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A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins*

  • Andy van Hateren
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,

    the Institute for Animal Health, Compton RG20 7NN, United Kingdom
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  • Rachel Carter
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,
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  • Alistair Bailey
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,
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  • Nasia Kontouli
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,
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  • Anthony P. Williams
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,
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  • Jim Kaufman
    Correspondence
    To whom correspondence may be addressed: Dept. of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom. Tel.: 44-1223-766423; Fax: 44-1223-333346
    Affiliations
    the Institute for Animal Health, Compton RG20 7NN, United Kingdom

    the Departments of Pathology and Veterinary Medicine, University of Cambridge, Cambridge CB2 1QP, United Kingdom
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  • Tim Elliott
    Correspondence
    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
    Affiliations
    From the Faculty of Medicine and Institute for Life Science, University of Southampton, Southampton SO16 6YD, United Kingdom,
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  • Author Footnotes
    * This work was supported by Cancer Research UK Program Grant C7056A (to T. E.).
Open AccessPublished:September 27, 2013DOI:https://doi.org/10.1074/jbc.M113.474031
      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.

      Introduction

      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 (
      • Sadasivan B.
      • Lehner P.J.
      • Ortmann B.
      • Spies T.
      • Cresswell P.
      Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP.
      ), ERp57 conjugated to tapasin (
      • Dick T.P.
      • Bangia N.
      • Peaper D.R.
      • Cresswell P.
      Disulfide bond isomerization and the assembly of MHC class I-peptide complexes.
      ), and calreticulin interacting with both MHC I and ERp57 to form a synergistically strong network of individually weak intermolecular interactions (
      • Van Hateren A.
      • James E.
      • Bailey A.
      • Phillips A.
      • Dalchau N.
      • Elliott T.
      The cell biology of major histocompatibility complex class I assembly. Towards a molecular understanding.
      ).
      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 (
      • Lewis J.W.
      • Elliott T.
      Evidence for successive peptide binding and quality control stages during MHC class I assembly.
      ). 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 (
      • Williams A.P.
      • Peh C.A.
      • Purcell A.W.
      • McCluskey J.
      • Elliott T.
      Optimization of the MHC class I peptide cargo is dependent on tapasin.
      ). This “editing” function ensures that MHC I becomes loaded with high affinity peptides, which allow prolonged cell surface expression (
      • Howarth M.
      • Williams A.
      • Tolstrup A.B.
      • Elliott T.
      Tapasin enhances MHC class I peptide presentation according to peptide half-life.
      ), rather than loading more prevalent low affinity peptides (
      • Williams A.P.
      • Peh C.A.
      • Purcell A.W.
      • McCluskey J.
      • Elliott T.
      Optimization of the MHC class I peptide cargo is dependent on tapasin.
      ,
      • Wearsch P.A.
      • Cresswell P.
      Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer.
      ). Mammalian MHC I alleles differ in their dependence upon tapasin for high affinity peptide loading (
      • Greenwood R.
      • Shimizu Y.
      • Sekhon G.S.
      • DeMars R.
      Novel allele-specific, post-translational reduction in HLA class I surface expression in a mutant human B cell line.
      ,
      • Peh C.A.
      • Burrows S.R.
      • Barnden M.
      • Khanna R.
      • Cresswell P.
      • Moss D.J.
      • McCluskey J.
      HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading.
      ). Whereas alleles like HLA-B*44:02 rely upon tapasin, alleles such as HLA-B*44:05 do not and efficiently self-load their peptide repertoire (
      • Williams A.P.
      • Peh C.A.
      • Purcell A.W.
      • McCluskey J.
      • Elliott T.
      Optimization of the MHC class I peptide cargo is dependent on tapasin.
      ).
      Two sites of interaction have been identified between tapasin and MHC I (
      • Van Hateren A.
      • James E.
      • Bailey A.
      • Phillips A.
      • Dalchau N.
      • Elliott T.
