Two polymorphisms facilitate differences in plasticity between two chicken major histocompatibility complex class I proteins

Major histocompatibility complex class I molecules (MHC I) present peptides to cytotoxic T-cells at the surface of almost all nucleated cells. The function of MHC I molecules is to select high affinity peptides from a large intracellular pool and they are assisted in this process by co-factor molecules, notably tapasin. In contrast to mammals, MHC homozygous chickens express a single MHC I gene locus, termed BF2, which is hypothesised to have co-evolved with the highly polymorphic tapasin within stable haplotypes. The BF2 molecules of the B15 and B19 haplotypes have recently been shown to differ in their interactions with tapasin and in their peptide selection properties. This study investigated whether these observations might be explained by differences in the protein plasticity that is encoded into the MHC I structure by primary sequence polymorphisms. Furthermore, we aimed to demonstrate the utility of a complimentary modelling approach to the understanding of complex experimental data. Combining mechanistic molecular dynamics simulations and the primary sequence based technique of statistical coupling analysis, we show how two of the eight polymorphisms between BF2*15:01 and BF2*19:01 facilitate differences in plasticity. We show that BF2*15:01 is intrinsically more plastic than BF2*19:01, exploring more conformations in the absence of peptide. We identify a protein sector of contiguous residues connecting the membrane bound ?3 domain and the heavy chain peptide binding site. This sector contains two of the eight polymorphic residues. One is residue 22 in the peptide binding domain and the other 220 is in the ?3 domain, a putative tapasin binding site. These observations are in correspondence with the experimentally observed functional differences of these molecules and suggest a mechanism for how modulation of MHC I plasticity by tapasin catalyses peptide selection allosterically

Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity.

The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC).This work was originally supported by core funding to the Basel Institute for Immunology (which was founded and supported by F. Hoffmann-La Roche & Co. Ltd., CH-4005 Basel, Switzerland), then by core funding to the Institute for Animal Health [now re-branded the Pirbright Institute, sponsored by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK] and finally by programme grant 089305 from the Wellcome Trust to JK.This is the author accepted manuscript. The final version is available from PNAS via http://dx.doi.org/10.1073/pnas.151185911

TAPBPR bridges UDP-glucose : glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

Funding Wellcome: Senior Research Fellowship 104647, Andreas Neerincx, Louise H Boyle Royal Society: University Research Fellowship, UF100371, Janet E Deane Cancer Research UK: Programme Grant, C7056A, Andy van Hateren, Tim Elliott Deutsche Forschungsgemeinschaft: SFB 685, Nico Trautwein, Stefan Stevanović Wellcome: PhD studentship, 089563, Clemens Hermann Wellcome: Strategic Award 100140, Robin Antrobus Wellcome: Programme grant, WT094847MA, Huan Cao Acknowledgements We are extremely grateful to Peter Cresswell and Najla Arshad (Yale University School of Medicine, New Haven, CT) for valuable advice, tapasin and TAP-specific antibody reagents, and the recombinant calreticulin proteins. We thank John Trowsdale (University of Cambridge, UK) for his mentorship and critical reading of this manuscript, and Jim Kaufman (University of Cambridge, UK) for useful discussions. We also thank Yi Cao (Cranfield University, UK) for MATLAB programming for densitometry analysis, and Mark Vickers and Sadie Henderson (Scottish National Blood Transfusion Services, UK) for permitting the use of and assistance with the Amersham WB system. The reagent ARP7099 FEC peptide pool was obtained from the Centre for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), and was donated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.Peer reviewedPublisher PD

TAPBPR alters MHC class I peptide presentation by functioning as a peptide exchange catalyst.

Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system.This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.755

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms ( SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds ( a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines - in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases

Visualising tapasin- and TAPBPR-assisted editing of major histocompatibility complex class-I immunopeptidomes

Which peptides are selected for presentation by major histocompatibility complex class-I (MHC-I) molecules is a key determinant of successful immune responses. Peptide selection is co-ordinated by the tapasin and TAP Binding PRotein (TAPBPR) proteins, which ensure MHC-I molecules preferentially acquire high-affinity-binding peptides. New structural analyses have offered insight into how tapasin achieves this function within the peptide-loading complex (PLC) (comprising the Transporter associated with Antigen Presentation (TAP) peptide transporter, tapasin–ERp57, MHC-I and calreticulin), and how TAPBPR performs a peptide editing function independently of other molecules. The new structures reveal nuances in how tapasin and TAPBPR interact with MHC-I, and how calreticulin and ERp57 complement tapasin to exploit the plasticity of MHC-I molecules to achieve peptide editing

Plasticity of empty major histocompatibility complex class I molecules determines peptide-selector function.

