Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6567-6571, July 1995 Medical Sciences Naturally processed peptides from two disease-resistance- associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DRI3 chain (molecular evolution/hepatitis B/malaria/epitope) MILES P. DAVENPORTtt, CHERYL L. QUINNt, ROMAN M. CHICZ§, BRIAN N. GREENS, ANTHONY C. WILLISII, WILLIAM S. LANEtt, JOHN I. BELLt, AND ADRIAN V. S. HILLt tMolecular Immunology Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom; §Department of Molecular and Cellular Biology and ttHarvard Microchemistry Facility, Harvard University, Cambridge, MA 02138; IFisons Instruments, Biotech MS, Tudor Road, Altrincham, Cheshire, WA14 5RZ, United Kingdom; and I'Medical Research Council Immunochemistry Unit, Department of Biochemistry, South Parks Road, Oxford, OXI 3QU, United Kingdom Communicated by Baruch S. Blumberg, Fox Chase Cancer Center, Philadelphia, PA, April 10, 1995 (received for review December 23, 1994) ABSTRACT HLA-DR13 has been associated with resis- tance to two major infectious diseases of humans. To inves- tigate the peptide binding specificity of two HLA-DR13 mol- ecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR,B chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA- DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define-binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly -> Val change at position 86. These data suggest a molecular basis for the differential associations of HLA- DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection. The genes of the major histocompatibility complex (MHC) code for cell surface glycoproteins important in the binding of peptides for recognition by T cells. Analysis of the crystal structure of MHC class I and class II molecules has demon- strated peptides bound in an extended conformation to the surface of these proteins (1). The amino acid variations between allelic variants encoded by a particular MHC locus are clustered around the peptide binding region of the molecule and are thought to alter the peptide binding specificity of the molecules. The binding specificity of MHC class II molecules has been analyzed by a variety of methods. Direct binding to MHC class II molecules has been measured by using synthetic peptides (2) and libraries of random peptides encoded in the coat protein of M13 bacteriophage (3). Endogenous peptides bound to class II molecules have been sequenced by MS (4) or Edman chemistry (5, 6). Analysis of these peptides reveals they are The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 12-20 amino acids long and sequence alignment suggests possible patterns of binding. Position 86 of the human HLA-DR,B chain (1386) is dimor- phic and the amino acids Gly and Val are found at this position. The large number of alleles that differ at this position and the differential disease associations of alleles differing only at this position indicate the functional importance of this dimorphism (7). The crystal structure of the HLA-DR1 molecule has shown that the Gly-,386 of DR1 interacts with the conserved hydrophobic anchor residue of class II binding peptides (1). However, as this is the only HLA-DR molecule for which the crystal structure has been solved, the effect of Val-1386 has not been determined. Studies of naturally processed peptides bound to several HLA-DR allelic proteins have also been performed (8, 9). HLA-DR molecules containing Gly-,B86 and Val-,386 have been analyzed in these studies that in some cases suggest preferences for aromatic or aliphatic anchor residues. However, to our knowledge, there has been no analysis of alleles differing only at 1386, and in unrelated proteins there is no clear correlation between aromatic or aliphatic anchor residue preference and the amino acid at (386 (9). In this study we examine the endogenous peptides eluted from the HIA-DRB1*1301 (DRB1*1301) and HLA-DRB1*1302 (DRB1*1302) molecules that differ only at position (386, with DRB1*1301 containing Val and DRB1*1302 containing Gly. We have chosen these DR13 alleles for detailed analysis because of their differential associations with two major infectious diseases of humans. In studies in West Africa both DR13 alleles were associated with clearance of hepatitis -B virus infection (10), whereas DRB1*1302 but not DRBI*1301 was associated with resistance to a clinical form of severe malaria (7). Individual peptides were sequenced by tandem MS or Edman degradation. These peptides and Ala-substituted vari- ants of them were then synthesized and used in in vitro binding assays to purified HLA-DR molecules. In addition, pool sequencing of eluted peptides was performed to gain a further view of amino acid preferences in particular positions. Our results identify a peptide binding motif for DRB1*1301 and * 1302 and indicate that the effect of the Gly/Val dimorphism is to alter the preference for particular hydrophobic anchor residues in naturally processed and presented peptides. MATERIALS AND METHODS HLA-DR and Peptide Purification. HLA-DR protein was prepared from the Epstein-Barr virus-transformed B-cell lines Abbreviations: MHC, major histocompatibility complex; (386, position 86 of the HLA-DR,3 chain; HA, hemagglutinin; (32m, (32-microglobulin; Ii, invariant chain. 4To whom reprint requests should be addressed. 6567 Downloaded by guest on October 7, 2020
