Natural peptides isolated from Gly86/Val86-containing variants of HLA-DR1, -DR11, -DR13, and -DR52.
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Amino Acid Sequence↗
Biomedical subjects
J W Drijfhout
Publications and source records attributed to J W Drijfhout.
Peptide binding characteristics of the coeliac disease-associated DQ(alpha1*0501, beta1*0201) molecule.
Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of the HLA-DQ2 (alpha1(*)0501, beta1(*)0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several size-variants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClalpha46 - 63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClalpha46 - 63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.
Amino Acid Sequence↗
The generation of SDS-stable HLA DR dimers is independent of efficient peptide binding.
MHC class II molecules can exist in two conformations which can be distinguished on the basis of their stability in SDS. The formation of SDS-stable dimers has been shown to correlate with persistent expression of antigen MHC class II-peptide complexes by murine antigen-presenting cells. HLA DR molecules contain either a Val or a Gly at position 86 of the beta chain, which is located in a conserved and prominent hydrophobic pocket in the peptide binding site. Here we show that Val86-containing DR molecules more frequently select peptides which induce the formation of SDS-stable dimers than Gly86 variants. Using analogues of the influence virus haemagglutinin epitope 307-319 we found that the replacement of the aromatic hydrophobic anchor residue (Tyr)at position 309 by amino acids with an aliphatic hydrophobic side chain resulted in the specific formation of high numbers of SDS-stable Val86-DR but not Gly86-DR dimers. These results indicate that the fit between the first anchor residue and the hydrophobic pocket around Dr beta 86 plays a critical role in the formation of SDS-stable DR dimers. Synthetic analogues of naturally processed DR-associated peptides displayed promiscuity in their capacity to bind to several DR specificities and in their ability to induce the SDS-stable conformation. However, no correlation was observed between binding capacity and the ability to induce the SDS-stable conformation. Since it has been shown that SDS stability can relate to the kinetics of peptide-MHC class II interactions, the definition of the requirements for the formation of SDS-stable HLA class II molecules may be important for the design of effective peptide-based immunomodulation protocols.
Amino Acid Sequence↗
The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.
The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.
Amino Acid Sequence↗
Purification and functional analysis of the Mycobacterium leprae thioredoxin/thioredoxin reductase hybrid protein.
In Mycobacterium leprae, thioredoxin and thioredoxin reductase are expressed from a single gene. This results in the expression of a hybrid protein with subunits attached to each other by a hydrophilic peptide linker. In all other organisms studied so far, thioredoxin (Trx) and thioredoxin reductase (TR) are expressed as two separate proteins. This raises the question of whether the hybrid protein is enzymatically active and, if so, whether TR reduces its own Trx partner or alternatively a heterologous Trx subunit. To address this question, the hybrid TR/Trx protein of M. leprae as well as the individual parts of the hybrid gene coding for either TR or Trx were overexpressed in Escherichia coli and purified. The purified proteins were tested for their ability to catalyze NADPH-dependent insulin disulfide reduction. Here we show that the M. leprae hybrid protein is indeed enzymatically active. Compared with the enzymatic activity of the separately expressed Trx and TR proteins, the hybrid protein is shown to be more efficient, particularly at low equimolar concentrations. This suggests that the hybrid protein of M. leprae is active by itself and that its activity involves intramolecular interactions between the TR and Trx domains. The activity of the hybrid protein increases when exogenous TR or Trx is added, indicating an additional role for intermolecular interactions.
Bacterial Proteins↗
Identification of a graft versus host disease-associated human minor histocompatibility antigen.
Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.
Amino Acid Sequence↗
Clip binds to HLA class II using methionine-based, allele-dependent motifs as well as allele-independent supermotifs.
The invariant chain (Ii) region that interacts with class II and inhibits premature peptide binding has been mapped to amino acids 82-107, known as CLIP. It is unclear whether CLIP binds directly to the class II peptide binding groove and thus competitively blocks binding of other peptides, or whether it binds to conserved class II sites and indirectly inhibits peptide binding by inducing conformational changes in class II. Here we show evidence that strongly suggests that CLIP binds within the peptide binding groove, as CLIP binds to various HLA-DR alleles using allele-dependent as well as allele-independent, methionine-based binding motifs. First, a core sequence of 12 amino acids was identified within CLIP which is required for optimal binding to DR1, DR2, DR3(17) and DR7. This sequence is composed of CLIP p88-99 (SKMRMATPLLMQ). By substitution analysis, all three methionine residues appeared to control CLIP binding to HLA-DR. However, whereas M90 controlled binding to all four alleles, M92 and M98 were of different importance for the various alleles: M92 is involved in CLIP binding to DR1 and DR3(17) but not to DR2 or DR7, and M98 controls CLIP binding to DR2, DR3(17) and DR7 but not DR1. Also, CLIP competes with known immunogenic peptides for class II binding in a manner indistinguishable from regular, class II binding competitor peptides. Finally, the dissociation rates of CLIP-class II complexed are similar to those of antigenic peptide-class II complexes. Thus, CLIP most likely binds to the class II peptide binding groove, since most allelic class II differences are clustered here. CLIP uses unconventional methionine anchor residues representing an allele-independent supermotif (M90) as well as allele-dependent motifs (M92 and M98).
