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International Immunology, Vol. 13, No. 10, 1275-1282, October 2001
© 2001 Japanese Society for Immunology

Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations

Michael R. Harris1,2, Lonnie Lybarger1, Nancy B. Myers1, Christine Hilbert1, Joyce C. Solheim3, Ted H. Hansen1 and Yik Y. L. Yu1

1 Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA
2 Department of Newborn Medicine, Children's Hospital, St Louis, MO 63110, USA
3 Epply Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA

Correspondence to: Correspondence to: T. H. Hansen


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and discussion
 References
 
MHC class I heavy chains assemble in the endoplasmic reticulum with ß2-microglobulin and peptide to form heterotrimers. Although full assembly is required for stable class I molecules to be expressed on the cell surface, class I alleles can differ significantly in their rates of, and dependencies on, full assembly. Furthermore, these differences can account for class I allele-specific disparities in antigen presentation to T cells. Recent studies suggest that class I assembly is assisted by an elaborate complex of proteins in the endoplasmic reticulum, collectively referred to as the peptide loading complex. In this report we take a mutagenesis approach to define how HLA-B27 molecules interact with the peptide loading complex. Our results define subtle differences between how B27 mutants interact with tapasin (TPN) and calreticulin (CRT) in comparison to similar mutations in other mouse and human class I molecules. Furthermore, these disparate interactions seen among class I molecules allow us to propose a spatial model by which all class I molecules interact with TPN and CRT, two molecular chaperones implicated in facilitating the binding of high-affinity peptide ligands.

Keywords: antigen binding, autoimmunity, chaperones, immunochemistry, MHC


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and discussion
 References
 
There is a striking association between the expression of HLA-B27 molecules and susceptibility to a group of closely related arthritic diseases collectively referred to as spondyloarthropathies (1). Although this association suggests direct involvement of HLA-B27 in disease pathogenesis, the mechanism remains unclear in spite of intense investigation (2–5). Most of these investigations have focused on identifying unique arthritogenic peptides that bind to HLA-B27 (6). The current dogma is that T cell responses to putative arthritogenic peptides may be initially induced by infection with Gram-negative bacteria such as Klebsiella, Chlamydia, Shigella or Yersinia (1). Subsequently, it has been proposed that these B27-restricted T cells are autoreactive such that they detect peptides derived from normal tissues such as the joint. Although this attractive model is consistent with considerable published evidence, arthritogenic peptides detected by B27-restricted T cells have not been identified. What is perhaps most surprising, is the failure to identify disease-related B27 peptide ligands in rodent models that have transgenic expression of HLA-B27 molecules and are susceptible to arthritic disease (3–5).

The failure to identify arthritic peptide ligands of B27 has resulted in more recent studies proposing that B27 molecules may have disease-related properties other than specific peptide binding. For example, Peh et al. (7) reported that B27 molecules are less dependent upon the chaperone tapasin (TPN) than other class I molecules. Poor interaction with TPN could result in poor peptide loading in the endoplasmic reticulum (ER) and subsequently a greater propensity for B27 to bind peptides at the cell surface. Alternatively, Mear et al. (8) reported that the unique B pocket architecture results in the misfolding and premature turnover of a portion of B27 molecules, by an unknown mechanism that could involve disparate chaperone interaction. Additionally, Allen et al. (9) reported that B27 heavy chains can be expressed as novel ß2-microglobulin (ß2m)-free heavy chain homodimers that have unique antigen presentation properties. In a perhaps related study, Khare et al. (10) reported that in a murine HLA-B27 transgenic model, ß2m-deficient mice develop arthritis and that disease can be blocked with mAb to free B27 heavy chains. These combined findings underscore the importance of defining the precise mechanism of B27 assembly and folding.

Although allele differences have been noted (11), prior to peptide binding most class I molecules are in physical association with the complex of transporter associated with antigen processing (TAP) (12–14), TPN (15–17), calreticulin (CRT) (15,17,18) and ERp57 (19–21). This complex, collectively referred to here as the peptide loading complex, has been implicated in facilitating class I folding and assembly by unknown mechanisms (22). Furthermore, the contribution of individual members of the peptide loading complex remains undefined, as does the manner in which they spatially associate with the class I molecule. To address these questions, we and others have shown that mutations at three sites in the class I H chain determine its association with TAP, TPN and CRT (14,23–28). Interestingly, each of these three sites is located in each of the three class I extracellular domains, {alpha}1, {alpha}2 and {alpha}3. However, due to an apparent requirement for cooperative binding (28,29), it has been difficult to unequivocally determine which chaperone interacts with which of the three sites on the class I H chain.

