register interest

Professor Chris A O'Callaghan

Research Area: Immunology
Technology Exchange: Bioinformatics, Cellular immunology, Computational biology, Crystallography, Flow cytometry, Microscopy (Confocal), Protein interaction and Transcript profiling
Scientific Themes: Immunology & Infectious Disease and Physiology, Cellular & Molecular Biology
Keywords: innate immunity, molecular immunology, protein-protein interactions, surface plasmon resonance, crystallography and ligand identification
Web Links:
A crystal of pure CLEC-2 protein for structural studies.

A crystal of pure CLEC-2 protein for structural studies.

Fluorescence microscopy demonstrating the intracellular location of HLA-F.

Fluorescence microscopy demonstrating the intracellular location of HLA-F.

CLEC-2 is present on platelets and immune cells and the colours highlight the charged areas of the molecule that interact with its ligand.

CLEC-2 is present on platelets and immune cells and the colours highlight the charged areas of the m ...

Our group is studying immunity and inflammation with a disease focus on atherosclerotic vascular disease. We are particularly interested in understanding the mechanisms underlying the immune/inflammatory aspects of the disease process with a view to identifying new approaches to treatment. We use a wide range of techniques including molecular and cellular biology, immunological assays, fluorescence techniques, recombinant protein production, structural biology, next generation sequencing and epigenetic analysis.

Name Department Institution Country
Professor Richard J Cornall FMedSci FRCP Centre for Cellular and Molecular Physiology University of Oxford United Kingdom
Pamela Bjorkman California Institute for Technology (Caltech) United States
Steve Watson University of Birmingham United Kingdom
Professor Paul Klenerman Experimental Medicine Division University of Oxford United Kingdom
Johannes Eble University of Frankfurt Germany
Professor Benedikt M Kessler Target Discovery Institute University of Oxford United Kingdom
Professor Paul Moss University of Birmingham United Kingdom
Del Besra University of Birmingham United Kingdom

Reschen ME, Gaulton KJ, Lin D, Soilleux EJ, Morris AJ, Smyth SS, O'Callaghan CA. 2015. Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding. PLoS Genet, 11 (4), pp. e1005061. Read abstract | Read more

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli. Hide abstract

McCarthy MT, O'Callaghan CA. 2014. PeaKDEck: a kernel density estimator-based peak calling program for DNaseI-seq data. Bioinformatics, 30 (9), pp. 1302-1304. Read abstract | Read more

Hypersensitivity to DNaseI digestion is a hallmark of open chromatin, and DNaseI-seq allows the genome-wide identification of regions of open chromatin. Interpreting these data is challenging, largely because of inherent variation in signal-to-noise ratio between datasets. We have developed PeaKDEck, a peak calling program that distinguishes signal from noise by randomly sampling read densities and using kernel density estimation to generate a dataset-specific probability distribution of random background signal. PeaKDEck uses this probability distribution to select an appropriate read density threshold for peak calling in each dataset. We benchmark PeaKDEck using published ENCODE DNaseI-seq data and other peak calling programs, and demonstrate superior performance in low signal-to-noise ratio datasets. Hide abstract

Reschen M, Kini U, Hood RL, Boycott KM, Hurst J, O'Callaghan CA. 2012. Floating-Harbor syndrome and polycystic kidneys associated with SRCAP mutation. Am J Med Genet A, 158A (12), pp. 3196-3200. Read abstract | Read more

Floating-Harbor syndrome (FHS) is a rare genetic disorder recently shown to be caused by mutations in the Snf2-related CREB-binding protein activator protein gene (SRCAP). It comprises three key clinical features of characteristic facies, expressive and receptive speech impairment and short stature. We report on a patient with this syndrome associated with early adult-onset hypertension and bilateral polycystic kidneys. Family screening for polycystic kidney disease was negative and mutations in polycystic kidney disease 1 and 2 genes (PKD1 and PKD2) were absent. Sequencing of the SRCAP gene demonstrated a de novo mutation matching one of the known FHS-associated mutations. The patient required treatment with anti-hypertensives and will require lifelong renal monitoring. We suggest this patient's presentation may be due to the pleiotropic effects of SRCAP mutations. Further, the protein encoded by SRCAP is known to interact with CREB-binding protein, the product of the gene mutated in Rubinstein-Taybi syndrome, which is associated with renal abnormalities. A literature review of the renal findings in patients with Floating-Harbor syndrome identified another patient with possible polycystic kidneys, two patients with early onset hypertension, and a young patient with a ruptured intracranial aneurysm, which can be a feature of classic adult polycystic kidney disease. Collectively, these findings suggest that all patients with Floating-Harbor syndrome should undergo regular blood pressure monitoring and screening for polycystic kidneys by ultrasound at the time of the FHS diagnosis with imaging to be repeated during adulthood if a childhood ultrasound was negative. Hide abstract

Lin D, Lavender H, Soilleux EJ, O'Callaghan CA. 2012. NF-κB regulates MICA gene transcription in endothelial cell through a genetically inhibitable control site. J Biol Chem, 287 (6), pp. 4299-4310. Read abstract | Read more

Endothelial cells form a barrier between blood and the underlying vessel wall, which characteristically demonstrates inflammatory damage in atherosclerotic disease. MICA is a highly polymorphic ligand for the activating immune receptor NKG2D and can be expressed on endothelial cells. We hypothesized that damaged vessel walls, such as those involved in atherosclerosis, might express MICA, which could contribute to the vascular immunopathology. Immune activation resulting from MICA expression could play a significant role in the development of vascular damage. We have demonstrated that TNFα up-regulates MICA on human endothelial cells. The up-regulation is mediated by NF-κB, and we have defined the regulatory control site responsible for this at -130 bp upstream of the MICA transcription start site. This site overlaps with a heat shock response element and integrates input from the two pathways. We have shown that in atherosclerotic lesions there is expression of MICA on endothelial cells. Using lentivirus-mediated gene delivery in primary human endothelial cells, we were able to inhibit the MICA response to TNFα with a truncated HSF1 that lacked a transactivation domain. This highlights the potential for transcription-based therapeutic approaches in atherosclerotic vascular disease to reduce immune-mediated endothelial and vessel wall damage. Hide abstract

Watson AA, Lebedev AA, Hall BA, Fenton-May AE, Vagin AA, Dejnirattisai W, Felce J, Mongkolsapaya J et al. 2011. Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling. J Biol Chem, 286 (27), pp. 24208-24218. Read abstract | Read more

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1-4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease. Hide abstract

Hughes CE, Pollitt AY, Mori J, Eble JA, Tomlinson MG, Hartwig JH, O'Callaghan CA, Fütterer K, Watson SP. 2010. CLEC-2 activates Syk through dimerization. Blood, 115 (14), pp. 2947-2955. Read abstract | Read more

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor. Hide abstract

Watson AA, Christou CM, James JR, Fenton-May AE, Moncayo GE, Mistry AR, Davis SJ, Gilbert RJ, Chakera A, O'Callaghan CA. 2009. The platelet receptor CLEC-2 is active as a dimer. Biochemistry, 48 (46), pp. 10988-10996. Read abstract | Read more

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer. Hide abstract

Christou CM, Pearce AC, Watson AA, Mistry AR, Pollitt AY, Fenton-May AE, Johnson LA, Jackson DG, Watson SP, O'Callaghan CA. 2008. Renal cells activate the platelet receptor CLEC-2 through podoplanin. Biochem J, 411 (1), pp. 133-140. Read abstract | Read more

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. Hide abstract

Mistry AR, O'Callaghan CA. 2007. Regulation of ligands for the activating receptor NKG2D. Immunology, 121 (4), pp. 439-447. Read abstract | Read more

The outcome of an encounter between a cytotoxic cell and a potential target cell depends on the balance of signals from inhibitory and activating receptors. Natural Killer group 2D (NKG2D) has recently emerged as a major activating receptor on T lymphocytes and natural killer cells. In both humans and mice, multiple different genes encode ligands for NKG2D, and these ligands are non-classical major histocompatibility complex class I molecules. The NKG2D-ligand interaction triggers an activating signal in the cell expressing NKG2D and this promotes cytotoxic lysis of the cell expressing the ligand. Most normal tissues do not express ligands for NKG2D, but ligand expression has been documented in tumour and virus-infected cells, leading to lysis of these cells. Tight regulation of ligand expression is important. If there is inappropriate expression in normal tissues, this will favour autoimmune processes, whilst failure to up-regulate the ligands in pathological conditions would favour cancer development or dissemination of intracellular infection. Hide abstract

Watson AA, Brown J, Harlos K, Eble JA, Walter TS, O'Callaghan CA. 2007. The crystal structure and mutational binding analysis of the extracellular domain of the platelet-activating receptor CLEC-2. J Biol Chem, 282 (5), pp. 3165-3172. Read abstract | Read more

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding. Hide abstract

O'Callaghan CA, Cerwenka A, Willcox BE, Lanier LL, Bjorkman PJ. 2001. Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60. Immunity, 15 (2), pp. 201-211. Read abstract | Read more

NKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with approximately 25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions. Hide abstract

Lepin EJ, Bastin JM, Allan DS, Roncador G, Braud VM, Mason DY, van der Merwe PA, McMichael AJ, Bell JI, Powis SH, O'Callaghan CA. 2000. Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur J Immunol, 30 (12), pp. 3552-3561. Read abstract | Read more

HLA-F is a human non-classical MHC molecule. Recombinant HLA-F heavy chain was refolded with 2-microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high-affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA-F. HLA-F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines and from HUT-78, a T cell line. HLA-F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA-F tetramers stain peripheral blood monocytes and B cells. HLA-F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA-F, suggest that HLA-F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells. Hide abstract

O'Callaghan CA, Tormo J, Willcox BE, Braud VM, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol Cell, 1 (4), pp. 531-541. Read abstract | Read more

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells. Hide abstract

Braud VM, Allan DS, O'Callaghan CA, Söderström K, D'Andrea A, Ogg GS, Lazetic S, Young NT et al. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391 (6669), pp. 795-799. Read abstract | Read more

The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A. Hide abstract


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