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In APCs, the Autologous Peptides Selected by the Diabetogenic I-A^g7 Molecule Are Unique and Determined by the Amino Acid Changes in the P9 Pocket¹

Anish Suri^*, Ilan Vidavsky, Koen van der Drift, Osami Kanagawa^*, Michael L. Gross and Emil R. Unanue^2,*

^* Department of Pathology and Immunology, and Center for Immunology, Washington University School of Medicine, St. Louis, MO 63110; and Department of Chemistry, Washington University, St. Louis, MO 63130

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A^g7 molecule, the P9 pocket of I-A^g7 plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A^g7 molecules or a mutant I-A^g7 molecule in which the 57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A^g7-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A^g7 were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A^g7 molecule. These peptides did not favor acidic residues toward the carboxyl terminus.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The role that histocompatibility molecules play in peptide selection is crucial to an understanding of T cell-mediated immune response. Peptide motifs that favor binding to a particular MHC genotype have been identified based on a variety of peptide libraries (1, 2), although the issue remains: how specific is the interaction and how broad or narrow is the repertoire of the major peptides that are selected from intracellular processing? The only direct approach to settle the issue of the degree of specificity in peptide selection is to examine chemically the peptides displayed by an MHC molecule of a given MHC genotype. From the early pioneering work of Rammensee and colleagues (3, 4), peptides isolated from class I MHC have shown some degree of sequence specificity for a given genotype (5, 6, 7, 8), although this preference has not been definitive regarding class II-associated peptides (9, 10, 11, 12). In part the problem with class II peptides is based on their size heterogeneity, which makes it very difficult to predict the residues responsible for selection. Regardless of the difficulties, this role of MHC in the specificity of peptide selection needs to be critically evaluated because it has important implications for immunogenicity and in particular in the context of organ-specific autoimmunity. Indeed, peptide selection should ultimately render certain individuals susceptible to disease. What are biochemical parameters that influence the peptides selected by a MHC molecule? And more importantly, how influential are the allele-specific sites of the peptide-binding groove in determining the nature of peptides that are selected? To answer these questions, we have chosen to investigate the peptides displayed by the diabetogenic class II MHC molecule I-A^g7.

Insulin-dependent diabetes mellitus is an organ-specific autoimmune disorder in which the T cells recognize and destroy the pancreatic islet cells (13). Both in the nonobese diabetic (NOD)³ mice (14) and in humans, diabetes is linked to the expression of certain disease-susceptible class II MHC alleles (15, 16, 17). The class II MHC molecule of NOD mice, I-A^g7, shares key biochemical features with the human class II MHC alleles, DQ2 and DQ8, known to be involved in clinical disease (16). In particular, I-A^g7 and the disease-susceptible human alleles express a non-Asp amino acid at position 57 of the -chain (I-A^g7 contains a Ser residue at 57), whereas most other MHC alleles contain a conserved Asp residue at this position (18, 19, 20). In case of I-A^g7, the critical role of 57Ser residue in disease susceptibility is highlighted by the following findings: 1) transgenic NOD mice expressing a modified I-A^g7, wherein the 57Ser has been changed to an Asp residue, are protected from diabetes (21, 22); 2) the expression of I-A^g7 in NOD mice or in H-2^g7 congenic B6 mice allows for a high incidence of self-reactive T cells in the periphery (23, 24); and 3) APCs expressing a modified I-A^g7 molecule, containing an Asp at 57 instead of the usual Ser residue, are unable to activate diabetogenic CD4⁺ T cells (25). Moreover, other studies analyzing the T cell response against the autoantigen glutamic acid decarboxylase have indirectly suggested that wild-type I-A^g7 and modified I-A^g7, expressing an Asp at 57, may select for distinct epitopes (26).

