The motif for peptide binding to the insulin-dependent diabetes mellitus-associated class II MHC molecule I-Ag7 validated by phage display library
Silvia Gregori1,
Elisa Bono1,
Fabio Gallazzi1,
Juergen Hammer2,
Leonard C. Harrison3 and
Luciano Adorini1
1 Roche Milano Ricerche, Via Olgettina 58, 20132 Milano, Italy
2 Hoffmann-La Roche Inc., Nutley, NJ 07110, USA
3 The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Correspondence to:
L. Adorini
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Abstract
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The MHC class II molecule I-Ag7 is essential for the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse but the requirements for peptide binding to I-Ag7 are still controversial. We have now isolated I-Ag7-binding phage from a large phage display library encoding random nonamer peptides. Ninety peptide-encoding regions of phage eluted from I-Ag7 were sequenced and >75% of the corresponding synthetic peptides bound to I-Ag7. Peptide alignment led to the identification of position-specific anchor residues. Hydrophobic (V and P) and positively charged (K) residues were highly enriched at P6 and positively charged (R and K), aromatic (Y) or hydrophobic (L) residues at P9. In addition, small amino acid residues (G and A) were enriched at P7 and G at P8. The primary anchors at P6 and P9 defining the phage-derived motif were present in most high-affinity I-Ag7-binding peptides from IDDM candidate antigens but only in
25% of peptides that were low-affinity binders or failed to bind to I-Ag7. A comparison of these results with the proposed motifs for peptide binding to I-Ag7 validates the one we have previously described.
Keywords: I-Ag7, insulin-dependent diabetes mellitus, non-obese diabetic mice, peptide-binding motif
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Introduction
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The non-obese diabetic (NOD) mouse spontaneously develops autoimmune diabetes and is a model of human insulin-dependent diabetes mellitus (IDDM) (1,2). A major IDDM susceptibility locus is closely linked to the class II MHC gene complex of NOD mice which encodes the I-Ag7 molecule (3) and at least one dose of NOD MHC is required for IDDM development (4). Although the molecular basis of the association of I-Ag7 with diabetes is still unclear (5), the ability of I-Ag7 to bind and present peptides to autoreactive T cells is considered critical to the development of IDDM. I-Ag7, found only in NOD and in Biozzi AB/H mice that are susceptible to chronic relapsing experimental allergic encephalomyelitis (6), has a ß chain characterized by a non-D residue at position 57 (7). Interestingly, D at I-Ag7ß57 decreases the spontaneous incidence of IDDM in NOD mice (8) and the presence of a non-D residue at ß57 in HLA-DQ was found to correlate with susceptibility to IDDM in humans (9,10).
Although the motifs for peptide binding to several class I and class II MHC molecules have been well defined (11), the rules that govern peptide binding to I-Ag7 are still controversial (1216). The studies performed to determine the peptide binding specificity of I-Ag7 have all used different approaches. Reich et al. (17) eluted and sequenced naturally processed peptides from I-Ag7 and concluded that binding may require an acidic residue at the C-terminus of the peptide. Amor et al. (18) analyzed pathogenic peptides from myelin oligodendrocyte glycoprotein and proteolipid protein that induce experimental allergic encephalomyelitis in Biozzi AB/H mice, and suggested a motif comprised of a series of basic, small and large hydrophobic residues within a core of 67 amino acid residues. Reizis et al. (19) analyzed the rules for peptide binding to I-Ag7 using an assay based on peptide competition for antigen presentation by fixed antigen-presenting cells (20). Their results suggested a binding motif based on a nonamer peptide with conserved anchor positions at P4, P6 and P9. A negatively charged residue at P9 was proposed to play a critical role. We have previously defined a motif for peptide binding to I-Ag7 using a sensitive competition assay for binding to purified, native MHC class II molecules (21). This led to the identification of two primary anchor positions: P6, characterized by large hydrophobic residues, and P9, at which aromatic or positively charged residues were preferred. In addition, two secondary anchor positions at P3 and P8 were identified.
