaques express specific MHC class I alleles in either elite controllers or normal progressors

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users.

Pathway-based analysis of genetic susceptibility to cervical cancer in situ: HLA-DPB1 affects risk in Swedish women

We have conducted a pathway-based analysis of genome-wide single-nucleotide polymorphism (SNP) data in order to identify genetic susceptibility factors for cervical cancer in situ. Genotypes derived from Affymetrix 500k or 5.0 arrays for 1076 cases and 1426 controls were analyzed for association, and pathways with enriched signals were identified using the SNP ratio test. The most strongly associated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were Asthma (empirical P=0.03), Folate biosynthesis (empirical P=0.04) and Graft-versus-host disease (empirical P=0.05). Among the 11 top-ranking pathways were 6 related to the immune response with the common denominator being genes in the major histocompatibility complex (MHC) region on chromosome 6. Further investigation of the MHC revealed a clear effect of HLA-DPB1 polymorphism on disease susceptibility. At a functional level, DPB1 alleles associated with risk and protection differ in key amino-acid residues affecting peptide-binding motifs in the extracellular domains. The results illustrate the value of pathway-based analysis to mine genome-wide data, and point to the importance of the MHC region and specifically the HLA-DPB1 locus for susceptibility to cervical cancer.

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1(*)0801-DQA1(*)0401/DQB1(*)0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-A(g7). Similar to HLA-DQ8 and H-2 I-A(g7), HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8- or I-A(g7) ligands, and several diabetes-specific peptides associated with DQ8 or I-A(g7) could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.