      The cell biology of major histocompatibility complex class I assembly. Towards a molecular understanding.
      ,
      • Lewis J.W.
      • Elliott T.
      Evidence for successive peptide binding and quality control stages during MHC class I assembly.
      ,
      • Dong G.
      • Wearsch P.A.
      • Peaper D.R.
      • Cresswell P.
      • Reinisch K.M.
      Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer.
      ,
      • Yu Y.Y.
      • Turnquist H.R.
      • Myers N.B.
      • Balendiran G.K.
      • Hansen T.H.
      • Solheim J.C.
      An extensive region of an MHC class I α2 domain loop influences interaction with the assembly complex.
      ,
      • Suh W.K.
      • Derby M.A.
      • Cohen-Doyle M.F.
      • Schoenhals G.J.
      • Früh K.
      • Berzofsky J.A.
      • Williams D.B.
      Interaction of murine MHC class I molecules with tapasin and TAP enhances peptide loading and involves the heavy chain α3 domain.
      ). 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 (
      • The MHC sequencing consortium
      Complete sequence and gene map of a human major histocompatibility complex.
      ). Few polymorphisms have been documented in tapasin or TAP, with no functional distinctions between alleles (
      • Copeman J.
      • Bangia N.
      • Cross J.C.
      • Cresswell P.
      Elucidation of the genetic basis of the antigen presentation defects in the mutant cell line. 220 reveals polymorphism and alternative splicing of the tapasin gene.
      ,
      • Furukawa H.
      • Kashiwase K.
      • Yabe T.
      • Ishikawa Y.
      • Akaza T.
      • Tadokoro K.
      • Tohma S.
      • Inoue T.
      • Tokunaga K.
      • Yamamoto K.
      • Juji T.
      Polymorphism of TAPASIN and its linkage disequilibria with HLA class II genes in the Japanese population.
      ,
      • Herberg J.A.
      • Sgouros J.
      • Jones T.
      • Copeman J.
      • Humphray S.J.
      • Sheer D.
      • Cresswell P.
      • Beck S.
      • Trowsdale J.
      Genomic analysis of the Tapasin gene, located close to the TAP loci in the MHC.
      ). 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 (
      • Kaufman J.
      • Milne S.
      • Göbel T.W.
      • Walker B.A.
      • Jacob J.P.
      • Auffray C.
      • Zoorob R.
      • Beck S.
      The chicken B locus is a minimal essential major histocompatibility complex.
      ,
      • Wong G.K.
      • Liu B.
      • Wang J.
      • Zhang Y.
      • Yang X.
      • Zhang Z.
      • Meng Q.
      • Zhou J.
      • Li D.
      • Zhang J.
      • Ni P.
      • Li S.
      • Ran L.
      • Li H.
      • Li R.
      • Zheng H.
      • Lin W.
      • Li G.
      • Wang X.
      • Zhao W.
      • Li J.
      • Ye C.
      • Dai M.
      • Ruan J.
      • Zhou Y.
      • Li Y.
      • He X.
      • Huang X.
      • Tong W.
      • Chen J.
      • Ye J.
      • Chen C.
      • Wei N.
      • Dong L.
      • Lan F.
      • Sun Y.
      • Yang Z.
      • Yu Y.
      • Huang Y.
      • He D.
      • Xi Y.
      • Wei D.
      • Qi Q.
      • Li W.
      • Shi J.
      • Wang M.
      • Xie F.
      • Zhang X.
      • Wang P.
      • Zhao Y.
      • Li N.
      • Yang N.
      • Dong W.
      • Hu S.
      • Zeng C.
      • Zheng W.
      • Hao B.
      • Hillier L.W.
      • Yang S.P.
      • Warren W.C.
      • Wilson R.K.
      • Brandstrom M.
      • Ellegren H.
      • Crooijmans R.P.
      • van der Poel J.J.
      • Bovenhuis H.
      • Groenen M.A.
      • Ovcharenko I.
      • Gordon L.
      • Stubbs L.
      • Lucas S.
      • Glavina T.
      • Aerts A.
      • Kaiser P.
      • Rothwell L.
      • Young J.R.
      • Rogers S.
      • Walker B.A.
      • van Hateren A.
      • Kaufman J.
      • Bumstead N.
      • Lamont S.J.
      • Zhou H.
      • Hocking P.M.
      • Morrice D.
      • de Koning D.J.
      • Law A.
      • Bartley N.
      • Burt D.W.
      • Hunt H.
      • Cheng H.H.
      • Gunnarsson U.
      • Wahlberg P.
      • Andersson L.
      • Kindlund E.
      • Tammi M.T.
      • Andersson B.
      • Webber C.
      • Ponting C.P.
      • Overton I.M.
      • Boardman P.E.
      • Tang H.
      • Hubbard S.J.
      • Wilson S.A.
      • Yu J.
      • Yang H.
      A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms.
      ). In contrast to most mammals, the highly polymorphic chicken TAP genes have distinct transport specificities that match the peptide motif of the single dominantly expressed MHC I (BF2) molecule (
      • Walker B.A.
      • Hunt L.G.
      • Sowa A.K.
      • Skjødt K.
      • Göbel T.W.
      • Lehner P.J.
      • Kaufman J.
      The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes.
      ). We have also found that chicken tapasin is highly polymorphic and moderately divergent in sequence (

