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. 2013 Nov 8;288(45):32797-32808.
doi: 10.1074/jbc.M113.474031. Epub 2013 Sep 27.

A mechanistic basis for the co-evolution of chicken tapasin and major histocompatibility complex class I (MHC I) proteins

Andy van Hateren  1 , Rachel Carter  2 , Alistair Bailey  2 , Nasia Kontouli  2 , Anthony P Williams  2 , Jim Kaufman  3 , Tim Elliott  4
Affiliations

A mechanistic basis for the co-evolution of chicken tapasin and major histocompatibility complex class I (MHC I) proteins

Andy van Hateren et al. J Biol Chem. .

Abstract

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.

Keywords: Antigen Presentation; Chicken MHC; Co-evolution; Evolution; Immunology; Major Histocompatibility Complex (MHC); Peptide Selection; Protein-Protein Interactions; Tapasin.

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Figures

FIGURE 1.
FIGURE 1.
Model 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 (25) 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.
FIGURE 2.
FIGURE 2.
In 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 3.
FIGURE 3.
In 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 Fig. 2. 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 Fig. 2f.
FIGURE 4.
FIGURE 4.
Surface 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.
FIGURE 5.
FIGURE 5.
Comparison of the modeling of the wild-type and double mutant sensorgrams via the heterologous ligand interaction model. a, BF2*1501; b, BF2*1901; c, BF2*1501-Double; d, BF2*1901-Double. The top panels show the binding of the indicated protein (concentrations between 0.375 and 6 μm) to Tapasin*12jun (with a binding level of 1035 RU) as a colored line, with the fit of the “heterologous ligand” interaction model shown as a black line. The bottom panels show how the total binding was attributed between the two components and the bulk effect. The binding of component 2 is taken to represent the specific binding of monomeric BF2 proteins to tapasin as discussed in the legend to Fig. 4 and under “Results.”
FIGURE 6.
FIGURE 6.
Comparison of maturation of BF2*15myc, BF2*19myc, BF2*19myc-Double (a), or BF2*15myc-Double (b) in TG15 cells. Only the bands corresponding to BF2myc molecules are shown from a representative experiment. EHS, molecular weight of endoglycosidase H-sensitive, or immature, BF2 molecules; EHR, molecular weight of endoglycosidase H-resistant, or mature, BF2 molecules. The maturation of BF2*15myc and BF2*19myc molecules has been compared in more than eight experiments using two different clones, and their maturation has been compared with that of BF2*19myc-Double in at least two experiments using two different clones and with that of BF2*15myc-Double in two experiments. Comparable results were observed in each experiment. The graphs depict the rate and extent of the acquisition of resistance of the BF2myc molecules to endoglycosidase H digestion averaged from two experiments.
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