In this study we introduced mutations into the B27 heavy chain at the three sites previously implicated in ER chaperone association. Our findings define unique properties of B27 in regard to ER chaperone associations, and strongly support a general model of how all class I molecules spatially associate with TPN and CRT.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and discussion
 References
 
mAb and antisera
The mAb 64-3-7 (IgG2b) is specific for open forms of Ld/Lq and other molecules tagged with this epitope (30–32). mAb ME-1 (IgG1) recognizes assembled HLA-B27 H chains (33) and mAb BBM1 (IgG2b) recognizes human ß2m (34). The anti-human TAP antibody (#1478) (25) and the anti-human TPN antibody (#1848) were generated in rabbits immunized with peptides derived from the N-terminal sequence of the respective human proteins. Anti-CRT antisera were purchased from either Stressgen (SPA-600, rabbit antibody; Victoria, BC, Canada) or Affinity Bioreagents (PA1-903, chicken antibody; Golden, CO).

Cell lines and flow cytometry
HeLa cells were maintained at 37°C in complete medium containing 10% bovine calf serum (Hyclone, Logan, UT). To analyze cells for surface expression of class I molecules, cells were stained with 20 µl of culture supernatant from 64-3-7 or ME-1 hybridomas by standard methods. The data collected on a FACSCalibur (Becton Dickinson, San Jose, CA) were expressed in the form of histograms as fluorescence values (x-axis) plotted against cell numbers (y-axis) using CellQuest Software (Becton Dickinson).

Mutagenesis and transfection
The HLA-B*2705 cDNA was kindly provided by Dr Joel Taurog (University of Texas Southwestern Medical Center at Dallas) and it was subcloned in the mammalian expression vector RSV.5.neo (35). All mutageneses were performed using the Quik Change Mutagenesis kit from Stratagene (San Diego, CA) as previously described (32) with the exception of the S132K mutant which was constructed in an identical fashion to that of the Ld loop mutants (28). The mutants were named by first indicating the wild-type residue followed by the amino acid position in the mature protein and then the mutated residue. The HeLa cells were transfected with Lipofectin (Life Technologies, Gaithersburg, MD) according to a modified protocol based on the manufacturer's instructions. The transfectants were selected in 0.6 mg/ml of active G418. HLA-B27 R48Q (etB27) molecules were transfected into the human B lymphoblastoid cell lines LCL721.221 and .220 (36) (kindly provided by Dr Thomas Spies, Fred Hutchinson Cancer Center, Seattle, WA) by electroporation using the Gene Pulser II system from BioRad (Hercules, CA).

Immunoprecipitations and Western blots
HeLa cells and HeLa transfectants were lysed in buffers containing 1% digitonin (Wako, Richmond, VA) in 10 mM Tris-buffered saline pH 7.4 (TBS) with 20 mM iodoacetamide and 0.2 mM of freshly added PSMF. Saturating amounts of the primary antibody were added to the lysis buffer and subjected to immunoprecipitations as previously described (25). Briefly, lysates were centrifuged to remove cell debris and nuclei and supernatants were added to Protein A–Sepharose CL-4B (Amersham Pharmacia, Uppsala, Sweden) for 60 min on ice and Protein A-bound material was washed in 0.1% digitonin in TBS. Immunoprecipitates were eluted from Protein A by boiling for 5 min in elution buffer consisting of 0.125 M Tris, pH 6.8, 2% SDS, 12% glycerol, 2% bromophenol blue (w/v). Samples were electrophoresed on either Tris–glycine or Tris–acetate, pre-poured gels (Invitrogen, Carlsbad, CA) and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). After overnight blocking in 10% milk, PBS/0.05% Tween 20, membranes were incubated in a dilution of antibody for 2 h, washed 3 times with PBS/0.05% Tween 20 and incubated for 1 h with biotin-conjugated goat anti-mouse IgG or anti-rabbit IgG (Caltag, San Francisco, CA) or rabbit anti-chicken/turkey IgG (Zymed, San Francisco, CA). Following three washes with PBS/0.05% Tween 20, membranes were incubated for 1 h with streptavidin-conjugated horseradish peroxidase (Zymed, San Francisco, CA), washed 3 times with PBS/0.3% Tween 20 and incubated with ECL chemiluminescent reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Western blots were then developed on BioMax film (Eastman Kodak, Rochester, NY).


