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To whom correspondence may be addressed: Faculty of Medicine, University of Southampton, Somers Cancer Sciences Bldg. MP824, Southampton SO16 6YD, United Kingdom. Tel.: 44-23-8079-6193; Fax: 44-23-8079-5152
MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.
Background: Tapasin edits the MHC I peptide repertoire and is highly polymorphic in birds but not mammals.
Results: Two chicken MHC I alleles differ in peptide binding properties and participate in an allele-specific interaction with tapasin.
Conclusion: Tapasin-MHC alleles have co-evolved by balancing interaction characteristics against MHC peptide-binding ability.
Significance: Variations in the functional attributes of tapasin and MHC I alleles determine effective antigen presentation.
MHC I molecules help protect the host against infection and cancer by binding and presenting peptides to cytotoxic T cells. The peptides displayed at the cell surface are usually of intracellular origin and are loaded into MHC I molecules in the endoplasmic reticulum (ER)
The abbreviations used are: ER, endoplasmic reticulum; TAP, transporter associated with antigen presentation; SPR, surface plasmon resonance; mP, millipolarization units; RU, response units; TAMRA, tetramethylrhodamine.
with the assistance of the proteins that constitute the peptide loading complex. The peptide loading complex assembles around the peptide transporter associated with antigen presentation (TAP) with MHC I tethered to TAP via tapasin (
MHC I molecules are thought to load an optimal peptide cargo in two stages, where a low affinity peptide cargo is initially bound, probably representing the most abundant peptides in the ER, and then exchanged for a higher affinity cargo (
). Tapasin enhances MHC I peptide loading in several ways: tapasin localizes and stabilizes unloaded MHC I molecules at the site of peptide import, and the interaction between tapasin and TAP stabilizes the TAP transporter, thus increasing peptide supply. Perhaps most significant is that tapasin increases not just the rate and extent of peptide loading but the discrimination that occurs between peptides for binding (
). The first involves the N-terminal tapasin domain, which is thought to interact with a loop underlying the short α2-1 helix of the MHC I peptide binding groove. The second involves the membrane-proximal tapasin domain, which is thought to interact with a loop in the MHC I α3 domain. The mechanism by which tapasin achieves high affinity peptide selection is poorly understood but may involve stabilization of MHC I conformations where iterative cycles of peptide binding and release occur that only high affinity peptides withstand. Variation in the dependence of MHC I alleles upon tapasin may reflect differences in the propensity for MHC I molecules to adopt conformations that facilitate peptide binding and exchange.
Tapasin is evolutionarily well conserved; however, the location of the gene within the MHC and its allelic diversity differs between mammals and non-mammals. In most mammals, the tapasin gene is in the extended class II region, far from the multiple MHC I loci (
). Thus, it seems likely that in most mammals, tapasin and TAP function universally for whichever MHC I molecules are expressed.
The best characterized non-mammalian MHC is that of the chicken, which has been described as minimal and essential, with the tapasin, TAP, and MHC I loci in a small and simple region virtually never disrupted by recombination (
A. van Hateren, C. Tregaskes, R. Carter, A. P. Williams, J. P. Jacob, T. Elliott, and J. Kaufman, manuscript in preparation.
Thus, it seems likely that different chicken haplotypes use functionally distinct combinations of TAP and tapasin alleles, with optimal peptide loading resulting from alleles of these proteins that have evolved within stable haplotypes to share complementary functional attributes.
We sought to test this hypothesis by comparing the functional attributes of the tapasin and MHC I alleles that are expressed in the B15 and B19 haplotypes. The dominantly expressed MHC I molecules in these haplotypes, BF2*1501 and BF2*1901, are very similar in sequence (
) but are expressed in haplotypes that encode different tapasin alleles. Thus, BF2*1501 is expressed with Tapasin*15, whereas BF2*1901 is expressed with Tapasin*12 (the tapasin allele found in both the B12 and B19 haplotypes).4 Intriguingly, two of the eight polymorphisms between BF2*1501 and BF2*1901 are at regions that are thought to bind tapasin directly. We therefore sought to determine how the peptide binding properties and the ability to bind tapasin differ between BF2*1501 and BF2*1901, the contribution that polymorphic amino acids thought to contact tapasin have on these functional attributes, and whether tapasin alleles possess different functional properties.
