Binding of 8-mer to 11-mer peptides carrying the anchor residues to slow assembling HLA class I molecules (HLA-B*5101)

 The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B^*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B^*5101 cells at 26 °C for 3 h. Analysis of the binding of these peptides to HLA-B^*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B^*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B^*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B^*5101 binding peptides to stabilize the HLA-B^*5101 molecules was markedly lower than that of HLA-B^*3501 binding peptides to stabilize the HLA-B^*3501 molecules. It is known that HLA-B^*5101 is a slow assembling molecule, while HLA-B^*3501 assembles rapidly. The results imply that the slow assembling of HLA-B^*5101 molecules results from the low affinity of peptides to HLA-B^*5101 molecules. Received: 14 August 1996 / Revised: 8 October 1996     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

Crucial role of N-terminal residue of binding peptides in recognition of the monoclonal antibody specific for the peptide-HLA-B5, -B35 complex

 The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B^*3501 and HLA-B^*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B^*3501 and -B^*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B^*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B^*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B^*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B^*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B^*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex. Received: 19 June 1997 / Revised: 27 August 1997     

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

 HLA-B^*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B^*3501-restricted antigens is the characterization of peptide-HLA-B^*3501 interactions. In this study, peptide-HLA-B^*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B^*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B^*3501 binding cleft. Received: 29 May 1996 / Revised: 5 August 1996     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.     

Two subtypes of HLA-B51 differing by substitution at position 171 of the α2 helix

Newly defined antigens of the B5, B35 cross-reacting group have been found in Japanese and North American Indians. Nucleotide sequencing of the alleles encoding the Japanese B5.35 antigen and the variant B5 antigen from the Piman Indians show them to be identical. This new allele, B ^* 5102, differs from B ^* 5101 by a single nucleotide substitution that changes residue 171 from histidine to tyrosine. Residue 171, which is part of the ₂ helix, is believed to contribute directly to peptide interaction in the A pocket of the binding groove and is either histidine or tyrosine in all HLA-A, B, C heavy chains. Tyrosine 171 is shared by B ^* 5102, B ^* 3501, B ^* 3502, and B ^* 5301 and must be responsible for the serological cross-reactivities of these molecules not shared with B ^* 5101. Stimulation of lymphocytes from a B ^* 5101 positive donor with B ^* 5102 positive cells failed to generate cytotoxic T cells with specificity for the difference between these molecules. However, one out of five clones of cytotoxic T cells raised against B ^* 5101 failed to lyse targets expressing B ^* 5102. Substitution of histidine for tyrosine at residue 171 affected recognition of HLA-B35-restricted human minor histocompatibility antigen-specific T cell clones.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M68964.     

Two distinct HLA-A * 0101-specific submotifs illustrate alternative peptide binding modes

 Previous studies have defined two different peptide binding motifs specific for HLA-A ^* 0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A ^* 0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A ^* 0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE₃ submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A ^* 0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A ^* 0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A ^* 0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A ^* 0101-restricted peptide epitopes. Received: 16 July 1996 / Revised: 18 September 1996     

The Bw4/Bw6 difference between HLA-B*0802 and HLA-B*0801 changes the peptides endogenously bound and the stimulation of alloreactive T cells

 HLA-B^*0801 is unique among HLA-B allotypes in having dominant amino acid anchors at positions 3 and 5 of the peptide-binding motif. HLA-B^*0802 is a variant of HLA-B^*0801 in which the Bw6 sequence motif is replaced by a Bw4 sequence motif. This change, involving substitutions at positions 77, 80, 81, 82, and 83 of the B^*08 heavy chain, is probably the result of a single evolutionary event of interallelic conversion. Moreover, the difference between B^*0802 and B^*0801 is sufficient to stimulate a cytotoxic T-cell response. To assess further the functional impact of the Bw4 motif on a B8 background, we compared the peptide-binding specificity of the B^*0801 and B^*0802 allotypes by sequencing the mixture of peptides endogenously bound to B^*0802 and 12 individual peptides purified from that mixture. The HLA-B^*0802 allotype, while able to bind some peptides bound by B^*0801, has a broader repertoire of endogenously bound peptides than B^*0801: the peptides bound by B^*0802 are more variable in length and exhibit greater diversity in the carboxyl-terminal amino acid which interacts with the F pocket. Received: 29 October 1997     

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.     

HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles

Recent data suggest that HLA-B locus alleles can evolve quickly in native South American populations. To investigate further this phenomenon of new HLA-B variants among Amerindians, we studied samples from another South American tribe, the Cayapa from Ecuador. We selected individuals for HLA-B molecular typing based upon their HLA class II typing results. Three new variants of HLA-B39 and one new variant of HLA-B15 were found in the Cayapa: HLA-B ^*3905, HLA-B^*3906, HLA-B^*3907, and HLA-B ^*1522. A total of thirteen new HLA-B alleles have now been found in the four South American tribes studied. Each of these four tribes studied, including the Cayapa, had novel alleles that were not found in any of the other tribes, suggesting that many of these new HLA-B alleles may have evolved since the Paleo-Indians originally populated South America. Each of these 13 new alleles contained predicted amino acid replacements that were located in the peptide binding site. These amino acid replacements may affect the sequence motif of the bound peptides, suggesting that these new alleles have been maintained by selection. New allelic variants have been found for all common HLA-B locus antigenic groups present in South American tribes with the exception of B48. In spite of its high frequency in South American tribes, no evidence for variants of B48 has been found in all the Amerindians studied, suggesting that B48 may have unique characteristics among the B locus alleles.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U14756 (HLA-B ^*1522), U15683 (HLA-B ^*3905), U15639 (HLA-B ^*3906), and U15640 (HLA-B ^*3907)The names listed for these sequences were officially assigned by the WHO nomenclature Committee in September 1994, B ^*3905, and November 1994, B ^*1522, B^*3906, and B ^*3907. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1994), names will be assigned to the new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report.     

The peptide binding specificity of HLA-B27 subtypes

Five HLA-B27 subtypes, B^*2701, B^*2703, B^*2704, B^*2705, and B^*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B^*2705. The peptide sequences were derived from human HSP89, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B ^* 2701), CH (B ^* 2703), WE1 (B ^* 2704), BTB (B ^* 2705), and LIE (B ^* 2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B^*2705 and a new motif H-R were accepted by B^*2703, B^*2704, and B^*2706, but not by B^*2701. However, other motifs, including known B^*2702 and/or B^*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.     

Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501

 A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8⁺ T-cell response. Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual’s CD8⁺ cytotoxic T lymphocytes will be presented by a second individual’s different class I molecules. Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules. In applying this strategy to HLA-B^*1501, we isolated milligram quantities of B^*1501-bound peptides and mapped them using mass spectrometry. Repeated analyses consistently assign the same peptide binding signature to B^*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures. Received: 3 October 1996 / Revised: 20 November 1996     

Molecular analysis of HLA-B39 subtypes

Serological studies have suggested the presence of a new HLA-B39 subtype (B39.2) in the Japanese population. To identify the new HLA-B39 subtype and compare it with an other HLA-B39 subtype (B39.1), the genes encoding HLA-B39.1 (B ^* 39013) and B39.2 (B ^* 3902) have been cloned from Japanese. We have sequenced these genes and completed the sequence of HLA-B39.1 (B ^*39011 ) gene from a Caucasian that was partially sequenced. Comparison of the sequence data revealed that B ^* 3902 and B ^* 39013 differ by three nucleotide substitutions which result in a two amino acids change at residues 63 and 67, while one silent substitution at codon 312 is found between B ^* 39011 and B ^* 39013. These results suggest that B ^* 3902 has evolved from B ^* 39013 rather than B ^* 39011.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94051 (HLA-B^*39013), M94052 (HLA-B^*39011), and M94053 (HLA-B^*3902).     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Complexity among constituents of the HLA-B*1501 peptide motif

 Analysis of peptides derived from HLA class I molecules indicates that thousands of unique peptides are bound by a single molecular type, and sequence examination of the pooled constituents yields a motif which collectively defines the peptides bound by a given class I molecule. Motifs resulting from pooled sequencing are then used to infer whether particular viral and tumor protein fragments might serve as class I-presented peptide therapeutics. Still undetermined from a pooled motif is the breadth or range of peptides in the population which are brought together to form the pooled motif, and it is therefore not yet known how representative of the population a pooled motif is. By employing hollow fiber bioreactors for large-scale production of HLA class I molecules, sufficient peptides are produced to investigate individual subsets of peptides comprising a motif. Edman sequencing and mass spectrometric analysis of peptides eluted from HLA-B^*1501 reveal that many peptide sequences fail to align with either the N- or C-terminal anchors predicted for the B^*1501 peptide motif through whole pool sequencing. These analyses further reveal auxiliary anchors not previously detected and peptides significantly larger and smaller than the predicted nonamer, ranging from 6 to 12 amino acids in length. These results demonstrate that constituents of the B^*1501 peptide pool vary markedly in comparison with one another and therefore in comparison with previously established B^*1501 motifs, and such complexity indicates that many of the peptide ligands presented to CTL cannot be predicted using class I consensus motifs as search criteria. Received: 7 October 1997 / Revised: 10 December 1997     

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

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

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995