   Results and discussion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
References
 
Approach
To detect its specific association with the peptide loading complex, we tagged the HLA-B27 molecule with the 64-3-7 epitope by introducing a single mutation (R48Q). The 64-3-7 epitope is only detected when H chains are in an open conformation and this epitope is not sterically blocked when class I is associated with the peptide loading complex (32). The 64-3-7 epitope is naturally present in only mouse Ld and Lq molecules, but has been successfully transferred into mouse Kd, Kb, M3 and Qa1b molecules with either one or two amino acid substitutions (32,37,38). Furthermore, epitope transfer has been found not to alter the peptide binding signature of each of these class I molecules. Thus, by introducing this epitope into B27 we could quantify the peptide loading of B27 molecules by determining the percent of B27 molecules expressed in an open versus folded conformation and then compare these findings to those obtained with other epitope-tagged class I molecules. Furthermore, tagged B27 molecules allowed us to compare how B27 interacts with components of the peptide loading complex, relative to other class I molecules.

As shown in Fig. 1Go(c), expression of epitope-tagged B27 (etB27) in human HeLa cells rendered a subset of surface B27 molecules detectable with mAb 64-3-7. In studies of other class I molecules, 64-3-7+ conformers at the cell surface were shown to arise after peptide dissociation (31,38). As expected, endogenous class I molecules expressed by HeLa cells and wild-type B27 molecules are not 64-3-7+ (Fig. 1a and bGo respectively), demonstrating that mAb 64-3-7 uniquely detects etB27 molecules in HeLa cell transfectants. Thus, this is a valid test system to specifically study the assembly of B27 molecules. Relative to the amount of fully assembled etB27 (ME1+) (33) there were relatively few open etB27 (64-3-7+). Indeed, on the surface of HeLa-etB27 cells, only 4% of etB27 molecules were detected in a open conformation. Similarly, on .221-etB27 cells the percent open was found to be 3% (not shown). By comparison, the percentage of surface open forms is 7% for Kd, 15% for Kb and 28% for Ld as observed on L cells or .221 cells (37). Furthermore, in data not shown the surface half-life of etB27 [like wild-type B27 (39)] was found to be >20 h, compared to reported half-lives of 2 and 10 h for Ld and Kb respectively (31 and unpublished data). These comparisons suggest that the overall quality of peptide loading of B27 is relatively good compared to these mouse class I molecules.



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  		Fig. 1. Surface expression of etB27 and etB27 mutants in HeLa cells. Flow cytometric analysis was performed on cells that were stained with ME-1 (solid line) or 64-3-7 (dashed line). The dotted line shows secondary antibody alone control. (a) Untransfected HeLa cells, (b) HeLa cells transfected with wild-type B27, (c) HeLa cells transfected with etB27 or (d–f) HeLa cells expressing etB27 carrying the indicated mutation(s). The percentage of surface-empty B27 was calculated as follows. First, the fluorescence values derived by the use of both primary and secondary antibodies were corrected by subtracting out the values obtained in the presence of secondary antibody alone. Second, the percentages of 64-3-7+ forms were derived by the following formula: (64-3-7/64-3-7 + ME-1)x100. The percentages calculated were 4% for etB27 (c), 25% for etB27 N86Q (d), 30% for etB27 S132K (e) and 22% for etB27 D227K,E229K (f). This experiment was repeated twice with similar results.

 
Importance of the N-linked glycan in the {alpha}1 domain
To address which regions of B27 are required for chaperone interactions in the ER, mutations were introduced into etB27 molecules at sites in the {alpha}1, {alpha}2 and {alpha}3 domains previously implicated in ER chaperone association with other class I molecules (14,23–28). Earlier studies using castanospermine (18) demonstrated that an N-linked oligosaccharide is involved in class I association with TAP/TPN/CRT, the class I peptide loading complex. Furthermore, the location of the N-linked glycan at N86 in the {alpha}1 domain is of critical importance, since mutation of the glycosylation site N176 residue in the {alpha}2 domain of a mouse class I molecule did not affect its ability to associate with the peptide loading complex (25). To determine the importance of the N86 attached glycan of HLA-B27 for association with the peptide loading complex, etB27 N86Q molecules were expressed in HeLa cells. Although etB27 N86Q molecules were expressed at the surface of HeLa cells, the quality of peptide loading was clearly impaired compared with etB27. As shown in Fig. 1Go(B), 25% of surface etB27 N86Q molecules were detected in a peptide-empty form (64-3-7+), whereas only 4% of etB27 molecules were detected in a peptide-empty form. As further evidence for peptide loading deficiencies, surface B27 N86Q molecules were found to be markedly inducible when cells were cultured at 25°C (data not shown), a hallmark of suboptimal peptide loading (40). As shown in Fig. 2Go(A), the amount of ß2m assembly was clearly impaired in N86Q compared with etB27 molecules. Even though the HeLa-etB27 and HeLa-etB27 N86Q cells expressed similar amounts of B27 H chain, the relative amount of N86Q H chain assembled with ß2m was significantly reduced. This reduced assembly was seen when precipitating ß2m and blotting for H chain with either mAb 64-3-7 (Fig. 2AGo) or when precipitating H chains with either mAb 64-3-7 or ME1 and blotting with anti-ß2m (Fig. 2CGo). Thus the N86Q mutation significantly impairs the quality of B27 as indicated by poor assembly with peptide and ß2m. It should be noted that this defect is not absolute, since folded/assembled N86Q molecules were detected in both lysates and at the cell surface.




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  		Fig. 2. Effects of individual etB27 mutation(s) on chaperone association. Lysates of HeLa cells transfected with the construct indicated on the top of the figure were immunoprecipitated and tested by Western blot analysis. (A) Respective lysates were immunoprecipitated with the reagent listed along the left side of the figure and each precipitate was then Western blotted with mAb 64-3-7 to specifically detect etB27 H chains. (B) Respective lysates were immunoprecipitated with 64-3-7 and then Western blotted to detect the associated ER protein listed along the left side of the figure. (C) Respective lysates were precipitated with the reagent listed along the left of the figure and then Western blotted with mAb BBM1 to detect human ß2m molecules. Antibodies used for this experiment were: anti-epitope tagged H chain (64-3-7), anti-BBM1 (47) anti-TAP (rabbit serum #1478), anti-TPN (rabbit serum #1848) and anti-CRT for (A) (SPA-600, StressGen Biotechnologies) and anti-CRT for (B) (PA1-903, Affinity BioReagents).

 
To determine if suboptimal assembly was indicative of aberrant associations with the peptide loading complex, etB27 and etB27 N86Q molecules were compared with regard to their associations with TAP, TPN and CRT. As shown in Fig. 2Go, lack of carbohydrate in the {alpha}1 domain resulted in a loss of association of etB27 molecules with TAP, TPN and CRT. Indeed, lack of association of etB27 N86Q with TAP/TPN/CRT was demonstrated by either precipitating etB27 H chains and blotting with antibody to the ER protein (Fig. 2BGo), or precipitating with antibody to the ER protein and blotting with mAb 64-3-7 to detect etB27 H chains (Fig. 2AGo). Thus like other mouse and human class I molecules studied so far, B27 is dependent upon N-linked glycosylation in the {alpha}1 domain for association with the assembly complex of TAP, TPN and CRT. Since CRT has been shown in other systems to have lectin-like activity, it seems likely that CRT specifically binds class I H chains via their {alpha}1 domain glycan (18). Failure to detect TAP and TPN in association with H chains carrying a mutation at N86 could be explained by CRT being a prerequisite for H chains to form stable complexes with TAP/TPN or by improper folding of class I molecules in the absence of carbohydrate addition.

To further characterize the impaired assembly of mutant N86Q H chains, peptides were added to lysates of HeLa-etB27 and HeLa-etB27 N86Q cells. For this experiment the HIV gag peptide (41) was used as a known B27 binder and the Dd tum– peptide (42) as a length-matched, control non-B27 binder. Incubation with the HIV peptide induced both the specific loss of ß2m-associated open forms of etB27 (Fig. 3AGo, left panel) and the specific gain of assembled etB27 molecules as detected with ME1 (Fig. 3BGo, left panel). By contrast no peptide-specific conversion was observed with mutant N86Q molecules (Fig. 3A and BGo, right panel). Thus, these findings demonstrate that the N86Q mutation clearly impairs peptide-induced H chain folding in cell lysates, an observation consistent with the role of the peptide loading complex in facilitating peptide binding.



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  		Fig. 3. Impaired peptide-induced folding of N86Q mutant B27 molecules compared to wild-type. Samples representing cell equivalents of etB27 or etB27 N86Q were lysed in 1% digitonin and incubated on ice for 2 h with 200 µM of a length-matched, non-B27 binding, control peptide (Ddtum-GPPHSNNFGY) (42), no peptide or 200 µM of B27 ligand HIV-gag (KRWIILGLNK) (41) as indicated along the top of the figure. Samples in (A) were precipitated with mAb 64-3-7 and the co-precipitating ß2m molecules were detected by immunoblotting with mAb BBM-1. Results in (A) demonstrate that peptide can specifically induce the loss of ß2m-associated open forms of wild-type B27 molecules, but not N86Q mutant molecules. Samples in (B) were precipitated with mAb ME-1 and the etB27 H chains were detected by blotting with mAb 64-3-7. Results in (B) demonstrate that peptide can specifically induce the gain of folded wild-type B27 H chains, but not N86Q mutant B27 H chains.

 
Importance of residues around D227 in the {alpha}3 domain
Previous studies of mouse Ld (14), Db (26) and Dd (27) molecules showed that mutations in the {alpha}3 domain around residue 227 disrupted their ability to associate with TAP, TPN and CRT. This residue is located on an exposed bridge that transects the {alpha}3 domain (Fig. 4Go). It is noteworthy that surface expression of Ld and Dd molecules was not appreciably reduced by single substitutions in the {alpha}3 domain (14,27). Furthermore, the double mutation of D227K and E229K of Ld was found to more completely disrupt association with TAP, TPN and CRT (43). Thus, to determine the role of this {alpha}3 site on B27 surface expression, etB27 D227K,E229K mutant molecules were expressed in HeLa cells. As shown in Fig. 1Go(f), etB27 molecules with the {alpha}3 mutations were expressed at a high level on the cell surface. Furthermore, the level of assembly with ß2m was very similar between etB27 D227K,E229K versus etB27 molecules (Fig. 2AGo). However, etB27 molecules with the {alpha}3 mutation had a higher percentage of peptide empty forms at the cell surface (22%) compared with etB27 molecules without the {alpha}3 mutation (4%) (shown in Fig. 1c and fGo). Furthermore, surface etB27 D227K,E229K molecules were found to be more accessible to exogenous peptide ligands (data not shown). Thus the expression defect of this {alpha}3 mutant appears to involve impaired peptide loading.



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  		Fig. 4. Location of the HLA-B27 mutations rendered as a ribbon diagram (49). The structure of B27 with a modeled peptide ligand was taken from Madden et al. (50). The putative CRT interaction site (N86) is shown in purple, whereas the putative TPN interactions sites (S132) and (D227/E229) are shown in orange. Other resides implicated in these latter two sites are shown in blue. From this perspective, ß2m (not shown) is behind the H chain. This figure was kindly generated by Michael J. Miley and Dr Daved H. Fremont.

 
To determine whether this impaired peptide loading was indicative of aberrant association with the peptide loading complex, etB27 D227K,E229K molecules were tested for their associations with TAP, TPN and CRT. As shown in reciprocal assays in Fig. 2Go(A and B), etB27 D227K,E229K molecules were found not to be associated with TAP, but were associated with CRT. Interestingly, in certain assays (e.g. Fig. 2AGo) etB27 D227K,E229K molecules were detected in association with TPN, whereas in other assays (e.g. Fig. 2BGo) no TPN association was detected. Our interpretation of these ambivalent findings is that this {alpha}3 mutation reduces association with TPN to near the threshold for detection in this assay. It is also important to note that this apparent weak association of etB27 D227K,E229K with TPN is not sufficient to promote its concomitant association with TAP. This finding suggests that class I molecules may have to achieve stable association with TPN via the {alpha}3 domain, for class I/TPN complexes to maintain association with TAP. This is an attractive model because it fits with studies of truncated forms of TPN demonstrating that membrane proximal regions of TPN determine its association with TAP (29). Furthermore, this model is consistent with results obtained with HLA-B44 and its disparate interactions with human versus mouse TPN (44). In any case, these findings with the etB27 D227K,E229K extend to a human class I molecule the importance of this site in the {alpha}3 domain for TAP/TPN association. Furthermore, the fact that {alpha}3 mutation of B27 can disrupt TPN association and not CRT association, supports the prediction that the {alpha}3 domain is a TPN interaction site (27).

Importance of residues around position 132 in the {alpha}2 domain
Initial evidence that a site in the {alpha}2 domain of the class I heavy chain determines its interaction with TAP/TPN/CRT came from studies of a human A2 molecule with an induced T134K mutation (23,24). Subsequent studies by us with the mouse Ld molecule showed that single substitutions in an extensive region of the {alpha}2 domain (residues 128–136) prevented its association with TAP/TPN/CRT (28). As shown in Fig. 4Go, this site is on a loop connecting a ß strand that next assumes a helical conformation. Furthermore, it should be noted that both A2 T134K (23,24) and Ld T132K (28) molecules also displayed reduced surface expression. To determine the importance of this site for the cell surface expression of B27 molecules, we expressed etB27 S132K molecules in HeLa cells. Surface expression of etB27 S132K molecules was impaired relative to etB27 molecules lacking the mutation in the {alpha}2 domain (Fig. 1eGo) and expression of this mutant was found to be cold-inducible (data not shown). Furthermore, etB27 S132K surface molecules had a higher percentage of peptide-empty forms expressed at the cell surface compared with etB27 molecules lacking the mutation in the {alpha}2 domain (30 versus 4% respectively) (shown in Fig 1e and cGo), suggesting suboptimal peptide loading in the ER.

To determine whether this mutation also affected association of B27 with members of the class I peptide loading complex, lysates of HeLa cells expressing etB27 S132K were analyzed by immunoprecipitation and Western blotting. As shown in reciprocal assays in Fig. 2Go(A and B), the S132K mutation ablated association of etB27 with TAP/TPN. Thus, this finding agreed with the aforementioned results obtained with A2 T134K (23,24) and Ld {alpha}2 mutants (28). Surprisingly, however, the S132K mutation did not affect the association of etB27 with CRT. This finding contrasts with earlier studies of A2 (45) and Ld (28) that demonstrated that mutations around 132–134 disrupt heavy chain association with CRT as well as TAP/tapasin. Thus B27, unlike A2 and Ld, can proficiently form stable complexes with CRT without simultaneously binding TAP/TPN. Therefore, there appear to be differences among class I molecules in regard to their dependency on CRT to maintain stable association with TPN/TAP. Furthermore, our findings with B27 indicate that the {alpha}2 residues 128–136 are not involved in H chain interaction with CRT. Therefore, H chain residues 128–136 are likely a TPN interaction site.

The initial association of CRT with HLA-B27 is dependent upon TPN
The above findings with {alpha}2 and {alpha}3 mutants indicate that CRT can associate with class I in the absence of TPN. This finding was unexpected, since studies of several other mouse and human class I molecules have demonstrated that CRT and TPN required cooperative binding to maintain high levels of steady state association with class I (28,29,41). However, reports by us and others suggest that limited mutagenesis may not totally ablate H chain interaction with TPN/TAP (26,40). Thus, to more rigorously test the TPN-dependency of CRT binding to class I, associations were compared in lysates of TPN-deficient .220-etB27 cells and TPN-positive .221-etB27 cells. As shown in Fig. 5Go, CRT was not detected in association with etB27 in .220 cells. Thus CRT association requires TPN to form stable complexes with B27. The discordance between the mutagenesis data and the .220-etB27 data is intriguing. Perhaps the weak interaction between mutant H chains and TPN is sufficient to allow CRT to join the complex. It is also possible that the relatively unique C67 residue in the B pocket of B27 is unpaired, and forms an abnormal disulfide bonded intermediate that retains CRT interaction in the lysate and not TPN interaction. In any case, our data show that the continuous presence of TPN is not required for CRT to remain bound to class I H chain. A speculative model consistent with these data, and recent reports of others, is that TPN is required for the initial binding of a suboptimal peptide used to maintain the integrity of the ligand binding site until a high-affinity peptide is available for binding (45). It could then be proposed that CRT can maintain interaction with the class I/low-affinity peptide complex in the absence of stable TPN interaction. Regardless of the validity of this model, the data presented here with .220-etB27 provide additional evidence that H chain mutations are leaky in their interaction with members of the peptide loading complex. Thus given the cooperative nature of the association of CRT and TPN with class I, mutations are clearly advantageous for defining specific sites of interaction.



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  		Fig. 5. CRT association with HLA-B27 is TPN-dependent. Lysates of .221-etB27 and .220-etB27 cells were precipitated with mAb ME-1 or 64-3-7 as indicated along the top. Respective precipitates were then blotted with anti-CRT (PA1-903; Affinity BioReagents), or anti-etB27 H chain (mAb 64-3-7) as indicated along the side. Note that CRT was only detected in TPN+ .221-etB27 cells and not TPN– .220-etB27 cells. Similar findings were seen in three separate experiments.

 
In summary, we show here that B27 molecules can be tagged with a novel epitope specific for their open conformation. Furthermore, using this approach we tested B27 molecules to determine whether they interact with the various components of the peptide loading complex in the ER in a manner similar to other human and mouse class I alleles. Like other mouse and human class I alleles studied thus far, HLA-B27 molecules appear to use at least three sites, one in each of the extracellular domains ({alpha}1, {alpha}2 and {alpha}3), to interact with components of the peptide loading complex (Fig. 4Go). Based on these findings with HLA-B27, we propose that TPN interacts with the {alpha}2 and {alpha}3 sites of the class I H chain, and that the {alpha}3 site may be more important for TPN-mediated TAP association. By contrast, the {alpha}2 site may be important for TPN monitoring an H chain conformational change when peptide binds. Indeed, a homologous loop on the opposing side of the H chain in the {alpha}1 domain is known to change conformation when peptide binds (32). Furthermore, the peptide induced change in the {alpha}2 site (residues 128–136) could be related to how the peptide specifically interacts with the F pocket of the peptide binding groove of the H chain as proposed by Tim Elliot (46). In this model the introduction of the C-terminal end of the peptide into the F pocket is accompanied by formation of hydrogen bonds that require the displacement of the {alpha}2 helix toward the cleft. This displacement could potentially alter the conformation of residues 128–136 that in turn induce H chain release from TPN. This model could explain why mutations or polymorphisms of HLA residues 115 and 116 (that are in or near the F pocket), have been found to influence class I association with the peptide loading complex (11,47,48). Based on additional findings reported here, we propose that CRT interacts via the {alpha}1 H chain glycan and not the {alpha}2 or {alpha}3 sites, and that CRT interaction with the H chain {alpha}1 glycan is TPN dependent initially, but may not require continuous TPN. From a spatial standpoint this is an attractive model, because the {alpha}2 and {alpha}3 sites are located on a common plane of the class I H chain (Fig. 4Go). Furthermore, residue N86 is spatially removed from the common plane shared by the {alpha}2 and {alpha}3 sites, thus allowing TPN and CRT to bind simultaneously to the class I heavy chain. Importantly, in this model both CRT and TPN are bound close to the peptide binding site of class I, whereby they could facilitate the binding of high-affinity ligands and/or monitor the release of class I from the loading complex once a high-affinity peptide has bound.


   Acknowledgments
 
We thank Dr Joel Taurog for the generous gift of the B*2705 cDNA, Michael J. Miley and Dr Daved Fremont for generating Fig. 4Go, and Susan Harris for Figs 2 and 3GoGo. This work was supported by National Institutes of Health Grants AI19876 and AI42793 (to T. H. H.), AI01498 (to M. R. H.), GM57428 and LB595/Cattlemen's Grant (to J. C. S.) and C. M. H, Y. Y. L. Y and L. L. were supported by training grant T32AI07163.


   Abbreviations
 
ß2m ß2-microglobulin
CRT calreticulin
CXN calnexin
ER endoplasmic reticulum
etB27 epitope-tagged B27
TAP transporter associated with antigen processing
TPN tapasin

   Notes
 
Transmitting editor: R. M. Steinman

Received , accepted 4 July 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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