Alleles↗
Detailed motifs for peptide binding to HLA-A*0201 derived from large random sets of peptides using a cellular binding assay.
Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A*0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A*0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at positions 4 for binding, and the deleterious effect of R at position 5 (in 9-mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A*0201 by computer analysis (see accompanying paper [56]).
Alleles↗
A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs.
Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths.
Amino Acid Sequence↗
An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells. Identification of conserved HIV-1 polymerase peptides binding to HLA-A*0301.
A peptide-binding assay employing the HLA class I molecules on intact human B cells is described. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment, after which the cells are incubated with a FL-labeled reference peptide together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACscan analysis. The assay is easy to perform because there is no need to purify HLA class I molecules, or to transfect cells with HLA class I molecules, and no radioactive label is used. Moreover, large panels of HLA-typed human B-cell lines are available as tools for peptide binding to a vast array of HLA molecules. The binding assay was optimized and validated with peptides of known binding capacity to either HLA-A*0201 or HLA-A*0301. The kinetics of peptide binding in this assay were shown to be comparable to that in assays employing soluble HLA class I molecules. Application of the assay in the search for potential HLA-A*0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five high-affinity binding peptides.
Amino Acid Sequence↗
Solid-phase synthesis of peptide haptens containing a cysteine-sulfur mustard adduct.
Solid-phase synthesis of peptide haptens containing 2-[2-(S-cysteinyl)ethanol has been achieved by solution-phase synthesis of a properly protected S-alkylated cysteine derivative and subsequent solid-phase incorporation.
Amino Acid Sequence↗
HLA-DR3 molecules can bind peptides carrying two alternative specific submotifs.
Three different HLA-DR3-specific peptide binding motifs have been proposed. These motifs shared a major hydrophobic anchor at the N-terminus, but differed in the C-terminal anchor residues. In the present study, the structural requirements for peptide binding to HLA-DR3 were examined in further detail by using quantitative HLA-DR3-specific binding assays and sets of single substitution analogues of DR3 binding peptides (Lol pollen amino acids 171-190 and sperm whale myoglobin amino acids 132-151). We found that the requirements for binding to HLA-DR3 vary among different DR3 binding peptides; the absence of an anchor or the presence of only a weak anchor residue at either position n or n + 3 can be compensated for by the presence of a strong, positively charged anchor residue at position n + 5. These results explain several of the previously reported differences between DR3-specific peptide binding motifs. To evaluate the predictive value of the thus-refined motif, the DR3 binding capacity of an overlapping set of peptides, spanning the entire sequence of the 65-kDa heat shock protein of Mycobacterium tuberculosis was investigated and correlated with the occurrence of the different DR3 motifs. A strong correlation was found between the presence of the refined DR3 motif and peptide binding to purified HLA-DR3 molecules.
Allergens↗
Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.
We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.
Alleles↗
Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing.
Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogenous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.
Amino Acid Sequence↗
Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.
Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.
Amino Acid Sequence↗
Analogues of peptide 9-21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies.
Several analogues of the amino acid sequence of peptide 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and Ev-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9-21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10-18 and peptide 9-17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9-21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.
Antibodies, Monoclonal↗
Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B, which is specifically recognized by antibodies from patients with leprosy.
Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions (AA) 206-230, 251-280, and 291-325. Sera of patients with active tuberculosis who responded to the native 30-kDa antigen did not recognize these peptides. The antibody-binding specificity to the defined B cell regions was evaluated in a blind study with 71 serum samples from patients and household contacts living in Ethiopia where leprosy is endemic. The peptide of AA 256-280 was recognized by 88% of LL patients, 15% of patients with tuberculoid leprosy, and none of the contacts. These findings suggest that there are major linear B cell epitopes on the M. leprae 30-kDa protein that are recognized by lepromin-negative LL patients, whereas lepromin-positive patients respond preferentially to conformational epitopes.
Amino Acid Sequence↗
Isolation of an HLA-A2.1 extracted human minor histocompatibility peptide.
Purified HLA-A2.1 molecules obtained by affinity chromatography of 6 x 10(10) Epstein Barr virus (EBV)-transformed B lymphocytes were used in an attempt to isolate the human HLA-A2.1-restricted minor histocompatibility (H) peptides H-Y and HA-2. Fraction 18 of the high-performance liquid chromatography (HPLC)-separated HLA-A2.1 peptide pool was found to contain the natural HA-2 peptide. An HA-2-specific, HLA-A2.1-restricted cytotoxic T lymphocyte clone lysed HLA-A2.1+ HA-2- EBV-transformed B lymphocyte cell lines reproducibly and in a concentration-dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H-Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA-2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA-2 from a peptide pool with the established pattern for binding to HLA-A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA-A2.1 binding peptide.
Amino Acid Sequence↗