Although there has been much discussion concerning the composition of peptides that prefer to interact with I-A^g7 (26, 27, 28, 29, 30, 31, 32, 33, 34, 35), the recent determination of the crystal structure of I-A^g7 clarified much by identifying peptide anchor residues that are favored at the binding sites of the MHC molecule (36, 37). When 57 is an Asp, an ion pair is established with Arg⁷⁶ of the -chain, and this ion pair promotes both the dimerization of the two chains and the structure of the P9 binding site (38, 39). Because this ion pair interaction is absent in I-A^g7, the P9 pocket is shallow and more open toward the C terminus of the peptide, and the unpaired 76Arg is available to interact with peptide residues (36, 37). Similar structural characteristics were also observed in a recent report by Wiley and colleagues (40) describing the crystal structure of the diabetes-susceptible human class II MHC, HLA-DQ8. However, a fundamental issue that remains unknown is the role of the P9 pocket, containing the 57Ser, in determining peptide selection by I-A^g7-expressing APCs. However, for many peptides the contribution of the P9 site to binding is not major (37), so the issue needs to be directly examined and tested experimentally.

We have chosen to analyze the peptides that are endogenously selected by APCs expressing either wild-type I-A^g7or a modified I-A^g7, wherein the critical 57Ser has been changed to an Asp (as well as the 56His to Pro; we term it I-A^g7PD). The two prior publications reporting the crystal structure of I-A^g7 showed that the 56His does not interact directly with the peptide (36, 37), while the 57Ser forms a hydrogen bonding network with the 76Arg involving an acidic P9 peptide anchor residue (36). Nonetheless, both 57Ser and 56His modify the conformation and position of the H1 helix, which contributes to the generation of a P9 pocket that is wider and open toward the C terminus (36, 37). Due to this, we replaced both 56His and 57Ser to the conserved Pro and Asp residues, respectively.

In our studies, the two APC lines are identical, except for the class II MHC molecule, which allows us to test whether the changes in the P9 pocket determine the nature of the selected peptides. We find that this is indeed the case. More importantly, because the selected peptides are the natural ones favored by I-A^g7, it enables the sequence motifs to be definitively established, information that should help in the search for diabetogenic peptides from pancreatic islet cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cell lines and Abs

The murine B cell lymphoma line M12.C3 was transfected with plasmids expressing I-A^g7 or I-A^g7PD to generate C3.G7 (expressing wild-type I-A^g7) or C3.G7PD (expressing I-A^g7PD) cell lines as previously described (27, 37, 41). Fusing the M12.C3 B cell lymphoma to splenic B cells expressing I-A^g7 generated another I-A^g7-bearing APC line, termed M12.G7. All of these different APC lines were cultured in DMEM supplemented with 10% calf serum. mAbs AG2.42.7 and AG2.27.5, which recognize I-A^g7 and I-A^g7PD respectively, were generated by M. B. Deck in our laboratory.

Purification of naturally processed peptides

C3.G7, M12.G7, or C3.G7PD lymphoma cells (2–8 x 10⁹) were lysed in PBS containing 40 mM MEGA 8 and MEGA 9 detergents, in the presence of protease inhibitors (1 mM PMSF, 10 mM iodoacetamide, and 40 µM leupeptin). After 1 h at 4°C, the lysate was spun in a centrifuge at 8000 rpm for 30 min following which class II MHC molecules were purified using AG2.42.7 mAb (for I-A^g7) or AG2.27.5 mAb (for I-A^g7PD) coupled to cyanogen bromide-activated Sepharose beads (Sigma-Aldrich, St. Louis, MO). Sepharose beads were loaded into disposable chromatography columns (Bio-Rad, Hercules, CA) and washed first with 40 ml of wash buffer-1 (10 mM MEGA 8 and MEGA 9 in PBS) and 40 ml of wash buffer-2 (2.5 mM MEGA 8 and MEGA 9 in PBS), and then extensively with PBS (100 ml) and distilled water (100 ml). I-A^g7- or I-A^g7PD-peptide complexes were eluted with 15 ml of 0.1% trifluoroacetic acid (pH 1.9). The eluted peptides were separated from MHC molecules using centriprep YM-10 (Millipore, San Jose, CA) (molecular mass cut-off of 10 kDa) membrane separation. Peptide extracts were then analyzed by tandem mass spectrometry (MS/MS) to identify the amino acid sequences of the naturally processed peptides isolated from I-A^g7 and I-A^g7PD.