DNA encoding amino acids 1–271 of the mature BF2*1501 and BF2*1901 proteins was amplified by PCR with primers introducing 5′ NdeI and 3′ NcoI sites and cloned into pET22b plasmid (Invitrogen). The Fos leucine zipper sequence (GGSGG linker, thrombin site, and Fos leucine peptide) was amplified by PCR from HLA-B*08-fos DNA (
) using primers introducing 5′ NcoI and 3′ HindIII sites and subsequently cloned into the BF2-containing plasmid. Site-directed mutants were produced by PCR methods analogous to those recommended in the QuikChange mutagenesis kit (Stratagene).
DNA encoding amino acids 1–271 of mature BF2*1501 and BF2*1901 proteins was amplified by PCR from BF2-fos DNA using primers that replaced the 3′ NcoI site with a stop codon followed by a HindIII site and cloned into pET22b plasmid.
Peptide-loaded BF2 Complexes
Peptide-loaded BF2-fos or BF2 complexes were obtained by refolding solubilized inclusion bodies of BF2-fos or BF2 heavy chains with chicken β2-microglobulin (as described (
) was replaced by PCR mutagenesis with an HA epitope. The modified Jun leucine zipper sequence (GGSGG linker, thrombin site, Jun leucine peptide, HA tag, and stop codon) was amplified by PCR using primers introducing 5′ EcoRI and 3′ XbaI sites and cloned into tapasin-containing pMT/BiP plasmid. Stable polyclonal transfectants of S2 cells were obtained by co-transfecting 1 μg of tapasin-jun DNA with 50 ng of pCoHygro plasmid (Invitrogen) using Fugene 6 (Roche Applied Science), and hygromycin selection. Transfectants were adapted to Express 5 serum-free medium (Invitrogen), and tapasin-jun expression was induced with 500 μm CuSO4. Supernatants were harvested 3 days later and frozen with 10% glycerol or purified immediately using anti-HA-agarose (Sigma) and eluted either by brief exposure to glycine, pH 2.5, with immediate neutralization or by the addition of HA peptide. Tapasin-jun was purified to >95%, as ascertained by SDS-PAGE, and was dialyzed against 25 mm Tris, pH 8, 150 mm sodium chloride, 50 mml-arginine, 50 mml-glutamic acid, 10% glycerol.
Biotinylated Tapasin-jun Proteins
PCR mutagenesis was used to insert a BirA motif (GLNDIFEAQKIEWHE) between the HA tag and stop codon of Tapasin*12-jun and Tapasin*15-jun. To enable in vivo biotinylation, the E. coli birA gene with the KDEL ER retention motif was amplified by PCR from pDisplay birA (
) using primers introducing a 5′ BglII site followed by a Myc epitope and a 3′ EcoRI site and was then cloned into pMT/BiP. Stable polyclonal co-transfectants of S2 cells were obtained by co-transfecting 0.5 μg of tapasin-jun DNA, 0.5 μg of myc-birA DNA, and 50 ng of pCoHygro plasmid. Protein expression was induced in Express 5 serum-free medium supplemented with 10 μm biotin.
