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Polymorphism at Position 97 in MHC Class I Molecules Affects Peptide Specificity, Cell Surface Stability, and Affinity for ₂-Microglobulin¹

Ruth A. Smith^2,*, Nancy B. Myers, Melanie Robinson^*, Ted H. Hansen and David R. Lee^3,*

^* Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212; and Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The two mouse MHC class I alleles, L^d and L^q, share complete amino acid sequence identity except in the 2 domain, where they differ at six positions. Despite their similarity, L^q has a stronger association with ₂-microglobulin (₂m), is expressed at higher levels on the cell surface, demonstrates an increased cell surface half-life, and has fewer open forms on the cell surface than L^d. To determine the basis for their phenotypic differences, L^d molecules containing chimeric L^d-L^q 2 domains were characterized, and these analyses implicated residue 97 (L^dTrp and L^qArg) as the polymorphic site responsible for the disparity in ₂m association between the two alleles. Single substitution analysis at this site (L^dW97R and L^qR97W) confirmed this. Furthermore, the L^dW97R mutant molecule has a longer cell surface half-life than either L^q or L^d, and fewer open forms of L^dW97R are observed on the cell surface. In addition, both L^dW97R and L^q possess decreased binding affinity for the L^d-restricted tum^- P91A_14–22 peptide compared with L^d. Collectively, these results and the known location of Trp⁹⁷ in the peptide binding cleft of L^d strongly suggest that the substitution of Arg for Trp⁹⁷ in L^d alters the peptide binding cleft, increasing its affinity for endogenous peptides, which results in greater cell surface stability and better retention of ₂m. Furthermore, these results imply that Trp⁹⁷ plays an important role in the ability of L^d to efficiently participate in alternative MHC class I Ag presentation pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mouse MHC class I molecule, L^d, has a number of features that distinguish it from most of its other mouse and human counterparts, the most prominent of which is its low cell surface expression (1, 2). Many of the other traits have been cited, at least in part, as the bases for the lower steady state expression of L^d on the cell surface. First, intracellular trafficking studies indicated that L^d assembles and trafficks slowly to the cell surface compared with many other mouse MHC class I molecules (2, 3). Second, the propensity of L^d to either relinquish endogenous mouse ₂-microglobulin (₂m)⁴ or exchange the latter for either bovine or human ₂m suggests that L^d has a low affinity for mouse ₂m (2, 4). The crystallographic structure of L^d indicates that it has considerably fewer contacts with ₂m than do other mouse class I molecules, such as K^b and D^b, for which similar structural analyses have been performed (5, 6, 7, 8, 9). Interestingly, the 1 and 2 domains of L^d have a novel orientation compared with other MHC class I structures, rendering it particularly deficient in the number of residues that interact with ₂m compared with K^b and D^b (5). Analysis of the chimeric D^d-L^d MHC class I molecule, D^dm1 suggested that the amino-terminal half of L^d (the 1 and 2 domains) was mostly responsible for its slow trafficking and assembly and weak ₂m association (2), in agreement with the structural studies cited above. Third, L^d has a shorter half-life on the cell surface (3, 10, 11), and a higher proportion of the total L^d molecules on the cell surface exists as open (mAb 64-3-7⁺) forms (3, 12). These latter findings suggest that peptide ligands associated with cell surface forms of L^d tend to dissociate comparatively quickly to generate transient open forms of L^d. This is the probably the main reason why L^d expression can be increased efficiently by the continuous incubation of cells in exogenous L^d-binding peptides (12, 13). The concept that peptide affinity can affect the cell surface stability of L^d is further supported by the observed differential stability of different L^d-peptide complexes on the cell surface (3, 10). Thus, it is presently unclear which of these traits, poor assembly and trafficking, low affinity for ₂m, or poor affinity for bound peptides and cell surface instability, are principally responsible for the low expression of L^d on the cell surface. Furthermore, the interdependency of these traits to each other is unclear.