Major histocompatibility complex class I (MHC I) proteins provide protection from intracellular pathogens and cancer via each of a cell's MHC I molecules binding and presenting a peptide to cytotoxic T lymphocytes. MHC I genes are highly polymorphic and can have significant diversity, with polymorphisms predominantly localised in the peptide-binding groove where they can change peptide-binding specificity. However, polymorphic residues may also determine other functional properties, such as how dependent MHC I alleles are on the peptide-loading complex for optimal acquisition of peptide cargo. We describe how differences in the peptide-binding properties of two MHC I alleles correlates with altered conformational flexibility in the peptide-empty state. We hypothesise that plasticity is an intrinsic property encoded by the protein sequence, and that co-ordinated movements of the membrane-proximal and membrane-distal domains collectively determines how dependent MHC I are on the peptide-loading complex for efficient assembly with high affinity peptides

The role of MHC I protein dynamics in tapasin and TAPBPR-assisted immunopeptidome editing

Major Histocompatibility Complex class I (MHC I) molecules are highly polymorphic, with allotypes differing in peptide binding preferences, and in their dependence upon tapasin for optimal peptide selection. The tapasin dependence of MHC allotypes is inversely correlated with their self-editing ability, and underpinned by conformational plasticity. Recently, TAPBPR has been shown to enhance MHC I assembly via a chaperone-like function, and by editing the peptide repertoire of some MHC I allotypes. Structural analysis has shown TAPBPR binding changes the conformation and dynamics of MHC I, with MHC protein dynamics likely to determine the prevailing TAPBPR function: generically enhancing MHC I assembly by stabilising highly dynamic peptide-empty MHC I; and by editing the peptide repertoire of highly dynamic MHC I allotypes

Tapasin-related protein restricts the MHC class I peptide repertoire by enhancing peptide exchange

Recently we have found the tapasin-related protein TAPBPR is an additional MHC I chaperone of unknown function in the antigen presentation system. In contrast to tapasin, TAPBPR is not an integral component of the peptide loading complex and is not required for peptide loading. Furthermore, we have found TAPBPR and tapasin bind in a similar orientation to the same face of MHC I.This raised the possibility that, like tapasin, TAPBPR could functionas an MHC I peptide “editor”.Here we demonstrate that TAPBPR expression has a significanteffect in shaping the peptide repertoire presented by MHC I. Usingin vitro peptide exchange assays we demonstrate that TAPBPRis able to increase peptide association and peptide dissociationfor both HLA-A*02 and -B*08. Therefore, like tapasin, TAPBPR enhances peptide exchange on MHC I. Interestingly, the luminal portion of TAPBPR alone, without the need for a leucine zipper,is sufficient to promote MHC I peptide exchange, suggesting that TAPBPR might have a higher affinity for MHC I than tapasin. Todetermine the in vivo effect of TAPBPR expression on the MHC Ipeptidome, we isolated pMHC I from WT and TAPBPR knockout cells, eluted the bound peptides and determined their amino acid sequence by mass spectrometry. This revealed a significantly broader repertoire of peptides presented on MHC I in the absence of TAPBPR (up to a 9.5 fold increase in the total number of pMHC), despite the fact that cell surface levels of MHC I were unaffected byTAPBPR depletion. This is in contrast to tapasin depletion where a significant decrease in the total number of pMHC was observed

Direct evidence for conformational dynamics in major histocompatibility complex class I molecules

Major histocompatibility complex class I molecules (MHC I) help protect jawed vertebrates by binding and presenting immunogenic peptides to cytotoxic T lymphocytes. Peptides are selected from a large diversity present in the endoplasmic reticulum. However, only a limited number of peptides complement the polymorphic MHC specificity determining pockets in a way that leads to high-affinity peptide binding and efficient antigen presentation. MHC I molecules possess an intrinsic ability to discriminate between peptides, which varies in efficiency between allotypes, but the mechanism of selection is unknown. Elucidation of the selection mechanism is likely to benefit future immune-modulatory therapies. Evidence suggests peptide selection involves transient adoption of alternative, presumably higher energy conformations than native peptide-MHC complexes. However, the instability of peptide-receptive MHC molecules has hindered characterization of such conformational plasticity. To investigate the dynamic nature of MHC, we refolded MHC proteins with peptides that can be hydrolyzed by UV light and thus released. We compared the resultant peptide-receptive MHC molecules with non-hydrolyzed peptide-loaded MHC complexes by monitoring the exchange of hydrogen for deuterium in solution. We found differences in hydrogen-deuterium exchange between peptide-loaded and peptide-receptive molecules that were negated by the addition of peptide to peptide-receptive MHC molecules. Peptide hydrolysis caused significant increases in hydrogen-deuterium exchange in sub-regions of the peptide-binding domain and smaller increases elsewhere, including in the α3 domain and the non-covalently associated β2-microglobulin molecule, demonstrating long-range dynamic communication. Comparing two MHC allotypes revealed allotype-specific differences in hydrogen-deuterium exchange, consistent with the notion that MHC I plasticity underpins peptide selection.</p