Fluorescence Polarization Experiments
The Affinity at Which KRLIGK*RY Is Bound by BF2-fos Molecules
A final concentration of 0.3 μm BF2*15fos or BF2*19fos molecules was exposed to ∼360-nm light for 20 min at 4 °C. Various concentrations of KRLIGK*RY (where K* represents 5′-TAMRA-labeled lysine) peptide were then added to aliquots of the empty BF2-fos molecules, each in a total volume of 60 μl. Fluorescence polarizations measurements were taken after being left at room temperature for ∼22 h using an Analyst AD (Molecular Devices) with 530-nm excitation and 580-nm emission filters and 561-nm dichroic mirror. Binding of KRLIGK*RY is reported in millipolarization units (mP) and is obtained from the equation, mP = 1000 × (S − G × P)/(S + G × P), where S and P are background-subtracted fluorescence count rates (S = polarized emission filter is parallel to the excitation filter; P = polarized emission filter is perpendicular to the excitation filter), and G (grating) is an instrument- and assay-dependent factor.
Association Rate Measurements
For association rate measurements (Fig. 2b), empty BF2-fos complexes were obtained by exposing purified BF2-fos complexes to ∼360-nm light for 20 min at 4 °C. 0.375 μm KRLIGK*RY was added to 0.65 μm empty BF2-fos complexes, and binding was followed. In Fig. 2, g and h, 0.026 μm KRLIGK*RY was added to 0.45 μm empty BF2-fos complexes, and KRLIGK*RY binding was followed in the presence or absence of 0.25 μm tapasin-jun proteins. In Fig. 3, a–c, 0.1 μm KRLIGK*RY was added to 0.3 μm empty BF2-fos complexes, and KRLIGK*RY binding was followed in the presence or absence of 0.25 μm Tapasin*21jun protein.
Dissociation Rate Measurements
For dissociation rate measurements, 0.5 μm empty BF2-fos molecules were allowed to bind 0.25 μm KRLIGK*RY, and then dissociation was followed after the addition of a 500× molar excess of KRLIGKRY with or without 0.25 μm tapasin-jun proteins. All experiments were conducted at room temperature in duplicate and used PBS supplemented with 0.5 mg/ml bovine γ-globulin (Sigma).
Dissociation data from ∼24 h was processed in GraphPad Prism using one-phase exponential decay non-linear regression. The rate of peptide dissociation was taken as the calculated half-life of dissociation. Tapasin specific activity was calculated in four steps: 1) to calculate “tapasin rate enhancement,” the half-life of uncatalyzed dissociation was divided by the half-life of tapasin-catalyzed dissociation; 2) the “tapasin bonus” was the tapasin rate enhancement minus 1 (to account for the uncatalyzed reaction being divided by uncatalyzed reaction producing a tapasin rate enhancement of 1); 3) “tapasin catalysis,” the number of units of tapasin catalysis, was calculated assuming that one unit of tapasin allows the catalyzed half-life to be reached in half the time of uncatalyzed dissociation; and 4) specific activity was calculated as the number of units of tapasin activity divided by the molar concentration of tapasin-jun. Paired two-tailed t tests (GraphPad Prism) were performed to ascertain whether differences between groups of results were statistically significant, with a p value of 0.05 denoting statistically significant differences.
Surface Plasmon Resonance (SPR) Assays
Binding studies were performed at 25 °C using a Biacore T100, streptavidin chips, and HBS EP+ buffer (GE Healthcare) at a 30 μl/min flow rate. Biotinylated Tapasin*12-jun and Tapasin*15-jun proteins were immobilized on different flow cells to densities that varied between experiments in the range of ∼200–1000 RU. Monomeric BF2 complexes were repurified by size exclusion chromatography in HBS or HBS EP+ buffer 1 day before SPR and stored at 4 °C. Repurified BF2 complexes were UV-exposed for 20 min at 4 °C immediately before SPR. Kinetic rate constants were calculated from five sequential injections (2 min each or in one experiment 2.5 min) of BF2 proteins within the range of 0.37–6 μm. Regeneration of the chip was performed with a 5–10-min injection of 50 μm KRLIGKRY peptide in HBS EP+ buffer supplemented with 0.5 m NaCl to remove tapasin-bound peptide-empty BF2 molecules. Sensorgrams were corrected for bulk refractive index changes and nonspecific binding using a blank flow cell. All data were double referenced using responses from blank injections with running buffer. There was no evidence in any experiment that the binding of BF2 to tapasin was mass transport-limited. Data were processed using BiaEvaluation software (GE Healthcare) using “heterologous ligand” interaction model fitting, which fit the data better than “one-to-one” interaction models. We assume that this is because the tapasin-jun preparations contained a small proportion of protein that bound BF2 protein nonspecifically. The kinetic rates reported were therefore corrected for this nonspecific binding as described in the legend for Fig. 4. Unpaired two-tailed t tests (GraphPad Prism) were performed to ascertain whether differences between groups of results were statistically significant, with a p value of 0.05 denoting statistically significant differences.