L^q is the most similar documented allele of L^d and differs from L^d by only six amino acid residues in the 2 domain (14, 15). Despite this similarity, L^q is expressed at higher levels on the cell surface (16) and displays an increased affinity for ₂m (16) and increased cell surface stability compared with L^d. To ascertain which of the 2 domain polymorphic residues between L^d and L^q determines these phenotypic differences between the two alleles and potentially the basis of those phenotypic differences, L^d constructs containing either chimeric L^d-L^q 2 domains or single-site reciprocal substitutions were made or obtained. The analyses of these chimeric and mutant L^d molecules are presented herein and suggest that Trp⁹⁷ in L^d has a major impact on peptide binding and the resulting association with ₂m and cell surface stability. Thus, this residue may play an important role in the involvement of L^d in alternative MHC class I Ag presentation pathways (17, 18).

	Materials and Methods

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Antibodies

The following mAbs with their given reactivities were used: 28-14-8 (L^d and L^q 3 domain of assembled and unassembled molecules) (12, 14, 19, 20, 21, 22), 30-5-7 (L^d 2 domain of assembled molecules) (12, 19, 21, 23), B22/249 (L^d and L^q 1 domain of assembled molecules) (24, 25), and 64-3-7 (L^d and L^q 1 domain of empty molecules) (12, 26, 27, 28).

Cell lines

The L^d transfectant had been generated previously in our laboratory (29) and contains the intact wild-type L^d gene transfected into DAP-3 cells. The L^q transfectant had been generated previously by transfection of DAP-3 with the intact cosmid 33.3 containing the L^q gene (14). The L^d/L^q chimeric molecules were generated using the PCR technique, splicing by overlap extension, developed by Pease and colleagues (30). Briefly, PCR was performed using the appropriate vector that contained either the 2 exon of L^d (p305) or L^q (p133). Primers were generated that could anneal to both L^d and L^q on either strand of the DNA in areas of the exon that were between combinations of the residues that differed between them. These primers were used in conjunction with primers that flanked the 2 exon to amplify segments of the exon that corresponded to L^d or L^q sequence; these primers contained terminal restriction endonuclease sites (29). Because primers that overlapped were used, a 5' segment of the L^d 2 exon could be annealed to a 3' segment of the L^q 2 exon, and PCR could be performed using the splicing by overlap extension technique (29, 30) to generate complete 2 exons that were hemi-exon or quarter-exon constructs of L^d and L^q depending on the location of the overlapping primer combination. The chimeric 2 exons were sequenced to verify exon shuffling and then ligated into vector constructs that contain the promoter, 5'-untranslated region, 1 exon, 3 exon, exons 5–8, 3'-untranslated region, and the 3'-flanking region from the L^d gene (29). All full-length constructs were partially sequenced to verify proper exon assembly. These constructs were cotransfected into DAP-3 (thymidine kinase^- L cell line) cells along with the thymidine kinase gene using calcium phosphate precipitation (31). Stable transfectants were selected using hypoxanthine-, aminopterin-, and thymidine-containing medium. The L^d W97R mutant was generated by site-directed mutagenesis using overlapping mutagenic primers (32). The L^q R97W mutant was generated as previously described (33).

Immunoprecipitations

Cells (1 x 10⁷) were incubated in cysteine^-, methionine^- MEM (Life Technologies, Grand Island, NY) with 10% FBS for 30 min at 37°C in 5% CO₂. A mixture (1.25 mCi) of ³⁵S-labeled cysteine and methionine (ICN, Irvine, CA) was added, and the cells were incubated at 37°C for 1 h. The radiolabeled cells were washed three times in cold PBS and then lysed in 150 mM sodium chloride, 50 mM Tris-HCl (pH 7.4), and 0.5% Nonidet P-40 with freshly added 0.2 mM PMSF for 30 min on ice. Each lysate was precleared twice with IgGsorb (The Enzyme Center, Malden, MA) at 4°C. Each precleared lysate was divided into aliquots for immunoprecipitation with the appropriate mAbs in the presence of freshly added PMSF. The immune complexes were captured using immobilized protein A (IPA; RepliGen, Cambridge, MA). The class I/mAb complexes were eluted from IPA in gel sample buffer (125 mM Tris-HCl (pH 6.8), 2% SDS, 12.5% glycerol, 1% 2-ME, and 0.2% bromophenol blue) at 100°C for 2 min. IPA was removed by centrifugation at 12,000 x g for 2 min. Samples of each aliquot were analyzed by electrophoresis on 12% polyacrylamide gels. The fixed and dried gels were exposed to film. Quantitation of the bands was performed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) equipped with ImageQuant software.

Flow cytometric analyses

For peptide binding studies, transfected L cell lines were incubated overnight in complete DMEM and 10% FBS in the presence of either the murine CMV (MCMV) pp89_168–176 (YPHFMPTNL) (34) or tum^- P91A_14–22 (TQNHRALDL) (10, 35) peptide at the indicated concentration or in the absence of exogenous peptide. Peptides were synthesized using 9-fluorenylmethyloxycarbonyl chemistry on a PE Applied Biosystems 432A peptide synthesizer (Foster City, CA). The cells were then stained with either mAb 64-3-7 or 28-14-8, and polyclonal goat anti-mouse IgG conjugated with FITC (ICN) before analysis on a FACScan (BD Biosciences, San Jose, CA).

Cell surface half-life analyses

Trypsinized L cell transfectants were incubated for 12–14 h in either complete DMEM (with 10% FBS) or Hybridoma SFM (serum-free; Invitrogen, Carlsbad, CA) at 1 x 10⁶ cells/ml. Brefeldin A (Epicentre, Madison, WI) was then added to a final concentration of 10 µg/ml, and the cells were divided into 3-ml aliquots and cultured in the wells of six-well plates at 37°C. At the indicated times, 3 ml (3 x 10⁶ cells) were removed from the wells and stored at 4°C. After all time points had been collected, the cells from each time point were further divided and stained with no primary Ab, mAb 28-14-8 or mAb B22/249 plus FITC-conjugated goat anti-mouse IgG. After analysis on a FACS Vantage (BD Biosciences), the mean fluorescence intensity (MFI) for each mAb and time point was determined, and the percentage of the initial MFI (time zero) was calculated and plotted vs time.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of the disparity between L^d and L^q in ₂m affinity to amino acid position 97

To determine which residues of L^d and L^q (Fig. 1A) are responsible for their disparate affinity for ₂m, chimeric constructs that contained different combinations of the L^d and L^q sequences in the 2 exon were created using PCR (Fig. 1B). The transfectants expressing the 2 hemi-exon-shuffled constructs were first analyzed and compared with transfectants expressing the wild-type L^d and L^q genes. There are four polymorphic residues between these two alleles in the amino-terminal half of the 2 domain (positions 95, 97, 107, and 116) and two in the carboxyl-terminal half (155 and 157; Fig. 1). Immunoprecipitates of the L^q/L^d (322; amino-terminal half of the 2 domain, L^q residues/carboxyl-terminal half, L^d residues) chimeric and wild-type L^q molecules contained considerably higher ratios of ₂m to H chains than did immunoprecipitates of the reciprocal L^d/L^q (323; amino-terminal half of the 2 domain, L^d residues/C-terminal half, L^q residues) chimeric and wild-type L^d molecules (data not shown). These results suggest that one or more of the four polymorphic residues (positions 95, 97, 107, and 116) in the amino-terminal half (in the -pleated sheet portion) of the 2 domain are responsible for the disparate ₂m association of L^d and L^q.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. A, The six amino acid residues, all located in the 2 domain, that differ between Ld and Lq for Ld (Leu95, Trp97, Gly107, Phe116, Tyr155, and Arg157) and for Lq (Ile95, Arg97, Trp107, Tyr116, His155, and Lys157) are shown in the context of the original x-ray crystal structure of an MHC class I molecule (14 49 ). B, Linear representation of the 2 domain structures in the chimeric and single-site substitution mutant Ld molecules examined. The arrows represent the locations of primers used in the synthesis of the chimeric 2 exons.