To clarify the motif for peptide binding to I-Ag7 we have used the phage display technique (22). This allows definition of sequence characteristics that account for peptide binding to I-Ag7 molecules through the screening of a large, highly diverse, random-peptide library expressed on the surface of the M13 filamentous phage. A major advantage of the phage display technology is the identification of an unbiased repertoire of I-Ag7-binding peptides. The results obtained better define general rules for peptide binding to I-Ag7 and validate the peptide-binding motif we have previously described.
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Methods
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Purification of the I-Ag7 molecule
The 4G4.7 B cell hybridoma (21) was derived by polyethylene glycol-induced fusion of NOD mouse T cell-depleted splenocytes with the HAT-sensitive A20.2J lymphoma line (23). It expresses I-Ag7, I-Ad, I-Eg7 and I-Ed molecules. I-Ag7 molecules were affinity-purified from detergent lysates of 4G4.7 cells by sequential adsorption to 34.1.4 and 14.4.4S, and desorption from OX-6 mAb respectively, as previously described (21). 14.4.4S is a mouse monoclonal IgG2a antibody recognizing the E
chain (24); 34.1.4 is a mouse monoclonal IgG1 antibody against the Eßd chain which does not bind to I-Eg7 (24) and OX-6 is a mouse monoclonal IgG1 antibody against an invariant chain determinant of rat Ia, which also recognizes I-Ag7 but not I-Ad (25). Approximately 20 mg of purified mAb was first bound to 5 ml of Protein ASepharose 4 Fastflow (Pharmacia, Uppsala, Sweden) and then chemically cross-linked to the Protein A with dimethyl pimelimidate dihydrochloride (Pierce, Rockford, IL) in sodium borate buffer, pH 9.0. After 40 min incubation at room temperature, the reaction was quenched by adding in 0.2 M ethanolamine, pH 8.0, for 60 min. The suspension was thoroughly washed in PBS and stored in PBS containing 0.02% NaN3. 4G4.7 cells were harvested by centrifugation, washed in PBS, resuspended at 108 cells/ml in lysis buffer and then allowed to stand at 4°C for 120 min. The lysis buffer was 0.05 M sodium phosphate, pH 7.5, containing 0.15 M NaCl, 1% (v/v) NP-40 detergent and the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 5 mM
-amino-n-caproic acid, and 10 µg/ml each of soybean trypsin inhibitor, antipain, pepstatin, leupeptin and chymotrypsin (all from Sigma, St Louis, MO). Lysates were cleared of nuclei and debris by centrifugation at 27,000 g for 30 min, and stored as such if not immediately processed further. Then 0.2 volumes of 5% sodium deoxycholate (DOC; Sigma) was then added to the postnuclear supernatant. After mixing at 4°C for 10 min, the supernatant was centrifuged at 100,000 g at 4°C for 120 min, carefully decanted and filtered through 0.45 µm nylon membrane. The lysate of 1011 4G4.7 cells was recycled overnight at 4°C on a 34.1.4Protein ASepharose column and then on 14.4.4S-Protein ASepharose to bind I-Eg7, and finally on OX-6Protein ASepharose to bind I-Ag7. The OX-6Protein ASepharose column was then washed with at least 20 volumes of buffer A, 5 volumes of buffer B and 5 volumes of buffer C. Buffer A was 0.05 M Tris, pH 8.0, 0.15 M NaCl, 0.5% NP-40, 0.5% DOC, 10% glycerol and 0.03% NaN3; buffer B was 0.05 M Tris, pH 9.0, 0.5 M NaCl, 0.5% NP-40, 0.5% DOC, 10% glycerol and 0.03% NaN3; buffer C was 2 mM Tris, pH 8.0, 1% octyl-ß-D-glucopyranoside (Sigma), 10% glycerol and 0.03% NaN3. Bound MHC molecules were eluted with 50 mM diethylamineHCl, pH 11.5 in 0.15 M NaCl, 1 mM EDTA, 1% octyl-ß-D-glucopyranoside, 10% glycerol and 0.03% NaN3, and immediately neutralized with 1 M Tris. Approximately 2 mg of I-Ag7 protein was purified from 1011 4G4.7 cells. In SDSPAGE, the majority (>95%) of the protein was resolved as two bands of mol. wt ~33,000 and ~28,000 which correspond to the
and ß subunits respectively of class II MHC molecules.