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Proc. Natl. Acad. Sci. USAVol. 92, pp. 6567-6571, July 1995Medical Sciences
Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequencemotifs and the effects of the dimorphism at position86 of the HLA-DRI3 chain
(molecular evolution/hepatitis B/malaria/epitope)
MILES P. DAVENPORTtt, CHERYL L. QUINNt, ROMAN M. CHICZ§, BRIAN N. GREENS, ANTHONY C. WILLISII,WILLIAM S. LANEtt, JOHN I. BELLt, AND ADRIAN V. S. HILLttMolecular Immunology Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom;§Department of Molecular and Cellular Biology and ttHarvard Microchemistry Facility, Harvard University, Cambridge, MA 02138; IFisons Instruments,Biotech MS, Tudor Road, Altrincham, Cheshire, WA14 5RZ, United Kingdom; and I'Medical Research Council Immunochemistry Unit,Department of Biochemistry, South Parks Road, Oxford, OXI 3QU, United Kingdom
Communicated by Baruch S. Blumberg, Fox Chase Cancer Center, Philadelphia, PA, April 10, 1995 (received for review December 23, 1994)
ABSTRACT HLA-DR13 has been associated with resis-tance to two major infectious diseases of humans. To inves-tigate the peptide binding specificity of two HLA-DR13 mol-ecules and the effects of the Gly/Val dimorphism at position86 of the HLA-DR,B chain on natural peptide ligands, thesepeptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only atthis position. The eluted peptides were subjected to poolsequencing or individual peptide sequencing by tandem MS orEdman microsequencing. Sequences were obtained for 23peptides from nine source proteins. Three pool sequences foreach allele and the sequences of individual peptides were usedto define-binding motifs for each allele. Binding specificitiesvaried only at the primary hydrophobic anchor residue, thedifferences being a preference for the aromatic amino acidsTyr and Phe in DRB1*1302 and a preference for Val inDRB1*1301. Synthetic analogues of the eluted peptidesshowed allele specificity in their binding to purified HLA-DR,and Ala-substituted peptides were used to identify the primaryanchor residues for binding. The failure of some peptideseluted from DRB1*1302 (those that use aromatic amino acidsas primary anchors) to bind to DRB1*1301 confirmed thedifferent preferences for peptide anchor residues conferred bythe Gly -> Val change at position 86. These data suggest amolecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malariaand clearance of hepatitis B virus infection.
The genes of the major histocompatibility complex (MHC)code for cell surface glycoproteins important in the binding ofpeptides for recognition by T cells. Analysis of the crystalstructure of MHC class I and class II molecules has demon-strated peptides bound in an extended conformation to thesurface of these proteins (1). The amino acid variationsbetween allelic variants encoded by a particularMHC locus areclustered around the peptide binding region of the moleculeand are thought to alter the peptide binding specificity of themolecules.The binding specificity of MHC class II molecules has been
analyzed by a variety of methods. Direct binding to MHC classII molecules has been measured by using synthetic peptides (2)and libraries of random peptides encoded in the coat proteinof M13 bacteriophage (3). Endogenous peptides bound to classII molecules have been sequenced by MS (4) or Edmanchemistry (5, 6). Analysis of these peptides reveals they are
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.
12-20 amino acids long and sequence alignment suggestspossible patterns of binding.
Position 86 of the human HLA-DR,B chain (1386) is dimor-phic and the amino acids Gly and Val are found at this position.The large number of alleles that differ at this position and thedifferential disease associations of alleles differing only at thisposition indicate the functional importance of this dimorphism(7). The crystal structure of the HLA-DR1 molecule hasshown that the Gly-,386 of DR1 interacts with the conservedhydrophobic anchor residue of class II binding peptides (1).However, as this is the only HLA-DR molecule for which thecrystal structure has been solved, the effect of Val-1386 has notbeen determined. Studies of naturally processed peptidesbound to several HLA-DR allelic proteins have also beenperformed (8, 9). HLA-DR molecules containing Gly-,B86 andVal-,386 have been analyzed in these studies that in some casessuggest preferences for aromatic or aliphatic anchor residues.However, to our knowledge, there has been no analysis ofalleles differing only at 1386, and in unrelated proteins there isno clear correlation between aromatic or aliphatic anchor residuepreference and the amino acid at (386 (9).