Cellular Mismatching Experiments
BF2-myc DNA Constructs
The Myc epitope with 5′ EcoRI and 3′ stop codon followed by an XbaI site was created by annealing oligonucleotides and cloned into pcDNA3.1+ plasmid (Invitrogen). DNA encoding full-length BF2*1501 or BF2*1901 proteins was amplified by PCR with primers introducing a 5′ HindIII site and replacing the stop codon with an EcoRI site and was cloned into the myc-containing pcDNA3.1+ plasmid. Transfections of TG15 cells (reticuloendotheliosis virus-transformed lymphocytes from a homozygous B15 chicken (
)) were performed using a Nucleofector (Amaxa). Stable transfectants were cloned from single cells under G418 selection.
35S Pulse-Chases, Myc Immunoprecipitation, and Endoglycosidase H Digestions
5 × 106 cells were incubated at 41 °C in a 5% CO2 incubator for 45–60 min in cysteine- and methionine-free RPMI supplemented with 10% dialyzed FCS. Cells were labeled by the addition of 100–200 μCi of 35S Promix (Amersham Biosciences) for 20–30 min. The chase was initiated by 10-fold dilution in prewarmed, CO2-equilibrated RPMI supplemented with 10% FCS, 2 mm cysteine, and 2 mm methionine or by brief centrifugation and resuspension in the above medium. Aliquots were taken as indicated and lysed in 100 μl of ice-cold radioimmune precipitation assay buffer (Sigma) supplemented with 10 mm iodoacetamide and 4 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Roche Applied Science). Lysates were centrifuged at 16,000 × g at 4 °C for 15 min to remove nuclei and other subcellular structures and were then precleared with 50 μl of 50% Sepharose 4B and rotated at 4 °C for at least 1 h. Precleared samples were mixed with 50 μl of washed myc-agarose (Sigma) for at least 1 h at 4 °C before extensive washing in lysis buffer. Proteins were eluted in 100 mm sodium acetate, pH 5.4, 0.02% SDS, 100 mm β-mercaptoethanol, 4 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 10 mm iodoacetamide; heated at 85 °C for 5 min; divided as indicated; and digested or mock-digested with 5 milliunits of endoglycosidase H (Roche Applied Science) at 37 °C overnight. Samples were separated by SDS-PAGE, fixed, soaked in Amplify (Amersham Biosciences), and exposed to phosphor screens.
The effect of tapasin on MHC I peptide loading and editing has been intensively researched (
). Studies of mammalian MHC I molecules show that some MHC I alleles are more effective at loading an optimal peptide cargo independently of tapasin, and thus there is diversity in the extent that tapasin enhances peptide loading. Our comparison of BF2*1501 and BF2*1901 showed that this is also the case in the chicken MHC, where BF2*1501 is more effective than BF2*1901 at loading peptides and undergoing peptide dissociation without tapasin, and consequently BF2*1501 benefits less from the action of tapasin. Thus, BF2*1501 and BF2*1901 bear certain similarities in their loading properties to HLA-B*44:05 and B*44:02, which may define the extremes of tapasin dependence in mammals. This suggests that efficient self-loading and significant tapasin enhancement of peptide loading are fundamentally opposing characteristics of MHC I function. There may have been several evolutionary pressures that led to MHC I alleles varying in their dependence on tapasin to achieve optimal peptide loading. For example, MHC alleles with the ability to self-load efficiently may have arisen in response to viral subversion of tapasin (
). Therefore, it is possible that tapasin has facilitated diversification of the MHC I peptide binding groove by allowing “new” MHC alleles to function even when the amino acid changes that allow them to present new epitopes might destabilize the peptide-receptive protein and render the new MHC allele unable to self-load. Tapasin may therefore allow the spread of such MHC alleles to confer survival advantage. Thus, although BF2*1501 and BF2*1901 are quite similar in sequence and in peptide-binding specificity, our findings suggest that these alleles have evolved to use different peptide loading mechanisms; whereas BF2*1901 appears to have evolved to benefit from the loading enhancement that tapasin affords, BF2*1501 appears to have evolved to self-load efficiently.