Phage display library construction and I-Ag7-binding phage selection
The M13 peptide library was constructed as described elsewhere (22,26). It consisted of 2x107 independent phage-displaying random nonamer peptides flanked by four glycine spacers at each side at the N-terminus phage protein III. Protein III is a coat protein of the filamentous phage M13 and the region containing the foreign peptide is known to be exposed on the phage surface (22). Approximately 2x109 phage were incubated with 50 pM biotinylated I-Ag7 molecules in a binding buffer containing 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2% NP-40 and 80 mM citric phosphate, pH 6.0. After 48 h of incubation at room temperature, BSA-blocked streptavidin on 4% beaded agarose was added and incubated for 10 min. The M13 phageI-Ag7 complexes were purified by washing the solid phase 8 times with 8 volumes of washing buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.2 % NP-40). The phage were eluted with 8 volumes of elution buffer (0.1 N glycineHCl, pH 2.2, 1 mg/ml BSA) for 10 min and neutralized with 0.5 volumes of 2 M Tris base. Up to 107 eluted phage were used to infect 500 µl of Escherichia coli XL-1 blue plating cells (0.8 at OD650) (Stratagene, La Jolla, CA). The infected cells were transferred to 7 ml LuriaBertani medium in a 25 cm2 tissue culture flask. After 1 h incubation (37°C, 200 r.p.m.), ampicillin was added to a final concentration of 20 µg/ml. The phage were amplified overnight, harvested and purified by precipitation twice with polyethylene glycol. After four rounds of successive selection and amplification, aliquots of the fourth elution were mixed with M13mP19 reference phage, and the enrichment of white versus blue plaques was determined by separately plating this mixture and the eluates of M13I-Ag7-binding phage on X-gal indicator plates. The enriched phage were directly isolated from these eluates and the peptide-encoding regions were sequenced using the T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden).
Peptide synthesis
Peptides were synthesized with a multiple peptide synthesizer (model 396; Advanced Chemtech, Louisville, KY) using Fmoc chemistry and solid-phase synthesis on Rink Amide resin. All acylation reactions were effected with 3-fold excess of activated Fmoc amino acids and a standard coupling time of 20 min was used. Cleavage and side chain deprotection was achieved by treating the resin with 90% trifluoroacetic acid, 5% thioanisole and 2.5% phenol/2.5% water. The indicator peptide for the binding assays was biotinylated before being cleaved from the resin by coupling two 6-aminocaproic acid spacers and one biotin molecule at the N-terminus sequentially, using the above described procedure. The GAD (15mer) and IA-2 (16mer) peptides were synthesized with free N- and C-termini by Fmoc chemistry and solid-phase synthesis (Chiron Mimotopes, Melbourne, Australia). Peptides were routinely
85% pure as analyzed by reverse-phase HPLC.
Peptide binding assay
Peptides were dissolved at 10 mM in DMSO and diluted into 25% DMSO/PBS for assay. The indicator peptide hen egg lysozyme (HEL)1023 was synthesized with two spacer residues and a biotin molecule at the N-terminus. Approximately 500 nM of biotinylated peptide and each test peptide, diluted 10-fold from 50 µM to 50 pM, were co-incubated with 200 ng of MHC class II protein in U-bottom polypropylene 96-well plates (Serocluster; Costar, Cambridge, MA) in binding buffer at room temperature. The binding buffer was 6.7 mM citric phosphate, pH 6.0 with 0.15 M NaCl, 2% NP-40, 2 mM EDTA and the protease inhibitors as used in the lysis buffer. After 48 h, each incubate was transferred to the corresponding well of an ELISA plate (Maxisorp; Nunc, Roskilde, Denmark) containing pre-bound OX-6 antibodies (10 µg/ml overnight at 4°C followed by washing). After incubation at 37°C for 2 h and washing, bound biotinylated peptideMHC complexes were detected colorimetrically at 405 nm with streptavidinalkaline phosphatase and p-nitrophenylphosphate. Competition curves were plotted and the peptide affinity for MHC molecules was expressed as the peptide concentration required to inhibit the binding of biotinylated-peptide by 50% (IC50).