In this study we examine the endogenous peptides eluted fromthe HIA-DRB1*1301 (DRB1*1301) and HLA-DRB1*1302(DRB1*1302) molecules that differ only at position (386, withDRB1*1301 containing Val and DRB1*1302 containing Gly. Wehave chosen these DR13 alleles for detailed analysis because oftheir differential associations with two major infectious diseasesof humans. In studies in West Africa both DR13 alleles wereassociated with clearance of hepatitis -B virus infection (10),whereas DRB1*1302 but not DRBI*1301 was associated withresistance to a clinical form of severe malaria (7).
Individual peptides were sequenced by tandem MS orEdman degradation. These peptides and Ala-substituted vari-ants of them were then synthesized and used in in vitro bindingassays to purified HLA-DR molecules. In addition, poolsequencing of eluted peptides was performed to gain a furtherview of amino acid preferences in particular positions. Ourresults identify a peptide binding motif for DRB1*1301 and* 1302 and indicate that the effect of the Gly/Val dimorphismis to alter the preference for particular hydrophobic anchorresidues in naturally processed and presented peptides.
MATERIALS AND METHODSHLA-DR and Peptide Purification. HLA-DR protein was
prepared from the Epstein-Barr virus-transformed B-cell lines
Abbreviations: MHC, major histocompatibility complex; (386, position 86of the HLA-DR,3 chain; HA, hemagglutinin; (32m, (32-microglobulin; Ii,invariant chain.4To whom reprint requests should be addressed.
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LG-2 (DRB1*0101) (11), HHKB (DRB1*1301 and DRB3*0101)(12), and WT-47 (DRB1*1302 and DRB3*0301) (13). Peptidesfor individual peptide sequencing by Edman degradation wereprepared as described by using a solubilized cell membranepreparation and an LB3.1 affinity column (9). For use in pooisequencing and tandem MS studies, cells were prepared by thefollowing modified method. Briefly, a 10-g cell pellet was lysed inphosphate-buffered saline containing 2% (vol/vol) Nonidet P-40,leupeptin (2 lkg/ml), pepstatin (2 ttg/ml), and 5 mM EDTA. Celllysates were centrifuged at 100,000 X g for 90 min at 40C thenloaded onto an L-243 affinity column. The colunmn was washedextensively and then material was eluted with 0.05 M diethyl-amine/150 mM NaCl/0.1% Nonidet P-40, pH 11.5. Eluate wasconcentrated on a Centricon-10 column (Amicon) before elutionof peptides with 0.1% trifluoroacetic acid and separation byreverse-phase HPLC. HLA-DR for peptide binding studies waspurified in the same manner with the exception that CHAPSdetergent was used instead of Nonidet P-40. The HLA-DRB3gene products were not removed from the DRBI *1301 and *1302gene products. However, since these are thought to represent aminor population (<10%) of HIA-DR molecules, they areunlikely to significantly influence results.
individual Peptide Sequencing. Sequencing of individualpeptides by Edman chemistry was performed as described (9).Briefly, HPLC fractions were analyzed by matrix-assisted laserdesorption time of flight MS (MALDI--TOF) using a Laser-MAT (Finnigan-MAT, San Jose, CA) and an a-cyano-4-
hydroxycinnamic acid matrix. HPLC fractions that appeared tocontain a single dominant peptide species were then se-quenced by automated Edman microsequencing using a pulsedliquid protein sequencer with on-line HPLC (model 477A;Applied Biosystems). Masses of sequenced peptides wereconfirmed by a second MALDI-TOF analysis using an internalmass standard. Peptide sequencing by tandem MS was per-formed using a Quattro 11 triple quadrupole instrument withan electrospray ionization source (Fisons Instruments, Lough-borough, U.K.). MS-MS spectra were manually interpreted(Fig. 1).
Pool Sequencing of Peptides. Pool sequencing of elutedpeptides was performed as described (6, 14). Briefly, reverse-phase HPLC profiles of eluted peptides from 5 X 109 cells wereanalyzed and dominant peaks were removed before the re-mainder were pooled and sequenced by using a pulsed liquidprotein sequencer (model 473A; Applied Biosystems). Cys wasnot quantitated. Sequencing was repeated three times by usingfresh cell preparations. The data were analyzed as described(15). Briefly, the concentrati'on of each amino acid at eachposition was expressed as the percentage of the total aminoacids detected at that position. This was then averaged over thethree runs for each HLA-DR molecule. We then took theoverall average value for each amino acid by averaging thevalues at all positions except the first. Values for each aminoacid at each position were then expressed as a proportion of theaverage value for that amino a'cid over the whole run.
FIG. 1. Fragment ion spectrum of the li-(66-80) peptide. The doubly protonated precursor ion [(M + 2 H)2+] of mass-to-charge ratio 916.8was subjected to collision-induced dissociation with argon gas. The sequence of li-(66-80) and the predicted masses of the singly charged fragmentions [b = H(NHCHRCO),,+ and y" = H2(NHCHRCO),,OH+] are shown above the observed spectrum. Observed fragment ions are underlined.The isobaric residues Ile/Leu and Gln/Lys were distinguished by comparison with the protein sequence database.
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Peptide Binding Studies. Peptides were synthesized by usingstandard Fmoc chemistry on an SMPS-350 synthesizer (Zins-ser Analytic, Berkshire, U.K.). Influenza hemagglutinin(HA)-(306-318), HLA-C-(56-70), and HLA-B7-(104-122)peptides were biotinylated by using biotinamidocaproate N-hydroxysuccinamide ester (Sigma). Binding of peptides toHLA-DR was measured by their ability to inhibit the bindingof a biotinylated peptide. Briefly, 1 jig of HLA-DR wasincubated with biotinylated peptide with or without inhibitorpeptide at 37°C for 24 h at pH 5. Solutions were neutralizedwith Tris*HCl (pH 7.5) before transfer to wells precoated withL-243 antibody. Binding of biotinylated peptide was detectedwith ExtrAvidin-horseradish peroxidase conjugate (Sigma)and developed with o-phenylenediamine.
RESULTSSequences of Peptides Eluted from DRB1*1301 and
DRB1*1302. The sequences of endogenous peptides elutedfrom DRB1*1301 and DRB1*1302 are shown in Table 1.Two-thirds of the sequences were derived from membrane-bound proteins (predominantly MHC proteins and their as-sociated molecules) and the remainder was from secretedproteins expected to be found in high concentrations extra-cellularly or in endosomes. The length of the peptides was14-24 amino acids and nested sets of peptides with C-terminaltruncations were commonly seen. The promiscuous CLIPpeptide [Ii-(97-120)] was associated with both alleles (Table1). Two peptides of exogenous origin were sequenced, andboth were apolipoproteins believed to be derived from the
Table 1. Sequences of peptides eluted from HLA-DRB1*1301and DRB1.* 1302
DRB1*1302LPKPPKPVSKMRMATPLLMQALPM Ii-(97-120)TTAYFLYQQQGRLDKt Ii-(66-80)NPGGYVAYSKAATVTGKLt TR-(215-232)NPGGYVAYSKAATVTG TR-(215-230)SSVITLNTNVGLYDQSDIAK ApoB-100-(3342-61)tSSVITLNTNVGLYDQSD ApoB-100-(3342-58)tDPTLDHHWHLWKKTYGKQYKE Cathepsin S-(21-42)DPTLDHHWHLWKKTYGKQYK Cathepsin S-(21-41)GPEYWDRETQKYKRQAQ HLA-C-(56-72)GPEYWDRETQKYKRQA HLA-C-(56-71)GPEYWDRETQKYKRQ HLA-C-(56-70)APRFKHLRKYTYNYEA ApoB-100-(43-58)tSequences were determined by electrospray ionization tandem MS
or automated Edman degradation. The HLA-B7 peptide is from aregion identical in HLA-B*0701 and *0702, and the HLA-C peptideis from a region common to all HLA-C variants. They are presumedto be derived from the HLA-B*0701 and HLA-C*0501 moleculespresent on the HHKB and WT-47 cells, respectively. Ii, invariantchain; 132m, /32-microglobulin; TR, transferrin receptor.tSequences from tandem MS (the isobaric residues Ile/Leu andLys/Asp were allocated by comparison with the protein database).4Presumed bovine source of protein due to sequence homology withhuman protein.
fetal calf serum used in the B-cell cultures. The nature andsource of peptides sequenced in this study are, therefore,broadly similar to peptides found associated with other class IImolecules (5, 9).
Pool Sequencing of Peptides Eluted from DRB1*1301 andDRB1*1302. The results of three pool sequences from eachDR molecule were analyzed for positions in which particularamino acid residues were enriched. The method of analysisused allows estimation of both an increase or decrease in therelative abundance of an amino acid at a particular cycle. Thestrong enrichment for Pro in position 2 in our samples has beennoted previously and ascribed to an effect of processing (14).Due to the variable N-terminal extensions of these peptidesfrom the binding groove of the HLA-DR molecule, aminoacids that are enriched as a result ofMHC binding rather thanprocessing tend to be enriched over two to four positions in thepool sequence analysis (14). The first enrichment of this kindseen is that for hydrophobic amino acids at positions 4-6. Thishas been proposed to be associated with the hydrophobicanchor residues of the peptide necessary for interaction withthe hydrophobic pocket of the HLA-DR molecule (1, 14). Byconvention, the position of this hydrophobic anchor (absoluteposition 4 or 5) is denoted X, and subsequent positions of thepeptide are numbered relative to it as X + n.The most striking difference between the pool sequences of
peptides eluted from the two HLA-DR molecules studied isfound in the enrichment of hydrophobic amino acids at the firsthydrophobic anchor region. Ile and Leu are enriched -2-foldin both pools, whereas Trp is not significantly increased ineither. However, Tyr is increased 1.7-fold in the DRB1*1302pool (Gly-f386), and not at all in the DRB1*1301 pool (Val-(386), and Phe follows a similar trend (2.3-fold increased in theDRB1* 1302 and only 1.3-fold in the * 1301 pool). On the otherhand, Val shows a weaker trend in the opposite direction, beingincreased by up to 1.7-fold in the DRB1*1301 and <1.2-foldin the DRB1*1302 pool. This suggests distinct preferences forparticular hydrophobic anchors at this position, consonantwith the interaction of (86 with this region of the peptide andthe possibility that Val-1386 may restrict the hydrophobicbinding pocket.
Subsequent positions of the pool sequence are very similarbetween the two alleles. Charged and polar residues appearenriched - 1.5-fold over the entire region X + 3 to X + 9(positions 8-13), although the peaks of relative abundance ofpolar and positively charged amino acids appear to occur atposition X + 5. Tyr appears to be enriched later at position X+ 8, and other nonpolar amino acids also appear enriched inthis region of the pool sequence.
Binding of Eluted Peptides to HLA-DR. Synthesis of theeluted peptide sequences and studies of their in vitro bindingto purified HLA-DR were performed by measuring the abilityof peptides to compete with a biotinylated peptide. Table 2shows the concentration of unlabeled peptide required toinhibit 50% of the binding of a biotinylated peptide (IC50). ForDRB1*1301 and *1302, the biotinylated peptides used werefrom sequences obtained from peptides eluted from thesemolecules [HLA-B7-(104-122) and HLA-C-(56-70) peptides,respectively]. For DR1, competition was performed againstthe HA-(306-318) peptide known to bind to this HLA-DRmolecule (16). These binding studies demonstrated that alleluted peptides bound to the HLA-DR molecule from whichthey were derived. Since binding studies utilized competitionagainst a single peptide for each variant, it is clear that allpeptides bound to the dominant DRB1 gene product and notthe minor DRB3 gene contaminant. In addition, the allelespecificity of peptide binding was demonstrated by the fact thatnone of the eluted peptides with the exception of the promis-cuous Ii-(97-120) were observed to bind to the unrelated classII molecule DR1. No binding was seen with the control A4KA5peptide to any HLA-DR molecule.
Synthetic peptides from sequences of eluted peptides listed in Fig.1, Influenza HA-(306-318), or A4KA5 were used to inhibit the bindingof biotinylated peptides to HLA-DRB1*0101, DRB1* 1301, andDRB1* 1302. The concentration required to cause a 50% inhibition ofbinding of biotinylated peptide (IC50) to the three HLA-DR moleculesis shown. The biotinylated peptides used for DRB1*0101, DRB1*1301,and DRB1*1302 were the HA-(306-318), HLA-B7-(104-122), andHLA-C-(56-70), respectively. The source of the peptide sequence isshown, except for HA-(306-318), which is known to bind promiscu-ously, and A4KA5, which is a negative control. The anchors identifiedby single Ala substitutions are underlined. Values where an unlabeledpeptide is inhibiting the binding of its biotinylated analogue are shownin boldface type. NA, not applicable.
Effect of Hydrophobic Anchor Residues on Binding toDifferent HLA-DR Molecules. It was observed that all peptideseluted from DRB1* 1301 were able to bind to DRB1* 1302 anddid so with similar affinity to the peptides derived fromDRB1*1302. In addition the hierarchy of binding affinities ofpeptides eluted from DRB1* 1301 was the same for binding toboth DRB1*1301 and DRB1*1302, suggesting that commonfactors contributed to the binding affinity in each case. Onlythree out of six DRB1*1302-derived peptides bound toDRB1*1301. Peptide analogues were synthesized with singleAla substitutions at likely primary anchor residues near the Nterminus of peptides. Binding studies demonstrated that mostsubstitutions altered the IC50 value of peptides by <2-fold.Substitutions that abrogated binding were designated as pri-mary anchor residues. It was noted that those peptides elutedfrom DRB1*1302 that were unable to bind DRB1*1301 uti-lized aromatic anchor residues. In contrast, the DRB1*1301-derived peptides had Ile, Leu, and Val as anchor residues, asdid two out of three of the DRB1*1302-derived peptides thatbound to DRB1 * 1301. Thus only one peptide with an aromaticanchor (Phe) was able to bind DRB1*1301. In addition,substitution of the Ile anchor residue of the DRB1*1301-derived peptide j32m-(4-17) with an aromatic anchor abro-gated binding to DRB1*1301 (data not shown).Comparison of Results from Pool and Individual Peptide
Sequencing. Comparison of the motif derived from poolsequencing with the sequences of the individual peptidesderived from each allele was performed. In 7 of 10 uniquesequences, Pro was observed at position 2 of the peptides.The position and nature of hydrophobic anchor residues also
supported the pool sequence results since aromatic anchorresidues were found in four of six of the DRB1*1302-derivedpeptides and in zero of three of the DRB1*1301-derivedpeptides (Fig. 2). The preference for polar and chargedresidues observed in the pool sequence over the region X + 3to X + 9 was also reflected in the individual peptide sequences.Position X + 5 may be the most important "pocket" for theseresidues, as all residues at this position were positively chargedor polar. The preference for Tyr at position X + 8 was alsoobserved in the individual peptides sequenced.
x
vhydrophobic
DRB1*1301(I/L/V)
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FIG. 2. Alignment of individual peptide sequences. The sequencesof individual peptides eluted from DRB1*1301 and DRB1*1302 arecompared with the motif identified by pool sequencing of peptideseluted from these molecules. Peptides are aligned by using the primaryhydrophobic anchor residues identified by binding studies with singleAla-substituted analogs. Boxes indicate proposed anchor positions forHLA-DR binding.
DISCUSSIONAnalysis of the binding and processing requirements forpeptides to be presented on the surface of antigen-presentingcells in association with MHC class II molecules providesdirect information on what peptides may be seen by CD4+ Tcells. A major aim of such analysis is the prediction of T-cellepitopes from pathogen-derived proteins or autoantigens. Theelucidation of the structure of the HLA-DR molecule hasrevealed the interactions between peptide amino acid residuesand the variable HLA-DRf3 molecule. (386 of HLA-DR wasshown to interact with the previously described hydrophobicanchor of the peptide ligand (9).The effects on the HLA-DR-peptide interaction of the
Gly/Val dimorphism at position 86 have been studied by invitro peptide binding (17) and indirect cellular assays (21).Experiments using naturally occurring Gly-,386 and Val-,B86allelic variants or mutants of DR7 and either promiscuous orpoly(Ala) peptides have suggested that replacement of Tyrwith Leu, or even Ser, as an anchor or modifications at otherpositions of the peptide may increase binding to Val-/386-containing proteins (17-19). These in vitro binding studies havenot always been consistent and are unable to address the extentto which the Gly/Val dimorphism might affect the relativeabundance of these different hydrophobic anchors amongnaturally processed peptides.The data presented in this study compare individual peptide
sequences and pool sequences of peptides eluted from twoHLA-DR13 molecules differing only at position 1386. Theseresults indicate that the binding motifs for the two proteinsvary only at the hydrophobic anchor position. The sharedpeptide preference for Ile and Leu contrasts with the prefer-ence for Tyr and Phe in DRB1* 1302 and the increasedpreference for Val in DRB1*1301. These preferences areobserved in both the pool and individual peptide sequencesand in the peptide binding data. Previous studies have hintedat a similar phenomenon by using in vitro binding studies withpolyalanine or promiscuous peptides (18, 20). However, hereinwe demonstrate that these preferences are observed withpeptides that bind in an allele-specific manner. Previousstudies with polyalanine or promiscuous peptides may repre-sent more artificial situations where there may be an overde-pendence on the contribution of the anchor residue to totalbinding energy of the peptide. Moreover, we show that theanchor preference of these HLA-DR13 alleles has a direct
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effect on the processing and presentation of peptides withinthe antigen-presenting cell and, therefore, is likely to play animportant role in determining which peptides from an anti-genic protein will be presented to the T cell.
In the eluted peptides, the enrichment for charged (espe-cially basic) and polar amino acids at positions X + 3 to X +9, with a peak around position X + 5, is common to bothHLA-DR13 molecules, as is the preference for Tyr or hydro-phobic amino acids around position X + 8. In the absence ofstructural information regarding these two molecules, it ispossible to speculate that relative to HLA-DRB1*0101, forwhich the structure has been determined, these two HLA-DR13 proteins contain several charge changes in the "pockets"involved in binding of HLA-DR1 to positions X + 3, X + 6,and X + 8 of the HA-(306-318) peptide (i.e., Trp-,9 -- Glu,Gln-,f70 -> Asp, Arg-,B71 -> Glu) (1).The question remains as to whether, as proposed (21),
Val-,86 allelic variants may see a subset of Gly-1386 allelicvariants, since they are unable to bind peptides with Tyranchors. In evolutionary terms, it is unclear what benefit wouldbe conferred by such a reduction, since this would limitrecognition of peptides from some pathogens. It is clearhowever from the number of Val-J86 allelic variants andrecent evidence for their continuing selection (22) that evo-lution must favor the coexistence of alleles encoding Gly-,86and Val-,86. If DRB1*1301 does present a strict subset ofpeptides presented by DRB1 * 1302, then its selection might beexplained by some benefit accruing from lack of peptidebinding, such as the decreased "hole in the repertoire" of Tcells due to thymic selection (23). An alternative hypothesis isthat, although the inability to bind peptides with Tyr anchorsprevents high-affinity peptides from binding, other previouslylower-affin"ity peptides may then replace them, particularlypeptides with Val anchors. Thus each of these variants mightbind some unique peptides as well as a large overlapping set.The HLA-DR13 alleles examined in this study have been
associated with protection from three major diseases caused byinfectious pathogens. HLA-DRB1 *1302 but not *1301 wasfound to be associated with resistance to a form of severemalaria caused by Plasmodium falciparum (7). Both HL>A-DRB1 *1301 and *1302 have recently been found to be asso-ciated with resistance to persistent hepatitis B virus infectionin the same population of West African children (10). Inanother study, both alleles were associated with protectionfrom cervical cancer induced by human papilloma virus in-fection (24). The characterization of natural ligands for bothDRB1*1301 and *1302 proteins suggests a possible molecularbasis for the differential association of these alleles withparticular diseases. For malaria, where a protective associationwas observed with only DRB1 *1302, we may speculate that animmunodominant P. falciparum epitope might contain a Tyr asa major anchor in the Gly-,386-containing pocket and that thisepitope cannot bind to DRB1* 1301 with a Val-f386-containingpocket. Conversely, the key protective epitopes in the hepatitisand papilloma viruses might use a hydrophobic anchor residue,such as Leu, that binds well to both DR13 molecules. Analysisof candidate epitopes for DR13 molecules in these importantpathogens should help to clarify the mechanisms underlyingthese HLA-disease associations and facilitate rational vaccinedesign.
We thank Violaine Bailey, Polina Klimovitsky, Harriet Maclehose,Karl McIntyre, and Renee Robinson for technical assistance andProfessor Andrew McMichael, Dr. Robert Urban, and Professor JackStrominger for advice and careful review of the manuscript. M.P.D. isa Wellcome Prize Student and received support from the LionelMurphy Foundation. A.V.S.H. is a Wellcome Trust Senior ClinicalFellow.
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