Reversed phase-HPLC and MS/MS analysis

Peptide fractions were analyzed by reversed phase-HPLC on a Zorbax C18 0.3 mm x 15 cm column (MicroTech, Sunnyvale, CA). Solvent A was 0.6% acetic acid in water; solvent B was 0.6% acetic acid in acetonitrile. Solvent B started at 3–15% in 5 min and from 15 to 60% at 0.8%/min. Eluent flow was 4.5 µl/min and 15 µl was injected using Rheodyne 7125 injector (Rheodyne, San Francisco, CA). HPLC pump was MicroTech Ultra plus. The flow from the pump was split before the injector at a 1:25 ratio using a homemade splitter. All of the column effluent was directed at the mass spectrometer.

Mass spectrometry (MS) and MS/MS were performed on a Finnigan LCQ-deca ion trap mass spectrometer with Xcalibur 1.1 software (Thermo Finnigan, San Jose, CA). For MS, scan range was m/z 700-1400 in profile mode. Every three micro scans were averaged to one scan. Acquisition was started 10 min after start of the liquid chromatography run. For MS/MS in dependent scan mode, parent ions were selected excluding isotopic peaks dynamically. Scan range was m/z 700-1400 in centroid mode for the MS part; in the MS/MS, scan range was from 30% of the parent to m/z 2000. Every three micro scans were averaged to one scan in profile mode. Parent ions were isolated with a 2.5 m/z wide window and collision energy was 28% of the maximum energy. Parent ions for MS/MS were selected automatically according to intensity by using a dependent scan mode. Multiple MS/MS runs were made on each peptide extract isolating first and second, and third and fourth most intense ions (in some cases even fifth and sixth, seventh and eighth).

MS/MS spectra were analyzed and peptide sequences were determined automatically using SEQUEST software provided by the instrument manufacturer (Thermo Finnigan, San Jose, CA) and MASCOT (Matrix Science, London, U.K.). All the automatically determined sequences were verified manually against the experimental MS/MS.

Peptides sequenced by the MS/MS run were detected in the MS run using the mass and retention time. Selected ion chromatograms were generated for each one. Relative amounts for each peptide were estimated by measuring the area under a chromatographic peak. The peptide with the largest area was assigned a value of 1 (maximum) and all other peptides were measured relative to this value. For peptides with multiple family members, the relative abundance values were added and were represented as a total for each family. No use of calibrating standards was made so the relative chromatographic area may differ from the actual relative amount due to different response factors. This difference is estimated to be less than an order of magnitude in our experience.

Peptide binding assays

Soluble I-A^g7 and I-A^g7PD were produced using the recombinant baculovirus system as previously described (37). Peptide binding assays were done under acidic (pH 5.5) or neutral (pH 7.5) conditions. Briefly, 0.5–1 µg of I-A^g7/class II-associated invariant chain peptide or I-A^g7PD/class II-associated invariant chain peptide was treated with 0.1 U of thrombin to cleave both the zipper tails and peptide linker (Novagen, Madison, WI) and simultaneously incubated with 0.125 pmol of I¹²⁵-radiolabeled reference (GKKVATTVHAGYG) peptide (labeled by the chloramine T method as described previously (42)) and increasing doses of unlabeled peptides in 40 mM MES and 150 mM sodium chloride (pH 5.5) or in 20 mM HEPES and 150 mM sodium chloride (pH 7.5). Binding reactions were incubated overnight at 25°C in 30- to 32-µl volumes. Complexes were purified from free peptide by gel filtration Bio-spin columns (Bio-Rad). The percentage of bound peptide was evaluated by gamma counting. Less than 0.5% of peptides nonspecifically passed through the Bio-spin columns. Individual binding results varied <20% from the averaged value.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Peptides extracted from I-A^g7 and I-A^g7PD from M12-derived APC lines

Table I indicates the endogenously processed peptides isolated from I-A^g7or I-A^g7PD of M12.C3 lines expressing one or the other class II MHC molecule (referred to as C3.G7 or C3.G7PD, respectively). Isolated peptides were identified by electrospray ionization MS and sequenced by MS/MS analysis. In brief, peptide ions for MS/MS were selected according to their intensity, and each peptide extract was analyzed by multiple MS/MS runs that automatically selected and identified first and second, third and fourth most intense ions (in some cases fifth and sixth, seventh and eighth most intense ions). Peptides varied in length from 13 to 30 residues, but most were from 14 to 17 residues. The composition of peptides was very different in the two lines.