The analyses of the above chimeric L^d molecules suggest that L^qIle⁹⁵, L^qArg⁹⁷, or both are critical for a strong association with ₂m. Positions 95 and 97 of L^d are Leu and Trp residues, respectively. The similarity of the residues of position 95 (Leu and Ile) and the dissimilarity of the residues at position 97 (Trp and Arg) in L^d and L^q suggested that the latter are responsible for their disparate ₂m association. To test whether the residue at position 97 is important in ₂m affinity, a mutant in which the L^qArg⁹⁷ residue was substituted into L^d to replace Trp⁹⁷ was constructed: L^dW97R (Fig. 1B). The reciprocal L^q mutant had been previously constructed by other investigators (33) and was obtained for these studies: L^qR97W (Fig. 1B). Again, immunoprecipitation studies of radiolabeled transfectant cell lysates using mAb 28-14-8, which recognizes both empty and peptide-associated forms of L^d and L^q, were performed. The results shown in Fig. 2, in which three independent immunoprecipitations were performed from the same cell lysate, indicate that the nature of the residue at position 97 in L^d and L^q dramatically impacts the association of these closely related class I alleles with ₂m. Clearly, L^d and L^qR97W associate weakly, whereas L^q and L^dW97R associate strongly, with radiolabeled mouse ₂m.

View larger version (76K): [in this window] [in a new window]   FIGURE 2. Analysis of the amount of 2m associated with immunoprecipitated Ld, Lq, and position 97-substituted mutants of each indicates that position 97 is responsible for the disparate 2m association of Ld and Lq. Cells of each type were labeled with [35S]cysteine and -methionine and then lysed. The lysate were divided into three aliquots, and each was immunoprecipitated individually in triplicate using mAb 28-14-8 and IPA. The immunoprecipitations were resolved by SDS-PAGE (12% gels), and the bands were detected by autoradiography. Quantitation of the H chain and 2m bands was performed using by PhosphorImager. The ratio of 2m to H chain was calculated for each lane, and the mean and SEM values for each set of triplicates are shown below each set.

L^dW97R has a longer cell surface half-life than either L^d or L^q

The cell surface stability of L^dW97R relative to L^d and L^q was examined both in medium containing FBS, a source of bovine ₂m (4), and under serum-free conditions. In the presence of a source of exogenous ₂m (FBS), L^d has a t_1/2 of 2.4 h (similar to previous reports (10, 11)), L^q has a t_1/2 of 4.6 h, and L^dW97R has a predicted t_1/2 of 8.8 h (Fig. 3A). In the absence of exogenous ₂m (serum-free conditions), the half-lives are somewhat shorter, as expected, but the same hierarchy is retained: L^d t_1/2 of 1.6 h, L^q t_1/2 of 3.2 h, and L^dW97R t_1/2 of 6.6 h (Fig. 3B). These results along with the previous results examining ₂m association indicate that a strong association of the class I H chain with ₂m correlates with a longer cell surface half-life, even in the presence of exogenous ₂m. The same hierarchy of cell surface stability among L^d, L^q, and L^dW97R observed in both the presence and the absence of exogenous ₂m argues that the cell surface stability of these molecules depends on factors other than ₂m affinity. Studies performed by Parker and colleagues (36) indicate that the class I affinity for peptide affects the dissociation of ₂m from the H chain and the overall stability of the complex. Thus, the differences in stability and ₂m affinity among the three molecules could reflect differences in endogenous peptide affinity. Furthermore, the results indicate that the residue at position 97 plays an important role in these properties.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. The cell surface stability of Ld, Lq, and LdW97R in medium containing FBS (A) or in serum-free medium (SFM; B). Brefeldin A (10 µg/ml final concentration) was added to the cultures of cells expressing the indicated transfected gene product at time zero. The cells were harvested at the indicated times and stored at 4°C until the last time point was harvested. After staining the cells for the transfected gene product with either mAb 28-14-8 (shown) or B22/249 (data not shown) and FITC-conjugated goat anti-mouse IgG, the MFI for each group of cells at each time was determined, and the percentage of the MFI compared with that at time zero was plotted vs time. This experiment was repeated once with similar results.

Previous studies had shown that a large proportion of open (mAb 64-3-7⁺) forms of L^d exist on the cell surface (3, 12, 37). These forms are thought to result from the loss of peptide from the completely assembled forms of L^d on the surface. Thus, the relatively short half-life of L^d on the cell surface probably results from its inability to retain peptide, resulting in a higher steady state level of open forms. To compare the relative proportions of open forms of L^d, L^q, and L^dW97R on the cell surface, the levels of open and total transfected gene products were examined in L cell transfectants expressing these molecules, as detected by flow cytometry using mAbs 64-3-7 and 28-14-8, respectively. The relative amounts of open molecules for each in the absence of exogenous peptide are shown in Fig. 4. More than 50% of L^d was found in open forms in the two experiments, whereas 30–36% of L^q was found in open forms. Only 5–7% of L^dW97R was found in this form. Thus, the increased stability of this mutant molecule on the cell surface results in a lower steady state level of open forms.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. The amounts of open forms of Ld, Lq, and LdW97R relative to the total numbers of those molecules on the cell surface. These numbers were calculated on the basis of the MFI as analyzed by flow cytometry using mAbs 64-3-7 and 28-14-8, respectively (plus FITC-conjugated goat anti-mouse IgG) on transfectants expressing each gene product. Before staining, the transfectants were incubated in the absence of exogenous peptide or in the presence of one of two Ld-binding peptides: MCMV pp89168–176 (A) or tum- P91A14–22 (B). The results shown in A and B are derived from separate experiments. The numbers over the open bars represent the relative amount of open form of that molecule after incubation in exogenous peptide compared with the relative amount of that open form after incubation in the absence of exogenous peptide. The numbers represent the ability of the peptide to decrease the relative number of open forms on the cell surface.

The overnight incubation of cells expressing L^d in comparatively high concentrations of the peptide ligands MCMV pp89_169–176 (100 µM) and tum^- P91A_14–22 (250 µM) significantly decreases the total number (data not shown) as well as the proportion of open forms on the cell surface (Fig. 4). For L^d, the relative numbers of open forms decreased from 0.76 to 0.03 after incubation in 100 µM MCMV pp89_168–176 peptide, indicating that the binding peptide had decreased the numbers of open forms to 4% of that observed on cells that were incubated in the absence of exogenous peptides (Fig. 4A). The tum^- P91_14–22 peptide (250 µM) decreased the relative numbers of open forms to 2% of that observed on cells that had been incubated in the absence of exogenous peptides (Fig. 4B). Similar to L^d, the MCMV pp89_168–176 peptide decreased the relative numbers of open forms of L^q to 8% of that observed on cells incubated in the absence of exogenous peptides (Fig. 4A). In contrast to L^d, the tum^- P91A_14–22 peptide was comparatively ineffective in decreasing the proportion of open forms of L^q, since 77% as many open forms were detected after incubation in this peptide (Fig. 4B). Thus, these results suggest that tum^- P91A_14–22, which uses an anchor motif distinct from the consensus P2 Pro anchor for binding to L^d (10), binds inefficiently to L^q, whereas the MCMV pp89_168–176 peptide with its consensus P2 Pro anchor binds comparably to L^q. Using more traditional peptide binding assays (12, 13), L^q was also observed to bind the MCMV pp89_168–176, but not to the tum^- P91A_14–22 peptide (data not shown). Five other L^d-restricted peptides have been shown to bind efficiently to L^q using both the traditional assay and the assay used here (data not shown): LCMV nucleoprotein 118–126 (38, 39), p29 (40), -galactosidase 876–884 (41), and the related peptides, p2Ca (42) and QL9 (both of these two related peptides use a nonconsensus anchor motif for binding to L^d) (43). Using the same assay as that employed above, the MCMV pp89_168–176 peptide was able to bind to L^dW97R on the basis of its ability to decrease the already small proportion of open L^dW97R molecules on the cell surface to 11% of that found on cell incubated in the absence of exogenous peptide (Fig. 4A). In contrast and similar to the situation with L^q, the L^d-restricted tum^- P91A_14–22 peptide only slightly decreased the proportion of open L^dW97R molecules on the cell surface (Fig. 4B) to 71% of the level found on cells incubated in the absence of exogenous peptide. These results suggest that the L^dW97R mutant molecule is more similar to L^q than to L^d in its peptide binding specificity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Examination of the L^d x-ray crystal structure (5, 6) raises an intriguing question concerning how Trp⁹⁷ negatively impacts on the association of L^d with ₂m. Trp⁹⁷ points up from the -pleated sheet that forms the floor of the peptide binding cleft (Fig. 1A). In fact, Trp⁹⁷ and several other residues form a ridge in the peptide binding cleft that forces the central portion of the bound peptide out of the L^d cleft (5, 6); these protruded peptide residues may interact with the TCR on T cells (6, 35). Thus, Trp⁹⁷ in L^d clearly does not directly interact with ₂m. When Arg was substituted for Trp⁹⁷ in the L^d structure using computer modeling, its density could be fit entirely within that of the original Trp, suggesting that, if Arg⁹⁷ were aligned in the same direction as Trp⁹⁷, it would not cause the strand, on which it is located, to deflect more proximal to the ₂m loops that lie underneath it (data not shown). Furthermore, as suggested from the peptide binding results (Fig. 4), the substitution of an Arg residue at position 97 in L^d may induce a change in the peptide binding groove that precludes efficient binding of the L^d ligand, tum^- P91A_14–22, to it. Thus, both the known structural information on the location of position 97 and the peptide binding results suggest that the residue at position 97 does not directly affect the association of these class I molecules with ₂m, but, rather, affects their association with peptide ligands, which may then indirectly affect their association with ₂m. The increased cell surface stability of L^q and L^dW97R (Fig. 3) and the presence of fewer open forms of these molecules (Fig. 4) compared with L^d suggest that Arg⁹⁷ has a dramatic impact on the peptide binding cleft, increasing the affinity and retention of peptide ligands by L^q and L^dW97R. Taken together, these studies argue that the residue at position 97 in L^d, L^q, and L^dW97R has a critical effect on peptide specificity and affinity, which subsequently affects the stability of folded forms and the proportion of open forms of these molecules on the cell surface as well as their retention of ₂m.

Based on x-ray crystal structures, ₂m has an unique orientation with L^d compared to that with other mouse class I molecules (5, 6). Balendiran et al. (5) suggested that the weak association between ₂m and L^d results from this suboptimal orientation and not directly from polymorphic L^d residues at ₂m contact sites. Moreover, L^d is particularly deficient in 1 and 2 domain interactions with ₂m compared with K^b and D^b (5). Furthermore, Speir et al. (6) noted that L^d has fewer hydrogen bonds and less hydrophobic surface area contact with ₂m than do K^b and D^b. Overall, the crystallographic studies suggest that the polymorphic residues in L^d affect its overall structure, resulting in less area of contact between it and ₂m. The substitution of Arg for Trp⁹⁷ in the L^dW97R mutant may increase its affinity for peptide ligands and allow it to achieve a conformation that maximizes the area of contact between it and ₂m.

On the other hand, it is feasible that the replacement of Trp⁹⁷ in L^d with an Arg residue in the L^dW97R mutant results in a conformational alteration in L^d that induces a more stable interaction between the mutant molecule and ₂m, independent of the interaction of L^dW97R with peptide ligands. The increased stability of this interaction might result in the observed increased cell surface stability of this class I complex. However, the change in peptide specificity of L^dW97R argues against this. Furthermore, the cell surface stability of MHC class I complexes has been suggested to be mostly dependent on the stability of the interaction of the class I molecule with its peptide ligand (36). While it could be argued that L^d differs from most MHC class I molecules and that its stability may be more dependent on its ability to retain ₂m, previous studies have shown that L^d complexes loaded with different peptides exhibit differential cell surface stability (3, 10), indicating that the affinity for peptide ligands is the critical factor in determining the stability of L^d. Thus, we favor the model in which the increased stability of L^dW97R is due to an enhanced affinity for associated endogenous peptides, thereby increasing the stability of its interaction with ₂m.

Ribaudo and Margulies (44) reported that substitution of a Val residue for Glu⁹ in L^d increased its affinity for ₂m. Analysis of an independently generated L^dE9V mutant (29) demonstrated that this mutant molecule has a moderately increased affinity for ₂m and a dramatically increased cell surface expression. The x-ray crystallographic studies of L^d (5, 6) indicate that Glu⁹, like Trp⁹⁷, is located in the peptide binding cleft of L^d and, in fact, participates in a peptide binding pocket (5). In contrast to the studies presented here, peptide binding and cell surface stability studies were not performed on the L^dE9V mutant molecules (29, 44). Although we cannot rule out that residues 9 and 97 impact on ₂m association in a peptide-independent manner, the studies presented here indicate that differences in peptide binding alone may be sufficient to determine their disparate association with ₂m.

The functional consequence of the comparative instability of L^d and the high proportion of open forms of L^d on the cell surface is an increased ability of L^d to bind exogenous peptides at the cell surface (12, 13). This is the basis of peptide binding assays for L^d, since, in the continuous presence of high concentrations of exogenous peptides capable of binding to L^d, the total number of L^d molecules increases, and the proportion of open forms decreases dramatically (Fig. 4). Thus, L^d would be able to participate more effectively in an alternative class I Ag presentation pathway in which regurgitated exogenous peptides bind at the cell surface for presentation to CD8⁺ T lymphocytes (17, 18, 45).

An alternative MHC class I pathway that is perhaps more physiologically relevant than the aforementioned regurgitation pathway has also been described (17, 18). In this alternative pathway open MHC class I molecules can bind peptides in endosomal compartments, followed by their egress to the cell surface (17, 18). The peptides are generated in endosomal compartments from phagocytosed exogenous proteins. The empty MHC class I molecules that bind peptides from exogenous Ags in endosomal compartments are derived from MHC class I molecules that have lost their endogenous peptides either within the endosomal compartment or on the cell surface before endocytosis. Interestingly, L^d has been shown to participate in these alternative pathways (46, 47) either by binding regurgitated peptides on the cell surface or via internalization of open forms from the cell surface. While other MHC class I molecules can also participate in these pathways (17, 45, 48), the comparative instability of trimeric L^d-peptide-₂m complexes may allow L^d to use these pathways more efficiently. The studies presented here suggest that in the context of the rest of the L^d structure, Trp⁹⁷ plays an important role in potentially allowing L^d to participate more effectively in these alternative pathways.

	Acknowledgments

 
We thank Karen Ehlert for secretarial assistance, Louise Barnett for flow cytometry, and Dr. Thomas Quinn (Department of Biochemistry, University of Missouri) for helping to analyze the published x-ray crystal structures of L^d.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grants AI31189 (to D.R.L.) and AI19687 (to T.H.H.) and National Institutes of Health Training Grant GM08396 (to R.A.S.).

² Current address: Department of Pathology, 50N Medical Drive, University of Utah Medical School, Salt Lake City, UT 84132-0001.

³ Address correspondence and reprint requests to Dr. David R. Lee, M616 Medical Sciences Building, One Hospital Drive, Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212. E-mail address: leedr{at}missouri.edu

⁴ Abbreviations used in this paper: ₂m, ₂-microglobulin; IPA, immobilized protein A; MFI, mean fluorescence intensity; MCMV, murine CMV.

Received for publication June 4, 2002. Accepted for publication July 12, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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