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Results
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Binding to I-Ag7 of peptides displayed by the M13 library
To determine whether I-Ag7 molecules were able to bind peptides displayed on the M13 phage surface, the HEL1022 sequence, known to bind to I-Ag7 with high affinity (21), was inserted at the N-terminus of the M13 phage protein III (Fig. 1A
). The peptide sequence was flanked by three or four glycine spacers to maximize the flexibility of the peptide insert. Phage encoding HEL1022 bound to I-Ag7 while phage from the unselected random-peptide-encoding M13 library did not, demonstrating that I-Ag7 can bind peptides displayed by the M13 phage library. The random-peptide M13 library was selected by four successive rounds of binding to biotinylated I-Ag7 molecules, elution and amplification, as detailed in Methods. To monitor the enrichment of I-Ag7-binding phage, the binding capacity of the eluted libraries was tested after each round of selection (Fig. 1B
). The I-Ag7-selected library, after four rounds of selection, showed an enrichment for binding to I-Ag7 similar to that obtained with the library composed of HEL1022-encoding phage, of ~25-fold compared to the M13mP19 reference phage library.

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Fig. 1. Selection by I-Ag7 molecules of specific peptides displayed on the phage surface. The N-terminal region of the modified M13 protein III constructs is shown in (A). Amino acid sequences corresponding to HEL1022 and to random peptides, where X is a random amino acid, are underlined. (B) Binding to I-Ag7 of phage display libraries containing HEL1022 or random-peptide inserts. The enrichment factor represents the enrichment in phage carrying I-Ag7-binding peptides compared with the reference phage M13mP19. The phage input was 1 billion for M13 libraries and 2 billion for M13mP19.
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I-Ag7-eluted phage encode I-Ag7-binding peptides
Phage eluted from the unselected random-peptide M13 library and from the I-Ag7-selected library after four rounds of screening were isolated and their peptide-encoding regions sequenced (Table 1
). All 90 peptide sequences were different, confirming the independent origin of the selected phage. To ascertain whether phage eluted from I-Ag7 contained peptides capable of binding to I-Ag7 molecules, the corresponding peptides were synthesized and tested in a competition assay for binding to purified I-Ag7. As control, peptides corresponding to 13 independent phage eluted from the unselected random-peptide M13 library were tested for binding to I-Ag7. None of these peptides bound with high affinity (IC50 < 1 µM) to I-Ag7 and only two of 13 bound with low affinity (IC50 110 µM) (Table 1
). Conversely, among the 90 synthetic peptides corresponding to I-Ag7-eluted phage, 52% bound to I-Ag7 with high affinity (IC50 < 1 µM), 24% with low affinity (IC50 110 µM) and 24% did not bind (IC50 > 10 µM) (Table 1
). These results indicate that the majority (76%) of synthetic peptides corresponding to sequences from I-Ag7-eluted phage did indeed bind to purified I-Ag7 molecules, whereas only 15% of phage eluted from the unselected random-peptide M13 library bound with low affinity.
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Table 1. Sequences and affinity for I-Ag7 of peptides eluted from the unselected M13 phage display library or from I-Ag7-enriched library
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Results in Fig. 2
validate the I-Ag7 peptide-binding assay. The biotinylated I-Ag7-binding peptide HEL1023 (21) bound very efficiently to I-Ag7, whereas the biotinylated heat shock protein (Hsp)116, a high-affinity binder to I-Eg7 (27), did not (Fig. 2A
). The competition assay for peptide binding to purified I-Ag7 molecules is sensitive and highly reproducible. In separate assays, the IC50 values for competition between biotinylated and unlabeled HEL1023 were between 400 and 500 nM (Fig. 2B
). As mentioned above, the phage display library was composed of nonamer peptides flanked by four glycine residues at the N- and C-termini to increase peptide flexibility. The synthetic peptides used in the binding assay corresponded to the nonamer peptide sequences. However, as shown in Fig. 2
(C), the addition of three glycine spacers did not affect binding affinity. For technical reasons we could not synthesize in a pure form peptides flanked by eight glycine spacers. Representative titration curves of phage-derived nonamer peptides are shown in Fig. 2
(D).