The same general features were found for the peptides isolated from I-A^g7PD molecules. In this work we examined 43 different peptides represented in 24 distinct families. These peptides had the same large gradient of relative abundance, 350-fold. In binding analysis 12 peptides were tested, of which 10 bound to I-A^g7PD while two did not bind (the same two that were also found among peptides from I-A^g7).

Among the peptides isolated from I-A^g7 or I-A^g7PD, there were no distinguishing features in the amino or carboxyl termini residues, or in the residue preceding the amino terminal residue, to suggest a particular preference or motif for proteolysis.

Most importantly, all peptides isolated from I-A^g7, except one, contained acidic residues at the carboxyl terminus (19 of 20 or 95% of all peptides contained an Asp or Glu among the last six C-terminal residues of the peptide). Fig. 1 shows the distribution of acidic residues among all peptides, regardless of their distribution in families. It indicates the striking differences between the I-A^g7- and I-A^g7PD-extracted peptides. Very strikingly, 14 of the 20 peptide families (70%), isolated from I-A^g7, contained more than one acidic residue, some in pairs, others separated by one or two amino acids. This was not the finding among peptides isolated from I-A^g7PD. In this case, none of the peptides had pairs of acidic residues.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Distribution of acidic amino acids along the C termini of peptides isolated from I-Ag7 or I-Ag7PD. I-Ag7-associated peptides were isolated from C3.G7 and M12.G7 APC lines, whereas I-Ag7PD-associated peptides were isolated from the C3.G7PD APC line. All peptides are included in the analyses. The last 10 C termini residues of each peptide were scored for the presence of either Glu or Asp at each position. The bar graphs depict the presence of acidic residues, shown as percent positive, for each position starting at the most C-terminal residue. The most C-terminal residue was assigned position 10; similarly, the second-most C-terminal residue was assigned position 9 and so on. As shown, peptides isolated from I-Ag7 exhibit the biased presence of acidic amino acids among the C-terminal residues when compared with peptides isolated from I-Ag7PD (especially at positions 10, 9, 8, 7 and 6).

We also examined 47 I-A^g7-bound peptides from a different cell line resulting from the fusion of the M12.C3 line with splenic B cells expressing I-A^g7 (referred to as M12.G7, Table I). The same results were found regarding the sequence of peptides that were favored. All of the peptide families selected by I-A^g7 contained acidic residues at their C termini (17 of 17 or 100%) (Fig. 1). In addition, like the peptides from C3.G7, 12 of 17 peptides (71%) from M12.G7 cells also contained more than one acidic residue, some in pairs, others separated by one or two amino acids.

Binding features of the peptides

The binding properties of some of the peptides are shown in Table III. The peptides extracted from I-A^g7 showed much preference to bind at pH 5.5 rather than at pH 7.5, an indication that protonation of the acidic residues was important in the binding. These I-A^g7-extracted peptides bound also to I-A^g7PD but with lesser strength; it was striking that their binding to I-A^g7PD was practically abolished at pH 7.5. Peptides that were isolated from I-A^g7PD had less preference for acidic pH in binding to I-A^g7PD as compared with those extracted from I-A^g7. The peptides extracted from I-A^g7PD also preferred to bind to it rather than to I-A^g7. The preference for binding peptide at acidic pH indicates the importance of processing in late vesicles that are acidic. The extent of physiological interactions at neutral pH on the cell surface is not known.

HEL 11–25. In the original HEL11–25 peptide, structural analysis documented that Gly²² was the amino acid at the P9 position. Others (31) speculated about a different motif but the immunological and structural analysis performed by us (37) indicated only one register, as shown in Fig. 2. The differences cannot be accounted for by technical variations related to pH (37). Single replacements of Gly with either Asp or Glu resulted in a modest increase in binding strength: from 9 to 5 and 3 µM, respectively (Fig. 2A). A sequence of three acidic residues as found in the dominant E2B112–126 peptide (Tables II and III) was replaced for residues 22 to 24 in the HEL11–25 peptide. Placing the Glu-Glu-Asp sequence for the natural Gly-Tyr-Ser resulted in a marked increase in binding, from 9 to 0.9 µM. Making a "Lys map" indicated the preferential use of the Glu²²: it was the only position where replacing with a Lys resulted in marked inhibition. We concluded that the preferred register is the one having the first Glu as P9. As indicated in the natural sequence, this register results in the Arg¹⁴, a preferred basic residue, at P1.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Dominant binding registers of peptides isolated from I-Ag7. Lys-scan analysis of peptides isolated from I-Ag7 to determine the dominant binding register as well as the preferred P9 acidic residue. First shown is the binding analysis of HEL11–25 peptide and some of its variant peptides. This experiment was done four times with identical results with <10% variation from the mean. The site for amino acid change is indicated by a square showing the one-letter code for the mutated residue. The dominant binding register for each peptide is underlined and the core amino acid residues forming this register are shown in bold. All binding analyses were conducted at pH 5.5 as described in Materials and Methods.

BSA 304–319. Fig. 2B shows three conceivable binding registers for this peptide. The amino acid change is indicated by a square showing the one-letter code for the mutated residue. In bold and underlined is the peptide sequence of the favored dominantbinding register, in this case "register 2." The substitution of Glu³¹⁵ for Lys had a minor effect while Asp³¹⁹ to Lys resulted in a more pronounced loss of binding. However, changing Glu³¹⁷ to a Lys had the most profound negative effect on binding, suggesting that the preferred binding register is the second one, the register using Glu³¹⁷ as P9. This was further ascertained by changing the Ala³¹⁴ to a Lys; we knew that basic residues are also extremely hindering at the P6 position (37). Indeed, the peptide with an Ala³¹⁴ to a Lys failed to bind to I-A^g7. Therefore, this last mutant peptide confirms that the dominant binding register is register 2, which uses Glu³¹⁷ as the P9 anchor residue and a Lys at P1, which is the favored residue at this site (37). Also important to note is that replacing Glu³¹⁷ (P9) to an Ala resulted in a >10-fold loss of binding which suggested that, in this peptide, the acidic P9 anchor residue positively contributed to peptide binding to I-A^g7. Surprisingly, changing the Asp³¹⁹ (P11) to a Lys also negatively affected binding. This observation was similar to our previous results with the HEL11–25 peptide wherein basic residues at P10 and P11 also resulted in a loss of binding (Fig. 2A). To dissect the cooperative role of acidic residues in peptide binding, we changed Glu³¹⁵, Glu³¹⁷, and/or Asp³¹⁹ to Ala. As shown in Fig. 2, changing Glu³¹⁵ or Asp³¹⁹ to an Ala did not affect binding much. However, changing either or both of these acidic residues along with the P9 Glu³¹⁷ to an Ala resulted in a marked loss of binding.

LAMP213–227. The Lys replacement at Glu²²³ had some effect on binding, but that of Asp²²⁴ was considerably inhibitory. As shown, in binding register 2, Asp²²⁴ at P9 would allow for a Lys to be used as P1. Binding register 1, using Glu²²³ as P9, would result in a Met at P4, a residue that is not favored. To confirm that the preferred binding register 2 indeed uses Asp²²⁴ as the P9 anchor, we changed the putative P6 Ala²²¹ to a hindering Lys residue. This mutation resulted in a >20-fold loss of binding. Unlike the BSA 304–319 peptide, changing the P9 Asp²²⁴ to an Ala had a minimal effect on binding. Changing either the Glu²²³ or Asp²²⁴ alone to an Ala also had a minimal effect on binding; however, changing both of them to Ala resulted in a 4-fold loss of binding.

FK-506 binding protein 51–65. Although substitutions of both acidics, Glu⁶¹ and Glu⁶², had some effect, the major effect was found with the substitution of Glu⁶² to a Lys. Binding register 2, using Glu⁶² at P9, is preferred because Glu⁶¹ at P9 would have placed Arg⁵⁸ as P6; basic residues at P6 profoundly inhibit binding. Again, changing the Gly⁵⁹ to a Lys generated a non-binder peptide, which supported the fact that Glu⁶² is the P9 anchor and Gly⁵⁹ is P6 for this peptide. Finally, substituting either the P9 Glu⁶² or the P8 Glu⁶¹ to an Ala did not affect binding, but changing both of these together to Ala allowed for a >6-fold loss of binding (Fig. 2C).

E2B112–126. Binding register 2 that uses Glu¹²⁴ as P9 was clearly preferred; that is, it was the one where the substitution to a Lys had a 30-fold decrease in binding. Note that binding registers 1 and 3 are probably not preferred because of the hindering effect of Lys at either P4 or P6. This was proven by the second series of mutant peptides where a Lys was placed at the Ile at residues 119 or 121, corresponding with the P4 or P6 sites in register 2; in this case there was marked inhibition of binding. As previously seen with most other peptides, replacing the P9 Glu¹²⁴ to an Ala had no effect on binding. Similarly, changing the P8 Glu¹²³ or the P10 Asp¹²⁵ to Ala did not affect binding. However, changing Glu¹²³, Glu¹²⁴, and Asp¹²⁵ (P8, P9, and P10, respectively) to Ala, as shown in Fig. 2, resulted in a >5-fold loss of binding.

In summary, these five examples justify the use of the Lys scan in pointing to the most favored registers used by the selected peptides. Multiple acidic residues at positions P8, P10, and/or P11 may cooperate with acidic P9 anchor residues to provide a binding advantage, which may in turn be a decisive biochemical parameter for peptide selection by I-A^g7.

Comments

We conclude that the P9 pocket of I-A^g7 is central in the selection of peptides, favoring sequences that contain acidic residues that interact at that site. We commented that some of the peptides contain more than one acidic residue; the examination of mutant peptides in which we placed hindering basic amino acids allowed us to map with certainty the residues that interact at P9. We conclude that either one of the acidic residues is preferably selected or both acidics cooperate, presumably in the interaction with 76Arg, as envisioned in the recent report by Corper et al. (36). In the other publication on I-A^g7-extracted peptides, two peptides were identified that contained C-terminal acidic residues, only one of which was studied in binding (34). We conclude that I-A^g7 molecules exhibit noted selectivity for peptides, as do other class II molecules, such as I-A^k, the other allele that we have studied (42, 43, 44, 45). Thus we do not find promiscuity or degeneracy in peptide selection as would be deduced from our previous papers (27, 28) and in a recent report by Stratmann et al. (46). Indeed, there were marked ranges in the amounts of peptides but an overall clear preference for those having a defined composition.

On a related note, two reports isolated and characterized naturally processed peptides selected by HLA-DQ8, the diabetes-susceptible human class II MHC allele (47, 48). Of these two, the first one suggested that peptides bound to DQ8 contained positively charged residues at P1 and Gly or Ala at P6 (47), while the later report indicated that negatively charged residues were preferred at the P1 and P9 positions (48). However, the primary drawbacks of these studies were as follows: 1) very few naturally processed peptides were identified (47, 48), 2) of these few peptides, some never bound to DQ8 and were thus considered artifacts (47), and 3) none of these reports described any detailed binding analysis which might have supported the proposed binding motifs (47, 48). Other binding studies testing HLA-DQ8 pointed toward the role of acidic residues at the P9 position (49).

Two other important conclusions can be made from our analyses. First, although the peptides that were identified did bind back to their respective class II molecules, we did not find a strict correlation between the degree of binding and the extent of selection. Such a relationship between those peptides that had the highest binding strength for I-A^g7 and their amounts displayed may not be expected; the amount displayed may depend upon the expression levels of various self-proteins and their relative accessibility to the class II MHC processing pathway. We do not have any information on the pool of these self-proteins available for class II MHC binding and selection, which makes it impossible to correlate amounts with selection. Using a controlled processing system, that of APCs cultured with HEL and quantifying the peptides displayed by the I-A^k molecules, we also found a several hundredfold difference in the amounts of epitopes displayed. In this case, peptides with the highest affinity tended to be the ones most heavily represented and contained a sequence motif that favored binding (50). Moreover, by examining the data we conclude that the specificity of peptides selected by I-A^g7 or I-A^g7PD was not related to their binding strength to one or the other class II MHC allele. In terms of IC₅₀ values, which correlates with the K_D, the differences between peptides bound to I-A^g7 and their amounts or, more importantly, their binding strength to I-A^g7PD was not sufficient to explain the basis for their selection of one vs the other class II molecule. Take E2B112–126 for one example: it is the highest peptide family represented in I-A^g7 and it binds well to it, but 10 fold less to I-A^g7PD. Yet it was not represented at all among the I-A^g7PD extracts in contrast to other peptides extracted from I-A^g7PD, which had binding affinities about the same as E2B112–126 and which were selected. From a biochemical viewpoint, I-A^g7 peptides may have faster off-rates when bound to I-A^g7PD, which could explain why I-A^g7-associated peptides are never selected by I-A^g7PD. Current ongoing studies in our laboratory are focused toward addressing these very issues.

Second, our results examining the physiological peptides presented by I-A^g7 are definitive in establishing the importance and value of C termini acidic residues that interact with the P9 pocket. Besides this, peptides isolated from I-A^g7 also prefer small to medium hydrophobic amino acids at the P4 and P6 positions (especially Ile or Leu at P4 and Ala, Ile or Leu at P6), which is in consensus with the structural features reported recently by Corper et al. (36) and Latek et al. (37). Numerous reports in the past described peptides associated with I-A^g7; however, many of them were in disagreement regarding the peptide motifs involved in the interaction, making it difficult to reach a consensus (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). In agreement with our finding, many of the epitopes derived from other autoantigens (26, 28, 29, 36, 37), including those implicated in diabetes, such as glutamic acid decarboxylase, insulin, and carboxypeptidase H, contained acidic residues at the P9 position and some even exhibited the presence of multiple acidic residues toward their C termini (26, 29). Whether these are represented among the physiological peptides displayed from islet cells will need to be evaluated.

Although I-A^g7 tends to have weaker interactions with peptides when compared with I-A^k (I-A^g7 in the micromolar range and I-A^k in the nanomolar), the present chemical analyses do not support such a loose interaction as the basis for selection of many autologous peptides and autoimmunity. Since our initial reports (27, 28), the issue was raised that I-A^g7 favored weak binding peptides with little discrimination and that these weak interactions may be the element favoring a large number of autoreactive T cells escaping central negative selection in the thymus. Whether a loose interaction between autologous peptides and I-A^g7 is a factor in autoreactivity remains a possibility that needs reevaluation.

At this point we favor that islet cell-derived peptides will have the features outlined in this study and be optimally selected, this being the primary reason why I-A^g7 predisposes toward the onset of diabetes. In other words, autoantigenic islet cell peptides can be efficiently selected by I-A^g7 but not by I-A^g7PD, and this biochemical property is entirely dependent upon the amino acid composition of the P9 pocket. To support this conclusion, one of the peptides we isolated from I-A^g7 (HSP164–183: TPEEIAQVATISANGDKDIG; Table I) was derived from the murine 60-kDa heat shock protein (HSP)-60, which has been previously implicated as an autoantigen in insulin-dependent diabetes mellitus (51, 52, 53, 54). The HSP164–183 epitope was found only among the naturally processed peptides isolated from I-A^g7 and not among the peptides isolated from I-A^g7PD. Thus, although the expression of HSP-60 is not islet cell specific, the selection of the HSP peptide is unique to I-A^g7 and is indicative of the crucial role of the P9 pocket in peptide selection by I-A^g7.

In summary, we demonstrate in this study that peptide selection from protein processing has an important component of specificity that is dependent on the chemical composition of the allele-specific site of the peptide-binding groove.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health and the Kilo Diabetes and Vascular Research Foundation (St. Louis, MO), and by the Pharmacia-Washington University agreement.

² Address correspondence and reprint requests to Dr. Emil R. Unanue, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: unanue{at}pathbox.wustl.edu

³ Abbreviations used in this paper: NOD, nonobese diabetic; MS, mass spectrometry; MS/MS, tandem MS; HEL, hen egg white lysozyme; HSP, heat shock protein.

Received for publication August 1, 2001. Accepted for publication November 27, 2001.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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