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2.  Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1 
The chicken classical MHC class I antigen YF1*7.1 was crystallized together with β2-microglobulin but without a peptide ligand. Crystals diffracted synchrotron radiation to 1.32 Å and belonged to the monoclinic space group P21.
YF1*7.1 is an allele of a polymorphic major histocompatibility complex (MHC) class I-like locus within the chicken Y gene complex. With the aim of understanding the possible role of the YF1*7.1 molecule in antigen presentation, the complex of YF1*7.1 heavy chain and β2-microglobulin was reconstituted and purified without a peptide. Crystals diffracted synchrotron radiation to 1.32 Å resolution and belonged to the monoclinic space group P21. The phase problem was solved by molecular replacement. A detailed examination of the structure may provide insight into the type of ligand that could be bound by the YF1*7.1 molecule.
doi:10.1107/S1744309109009026
PMCID: PMC2664777  PMID: 19342797
YF1*7.1; chicken Rfp-Y; MHC class I antigens
3.  Structure of a Classical MHC Class I Molecule That Binds “Non-Classical” Ligands 
PLoS Biology  2010;8(12):e1000557.
The chicken MHC YF1*7.1 X-ray structures reveal that this protein binds lipids and thus represents a "hybrid" class I complex with features of classical as well as non-classical MHC molecules.
Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β2-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.
Author Summary
Proteins encoded by the major histocompatibility complex (MHC) play crucial roles in vertebrate immune systems, presenting pathogen-derived protein fragments to receptors on effector cells. In contrast, some non-classical MHC class I proteins such as CD1 molecules possess a hydrophobic groove that allows them to display lipids. Chicken MHC-Y is a genetic region outside the core MHC that harbors several immune-related genes, among them YF1*7.1, which encodes a protein whose structure we solved in this study. YF1*7.1 is an MHC class I molecule that exhibits the architecture typical of classical MHC class I antigens but possesses a hydrophobic binding groove that binds non-peptidic ligands. By using lipid-binding assays, we show that this molecule can indeed bind lipids. Therefore, YF1*7.1 bridges, at least in structural terms, the traditional gap between classical and non-classical MHC class I molecules. Lipid-binding YF1 proteins might serve the chicken to enlarge its otherwise very small repertoire of antigen-presenting MHC class I molecules. Furthermore, comparative analyses of the two protein subunits of classical MHC molecules revealed a structural feature in chickens that appears to be shared by non-mammalian but not by mammalian vertebrates. This unique feature is indicative of a structure-dependent co-evolution of two genetically unlinked genes in non-mammalian species.
doi:10.1371/journal.pbio.1000557
PMCID: PMC2998441  PMID: 21151886
4.  Self/nonself perception, reproduction and the extended MHC 
Self Nonself  2010;1(3):176-191.
Self/nonself perception governs mate selection in most eukaryotic species. It relies on a number of natural barriers that act before, during and after copulation. These hurdles prevent a costly investment into an embryo with potentially suboptimal genetic and immunological properties and aim at discouraging fertilization when male and female gametes exhibit extensive sharing of alleles. Due to the fact that several genes belonging to the extended major histocompatibility complex (xMHC) carry out crucial immune functions and are the most polymorphic within vertebrate genomes, it is likely that securing heterozygosity and the selection of rare alleles within this gene complex contributes to endowing the offspring with an advantage in fighting infections. Apart from MHC class I and II antigens, the products of several other genes within the xMHC are candidates for participating in mate choice, especially since the respective loci are subject to long-range linkage disequilibrium which may aid to preserve functionally connected alleles within a given haplotype. Among these loci are polymorphic odorant receptor genes that are expressed not only in the olfactory epithelium, but also within male reproductive tissues. They may thus not only be of importance in olfaction-influenced mate choice, by recognizing MHC-dependent individual-specific olfactory signals, but could also guide spermatozoa along chemical gradients to their target, the oocyte. By focusing on the human HLA complex and genes within its vicinity, we show here that the products of several xMHC-specified molecules might be involved in self/nonself perception during reproduction. Although the molecular details are often unknown, the existence of highly diverse, yet intertwined pre- and post-copulatory barriers suggests that xMHC-encoded proteins may be important for various stages of mate choice, germ cell development, as well as embryonic and foetal life in mammals and other vertebrates. Many of these genes should thus be regarded as crucial not only within the immune system, but also in reproduction.