      Van Hateren, A., (2006) Function of chicken tapasin in MHC class I antigen presentation. Ph.D. thesis, University of Southampton, Southampton, UK.

      ).
      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 (
      • Shaw I.
      • Powell T.J.
      • Marston D.A.
      • Baker K.
      • van Hateren A.
      • Riegert P.
      • Wiles M.V.
      • Milne S.
      • Beck S.
      • Kaufman J.
      Different evolutionary histories of the two classical class I genes BF1 and BF2 illustrate drift and selection within the stable MHC haplotypes of chickens.
      ) (Table 1 and Fig. 1) and bind very similar peptides on the cell surfaces (
      • Wallny H.J.
      • Avila D.
      • Hunt L.G.
      • Powell T.J.
      • Riegert P.
      • Salomonsen J.
      • Skjødt K.
      • Vainio O.
      • Vilbois F.
      • Wiles M.V.
      • Kaufman J.
      Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens.
      ,
      • Kaufman J.
      • Völk H.
      • Wallny H.J.
      A “minimal essential Mhc” and an “unrecognized Mhc.” Two extremes in selection for polymorphism.
      ) 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.
      TABLE 1BF2 amino acid polymorphisms
      Amino acid position, domainBF2*1501BF2*1901
      22, α1TyrPhe
      69, α1ThrSer
      79, α1ThrIle
      95, α2LeuTrp
      111, α2SerArg
      113, α2AspTyr
      126, α2AspGly
      220, α3GlnArg
      Figure thumbnail gr1
      FIGURE 1Model of BF2*1501, depicting the location of the eight amino acids that differ between BF2*1501 and BF2*1901. The BF2*1501 structure is based upon the BF2*2101 structure (
      • Koch M.
      • Camp S.
      • Collen T.
      • Avila D.
      • Salomonsen J.
      • Wallny H.J.
      • van Hateren A.
      • Hunt L.
      • Jacob J.P.
      • Johnston F.
      • Marston D.A.
      • Shaw I.
      • Dunbar P.R.
      • Cerundolo V.
      • Jones E.Y.
      • Kaufman J.
      Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding.
      ) and is shown in a space-filling format with polymorphic amino acids shown in green. β2-Microglobulin (β2m) is shown in gray in a ribbon format, and peptide is shown in dark gray. a, side view; b, view from above the peptide binding groove. Polymorphic residue 22 is buried beneath the α1 helix. The side chains of residues 95 and 111 are on separate β strands with their side chains orientated toward each other.

      EXPERIMENTAL PROCEDURES

      Production of BF2 and Tapasin-jun Proteins

      BF2-fos Proteins

      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 (
      • Chen M.
      • Bouvier M.
      Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.
      ) 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).

      BF2 Proteins

      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 (
      • Koch M.
      • Camp S.
      • Collen T.
      • Avila D.
      • Salomonsen J.
      • Wallny H.J.
      • van Hateren A.
      • Hunt L.
      • Jacob J.P.
      • Johnston F.
      • Marston D.A.
      • Shaw I.
      • Dunbar P.R.
      • Cerundolo V.
      • Jones E.Y.
      • Kaufman J.
      Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding.
      )) and UV-labile peptide KRLIGjRY (
      • Rodenko B.
      • Toebes M.
      • Hadrup S.R.
      • van Esch W.J.
      • Molenaar A.M.
      • Schumacher T.N.
      • Ovaa H.
      Generation of peptide-MHC class I complexes through UV-mediated ligand exchange.
      ) (Peptide Synthetics; j represents 3-amino-3-(2-nitro) phenyl-propionic acid) as in Ref.
      • Garboczi D.N.
      • Hung D.T.
      • Wiley D.C.
      HLA-A2-peptide complexes. Refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.
      .

      Tapasin-jun Proteins

      DNA encoding amino acids 23–397 of the primary sequence of chicken tapasin alleles (Tapasin*02, AM403065; Tapasin*12, AM403068; Tapasin*14, AM403069; Tapasin*15, AM403070; Tapasin*21, AM403072 (

      Van Hateren, A., (2006) Function of chicken tapasin in MHC class I antigen presentation. Ph.D. thesis, University of Southampton, Southampton, UK.

      )) was amplified by PCR from cDNA using primers introducing 5′ BglII and 3′ EcoRI sites and cloned into pMT/BiP (Invitrogen). The His6 tag present in the Jun leucine zipper of human tapasin-jun (
      • Chen M.
      • Bouvier M.
      Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.
      ) 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 mm l-arginine, 50 mm l-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 (
      • Howarth M.
      • Ting A.Y.
      Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.
      ) 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.
      Figure thumbnail gr2
      FIGURE 2In vitro analysis of MHC I peptide binding characteristics. a, affinity at which KRLIGK*RY is bound by BF2*15fos or BF2*19fos molecules. The BF2-fos molecules were rendered empty by exposure to UV light and then allowed to bind different concentrations of KRLIGK*RY. Fluorescence polarization measurements were taken after ∼22 h at room temperature. Binding of KRLIGK*RY is reported in mP. Unbound KRLIGK*RY is assumed to have an mP level of 50. b, binding of KRLIGK*RY to empty BF2*15fos or BF2*19fos. c, dissociation of KRLIGK*RY. Excess unlabeled peptide (Comp) or buffer (None) was added to BF2*15fos or BF2*19fos loaded with KRLIGK*RY. d and e, comparison of catalyzed dissociation. Buffer (None) or excess unlabeled peptide with (Comp+Tpn), or without tapasin-jun (Comp) was added to BF2*15fos (d) or BF2*19fos (e) loaded with KRLIGK*RY. Tapasin*12-jun was paired with BF2*19fos, and Tapasin*15jun was paired with BF2*15fos. The data shown are from a representative experiment. f, the half-life of KRLIGK*RY dissociation measured over ∼1 day. Individual results (dots) from 10–11 experiments are shown with the (mean) average depicted as a bar. Details of calculations are provided under “Experimental Procedures.” Statistically significant differences (i.e. p < 0.05) between the indicated results are bracketed. g and h, binding of KRLIGK*RY to empty BF2*15fos (g) or BF2*19fos (h) molecules in the presence or absence of tapasin-jun. The data shown are from a representative experiment. i, tapasin-jun allele enhancement of KRLIGK*RY dissociation. Dissociation assays were conducted as in d and e, using the indicated proteins. Tapasin specific activity was calculated as described under “Experimental Procedures,” with individual results (dots) and (mean) average specific activity (bars) from four experiments.
      Figure thumbnail gr3
      FIGURE 3In vitro analysis of the peptide binding characteristics of mutant BF2-fos molecules. a, binding of KRLIGK*RY to empty WT or double mutants of BF2*15fos or BF2*19fos. b and c, binding of KRLIGK*RY to empty WT or double mutants of BF2*19fos (b) or BF2*15fos (c) molecules in the presence or absence of Tapasin*21jun. A representative experiment is shown. d and e, dissociation assays were conducted using WT and position 126 and 220 double or single mutants of BF2*15fos or BF2*19fos with or without tapasin-jun as in . Individual results (dots) and (mean) averages (bars) from 10–12 experiments are shown. Statistically significant differences (i.e. p < 0.05) between the indicated results are bracketed. d, the extent to which tapasin-jun enhanced KRLIGK*RY dissociation for each BF2-fos molecule (calculated as “tapasin bonus” under “Experimental Procedures”) was normalized to the calculated “tapasin bonus” of BF2*15fos in each experiment. The same tapasin-jun allele was used for all BF2-fos molecules within each experiment, but different tapasin-jun alleles were used in some experiments, with the majority of experiments using Tapasin*21jun (mismatched for both BF2-fos alleles), with no difference observed between tapasin-jun alleles. The experiments that included single mutants used just Tapasin*21jun for all BF2-fos molecules and fewer experiments: BF2*15fos D126G = 3, BF2*15fos Q220R = 6, BF2*19fos G126D = 5, BF2*19fos R220Q = 5. It is likely that the small magnitude of tapasin bonus that BF2*15fos experiences may confound the calculation of statistical significance from replicate experiments. e, the half-life of KRLIGK*RY dissociation for each BF2-fos molecule in the absence of tapasin measured over ∼1 day, shown as in f.

      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.
      Figure thumbnail gr4
      FIGURE 4Surface plasmon resonance assays. a, sensorgram of 20 μm empty BF2*1501 binding to Tapasin*15jun (with a binding level of 2000 RU) during a 2-min injection, slow dissociation following the injection, and faster dissociation induced by injection of 50 μm KRLIGKRY peptide coincident with the arrowhead. All data are reference flow cell-corrected. b, sensorgram showing binding of empty BF2*1501-Double (concentrations between 0.375 and 6 μm) to Tapasin*12jun (with a binding level of 1035 RU) as a red line, with the fit of the “heterologous ligand” interaction model shown as a black line. c, the “heterologous ligand” interaction model fitted to the sensorgrams (including the example shown in b) was usually attributed to one slow, high affinity component (component 1), and a faster but lower affinity component (component 2), along with a “bulk effect” contribution resulting from mismatching of the refractive indices of the running buffer and sample. We anticipate that the faster but lower affinity component (component 2) represents the specific binding of monomeric BF2 proteins to tapasin, because this component appears most similar to binding profiles that were modeled by 1:1 interaction models (data not shown) and with the expected affinity of the tapasin-MHC interaction. The slow, higher affinity component (component 1) might represent a proportion of aggregated, denatured, or inactive tapasin protein that might bind MHC proteins nonspecifically and dissociate slowly. The proportion of BF2 proteins that could be loaded with peptide was consistent with the measured total protein concentration and was comparable between the different BF2 proteins examined within each experiment (data not shown). d–f, the interaction characteristics of empty WT or double mutants (labeled dbl) of BF2*1501 or BF2*1901 binding to Tapasin*12jun or Tapasin*15jun. The kinetic attributes measured were the association rate constant (ka; d) and the dissociation rate constant (kd; e). The equilibrium dissociation constant (KD; f) calculated from the dissociation rate constant divided by the association rate constant is presented as in d and e. The data in d–f were combined from three independent experiments with a variable number of replicates of each protein combination performed within each experiment (between 1 and 4 repeats). The data constitute those that were modeled satisfactorily by the heterologous ligand interaction model, where the fast, low affinity interaction characteristics are reported, and are presented as a bar chart with the S.D. value indicated by error bars. Statistically significant differences (i.e. p < 0.05) between the indicated results are bracketed.

      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 (
      • Walker B.A.
      • Hunt L.G.
      • Sowa A.K.
      • Skjødt K.
      • Göbel T.W.
      • Lehner P.J.
      • Kaufman J.
      The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes.
      )) 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.

      DISCUSSION

      The effect of tapasin on MHC I peptide loading and editing has been intensively researched (
      • Sadasivan B.
      • Lehner P.J.
      • Ortmann B.
      • Spies T.
      • Cresswell P.
      Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP.
      ,
      • Williams A.P.
      • Peh C.A.
      • Purcell A.W.
      • McCluskey J.
      • Elliott T.
      Optimization of the MHC class I peptide cargo is dependent on tapasin.
      ,
      • Howarth M.
      • Williams A.
      • Tolstrup A.B.
      • Elliott T.
      Tapasin enhances MHC class I peptide presentation according to peptide half-life.
      ,
      • Wearsch P.A.
      • Cresswell P.
      Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer.
      ,
      • Chen M.
      • Bouvier M.
      Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.
      ). 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 (
      • Bennett E.M.
      • Bennink J.R.
      • Yewdell J.W.
      • Brodsky F.M.
      Cutting edge. Adenovirus E19 has two mechanisms for affecting class I MHC expression.
      ,
      • Lybarger L.
      • Wang X.
      • Harris M.R.
      • Virgin 4th, H.W.
      • Hansen T.H.
      Virus subversion of the MHC class I peptide-loading complex.
      ). However, it is also clear that pathogen sequence diversification has driven increased MHC I polymorphism (
      • Apanius V.
      • Penn D.
      • Slev P.R.
      • Ruff L.R.
      • Potts W.K.
      The nature of selection on the major histocompatibility complex.
      ). 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 (
      • Hee C.S.
      • Beerbaum M.
      • Loll B.
      • Ballaschk M.
      • Schmieder P.
      • Uchanska-Ziegler B.
      • Ziegler A.
      Dynamics of free versus complexed β2-microglobulin and the evolution of interfaces in MHC class I molecules.
      ). 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 (
      • Chen M.
      • Bouvier M.
      Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.
      ), 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 (

      Van Hateren, A., (2006) Function of chicken tapasin in MHC class I antigen presentation. Ph.D. thesis, University of Southampton, Southampton, UK.

      ).4
      Our analysis of empty BF2 molecules binding KRLIGK*RY showed that tapasin increased the number of peptide-loaded BF2 molecules, consistent with functions attributed to tapasin previously (
      • Williams A.P.
      • Peh C.A.
      • Purcell A.W.
      • McCluskey J.
      • Elliott T.
      Optimization of the MHC class I peptide cargo is dependent on tapasin.
      ,
      • Wearsch P.A.
      • Cresswell P.
      Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer.
      ,
      • Schoenhals G.J.
      • Krishna R.M.
      • Grandea 3rd, A.G.
      • Spies T.
      • Peterson P.A.
      • Yang Y.
      • Früh K.
      Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.
      ,
      • Rizvi S.M.
      • Raghavan M.
      Mechanisms of function of tapasin, a critical major histocompatibility complex class I assembly factor.
      ,
      • Zarling A.L.
      • Luckey C.J.
      • Marto J.A.
      • White F.M.
      • Brame C.J.
      • Evans A.M.
      • Lehner P.J.
      • Cresswell P.
      • Shabanowitz J.
      • Hunt D.F.
      • Engelhard V.H.
      Tapasin is a facilitator, not an editor, of class I MHC peptide binding.
      ). 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 (
      • Lewis J.W.
      • Elliott T.
      Evidence for successive peptide binding and quality control stages during MHC class I assembly.
      ).
      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 × 106 m, 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 (
      • Chen M.
      • Bouvier M.
      Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection.
      ). 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 (
      • Kaufman J.
      • Milne S.
      • Göbel T.W.
      • Walker B.A.
      • Jacob J.P.
      • Auffray C.
      • Zoorob R.
      • Beck S.
      The chicken B locus is a minimal essential major histocompatibility complex.
      ). 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 (
      • Kaufman J.
      • Milne S.
      • Göbel T.W.
      • Walker B.A.
      • Jacob J.P.
      • Auffray C.
      • Zoorob R.
      • Beck S.
      The chicken B locus is a minimal essential major histocompatibility complex.
      ,
      • Kaufman J.
      • Völk H.
      • Wallny H.J.
      A “minimal essential Mhc” and an “unrecognized Mhc.” Two extremes in selection for polymorphism.
      ), which have co-evolved to balance tapasin-BF2 interaction characteristics against MHC I peptide binding properties.

      Acknowledgments

      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.

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