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Fig. 2. Binding assays to purified I-Ag7 molecules. (A) Binding of biotinylated HEL1023 or Hsp116 to purified I-Ag7 molecules. (B) Inhibition by unlabeled HEL1023 of biotinylated HEL1023 binding to I-Ag7. Three different competition curves from independent assays are shown. (C) Binding of a nonamer peptide versus the same peptide flanked by glycine residues. (D) Representative titration curves of phage-derived nonamer peptides. See Methods for assay details.
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Alignment of I-Ag7-bound peptides and phage-derived motif
Two well-characterized peptide-binding motifs for I-Ag7 have been proposed. Reizis et al. (19) derived a motif of nine residues with primary anchor positions at P4, P6 and P9. At P4 the aliphatic residue L was favored, whereas aromatic Y and small A and G residues were accepted. At P6 a strong preference for the small residues A and T was observed, and at P9 the negatively charged E and D were favored. Secondary anchor positions were identified at P1 and P7. In contrast, Harrison et al. (21) derived a peptide-binding motif for I-Ag7 composed of two primary anchors at P6 and P9 and two secondary anchors at P3 and P8. At P6 large hydrophobic (L, V, I and M) and at P9 positively charged (R and K) or aromatic (Y and F) residues were preferred. At P3 and P8 specific amino acids were not tolerated.
We analyzed the 73 I-Ag7-binding peptide sequences selected by phage display (Table 1
) to identify which of the two motifs was most suitable for peptide alignment. We first inspected the peptide sequences for the charged residues observed at P9 according to Reizis et al. (19) or Harrison et al. (21). Among the phage-derived peptides, 45% contained E or D and 79% R or K. Therefore, less than half of the I-Ag7-binding peptides identified by the phage display library have in their sequence a negatively charged residue as proposed by Reizis et al. (19), whereas about 80% have a charged residues as proposed by Harrison et al. (21). We then analyzed the peptide sequences by considering tolerated residues at primary anchor positions (Fig. 3
). Reizis et al. (19) proposed that three primary anchors at P4, P6 and P9 are important for peptide binding, but when the three anchors were combined only one of 73 peptides contained tolerated residues at all three positions. Since there is consensus that P9 is a critical anchor (19,21), for the purpose of comparison two possibilities for primary anchor positions were considered, either P4 and P9 or P6 and P9. Both possibilities were compared to the primary anchors, P6 and P9, suggested by Harrison et al. (21). The number of peptides identified by the phage display library with tolerated residues at P4 and P9 (10%) or P6 and P9 (12%) according to the Reizis motif was much lower compared to the Harrison motif (77%) (Fig. 3
). Therefore, we aligned the I-Ag7-binding peptides eluted from the phage display library according to the latter motif, i.e. at least one well-tolerated and one weakly-tolerated residue at P6 and P9 (21). According to these criteria, we could align 56 of 73 (77%) phage-derived I-Ag7-binding peptides (Table 2
). This alignment revealed a position-specific enrichment of specific amino acid residues (Fig. 4
). At P6 an enrichment of hydrophobic residues (V, P and L) was apparent, whereas at P9 positively charged (R and K), aromatic (Y) or hydrophobic (L) residues were enriched. At these positions residues not tolerated according to the Harrison motif were absent (Fig. 4
). At P8, G, a well-tolerated residue at this position according to Harrison et al. (21), was also enriched. Furthermore, we identified an additional position, P7, at which small residues (A and G) were more frequent.

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Fig. 3. Presence of anchor residues in I-Ag7-binding peptides. Percentage of phage-derived I-Ag7-binding peptides which contain residues tolerated at anchor positions according to the motifs described by Reizis (19) or Harrison (21).
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Fig. 4. Position-specific enrichment of particular amino acid residues within phage-derived I-Ag7-binding peptides. The percentages of individual amino acids at different positions (P1P9) were determined after the alignment described in Table 2 . Residues are grouped as aromatic (Y, W and F), hydrophobic (L, M, I, V and P), positively charged (R and K), hydrophilic (H, Q, N and C), negatively charged (D and E), OH-containing (S and T) and small (A and G). Residues with a frequency higher than 8% (upper line) were classified as well-tolerated and residues between 5 (lower line) and 8% as weakly-tolerated (see Table 3 ).