doi:10.4161/self.1.3.12736
PMCID: PMC3047782  PMID: 21487476
cryptic female choice; extended major histocompatibility complex; gene polymorphism; human leukocyte antigen complex; mate choice; odorant receptor; reproduction; self/nonself discrimination
5.  Neuronal MHC Class I Molecules are Involved in Excitatory Synaptic Transmission at the Hippocampal Mossy Fiber Synapses of Marmoset Monkeys 
Several recent studies suggested a role for neuronal major histocompatibility complex class I (MHCI) molecules in certain forms of synaptic plasticity in the hippocampus of rodents. Here, we report for the first time on the expression pattern and functional properties of MHCI molecules in the hippocampus of a nonhuman primate, the common marmoset monkey (Callithrix jacchus). We detected a presynaptic, mossy fiber-specific localization of MHCI proteins within the marmoset hippocampus. MHCI molecules were present in the large, VGlut1-positive, mossy fiber terminals, which provide input to CA3 pyramidal neurons. Furthermore, whole-cell recordings of CA3 pyramidal neurons in acute hippocampal slices of the common marmoset demonstrated that application of antibodies which specifically block MHCI proteins caused a significant decrease in the frequency, and a transient increase in the amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in CA3 pyramidal neurons. These findings add to previous studies on neuronal MHCI molecules by describing their expression and localization in the primate hippocampus and by implicating them in plasticity-related processes at the mossy fiber–CA3 synapses. In addition, our results suggest significant interspecies differences in the localization of neuronal MHCI molecules in the hippocampus of mice and marmosets, as well as in their potential function in these species.
Electronic supplementary material
The online version of this article (doi:10.1007/s10571-010-9510-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s10571-010-9510-3
PMCID: PMC2912721  PMID: 20232136
Calbindin; CA3; GFAP; Piccolo; Neuronal plasticity; PSD95; sEPSC; VGAT; VGlut1; VGlut 2
6.  Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1. Addendum 
Addendum to Hee et al. [Acta Cryst. (2009), F65, 422–425].
Additional funding is acknowledged by the authors of Hee et al. [Acta Cryst. (2009), F65, 422–425].
doi:10.1107/S1744309110001089
PMCID: PMC2815697
YF1*7.1; chicken Rfp-Y; MHC class I antigens; addendum
7.  Assessment of transmission distortion on chromosome 6p in healthy individuals using tagSNPs 
European Journal of Human Genetics  2009;17(9):1182-1189.
The best-documented example for transmission distortion (TD) to normal offspring are the t haplotypes on mouse chromosome 17. In healthy humans, TD has been described for whole chromosomes and for particular loci, but multiple comparisons have presented a statistical obstacle in wide-ranging analyses. Here we provide six high-resolution TD maps of the short arm of human chromosome 6 (Hsa6p), based on single-nucleotide polymorphism (SNP) data from 60 trio families belonging to two ethnicities that are available through the International HapMap Project. We tested all approximately 70 000 previously genotyped SNPs within Hsa6p by the transmission disequilibrium test. TagSNP selection followed by permutation testing was performed to adjust for multiple testing. A statistically significant evidence for TD was observed among male parents of European ancestry, due to strong and wide-ranging skewed segregation in a 730 kb long region containing the transcription factor-encoding genes SUPT3H and RUNX2, as well as the microRNA locus MIRN586. We also observed that this chromosomal segment coincides with pronounced linkage disequilibrium (LD), suggesting a relationship between TD and LD. The fact that TD may be taking place in samples not selected for a genetic disease implies that linkage studies must be assessed with particular caution in chromosomal segments with evidence of TD.
doi:10.1038/ejhg.2009.16
PMCID: PMC2986600  PMID: 19259136
transmission distortion; linkage disequilibrium; human chromosome 6p; SUPT3H; MIRN586; RUNX2
8.  Expression, purification and preliminary X-ray crystallographic analysis of the human major histocompatibility antigen HLA-B*1402 in complex with a viral peptide and with a self-peptide 
The crystallization of HLA-B*1402 in complex with two peptides is reported.
The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P21 (pLMP2) and P212121 (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 Å resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.
doi:10.1107/S1744309107029077
PMCID: PMC2335130  PMID: 17620730
HLA-B14 subtypes; major histocompatibility complex polymorphism; HLA-B*1402; subtype-dependent peptide-binding modes; ankylosing spondylitis
9.  Association of Smoking Behavior with an Odorant Receptor Allele Telomeric to the Human Major Histocompatibility Complex 
Genetic testing  2008;12(4):481-486.
Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d’Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971T) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a 97Thr → Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971T allele (p = 1.05×10−2), with higher significance than with A1-B8-DR3 (p = 2.38×10−2). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971T polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women.
doi:10.1089/gte.2008.0029
PMCID: PMC2635552  PMID: 18939942
10.  X-ray diffraction analysis of crystals from the human major histocompatibility antigen HLA-B*2706 in complex with a viral peptide and with a self-peptide 
The crystallization of HLA-B*2706 in complex with two peptides is reported.
The human leukocyte antigen (HLA) alleles HLA-B*2704 and HLA-B*2706 show an ethnically restricted distribution and are differentially associated with ankylosing spondylitis, with HLA-B*2706 lacking association with this autoimmune disease. However, the products of the two alleles differ by only two amino acids, at heavy-chain residues 114 (His in HLA-B*2704; Asp in HLA-B*2706) and 116 (Asp in HLA-B*2704; Tyr in HLA-B*2706). Both residues could be involved in contacting amino acids of a bound peptide, suggesting that peptides presented by these subtypes play a role in disease pathogenesis. Two HLA-B*2706–peptide complexes were crystallized using the hanging-drop vapour-diffusion method with PEG as precipitant. Data sets were collected to resolutions of 2.70 Å (viral peptide pLMP2, RRRWRRLTV; space group P212121) and 1.83 Å (self-peptide pVIPR, RRKWRRWHL; space group P21). Using HLA-B*2705 complexed with the pGR peptide (RRRWHRWRL) as a search model, unambiguous molecular-replacement solutions were found for both HLA-B*2706 complexes.
doi:10.1107/S1744309105037966
PMCID: PMC1978159  PMID: 16511245
HLA-B27 subtypes; major histocompatibility complex polymorphism; HLA-B*2706; subtype-dependent peptide-binding modes; ankylosing spondylitis
11.  Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide 
Crystallization of HLA-B*2704 in complex with two peptides.
The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P212121. Data sets were collected to 1.60 Å (complex with the self-peptide pVIPR) or to 1.90 Å (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.
doi:10.1107/S1744309105029234
PMCID: PMC1991317  PMID: 16511201
HLA-B27 subtypes; HLA-B*2704; subtype-dependent peptide-binding modes; ankylosing spondylitis; polymorphism
12.  Purification, crystallization and preliminary X-ray diffraction analysis of the human major histocompatibility antigen HLA-B*2703 complexed with a viral peptide and with a self-peptide 
The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. A pocket of the molecule, two HLA-B*2703–peptide complexes were crystallized and data sets were collected to high resolution using synchrotron radiation.
The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystals belong to space group P21 (pVIPR peptide) or P212121 (pLMP2 peptide). Data sets were collected to 1.55 Å (B*2703–pVIPR) or 2.0 Å (B*2703–pLMP2) resolution using synchrotron radiation. With B*2705–pVIPR as a search model, a clear molecular-replacement solution was found for both B*2703 complexes.
doi:10.1107/S1744309105007438
PMCID: PMC1952442  PMID: 16511044
HLA-B*2703; HLA-B27 subtypes; subtype-dependent peptide-binding modes; ankylosing spondylitis; residue 59 polymorphism
13.  Dual, HLA-B27 Subtype-dependent Conformation of a Self-peptide 
The products of the human leukocyte antigen subtypes HLA-B*2705 and HLA-B*2709 differ only in residue 116 (Asp vs. His) within the peptide binding groove but are differentially associated with the autoimmune disease ankylosing spondylitis (AS); HLA-B*2705 occurs in AS-patients, whereas HLA-B*2709 does not. The subtypes also generate differential T cell repertoires as exemplified by distinct T cell responses against the self-peptide pVIPR (RRKWRRWHL). The crystal structures described here show that pVIPR binds in an unprecedented dual conformation only to HLA-B*2705 molecules. In one binding mode, peptide pArg5 forms a salt bridge to Asp116, connected with drastically different interactions between peptide and heavy chain, contrasting with the second, conventional conformation, which is exclusively found in the case of B*2709. These subtype-dependent differences in pVIPR binding link the emergence of dissimilar T cell repertoires in individuals with HLA-B*2705 or HLA-B*2709 to the buried Asp116/His116 polymorphism and provide novel insights into peptide presentation by major histocompatibility antigens.
doi:10.1084/jem.20031690
PMCID: PMC2211767  PMID: 14734527
X-ray structure; major histocompatibility antigen; peptide binding modes; ankylosing spondylitis; residue 116

Results 1-13 (13)