Interestingly, we found that in the absence of tapasin, the rate at which KRLIGK*RY dissociated from the position 220 mutants of both BF2 alleles was significantly diminished in comparison with the WT molecules. However, we did not find such alterations for the double position 126 and 220 mutants. This suggests that a functional relationship exists between position 220 and the peptide-binding domain and position 126 in particular, which helps to define intrinsic peptide binding properties. It seems likely that β2-microglobulin may also participate in this intramolecular communication (
). Also supporting the concept that peptide binding properties are collectively defined by the whole MHC molecule is the finding that exchanging positions 126 and 220 improved the rate at which empty BF2*19fos-Double molecules bound peptide without tapasin.
Our in vitro analysis of the double mutants suggests that BF2 positions 126 and 220 exert significant influence on the ability of tapasin to function. We found that tapasin enhancement of KRLIGK*RY dissociation was greatest for both BF2-fos alleles when the residues derived from BF2*1501 were present (i.e. BF2*15fos and BF2*19fos-Double) and that both positions 126 and 220 independently contribute to influence the extent of tapasin function. The finding that tapasin function was influenced by BF2 position 220 strongly suggests that the membrane-proximal interaction between tapasin and BF2 allows tapasin to exert allosteric control of the peptide binding groove in order to enhance peptide dissociation. We suggest that the membrane-proximal MHC I-tapasin interaction allows the MHC I peptide binding domain to become more ordered and more readily adopt conformations conducive to enhancing peptide dissociation. This finding is consistent with previous observations, including the ablation of tapasin-jun function for the HLA-B*08fos E222K mutant (
), which collectively suggest that even when artificially tethered, the membrane-proximal tapasin-MHC interaction is of key significance. Interestingly, there is a prominent cluster of polymorphisms in the immunoglobulin domain of chicken tapasin that may influence this membrane-proximal interaction in an allele-specific manner (
). We did not find any evidence that tapasin catalyzed the rate at which peptides were bound by empty BF2 molecules, although we were unable to observe the earlier stages of the peptide binding reactions where catalysis may be most evident. It was, however, clear that tapasin allowed peptide binding reactions to reach equilibrium earlier. Combined with the finding that dissociation of KRLIGK*RY from BF2 molecules was catalyzed by tapasin, we suggest that tapasin-mediated peptide editing may result from tapasin minimizing the time that MHC molecules are unreceptive to peptide loading combined with increasing peptide dissociation rates, allowing many peptides to be sampled, consistent with the proposed “two-step” loading process (
Direct binding assays confirmed that peptide-deficient MHC I molecules are the preferred substrates of tapasin. Binding affinities between empty BF2 molecules and tapasin varied between 0.1 and 1.5 × 106m, depending on the combination of alleles. Thus, the tapasin-MHC I interaction is relatively weak, consistent with suggestions from previous studies and the requirement for leucine zipper sequences to be used in vitro (
). We found BF2*1501 and BF2*1901 differ in their interactions with tapasin, with empty BF2*1901 molecules binding tapasin faster than BF2*1501, forming higher affinity interactions than empty BF2*1501 molecules. If the peptides that become bound by MHC I molecules are of high affinity, peptide-loaded MHC I molecules are poor substrates for tapasin, and such molecules will quickly dissociate from the peptide loading complex. However, if peptides are bound and then rapidly released, empty MHC I molecules either remain bound by tapasin or rebind tapasin to start another cycle. Thus, MHC I alleles like BF2*1901 that bind tapasin with fast interaction kinetics might experience more peptide sampling cycles, allowing greater potential for tapasin to catalyze peptide exchange.