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Presence of the phage-derived motif in I-Ag7 binding peptides from IDDM-associated antigens
To validate the phage-derived binding motif, we tested overlapping peptides encompassing the sequences of candidate antigens in IDDM, Mycobacterium tuberculosis (MT)-hsp 65 kDa (amino acids 1540), the extracytoplasmic domain of human putative tyrosine phosphatase IA-2 (hIA-2) (amino acids 601979) and human glutamic acid decarboxylase (hGAD) 65 kDa (amino acids 1585) (28), for binding to I-Ag7. The peptide sequences of the IDDM-associated antigens that bind to I-Ag7 with high affinity (IC50 < 1 µM) are shown in Table 4
. Based on the frequency matrix obtained after alignment of the peptides eluted from I-Ag7 (Fig. 4
), we determined the threshold frequency of specific residues at P6 and P9 anchor positions which would result in the selective presence of the motif among I-Ag7-binding peptides (Fig. 5
). A single residue was classified as well tolerated or weakly tolerated according to its frequency. Using the threshold well tolerated >8% and weakly tolerated 58% we observed an increase in the frequency of the I-Ag7-binding peptides from IDDM-associated antigens possessing the motif, whereas this frequency remained low among low-affinity and non-binders (Fig. 5
). Therefore, this threshold was used to define a phage-derived peptide binding motif for I-Ag7 (Table 3
). The phage-derived motif was present in 90% of high-affinity binders (IC50 < 1 µM) from Mt-hsp 65 kDa, in 73% from hIA-2, in 67% from hGAD 65 and in 87% from mGAD 65 (Table 4
). Conversely, the motif was present in 24% of peptides from Mt-hsp 65 kDa, in 25% from hIA-2 and in 16% from hGAD 65 that failed to bind (IC50 > 10 µM) or bound with low affinity (IC50 110 µM) to I-Ag7 (Fig. 5
). In addition, mGAD 65 peptides binding with high affinity to I-Ag7 (247261, 0.63 µM; 509523, 0.54 µM; 524538 0.6 µM), as well as mGAD 65 peptides reported elsewhere to bind to I-Ag7 (29), were examined for the presence of a peptide-binding motif (Table 4
). A comparison of phage-derived, Harrison and Reizis motifs with respect to their capacity to predict peptide binding to I-Ag7 is included in Table 4
. The phage-derived and Harrison motifs are almost equally efficient in predicting peptide binding to I-Ag7, 80 and 90% respectively. In contrast, the motif defined by Reizis et al. (19), even considering only the two primary anchors at P6 and P9, predicts only ~50% of peptides binding with high affinity to I-Ag7.
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Table 4. Presence of the phage-derived compared to other motifs in high-affinity I-A97-binding peptides from IDDM-associated antigens
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Fig. 5. Threshold frequency of particular residues at P6 and P9 anchor positions according to the phage-derived motif. I-Ag7 high-affinity (IC50 < 1 µM), low-affinity (IC50 110 µM) or non-binding (IC50 > 10 µM) peptides from the IDDM candidate antigens Mt-hsp 65 kDa, hIA-2 and hGAD 65 were inspected to identify the presence of the phage-derived motif.
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Discussion
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This study aimed to refine and validate the motif for peptide binding to I-Ag7, the MHC class II molecule required for IDDM development in NOD mice. An unbiased repertoire of I-Ag7-binding peptides was identified by sequencing phage-encoded peptides eluted from I-Ag7 molecules after four rounds of enrichment of a large M13 phage display library encoding random nonamer peptides. We estimated our library to be a mixture of fusion phage displaying ~2x107 different nonamers, a number that should be representative of the universe of all possible class II-binding peptides (30). Peptides selected by phage display library screening would thus represent the spectrum of peptides binding to I-Ag7 from an unbiased peptide repertoire. The phage-derived binding motif was characterized by an enrichment of hydrophobic amino acids (V and P) and positively charged residues (K) at P6 and of positively charged (R and K), aromatic (Y) or hydrophobic (L) residues at P9. In addition, the frequency of specific residues was also increased at other positions. If only the well-tolerated residues are considered, the phage-derived motif is rather stringent, although multiple residues are allowed at different positions. Only V at P6 and R at P9 showed a frequency >25%, in contrast with stronger amino acid preferences exhibited by phage-derived peptides binding to DR1, DR4 or DR11 molecules (31). This may be due to the dominant P1 anchor of peptides binding to DR (32), which is absent in DQ molecules (33). DQ-like molecules such as I-A also appear to require overall less dominant anchors, allowing for higher residue flexibility.
At least four different I-Ag7 binding motifs have been proposed (1719,21) which differ in the number and specificity of putative anchor residues (see Table 5
). Reich et al. (17) sequenced naturally processed self-peptides eluted from I-Ag7 and found an acidic residue at the C-terminal region of several I-Ag7-binding peptides. The importance of a negatively charged residue near the C-terminus of I-Ag7-bound peptides was also proposed by Reizis et al. (19). In addition to this main anchor at P9, they proposed primary anchor residues at P4 and P6. Conversely, Amor et al. (18) suggested a binding motif shared by encephalitogenic peptides in the Biozzi AB/H mice, which also express I-Ag7 (6). This motif contained basic or positively charged (H, R or K) and hydrophobic (I or L), residues within a 67 amino acid core. We previously described a binding motif for I-Ag7, defined by analyzing the binding of single-residue-substituted analogues of a high-affinity binding HEL peptide to purified I-Ag7 molecules (21). This motif is characterized by two primary anchors at P6 and P9 and two secondary anchors at P3 and P8. At P6 large hydrophobic (V, L, I and M) and at P9 positively charged (R and K) or aromatic (Y and F) residues were required for binding.
A comparison of the different I-Ag7-binding motifs proposed reveals that at P6 small residues (G and A) are tolerated according to Amor et al. (18); S, N, T, V and A according to Reizis et al. (19) and large hydrophobic residues (V, L, I and M) according to Harrison et al. (21) (Table 5
). The phage-derived motif shows an enrichment of hydrophobic (V, P, L and I) and positively charged (K and R) amino acid residues at this position, consistent with the Harrison motif. The characteristics of anchor residues at P9 are even more controversial, because residues with opposite charges have been proposed. Negatively charged residues (E and D) were observed by Reich et al. (17) and by Reizis et al. (19), whereas positively charged (R and K) or aromatic (Y and F) residues were found by Harrison et al. (21). The phage-derived motif, characterized by an enrichment of positively charged, primarily R, and aromatic (Y) amino acid residues at P9, is similar to that described by Harrison (Table 5
). In particular, among the phage-derived peptides, 45% containedin any positionD or E and 79% R or K. Thus, a key residue in the Reisiz motif is present in <50% of phage peptides binding to I-Ag7. When two primary anchors are considered, including also the weakly tolerated residues, only 1012% of phage-derived peptides possess the motif proposed by Reisiz but 77% contain the motif proposed by Harrison. The similarity between the phage-derived and the Harrison motifs extends to residues found at secondary anchor positions, P3 and P8. We also identified an additional position, P7, at which small residues (A and G) were more frequent. This position was also proposed by Reizis et al. (19) as a secondary anchor position, but the residues they found to be enriched at P7 include neither A nor G. We have recently defined a binding motif for I-Eg7, the MHC class II molecule which protects NOD-E
transgenic mice from IDDM (27). This motif is characterized by a negatively charged anchor residue at P4. As described for several I-A and I-E alleles (34), I-Ag7 and I-Eg7 molecules also bind different peptide repertoires (27), further arguing against a peptide-binding motif for I-Ag7 which includes negatively charged residues.
The discrepancies over anchor positions could reflect differences in the assays or conditions used to define motifs for I-Ag7-binding peptides. Reich et al. (17) analyzed peptides eluted from I-Ag7 molecules. Motifs based on naturally processed peptides will not only reflect peptide-binding specificity, but also the processing of the protein (35). In addition, definition of specific motifs by alignment of eluted peptides can be difficult because of peptide size heterogeneity (36,37). Amor et al. (18) defined their motif from the sequences of only a few encephalitogenic peptides, which are not necessarily high-affinity binders (38). Reizis et al. (19) measured peptide binding to I-Ag7 by competitive inhibition of T cell proliferation to a reference peptide presented by fixed antigen-presenting cells, but this assay may be less sensitive and does not allow a precise quantitative assessment of peptideMHC interactions. Harrison et al. (21) defined a motif for peptide binding to I-Ag7 by truncation and substitution analysis of a high-affinity binding peptide. Possibly, the motif determined by this approach could have been constrained by effects of non-anchor residues.
The different pH values at which peptide binding was measured could also have biased the results. Reizis et al. (19) measured peptide binding to fixed antigen-presenting cells at pH 5.2, phage-derived peptides were routinely measured at pH 6 (present results) and Harrison et al. (21) used pH 7 in their competition assays for peptide binding to I-Ag7. In our binding assays to purified I-Ag7 no binding was seen at pH 5, whereas similar binding affinities were measured at pH 6 and 7 (S. Gregori, unpublished). Peptide binding at pH 6 or 7 is likely to be more representative of physiological peptide binding conditions at the cell surface. Interestingly, complexes of I-Ag7 and CLIP formed at a neutral pH but rapidly dissociated at pH 5 (15). The lower pH used by Reizis et al. (19) would favor binding of acidic residues (D and E) at P9 by protonating Hs at ß9 and ß56, which together with ß57S,
80R and ß55R form pocket 9 in I-Ag7 (19). The Harrison motif (21) was the most compatible with peptides eluted from the phage display library, a relatively unbiased approach with which to establish general rules for peptide binding to MHC molecules (39). In addition, the phage display-derived predicts three of five peptides and the Harrison motif four of five peptides eluted by Reich et al. from I-Ag7 molecules (17).
Specific allelic forms of the class II MHC molecule are the most important genetic elements associated with IDDM and the D/non-D polymorphism of ß57 has been suggested to play a key role in determining resistance or susceptibility respectively (9,40). The IDDM-associated class II MHC molecules, HLA-DQ2 and HLA-DQ8 in humans, RT1.B1 in the BB rat, and I-Ag7 in the NOD mouse, share a unique ß chain characterized by a non-D residue at position 57, usually S or A (9). The S residue at ß57 was predicted by molecular modeling to be involved in forming P9 in the binding groove of the I-Ag7 molecule and to interact with the side chain of the residue at P9 in the binding peptide (19). The polymorphism at ß57 is suggested to affect peptide binding specificity (41) and peptides binding to DQ molecules that carry a non-D residue at position ß57 show a specific preference for negatively charged residues at P9 (13,42,43). In contrast, HLA-DQ2 molecules, which are also associated with IDDM and share a non-D residue at position ß57, prefer large hydrophobic residues at P9 with no particular preference for binding peptides with negatively charged side chains at this position (44), although a positively charged residue at P9 was found to be deleterious for binding (45). Thus, the presence of a non-D residue at position ß57 does not necessarily predict the characteristics of anchor residues at P9. Indeed, molecular modeling of I-Ag7peptide interaction was consistent with a peptide motif characterized by a hydrophobic residue at P6 and an aromatic or positively charged residue at P9 (46,47).
In conclusion, using a phage display library we have validated a peptide-binding motif for I-Ag7 molecules characterized by hydrophobic residues at P6 and positively charged, aromatic or hydrophobic residues at P9. This is superior to the Reizis motif in predicting I-Ag7-binding peptides. Its capacity to identify potential epitopes involved in IDDM is indicated by the high sensitivity (6790%) and specificity (7584%) for high-affinity I-Ag7-binding peptides derived from the IDDM candidate antigens Mt-hsp 65 kDa, hIA-2 and GAD 65.
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Abbreviations
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DOC sodium deoxycholate |
HEL hen egg lysozyme |
hGAD 65 human glutamic acid decarboxylase 65 kDa |
hIA-2 human putative tyrosine phosphatase IA-2 |
Hsp heat shock protein |
IDDM insulin-dependent diabetes mellitus |
Mt-hsp 65 kDa Mycobacterium tuberculosis hsp 65 kDa |
NOD non-obese diabetic |
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Notes
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Transmitting editor: G. Doria
Received 3 November 1999,
accepted 27 December 1999.
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