Our analysis of the maturation of myc-tagged BF2 molecules expressed in B15 cells showed that BF2 maturation can be compromised by the mismatching of tapasin and BF2 alleles. The impaired maturation of BF2*19myc molecules was surprising, given our in vitro results, suggesting that tapasin alleles function in an allele-dependent way in vivo and/or that BF2-tapasin alleles interact in an allele-dependent manner in vivo. It is tempting to speculate that allelic differences in tapasin binding or function may only become apparent when tapasin is conjugated to ERp57, although chicken tapasin lacks a residue equivalent to cysteine 95, to which ERp57 conjugates in mammals (
). Importantly, we found that exchange of positions 126 and 220 between BF2 alleles influenced maturation efficiency. This in vivo mismatching of tapasin and BF2 alleles and the influence of BF2 positions 126 and 220 on maturation efficiency provide the best support for the notion that chicken tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading.
We suggest that in different MHC haplotypes, the tapasin and BF2 alleles have co-evolved to balance a number of functional attributes in different ways. Thus, in haplotypes like B19, where the MHC alleles have poor self-loading ability and whose amino acids at positions 126 and 220 confer poor tapasin functionality in comparison with their BF2*1501 counterparts, these BF2 alleles appear to have evolved to rely upon tapasin for efficient peptide loading; thus, these MHC I alleles bind tapasin quickly and with high affinity. This therefore allows plentiful opportunity for tapasin to load and edit the peptide cargo. In contrast, in other MHC haplotypes, such as B15, where the BF2 alleles are intrinsically more efficient at loading an optimal peptide cargo and whose amino acids at BF2 positions 126 and 220 confer greater tapasin functionality, there is likely to have been less pressure for the tapasin and BF2 interaction to evolve to interact rapidly and with high affinity, and there is less scope for tapasin to enhance peptide loading efficiency.
In conclusion, the BF2 alleles of the B15 and B19 haplotypes have different intrinsic peptide binding properties. These BF2 alleles also differ in their interactions with tapasin and in the extent that they benefit from tapasin. Our comparison of WT BF2 molecules and position 126 and 220 mutants shows that these polymorphisms are relevant for tapasin function and can profoundly influence binding to tapasin. We found BF2 position 220, located in the MHC I α3 domain, to be a key determinant of both intrinsic peptide binding efficiency and the ability of tapasin to function, suggesting that tapasin exerts allosteric control on MHC I peptide binding properties through this site. We found that when tapasin and BF2 alleles were mismatched in vivo that MHC I maturation could be severely impaired but that exchange of polymorphic tapasin contacts at positions 126 and 220 between the BF2 alleles alleviated this impairment. Collectively, we believe that this evidence supports the notion that efficient peptide loading of chicken MHC I requires tapasin and BF2 alleles from the same haplotype, as previously proposed (
), which have co-evolved to balance tapasin-BF2 interaction characteristics against MHC I peptide binding properties.
We thank Dr. Patrick Duriez and Leon Douglas of the Cancer Research UK Core Protein Production Facility for assistance with protein purification, Drs. Ian Mockridge and Ruth French for SPR expertise, Professor Joern Werner for helpful advice, Professor Marlene Bouvier for providing HLA-B*08-fos and human tapasin-jun, Dr. Mark Howarth for the gift of pDisplay birA, and Dr. Nick van Hateren for help producing figures and other invaluable help and advice. We thank Dr. Denise Boulanger for critical reading of the manuscript.
Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP.