Prof Simon J Davis

Research Area: Immunology
Technology Exchange: Bioinformatics, Crystallography, Microscopy (Confocal) and Protein interaction
Keywords: T cell, triggering, protein structure, protein interactions and cell surface
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The T-cell Biology Group (Summer 2007)

The T-cell Biology Group (Summer 2007)

Cover submitted to accompany a systematic survey of the protein composition of the T cell surface

Cover submitted to accompany a systematic survey of the protein composition of the T cell surface

Cover submiited to accompany a review of the fate of the kinetic-segregation model of TCR triggering, 10 years after its initial publication

Cover submiited to accompany a review of the fate of the kinetic-segregation model of TCR ...

Our earliest work was focussed on understanding how cell-cell recognition proteins achieve weak, specific recognition. This involved crystallographic and mutational studies of cell adhesion molecules and co-stimulatory proteins. We also used transcriptomic approaches to identify what has since turned out to be the complete set of T cell-specific proteins expressed at the T-cell surface. This was undertaken because we felt that it was important to know how many surface proteins remained to be discovered, so that sensible and largely complete theories of cell surface function could be formulated. Having identified all the components, a major emphasis now is to understand how the proteins are organized at the resting cell surface, which is very controversial. For this we use single-molecule, fluorescence-based imaging methods developed by Professor David Klenerman at the Department of Chemistry, Cambridge University, and other approaches. We have shown that the T-cell receptor forms monovalent complexes and produced data undermining the popular notion that G protein-coupled receptors invariably dimerize.

In 1996, with PA van der Merwe, we proposed a counter-intuitive explanation for how some of the most important receptors in the immune system, including the T-cell receptor, are “triggered” by their ligands, a theory called the “kinetic-segregation” model (go towww.t-cellbiology.org/ks_model). We have now extended the concept to receptor triggering by “superagonistic” antibodies (seewww.t-cellbiology/antibody). Our present goals are (1) to show that the kinetic segregation model does indeed explain T-cell receptor triggering, and (2) to use the idea to develop new types of therapeutic antibodies. The signalling concept is being tested using structural approaches and super-resolution imaging techniques, such as dSTORM. For this, the behaviour of T-cell surface proteins is being studied at contacts with glass surfaces and supported lipid bilayers, in collaboration with Professor Klenerman. New, potentially therapeutic superagonistic antibodies are being developed and licensed to industry in collaboration with Professor Richard Cornall.

Name Department Institution Country
Professor Richard J Cornall FMedSci FRCP Centre for Cellular and Molecular Physiology Oxford University, Henry Wellcome Building for Molecular Physiology United Kingdom
Prof David Klenerman FRS Chemistry University of Cambridge United Kingdom
Prof P. Anton van der Merwe School of Pathology University of Oxford United Kingdom
Prof Lars Fugger Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor E. Yvonne Jones FRS FMedSci Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Prof Vincenzo Cerundolo Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor David Stuart FRS Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Chang VT, Fernandes RA, Ganzinger KA, Lee SF, Siebold C, McColl J, Jönsson P, Palayret M, Harlos K, Coles CH et al. 2016. Initiation of T cell signaling by CD45 segregation at 'close contacts'. Nat Immunol, 17 (5), pp. 574-582. | Show Abstract | Read more

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Weimann L, Ganzinger KA, McColl J, Irvine KL, Davis SJ, Gay NJ, Bryant CE, Klenerman D. 2013. A quantitative comparison of single-dye tracking analysis tools using Monte Carlo simulations. PLoS One, 8 (5), pp. e64287. | Show Abstract | Read more

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.

Zinselmeyer BH, Heydari S, Sacristán C, Nayak D, Cammer M, Herz J, Cheng X, Davis SJ, Dustin ML, McGavern DB. 2013. PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis. J Exp Med, 210 (4), pp. 757-774. | Show Abstract | Read more

Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8(+) and CD4(+) T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8(+) and CD4(+) T cells. Unexpectedly, therapeutic blockade of PD-1-PD-L1 restored CD8(+) T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8(+) T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-γ. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8(+) T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.

Cheng X, Veverka V, Radhakrishnan A, Waters LC, Muskett FW, Morgan SH, Huo J, Yu C, Evans EJ, Leslie AJ et al. 2013. Structure and interactions of the human programmed cell death 1 receptor. J Biol Chem, 288 (17), pp. 11771-11785. | Show Abstract | Read more

PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

Klenerman D, Shevchuk A, Novak P, Korchev YE, Davis SJ. 2013. Imaging the cell surface and its organization down to the level of single molecules. Philos Trans R Soc Lond B Biol Sci, 368 (1611), pp. 20120027. | Show Abstract | Read more

Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.

Ikemizu S, Chirifu M, Davis SJ. 2012. IL-2 and IL-15 signaling complexes: different but the same. Nat Immunol, 13 (12), pp. 1141-1142. | Read more

Gregory AP, Dendrou CA, Attfield KE, Haghikia A, Xifara DK, Butter F, Poschmann G, Kaur G, Lambert L, Leach OA et al. 2012. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature, 488 (7412), pp. 508-511. | Show Abstract | Read more

Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn’s disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.

Fernandes RA, Shore DA, Vuong MT, Yu C, Zhu X, Pereira-Lopes S, Brouwer H, Fennelly JA, Jessup CM, Evans EJ et al. 2012. T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements. J Biol Chem, 287 (16), pp. 13324-13335. | Show Abstract | Read more

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.

Felce JH, Davis SJ. 2012. Unraveling receptor stoichiometry using bret. Front Endocrinol (Lausanne), 3 (JUL), pp. 86. | Read more

Fernandes RA, Huo J, Lui Y, Felce JH, Davis SJ. 2012. On the Control of TCR Phosphorylation. Front Immunol, 3 (MAY), pp. 92. | Read more

Oliveira MI, Gonçalves CM, Pinto M, Fabre S, Santos AM, Lee SF, Castro MA, Nunes RJ, Barbosa RR, Parnes JR et al. 2012. CD6 attenuates early and late signaling events, setting thresholds for T-cell activation. Eur J Immunol, 42 (1), pp. 195-205. | Show Abstract | Read more

The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

James JR, McColl J, Oliveira MI, Dunne PD, Huang E, Jansson A, Nilsson P, Sleep DL, Gonçalves CM, Morgan SH et al. 2011. The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins. J Biol Chem, 286 (37), pp. 31993-32001. | Show Abstract | Read more

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Bamberger M, Santos AM, Gonçalves CM, Oliveira MI, James JR, Moreira A, Lozano F, Davis SJ, Carmo AM. 2011. A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase. J Biol Chem, 286 (35), pp. 30324-30336. | Show Abstract | Read more

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

Yu C, Crispin M, Sonnen AF, Harvey DJ, Chang VT, Evans EJ, Scanlan CN, Stuart DI, Gilbert RJ, Davis SJ. 2011. Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells. Acta Crystallogr Sect F Struct Biol Cryst Commun, 67 (Pt 7), pp. 785-789. | Show Abstract | Read more

Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.

Brackenridge S, Evans EJ, Toebes M, Goonetilleke N, Liu MK, di Gleria K, Schumacher TN, Davis SJ, McMichael AJ, Gillespie GM. 2011. An early HIV mutation within an HLA-B*57-restricted T cell epitope abrogates binding to the killer inhibitory receptor 3DL1. J Virol, 85 (11), pp. 5415-5422. | Show Abstract | Read more

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

Yu C, Sonnen AF, George R, Dessailly BH, Stagg LJ, Evans EJ, Orengo CA, Stuart DI, Ladbury JE, Ikemizu S et al. 2011. Rigid-body ligand recognition drives cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor triggering. J Biol Chem, 286 (8), pp. 6685-6696. | Show Abstract | Read more

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).

Klenerman D, Korchev YE, Davis SJ. 2011. Imaging and characterisation of the surface of live cells. Curr Opin Chem Biol, 15 (5), pp. 696-703. | Show Abstract | Read more

Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.

Davis SJ, van der Merwe PA. 2011. Lck and the nature of the T cell receptor trigger. Trends Immunol, 32 (1), pp. 1-5. | Show Abstract | Read more

Exactly how ligand binding 'triggers' T cell receptor (TCR) phosphorylation is unclear. It has been proposed that ligand engagement by the TCR somehow activates the Src kinase Lck, which in turn phosphorylates the receptor. Recent data, however, suggest instead that a significant fraction of the Lck in resting T cells is already activated and that the proportion of active Lck does not change during the early stages of T cell activation. We argue that, caveats notwithstanding, these new observations offer support for the 'kinetic-segregation' model of TCR triggering, which involves spatial reorganization of signalling proteins upon ligand binding and requires a fraction of Lck to be active in resting T cells.

Jansson A, Davis SJ. 2011. Quantitative analysis predicts the relative therapeutic efficacy of different forms of CTLA4Ig Molecular Immunology,

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Fernandes RA, Yu C, Carmo AM, Evans EJ, van der Merwe PA, Davis SJ. 2010. What Controls T Cell Receptor Phosphorylation? CELL, 142 (5), pp. 668-669. | Read more

Sonnen AF, Yu C, Evans EJ, Stuart DI, Davis SJ, Gilbert RJ. 2010. Domain metastability: a molecular basis for immunoglobulin deposition? J Mol Biol, 399 (2), pp. 207-213. | Show Abstract | Read more

We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits.

van der Merwe PA, Dunne PD, Klenerman D, Davis SJ. 2010. Taking T cells beyond the diffraction limit. Nat Immunol, 11 (1), pp. 51-52. | Read more

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. | Show 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.

Crispin M, Chang VT, Harvey DJ, Dwek RA, Evans EJ, Stuart DI, Jones EY, Lord JM, Spooner RA, Davis SJ. 2009. A human embryonic kidney 293T cell line mutated at the Golgi alpha-mannosidase II locus. J Biol Chem, 284 (32), pp. 21684-21695. | Show Abstract | Read more

Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.

Gonçalves CM, Castro MA, Henriques T, Oliveira MI, Pinheiro HC, Oliveira C, Sreenu VB, Evans EJ, Davis SJ, Moreira A, Carmo AM. 2009. Molecular cloning and analysis of SSc5D, a new member of the scavenger receptor cysteine-rich superfamily. Mol Immunol, 46 (13), pp. 2585-2596. | Show Abstract | Read more

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30kb of genomic DNA, and contains fourteen exons producing a 4.8kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.

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22

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Dunne PD, Fernandes RA, McColl J, Ji WY, James JR, Davis SJ, Klenerman D. 2009. DySCo: Quantitating associations of membrane proteins using two-color single-molecule tracking Biophysical Journal, 97 (4), | Show Abstract | Read more

We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small. © 2009 by the Biophysical Society.

Evans EJ, Hene L, Vuong M, Abidi HS, Davis SJ. 2009. Transcriptome-based identification of candidate membrane proteins. Methods Mol Biol, 528 pp. 37-56. | Show Abstract | Read more

A full understanding of leukocyte responses to external stimuli requires knowledge of the full complement of proteins found on their surfaces. Systematic examination of the mammalian cell surfaces at the protein level is hampered by technical difficulties associated with proteomic analysis of so many membrane proteins and the large amounts of starting material required. The use of transcriptomic analyses avoids challenges associated with protein stability and separation and enables the inclusion of an amplification step; thus allowing the use of cell numbers applicable to the study of sub populations of, for example, primary lymphocytes. Here we present a transcriptomic methodology based on Serial Analysis of Gene Expression (SAGE) to recover an essentially complete and quantitative profile of mRNA species in a particular cell. We discuss how, using bioinformatic tools accessible to standard desktop computers, plasma membrane proteins can be identified in silico, from this list. While we describe the use of this approach to characterise the cell surface protein complement of a resting CD8(+) T-cell clone, it is theoretically applicable to any cell surface, where a suitable pure population of cells is available.

Abidi SH, Dong T, Vuong MT, Sreenu VB, Rowland-Jones SL, Evans EJ, Davis SJ. 2008. Differential remodeling of a T-cell transcriptome following CD8- versus CD3-induced signaling. Cell Res, 18 (6), pp. 641-648. | Show Abstract | Read more

CD8 engagement with class I major histocompatibility antigens greatly enhances T-cell activation, but it is not clear how this is achieved. We address the question of whether or not the antibody-mediated ligation of CD8 alone induces transcriptional remodeling in a T-cell clone, using serial analysis of gene expression. Even though it fails to induce overt phenotypic changes, we find that CD8 ligation profoundly alters transcription in the T-cell clone, at a scale comparable to that induced by antibody-mediated ligation of CD3. The character of the resulting changes is distinct, however, with the net effect of CD8 ligation being substantially inhibitory. We speculate that ligating CD8 induces weak, T-cell receptor (TCR)-mediated inhibitory signals reminiscent of the effects of TCR antagonists. Our results imply that CD8 ligation alone is incapable of activating the T-cell clone because it fails to fully induce NFAT-dependent transcription.

Schimanski LM, Drakesmith H, Talbott C, Horne K, James JR, Davis SJ, Sweetland E, Bastin J, Cowley D, Townsend AR. 2008. Ferroportin: lack of evidence for multimers. Blood Cells Mol Dis, 40 (3), pp. 360-369. | Show Abstract | Read more

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.

James JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D. 2007. Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells. Proc Natl Acad Sci U S A, 104 (45), pp. 17662-17667. | Show Abstract | Read more

The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the "association quotient," Q. In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCRbeta Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CD86, indicating that the dominant form of the TCR comprises single alphabeta heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells.

Chirifu M, Hayashi C, Nakamura T, Toma S, Shuto T, Kai H, Yamagata Y, Davis SJ, Ikemizu S. 2007. Crystal structure of the IL-15-IL-15Ralpha complex, a cytokine-receptor unit presented in trans. Nat Immunol, 8 (9), pp. 1001-1007. | Show Abstract | Read more

Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8(+) T cells and regulatory T cells, respectively, bind receptor complexes that share beta- and gamma-signaling subunits. Receptor specificity is provided by unique, nonsignaling alpha-subunits. Whereas IL-2 receptor-alpha (IL-2Ralpha) is expressed together in cis with the beta- and gamma-subunits on T cells and B cells, IL-15Ralpha is expressed in trans on antigen-presenting cells. Here we present a 1.85-A crystal structure of the human IL-15-IL-15Ralpha complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15-IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15-IL-15Ralpha and IL-2-IL-2Ralpha complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context of its unique receptor alpha-chain.

Aricescu AR, Siebold C, Choudhuri K, Chang VT, Lu W, Davis SJ, van der Merwe PA, Jones EY. 2007. Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. Science, 317 (5842), pp. 1217-1220. | Show Abstract | Read more

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.

James JR, Davis SJ. 2007. Experimental challenge to a 'rigorous' BRET analysis of GPCR oligomerization - James and Davis reply NATURE METHODS, 4 (8), pp. 601-601. | Read more

Petrovas C, Price DA, Mattapallil J, Ambrozak DR, Geldmacher C, Cecchinato V, Vaccari M, Tryniszewska E, Gostick E, Roederer M et al. 2007. SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection. Blood, 110 (3), pp. 928-936. | Show Abstract | Read more

Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8+ T cells and its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8+ T cells expressed a PD-1(high) phenotype, independent of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1(high)CD8+ T cells produced IFN-gamma, TNF-alpha, and IL-2 under cognate peptide stimulation. While CD8+ T cells that proliferated in response to antigen had a PD-1(high) phenotype, it was determined that there was a reduced proliferative capacity of PD-1(high) compared with PD-1(low) SIV-specific CD8+ T cells. PD-1(high) SIV-specific CD8+ T cells were highly susceptible to cell death leading to loss of such cells after in vitro stimulation. Thus, PD-1 is a negative regulator of SIV-specific CD8+ T cells, operating predominantly through the induction of cell death. Manipulation of the interaction of PD-1 with its ligands could thus potentially restore the CD8+ T-cell responses in SIV infection.

Schimanski LM, Drakesmith H, Talbot C, Horne K, James JR, Sweetland E, Bastin JM, Cowley D, Davis SJ, Townsend ARM. 2007. Wild-type ferroportin is not impeded in maturation, cell surface expression or iron export function by mutant ferroportin, and bio-luminescence resonance energy transfer (BRET) experiments indicate monomeric form in cell membranes AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 529-529.

Crispin M, Aricescu AR, Chang VT, Jones EY, Stuart DI, Dwek RA, Davis SJ, Harvey DJ. 2007. Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation. FEBS Lett, 581 (10), pp. 1963-1968. | Show Abstract | Read more

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.

Kearney A, Avramovic A, Castro MA, Carmo AM, Davis SJ, van der Merwe PA. 2007. The contribution of conformational adjustments and long-range electrostatic forces to the CD2/CD58 interaction. J Biol Chem, 282 (18), pp. 13160-13166. | Show Abstract | Read more

CD2 is a T cell surface molecule that enhances T and natural killer cell function by binding its ligands CD58 (humans) and CD48 (rodents) on antigen-presenting or target cells. Here we show that the CD2/CD58 interaction is enthalpically driven and accompanied by unfavorable entropic changes. Taken together with structural studies, this indicates that binding is accompanied by energetically significant conformational adjustments. Despite having a highly charged binding interface, neither the affinity nor the rate constants of the CD2/CD58 interaction were affected by changes in ionic strength, indicating that long-range electrostatic forces make no net contribution to binding.

Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson MG, Watson SP. 2007. Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost, 5 (5), pp. 1026-1033. | Show Abstract | Read more

BACKGROUND: Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets. OBJECTIVES: To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions. METHODS AND RESULTS: By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) gamma-chain. CONCLUSIONS: The present results directly demonstrate GPVI-FcR gamma-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI et al. 2007. Glycoprotein structural genomics: solving the glycosylation problem. Structure, 15 (3), pp. 267-273. | Show Abstract | Read more

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Nettleship JE, Aplin R, Aricescu AR, Evans EJ, Davis SJ, Crispin M, Owens RJ. 2007. Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry. Anal Biochem, 361 (1), pp. 149-151. | Read more

James JR, Davis SJ. 2007. BRET analysis of GPCR oligomerization: newer does not mean better - Reply NATURE METHODS, 4 (1), pp. 4-4. | Read more

Hene L, Sreenu VB, Vuong MT, Abidi SH, Sutton JK, Rowland-Jones SL, Davis SJ, Evans EJ. 2007. Deep analysis of cellular transcriptomes - LongSAGE versus classic MPSS. BMC Genomics, 8 (1), pp. 333. | Show Abstract | Read more

BACKGROUND: Deep transcriptome analysis will underpin a large fraction of post-genomic biology. 'Closed' technologies, such as microarray analysis, only detect the set of transcripts chosen for analysis, whereas 'open' e.g. tag-based technologies are capable of identifying all possible transcripts, including those that were previously uncharacterized. Although new technologies are now emerging, at present the major resources for open-type analysis are the many publicly available SAGE (serial analysis of gene expression) and MPSS (massively parallel signature sequencing) libraries. These technologies have never been compared for their utility in the context of deep transcriptome mining. RESULTS: We used a single LongSAGE library of 503,431 tags and a "classic" MPSS library of 1,744,173 tags, both prepared from the same T cell-derived RNA sample, to compare the ability of each method to probe, at considerable depth, a human cellular transcriptome. We show that even though LongSAGE is more error-prone than MPSS, our LongSAGE library nevertheless generated 6.3-fold more genome-matching (and therefore likely error-free) tags than the MPSS library. An analysis of a set of 8,132 known genes detectable by both methods, and for which there is no ambiguity about tag matching, shows that MPSS detects only half (54%) the number of transcripts identified by SAGE (3,617 versus 1,955). Analysis of two additional MPSS libraries shows that each library samples a different subset of transcripts, and that in combination the three MPSS libraries (4,274,992 tags in total) still only detect 73% of the genes identified in our test set using SAGE. The fraction of transcripts detected by MPSS is likely to be even lower for uncharacterized transcripts, which tend to be more weakly expressed. The source of the loss of complexity in MPSS libraries compared to SAGE is unclear, but its effects become more severe with each sequencing cycle (i.e. as MPSS tag length increases). CONCLUSION: We show that MPSS libraries are significantly less complex than much smaller SAGE libraries, revealing a serious bias in the generation of MPSS data unlikely to have been circumvented by later technological improvements. Our results emphasize the need for the rigorous testing of new expression profiling technologies.

James JR, Davis SJ. 2007. Reply to: Experimental challenge to a 'rigorous' BRET analysis of GPCR oligomerization Nature Methods, 4 (8), pp. 601-601. | Read more

Cited:

57

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James JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D. 2007. Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells Proceedings of the National Academy of Sciences of the United States of America, 104 (45), pp. 17662-17667. | Show Abstract | Read more

The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRαβ heterodimers that bind antigen and cluster of differentiation (CD) 3εδ, εγ, and ζζ dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, αβ heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the "association quotient," Q. In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCRβ Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CD86, indicating that the dominant form of the TCR comprises single αβ heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells. © 2007 by The National Academy of Sciences of the USA.

James JR, Oliveira MI, Carmo AM, Iaboni A, Davis SJ. 2006. A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods, 3 (12), pp. 1001-1006. | Show Abstract | Read more

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

Aricescu AR, Assenberg R, Bill RM, Busso D, Chang VT, Davis SJ, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U et al. 2006. Eukaryotic expression: developments for structural proteomics. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1114-1124. | Show Abstract | Read more

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Evans EJ, Castro MA, O'Brien R, Kearney A, Walsh H, Sparks LM, Tucknott MG, Davies EA, Carmo AM, van der Merwe PA et al. 2006. Crystal structure and binding properties of the CD2 and CD244 (2B4)-binding protein, CD48. J Biol Chem, 281 (39), pp. 29309-29320. | Show Abstract | Read more

The structural analysis of surface proteins belonging to the CD2 subset of the immunoglobulin superfamily has yielded important insights into transient cellular interactions. In mice and rats, CD2 and CD244 (2B4), which are expressed predominantly on T cells and natural killer cells, respectively, bind the same, broadly expressed ligand, CD48. Structures of CD2 and CD244 have been solved previously, and we now present the structure of the receptor-binding domain of rat CD48. The receptor-binding surface of CD48 is unusually flat, as in the case of rat CD2, and shares a high degree of electrostatic complementarity with the equivalent surface of CD2. The relatively simple arrangement of charged residues and this flat topology explain why CD48 cross-reacts with CD2 and CD244 and, in rats, with the CD244-related protein, 2B4R. Comparisons of modeled complexes of CD2 and CD48 with the complex of human CD2 and CD58 are suggestive of there being substantial plasticity in the topology of ligand binding by CD2. Thermodynamic analysis of the native CD48-CD2 interaction indicates that binding is driven by equivalent, weak enthalpic and entropic effects, in contrast to the human CD2-CD58 interaction, for which there is a large entropic barrier. Overall, the structural and biophysical comparisons of the CD2 homologues suggest that the evolutionary diversification of interacting cell surface proteins is rapid and constrained only by the requirement that binding remains weak and specific.

Crispin M, Harvey DJ, Chang VT, Yu C, Aricescu AR, Jones EY, Davis SJ, Dwek RA, Rudd PM. 2006. Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. Glycobiology, 16 (8), pp. 748-756. | Show Abstract | Read more

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.

Cited:

187

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Davis SJ, van der Merwe PA. 2006. The kinetic-segregation model: TCR triggering and beyond NATURE IMMUNOLOGY, 7 (8), pp. 803-809. | Show Abstract | Read more

How the T cell receptor engages antigen is known, but not how that 'triggers' intracellular signaling. The first direct support for a mechanism based on the spatial reorganization of signaling proteins, proposed 10 years ago and referred to as the 'kinetic-segregation' model, is now beginning to emerge, along with indications that it may also apply to the triggering of nonclonotypic receptors. We describe here the development of the model, review new data and suggest how the model fits a broader conceptual framework for receptor triggering. We also consider the capacity of the model, versus that of other proposals, to account for the established features of TCR triggering. © 2006 Nature Publishing Group.

Davis SJ, van der Merwe PA. 2006. The kinetic-segregation model: TCR triggering and beyond. Nat Immunol, 7 (8), pp. 803-809. | Show Abstract | Read more

How the T cell receptor engages antigen is known, but not how that 'triggers' intracellular signaling. The first direct support for a mechanism based on the spatial reorganization of signaling proteins, proposed 10 years ago and referred to as the 'kinetic-segregation' model, is now beginning to emerge, along with indications that it may also apply to the triggering of nonclonotypic receptors. We describe here the development of the model, review new data and suggest how the model fits a broader conceptual framework for receptor triggering. We also consider the capacity of the model, versus that of other proposals, to account for the established features of TCR triggering.

Ranasinghe SR, Abidi SH, Rowland-Jones SL, McMichael AJ, Dong T, Davis SJ. 2005. Functional analysis of candidate CD8+T cell-derived anti-HIV factors IMMUNOLOGY, 116 pp. 80-80.

Evans EJ, Jansson A, Yu C, Nilsson P, Sorensen P, Stuart DI, Davis SJ. 2005. Synaptic co-stimulatory complex formation and receptor triggering IMMUNOLOGY, 116 pp. 5-5.

Evans EJ, Esnouf RM, Manso-Sancho R, Gilbert RJC, James JR, Yu C, Hyning T, Ikemizu S, Stuart DI, Davis SJ. 2005. The structure and cross-reactivity of CD28 IMMUNOLOGY, 116 pp. 54-54.

Jansson A, Barnes E, Klenerman P, Harlén M, Sørensen P, Davis SJ, Nilsson P. 2005. A theoretical framework for quantitative analysis of the molecular basis of costimulation. J Immunol, 175 (3), pp. 1575-1585. | Show Abstract

We present a theoretical framework for simulating the synaptic accumulation of the costimulatory molecules CD28, CTLA-4, B7-1, and B7-2, based on a system of mean-field, ordinary differential equations, and rigorous biophysical and expression data. The simulations show that binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects all profoundly influence complex formation at cellular interfaces. B7-2 engages 33-fold more CD28 than CTLA-4 at the synapse in contrast to B7-1, which ligates approximately 7-fold more CTLA-4 than CD28. Although B7-1 completely dominates interactions with CTLA-4, forming linear arrays of 7-18 receptor-ligand pairs, CTLA-4 is fully engaged by B7-2 when B7-1 is absent. Additional simulations reveal the sensitivity of CD28 interactions to modeled transport processes. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be due to the differential binding properties of B7-1 and B7-2 only. We discuss the apparent redundancy of B7-1 in the context of a potentially dynamic synaptic microenvironment, and in light of functions other than the direct enhancement of T cell inhibition by CTLA-4.

James ES, Harney S, Wordsworth BP, Cookson WO, Davis SJ, Moffatt MF. 2005. PDCD1: a tissue-specific susceptibility locus for inherited inflammatory disorders. Genes Immun, 6 (5), pp. 430-437. | Show Abstract | Read more

Variation in genes encoding costimulatory molecules expressed on lymphocytes has been expected to contribute to the genetic component of inflammatory disease, but only the gene encoding the inhibitory protein, CTLA-4, seems consistently to confer disease susceptibility. Studies in murine models implicate the inhibitory product of the pd1 gene, programmed death-1, in the maintenance of peripheral tolerance to self-antigens. We identify 22 single-nucleotide polymorphisms (SNPs) in the equivalent human gene, PDCD1, a number of which show significant associations with the specific immunoglobulin E response to grass allergens in atopic individuals. Stepwise analyses indicate that four of the disease-associated SNPs have independent effects. The two most common haplotypes show positive and negative associations but rarer haplotypes are also likely to be of influence. In a case-control study, multiple regression analysis of genotypic data implies that PDCD1 also confers susceptibility to rheumatoid arthritis. Along with work linking PDCD1 with susceptibility to another autoimmune condition, systemic lupus erythematosus, our data identify PDCD1 as a second immunomodulatory gene with pleiotropic effects in human disease. Genes encoding negative regulators may generally confer a significant fraction of the genetic risk associated with inherited inflammatory disorders.

Evans EJ, Esnouf RM, Manso-Sancho R, Gilbert RJ, James JR, Yu C, Fennelly JA, Vowles C, Hanke T, Walse B et al. 2005. Crystal structure of a soluble CD28-Fab complex. Nat Immunol, 6 (3), pp. 271-279. | Show Abstract | Read more

Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the related, inhibitory homodimer CTLA-4. Cryo-electron microscopy-based comparisons of complexes of CD28 with mitogenic and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes the initial structural characterization of the CD28-CTLA-4-CD80-CD86 signaling system.

Davis SJ, van der Merwe PA. 2003. TCR triggering: co-receptor-dependent or -independent? Trends Immunol, 24 (12), pp. 624-626. | Read more

Love CA, Harlos K, Mavaddat N, Davis SJ, Stuart DI, Jones EY, Esnouf RM. 2003. The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D. Nat Struct Biol, 10 (10), pp. 843-848. | Show Abstract | Read more

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.

Evans EJ, Hene L, Sparks LM, Dong T, Retiere C, Fennelly JA, Manso-Sancho R, Powell J, Braud VM, Rowland-Jones SL et al. 2003. The T cell surface--how well do we know it? Immunity, 19 (2), pp. 213-223. | Show Abstract | Read more

The overall degree of complexity of the T cell surface has been unclear, constraining our understanding of its biology. Using global gene expression analysis, we show that 111 of 374 genes encoding well-characterized leukocyte surface antigens are expressed by a resting cytotoxic T cell. Unexpectedly, of 97 stringently defined, T cell-specific transcripts with unknown functions that we identify, none encode proteins with the modular architecture characteristic of 80% of leukocyte surface antigens. Only two encode proteins with membrane topologies found exclusively in cell surface molecules. Our analysis indicates that the cell type-specific composition of the resting CD8+ T cell surface is now largely defined, providing an insight into the overall compositional complexity of the mammalian cell surface and a framework for formulating systematic models of T cell surface-dependent processes, such as T cell receptor triggering.

Merry AH, Gilbert RJ, Shore DA, Royle L, Miroshnychenko O, Vuong M, Wormald MR, Harvey DJ, Dwek RA, Classon BJ et al. 2003. O-glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8. J Biol Chem, 278 (29), pp. 27119-27128. | Show Abstract | Read more

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.

Lühder F, Huang Y, Dennehy KM, Guntermann C, Müller I, Winkler E, Kerkau T, Ikemizu S, Davis SJ, Hanke T, Hünig T. 2003. Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists. J Exp Med, 197 (8), pp. 955-966. | Show Abstract | Read more

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): "conventional," TCR signaling-dependent costimulatory mAbs and "superagonistic" mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C"D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C"D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C"D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor kappaB pathway without inducing phosphorylation of either TCRzeta or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.

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Brown J, Walter TS, Carter L, Abrescia NGA, Aricescu AR, Batuwangala TD, Bird LE, Brown N, Chamberlain PP, Davis SJ et al. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 315-318. | Show Abstract | Read more

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

Davis SJ, Ikemizu S, Evans EJ, Fugger L, Bakker TR, van der Merwe PA. 2003. The nature of molecular recognition by T cells. Nat Immunol, 4 (3), pp. 217-224. | Show Abstract | Read more

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

van der Merwe PA, Davis SJ. 2003. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol, 21 (1), pp. 659-684. | Show Abstract | Read more

Over the past decade, key protein interactions contributing to T cell antigen recognition have been characterized in molecular detail. These have included interactions involving the T cell antigen receptor (TCR) itself, its coreceptors CD4 and CD8, the accessory molecule CD2, and the costimulatory receptors CD28 and CTLA-4. A clear view is emerging of how these molecules interact with their ligands at the cell-cell interface. Structural and binding studies have confirmed that the proteins span small but comparable distances and that, overall, they interact very weakly. However, there have been important surprises as well: that TCR interactions with peptide-MHC are topologically constrained and characterized by considerable conformational flexibility at the binding interface; that coreceptors engage peptide-MHC with extraordinarily fast kinetics and at angles apparently precluding direct interactions with the TCR bound to the same peptide-MHC; that the structural mechanisms allowing recognition by costimulatory and accessory molecules to be weak and yet specific are very heterogeneous; and that because of differences in both binding affinity and stoichiometry, there is enormous variation in the stability of the various costimulatory receptor/ligand complexes. These studies provide the necessary framework for exploring how these molecular interactions initiate T cell activation.

Davis SJ, Van Der Merwe PA, Randriamampita C, Delon J, Trautmann A. 2003. TCR triggering: Co-receptor-dependent or -independent? [2] (multiple letters) Trends in Immunology, 24 (12), pp. 624-627. | Read more

Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, Stuart DI, van der Merwe PA, Davis SJ. 2002. The interaction properties of costimulatory molecules revisited. Immunity, 17 (2), pp. 201-210. | Show Abstract | Read more

B7-1 and B7-2 are generally thought to have comparable structures and affinities for their receptors, CD28 and CTLA-4, each of which is assumed to be bivalent. We show instead (1) that B7-2 binds the two receptors more weakly than B7-1, (2) that, relative to its CTLA-4 binding affinity, B7-2 binds CD28 2- to 3-fold more effectively than B7-1, (3) that, unlike B7-1, B7-2 does not self-associate, and (4) that, in contrast to CTLA-4 homodimers, which are bivalent, CD28 homodimers are monovalent. Our results indicate that B7-1 markedly favors CTLA-4 over CD28 engagement, whereas B7-2 exhibits much less bias. We propose that the distinct structures and binding properties of B7-1 and B7-2 account for their overlapping but distinct effects on T cell responses.

Wheeler SF, Rudd PM, Davis SJ, Dwek RA, Harvey DJ. 2002. Comparison of the N-linked glycans from soluble and GPI-anchored CD59 expressed in CHO cells. Glycobiology, 12 (4), pp. 261-271. | Show Abstract | Read more

The N-linked glycosylation of recombinant human CD59, expressed in Chinese hamster ovary (CHO) cells with and without a membrane anchor, was compared to examine the effect of the anchor on glycan processing. N-Linked glycans were released with peptide-N-glycosidase F (PNGase F) within gel from SDS-PAGE-isolated soluble and glycosylphosphatidylinositol (GPI)-anchored human CD59 expressed in CHO cells. The anchored form contained core-fucosylated neutral and sialylated bi-, tri-, and tetraantennary glycans with up to four N-acetyllactosamine extensions. Exoglycosidase digestions and analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were used to define the relative amounts of the bi-, tri-, and tetraantennary glycans and to investigate the distribution of N-acetyllactosamine extensions between their antennae. Biantennary structures accounted for about 60% of the glycans, 30% of the triantennary structures, and about 10% of the tetraantennary structures. For tri- and tetraantennary glycans, those with extended antennae were found to be more abundant than those without extensions. The soluble form of CD59, expressed in CHO cells without the GPI anchor signal sequence, consisted almost entirely (97%) of biantennary glycans, of which 81% were unmodified, 17% contained one N-acetyllactosamine extension, and 2% contained two extensions. No compounds with longer extensions were found. A MALDI spectrum of the intact glycoprotein showed a distribution of glycans that matched those released with PNGase F. In addition, the protein was substituted with several small glycans, such as HexNAc, HexNAc-->Fuc, and HexNAc-->HexNAc, probably as the result of degradation of the mature N-linked glycans. The results show that the presence of the anchor increases the extent of glycan processing, possibly as the result of longer exposure to the glycosyltransferases or to a closer proximity of the protein to these enzymes.

van Der Merwe PA, Davis SJ. 2002. Immunology. The immunological synapse--a multitasking system. Science, 295 (5559), pp. 1479-1480. | Read more

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van der Merwe PA, Davis SJ. 2002. The immunological synapse - a multitasking system SCIENCE, 295 (5559), pp. 1479-1480. | Read more

Bromley SK, Iaboni A, Davis SJ, Whitty A, Green JM, Shaw AS, Weiss A, Dustin ML. 2001. The immunological synapse and CD28-CD80 interactions. Nat Immunol, 2 (12), pp. 1159-1166. | Show Abstract | Read more

According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28.

Fennelly JA, Tiwari B, Davis SJ, Evans EJ. 2001. CD2F-10: a new member of the CD2 subset of the immunoglobulin superfamily. Immunogenetics, 53 (7), pp. 599-602. | Show Abstract | Read more

The CD2 subset of the immunoglobulin superfamily consists of a rapidly expanding family of leukocyte cell surface receptors, at least five of which (CD2, CD48, CD58, CD150, and CD244) are involved in lymphocyte activation as either receptors or ligands. Completion of the draft sequence of the human genome offers the possibility of systematically identifying the full set of proteins and interactions of this important family. Here we describe the identification and characterization of the first new member of the subset, CD2F-10, found exclusively by genome searching.

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Davis SJ, van der Merwe PA. 2001. The immunological synapse: required for T cell receptor signalling or directing T cell effector function? CURRENT BIOLOGY, 11 (8), pp. R289-R290. | Read more

Davis SJ, Ikemizu S, Collins AV, Fennelly JA, Harlos K, Jones EY, Stuart DI. 2001. Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1. Acta Crystallogr D Biol Crystallogr, 57 (Pt 4), pp. 605-608. | Show Abstract | Read more

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Stamper CC, Zhang Y, Tobin JF, Erbe DV, Ikemizu S, Davis SJ, Stahl ML, Seehra J, Somers WS, Mosyak L. 2001. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature, 410 (6828), pp. 608-611. | Show Abstract | Read more

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 A resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.

Pellicci DG, Kortt AA, Sparrow LG, Hudson PJ, Sorensen HV, Davis SJ, Classon BJ. 2000. Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein. J Immunol Methods, 246 (1-2), pp. 149-163. | Show Abstract | Read more

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

Mavaddat N, Mason DW, Atkinson PD, Evans EJ, Gilbert RJ, Stuart DI, Fennelly JA, Barclay AN, Davis SJ, Brown MH. 2000. Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity. J Biol Chem, 275 (36), pp. 28100-28109. | Show Abstract | Read more

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

Vorup-Jensen T, Petersen SV, Hansen AG, Poulsen K, Schwaeble W, Sim RB, Reid KB, Davis SJ, Thiel S, Jensenius JC. 2000. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2. J Immunol, 165 (4), pp. 2093-2100. | Show Abstract

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Brodie D, Collins AV, Iaboni A, Fennelly JA, Sparks LM, Xu XN, van der Merwe PA, Davis SJ. 2000. LICOS, a primordial costimulatory ligand? Curr Biol, 10 (6), pp. 333-336. | Show Abstract | Read more

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.

Anton van der Merwe P, Davis SJ, Shaw AS, Dustin ML. 2000. Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. Semin Immunol, 12 (1), pp. 5-21. | Show Abstract | Read more

T cell antigen recognition is accompanied by cytoskeletal polarization towards the APC and large-scale redistribution of cell surface molecules into 'supramolecular activation clusters' (SMACs), forming an organized contact interface termed the 'immunological synapse' (IS). Molecules are arranged in the IS in a micrometer scale bull's eye pattern with a central accumulation of TCR/peptide-MHC (the cSMAC) surrounded by a peripheral ring of adhesion molecules (the pSMAC). We propose that segregation of cell surface molecules on a much smaller scale initiates TCR triggering, which drives the formation of the IS by active transport processes. IS formation may function as a checkpoint for full T cell activation, integrating information on the presence and quality of TCR ligands and the nature and activation state of the APC.

Ikemizu S, Gilbert RJ, Fennelly JA, Collins AV, Harlos K, Jones EY, Stuart DI, Davis SJ. 2000. Structure and dimerization of a soluble form of B7-1. Immunity, 12 (1), pp. 51-60. | Show Abstract | Read more

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Butters TD, Sparks LM, Harlos K, Ikemizu S, Stuart DI, Jones EY, Davis SJ. 1999. Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization. Protein Sci, 8 (8), pp. 1696-1701. | Show Abstract | Read more

Heterologous gene expression in either (1) the glycosylation-defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 A, c = 51.4 A alpha = beta = 90 degrees , gamma = 120 degrees , that diffract to a maximum resolution of 1.8 A.

Rudd PM, Wormald MR, Harvey DJ, Devasahayam M, McAlister MS, Brown MH, Davis SJ, Barclay AN, Dwek RA. 1999. Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells. Glycobiology, 9 (5), pp. 443-458. | Show Abstract | Read more

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.

Ikemizu S, Sparks LM, van der Merwe PA, Harlos K, Stuart DI, Jones EY, Davis SJ. 1999. Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution. Proc Natl Acad Sci U S A, 96 (8), pp. 4289-4294. | Show Abstract | Read more

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-A resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC'C" beta-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell-cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein-protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies.

Wyer JR, Willcox BE, Gao GF, Gerth UC, Davis SJ, Bell JI, van der Merwe PA, Jakobsen BK. 1999. T cell receptor and coreceptor CD8 alphaalpha bind peptide-MHC independently and with distinct kinetics. Immunity, 10 (2), pp. 219-225. | Show Abstract | Read more

The T cell surface glycoprotein CD8 enhances T cell antigen recognition by binding to MHC class I molecules. We show that human CD8 alphaalpha binds to the MHC class I molecule HLA-A2 with an extremely low affinity (Kd approximately 0.2 mM at 37 degrees C) and with kinetics that are between 2 and 3 orders of magnitude faster than reported for T cell receptor/peptide-MHC interactions. Furthermore, CD8 alphaalpha had no detectable effect on a T cell receptor (TCR) binding to the same peptide-MHC class I complex. These binding properties provide an explanation as to why the CD8/MHC class I interaction is unable to initiate cell-cell adhesion and how it can enhance TCR recognition without interfering with its specificity.

Davis SJ, Ikemizu S, Wild MK, van der Merwe PA. 1998. CD2 and the nature of protein interactions mediating cell-cell recognition. Immunol Rev, 163 (1), pp. 217-236. | Show Abstract | Read more

Rapid progress has recently been made in characterising the structures of leukocyte cell-surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co-receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell-cell recognition. As an example of biological recognition, the defining characteristic of cell-cell contact is that it involves the simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell-cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand-binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface-shape complementarity, is the dominant feature of specific, low-affinity protein recognition at the cell surface by CD2. The crystallographic analysis of the CD2-binding domain of CD58 strongly supports this concept.

Davis SJ, Davies EA, Tucknott MG, Jones EY, van der Merwe PA. 1998. The role of charged residues mediating low affinity protein-protein recognition at the cell surface by CD2. Proc Natl Acad Sci U S A, 95 (10), pp. 5490-5494. | Show Abstract | Read more

Insights into the structural basis of protein-protein recognition have come principally from the analysis of proteins such as antibodies, hormone receptors, and proteases that bind their ligands with relatively high affinity (Ka approximately 10(9) M-1). In contrast, few studies have been done on the very low affinity interactions mediating cell adhesion and cell-cell recognition. As a site of protein-protein recognition, the ligand binding face of the T lymphocyte cell-cell recognition molecule, CD2, which binds its ligands 10(4)- to 10(5)-fold more weakly than do antibodies and proteases, is unusual in being both very flat and highly charged. An analysis of the effect of mutations and ionic strength on CD2 binding to its ligand, CD48, indicates that these charged residues contribute little, if any, binding energy to this interaction. However, the loss of these charged residues is shown to markedly reduce ligand-binding specificity. Thus, the charged residues increase the specificity of CD2 binding without increasing the affinity. This phenomenon is likely to result from a requirement for electrostatic complementarity between charged binding surfaces to compensate for the removal, upon binding, of water interacting with the charged residues. It is proposed that this mode of recognition is highly suited to biological interactions requiring a low affinity because it uncouples increases in specificity from increases in affinity.

Vorup-Jensen T, Davis SJ, Poulsen K, Schwaeble W, Stover CM, Sim R, Reid KBM, Petersen SV, Thiel S, Jensenius JC. 1998. Studies of recombinant MBL and MASP-2: Is MASP-2 self-activating? MOLECULAR IMMUNOLOGY, 35 (6-7), pp. 409-409. | Read more

Rudd PM, Morgan BP, Wormald MR, Harvey DJ, van den Berg CW, Davis SJ, Ferguson MA, Dwek RA. 1997. The glycosylation of the complement regulatory protein, human erythrocyte CD59. J Biol Chem, 272 (11), pp. 7229-7244. | Show Abstract | Read more

Human erythrocyte CD59 contains N- and O-glycans and a glycosylphosphatidylinositol (GPI) anchor, all of which have been analyzed in this study. The anchor consists principally of the minimum core glycan sequence Manalpha1-2Manalpha1-6Manalpha1-4GlcN-linked to a phosphatidylinositol moiety with the structure sn-1-O-alkyl(C18:0 and C18:1)-2-O-acyl(C20:4)glycerol-3-phospho-1-(2-O-palmitoyl(C16:0))myo- inositol. This structure is essentially identical to that of human erythrocyte cholinesterase (Deeg, M. A., Humphrey, D. R., Yang, S. H. , Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580). This first comparison of GPI anchors from different proteins expressed in the same tissue suggests that human reticulocytes produce only one type of anchor structure. The N- and O-glycans were sequenced using a novel approach involving digestion of the total glycan pool with multiple enzyme arrays. The N-glycan pool contained families of bi-antennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. The predominant O-glycans were NeuNAcalpha2-3Galbeta1-3GalNAc and Galbeta1-3[NeuNAcalpha2-3]GalNAc. Inspection of a molecular model of CD59, based on the NMR solution structure of the extracellular domain and the structural data from this study, suggested several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases. This work completes the initial structural analysis of CD59, providing the most complete view of any cell surface glycoprotein studied to date.

van der Merwe PA, Bodian DL, Daenke S, Linsley P, Davis SJ. 1997. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med, 185 (3), pp. 393-403. | Show Abstract | Read more

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell-APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

Bodian DL, Davis SJ, Morgan BP, Rushmere NK. 1997. Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59. J Exp Med, 185 (3), pp. 507-516. | Show Abstract | Read more

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Davis SJ, vanderMerwe PA. 1996. CD2: An exception to the immunoglobulin superfamily concept? SCIENCE, 273 (5279), pp. 1241-1242. | Read more

McAlister MS, Mott HR, van der Merwe PA, Campbell ID, Davis SJ, Driscoll PC. 1996. NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48. Biochemistry, 35 (19), pp. 5982-5991. | Show Abstract | Read more

The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2).

Davis SJ, van der Merwe PA. 1996. The structure and ligand interactions of CD2: implications for T-cell function. Immunol Today, 17 (4), pp. 177-187. | Show Abstract | Read more

Considerable progress has recently been made in understanding the structure and ligand interactions of the T-cell antigen CD2, to the extent that CD2 is now a useful paradigm for considering the structural basis of cell-cell recognition. Here, Simon Davis and Anton van der Merwe review the new data and consider their implications for T-cell function in the context of CD2-knockout experiments.

Fischer PB, Collin M, Karlsson GB, James W, Butters TD, Davis SJ, Gordon S, Dwek RA, Platt FM. 1995. The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding. J Virol, 69 (9), pp. 5791-5797. | Show Abstract

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.

Davis SJ, Davies EA, Barclay AN, Daenke S, Bodian DL, Jones EY, Stuart DI, Butters TD, Dwek RA, van der Merwe PA. 1995. Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent. J Biol Chem, 270 (1), pp. 369-375. | Show Abstract | Read more

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

van der Merwe PA, McNamee PN, Davies EA, Barclay AN, Davis SJ. 1995. Topology of the CD2-CD48 cell-adhesion molecule complex: implications for antigen recognition by T cells. Curr Biol, 5 (1), pp. 74-84. | Show Abstract | Read more

BACKGROUND: The T-lymphocyte cell-surface molecule, CD2, was the first heterophilic cell-adhesion molecule to be discovered and has become an important paradigm for understanding the structural basis of cell adhesion. Interaction of CD2 with its ligands. CD58 (in humans) and CD48 (in mice and rats), contributes to antigen recognition by T cells. CD2, CD48 and CD58 are closely related members of the immunoglobulin superfamily and their extracellular regions are predicted to have very similar structures. The three-dimensional crystal structure of this region of CD2 has been determined, revealing two immunoglobulin domains with the ligand-binding site situated on an exposed beta sheet in the membrane-distal domain. This GFCC'C" beta sheet is also involved in a homophilic 'head-to-head' interaction in the CD2 crystal lattice, which has been proposed to be a model for the interactions of CD2 with its ligands. RESULTS: We show that the CD2-binding site on rat CD48 lies on the equivalent beta-sheet of its membrane-distal immunoglobulin domain. By making complementary mutations, we have shown that two charged residues in the CD48 ligand-binding site interact directly with two oppositely charged residues in CD2's ligand-binding site. These results indicate that the amino-terminal immunoglobulin domains of CD2 and CD48 bind each other in the same orientation as the CD2-CD2 crystal lattice interaction, strongly supporting the suggestion that CD2 interacts head-to-head with its ligand. Modelling CD48 onto the CD2 structure reveals that the CD2-CD48 complex spans approximately the same distance (134 A) as predicted for the complex between the T-cell receptor and the peptide-bound major histocompatibility complex (MHC) molecule. CONCLUSIONS: Our results, together with recent structural studies of CD2, provide the first indication of the specific topology of a cell-adhesion molecule complex. The similar dimensions predicted for the CD2-CD48 complex and the complex between the T-cell receptor and the peptide-bound MHC molecule suggest that one of the functions of CD2 may be to position the plasma membranes of the T cell and the antigen-presenting (or target) cell at the optimal distance for the low-affinity interaction between the T-cell receptor and the peptide-bound MHC molecule.

van der Merwe PA, Barclay AN, Mason DW, Davies EA, Morgan BP, Tone M, Krishnam AK, Ianelli C, Davis SJ. 1994. Human cell-adhesion molecule CD2 binds CD58 (LFA-3) with a very low affinity and an extremely fast dissociation rate but does not bind CD48 or CD59. Biochemistry, 33 (33), pp. 10149-10160. | Show Abstract | Read more

CD2 is a T lymphocyte cell-adhesion molecule (CAM) belonging to the immunoglobulin superfamily (IgSF) which mediates transient adhesion of T cells to antigen-presenting cells and target cells. Reported ligands for human CD2 include the structurally-related IgSF CAMs CD58 (LFA-3) and CD48 as well as, more controversially, the unrelated cell-surface glycoprotein CD59. Using surface plasmon resonance technology, which avoids several pitfalls of conventional binding assays, we recently reported that rat CD2 binds rat CD48 with a very low affinity (Kd 60-90 microM) and dissociates rapidly (koff > or = 6 s-1) [van der Merwe, P. A., Brown, M. H., Davis, S. J., & Barclay, A. N. (1993) EMBO J. 12, 4945-4954]. In contrast, a study using conventional equilibrium binding methods reported a much higher affinity (Kd 0.4 microM) for human CD2 binding CD58 which suggested that the weak binding of rat CD2 to CD48 may not represent a typical CAM interaction. In the present study we have used surface plasmon resonance to obtain definitive affinity and kinetic data on the interactions of a soluble, recombinant form of human CD2 with soluble forms of CD58, CD48, and CD59. Binding of CD2 to CD58 was readily detected but we were unable to detect any direct interaction between CD2 and either CD59 or CD48 under conditions in which very low affinity interactions (Kd approximately 0.5 mM) would have been detected. In contrast to previous reports we found that human CD2 bound CD58 with a very low affinity (Kd 9-22 microM) and dissociated with an extremely fast dissociation rate constant (koff > or = 4 s-1). The association rate constant (kon) could not be measured directly but was calculated to be > or = 400,000 M-1s-1. Taken together, these results provide conclusive evidence that CAM interactions can have very low affinities and extremely fast dissociation rate constants.

Bodian DL, Jones EY, Harlos K, Stuart DI, Davis SJ. 1994. Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution. Structure, 2 (8), pp. 755-766. | Show Abstract | Read more

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Kieffer B, Driscoll PC, Campbell ID, Willis AC, van der Merwe PA, Davis SJ. 1994. Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins. Biochemistry, 33 (15), pp. 4471-4482. | Show Abstract | Read more

The cell surface antigen CD59 is an inhibitor of complement-mediated lysis and a member of the Ly6 superfamily (Ly6SF) of cysteine-rich cell-surface molecules whose sequences are related to those of snake venom neurotoxins. The three-dimensional solution structure of a recombinant form of the extracellular region of the molecule (residues 1-70 of the mature protein; sCD59) has been solved by 2D NMR methods. sCD59 is a relatively flat, disk-shaped molecule consisting of a two-standed beta-sheet finger loosely packed against a protein core formed by a three-stranded beta-sheet and a short helix. Structure calculations allowed an unambiguous assignment of the disulfide-bonded cysteine pairs as 3-26, 6-13, 19-39, 45-63, and 64-69. The topology of sCD59 is similar to that of the snake venom neurotoxins and consistent with an evolutionary relationship existing between the Ly6SF and the neurotoxins.

VANDERMERWE P, BROWN M, DAVIS S, BARCLAY A. 1994. AFFINITY AND KINETIC-ANALYSIS OF THE INTERACTION OF THE CELL-ADHESION MOLECULE CD2 WITH ITS LIGANDS CD48 AND CD58 JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 257-257.

Tavernor AS, Kydd JH, Bodian DL, Jones EY, Stuart DI, Davis SJ, Butcher GW. 1994. Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements. Eur J Biochem, 219 (3), pp. 969-976. | Show Abstract | Read more

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

van der Merwe PA, Brown MH, Davis SJ, Barclay AN. 1993. Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48. EMBO J, 12 (13), pp. 4945-4954. | Show Abstract

CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.

Van der Merwe PA, Brown MH, Davis SJ, Barclay AN. 1993. Measuring very low affinity interactions between immunoglobulin superfamily cell-adhesion molecules. Biochem Soc Trans, 21 (4), pp. 340S.

Barclay AN, Brady RL, Davis SJ, Lange G. 1993. CD4 and the immunoglobulin superfamily. Philos Trans R Soc Lond B Biol Sci, 342 (1299), pp. 7-12. | Show Abstract | Read more

The CD4 membrane glycoprotein was one of the first cell surface antigens to be identified using monoclonal antibodies. It was shown to have a central role in the control of the recognition of foreign proteins by T lymphocytes and later as a receptor for the human immunodeficiency virus (HIV). The analysis of the amino acid sequence of CD4 showed that the extracellular region comprised four regions with sequence similarities to immunoglobulin domains. The structure of domains 3 and 4 of CD4 has been determined by X-ray crystallography and, like domains 1 and 2 previously determined, these have typical immunoglobulin-like folds. The results are discussed with respect to the identification of other domains with immunoglobulin-like folds from amino acid sequence data, and the evolution of CD4.

Dwek RA, Ashford DA, Edge CJ, Parekh RB, Rademacher TW, Wing DR, Barclay AN, Davis SJ, Williams AF. 1993. Glycosylation of CD4 and Thy-1. Philos Trans R Soc Lond B Biol Sci, 342 (1299), pp. 43-50. | Show Abstract | Read more

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with mutant molecules with only single glycosylation sites and with a truncated form containing only the two NH2-terminal domains, indicate that independent processing occurs at each glycosylation site and that domain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Davis SJ, James WS, Schockmel GA, Simon JH, Somoza C. 1993. The recognition of chimeras of rat and human CD4 by HIV-1 gp120 and by monoclonal antibodies. Philos Trans R Soc Lond B Biol Sci, 342 (1299), pp. 75-81. | Show Abstract | Read more

The use of chimeras of rat and human CD4 to probe the HIV-1 gp120 and antibody binding properties of CD4 is reviewed. Short segments of human CD4 sequence were substituted for the equivalent regions of rat CD4 which does not bind gp120, and analysis of the properties of these chimeras established: (i) that residues 33-58 of the NH2-terminal domain of human CD4 encompass the high-affinity gp120 binding site; and (ii) that chimeras containing residues 33-62 mediate HIV-1 infection. The chimera-binding specificities of gp120 and a large panel of anti-CD4 antibodies were also determined. This allowed a critical test of the popular notion that receptor mimics appear at high frequency among antibodies elicited by immunization with receptor ligands and that anti-idiotypic antibodies can be used to identify novel receptors. The data suggest that such mimics appear infrequently, if at all, a result which is consistent with the failure of the anti-idiotype approach to identify new genes encoding receptors with prescribed functions.

van der Merwe PA, McPherson DC, Brown MH, Barclay AN, Cyster JG, Williams AF, Davis SJ. 1993. The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2. Eur J Immunol, 23 (6), pp. 1373-1377. | Show Abstract | Read more

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.

Brady RL, Dodson EJ, Dodson GG, Lange G, Davis SJ, Williams AF, Barclay AN. 1993. Crystal structure of domains 3 and 4 of rat CD4: relation to the NH2-terminal domains. Science, 260 (5110), pp. 979-983. | Show Abstract | Read more

The CD4 antigen is a membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigens and is also a receptor for the human immunodeficiency virus. the extracellular portion of CD4 is predicted to fold into four immunoglobulin-like domains. The crystal structure of the third and fourth domains of rat CD4 was solved at 2.8 angstrom resolution and shows that both domains have immunoglobulin folds. Domain 3, however, lacks the disulfide between the beta sheets; this results in an expansion of the domain. There is a difference of 30 degrees in the orientation between domains 3 and 4 when compared with domains 1 and 2. The two CD4 fragment structures provide a basis from which models of the overall receptor can be proposed. These models suggest an extended structure comprising two rigid portions joined by a short and possibly flexible linker region.

Simon JH, Somoza C, Schockmel GA, Collin M, Davis SJ, Williams AF, James W. 1993. A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection. J Exp Med, 177 (4), pp. 949-954. | Show Abstract | Read more

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.

Ashford DA, Alafi CD, Gamble VM, Mackay DJ, Rademacher TW, Williams PJ, Dwek RA, Barclay AN, Davis SJ, Somoza C. 1993. Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells. J Biol Chem, 268 (5), pp. 3260-3267. | Show Abstract

The rat and human forms of the T-cell surface glycoprotein CD4 share a common glycosylation site at the Asn270/271 position but differ with respect to the locations of the second glycosylation sites at Asn159 (rat) and Asn300 (human). The glycosylation of soluble recombinant forms of human and rat CD4 (sCD4) expressed in Chinese hamster ovary cells has been characterized. The most obvious differences between the rat and human sCD4 oligosaccharides were the greater abundance of oligomannose and hybrid oligosaccharides on rat sCD4 and the presence of oligosaccharides carrying a terminal alpha-galactose residue on human sCD4. This is the first report of the occurrence of alpha-galactose residues on a glycoprotein expressed in Chinese hamster ovary cells. Comparison of mutant rat sCD4 molecules with single glycosylation sites and glycopeptides indicated that site-specific and independent processing occurred at each glycosylation site. The glycosylation at the conserved site at Asn270 of rat sCD4 was identical to that seen for the equivalent site in human sCD4, and the oligomannose and hybrid structures were restricted to the nonconserved site at Asn159 in rat sCD4. However, there was more oligosaccharide processing at this site in a truncated form of rat sCD4 consisting of the two NH2-terminal domains. These results indicate that not only the local three-dimensional structure but also domain interactions can influence the processing at individual glycosylation sites.

Davis SJ, Puklavec MJ, Ashford DA, Harlos K, Jones EY, Stuart DI, Williams AF. 1993. Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD2. Protein Eng, 6 (2), pp. 229-232. | Read more

Davis SJ. 1993. Mimicking ligands Nature, 361 (6409), pp. 212-212. | Read more

Jones EY, Davis SJ, Williams AF, Harlos K, Stuart DI. 1992. Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2. Nature, 360 (6401), pp. 232-239. | Show Abstract | Read more

The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.

Davis SJ, Schockmel GA, Somoza C, Buck DW, Healey DG, Rieber EP, Reiter C, Williams AF. 1992. Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors. Nature, 358 (6381), pp. 76-79. | Show Abstract | Read more

It has been proposed that antibodies can mimic the binding of a receptor to its ligand and that anti-idiotype antibodies raised against such antibodies can be used to identify the receptor. A large number of antibodies have been raised against CD4, the receptor on T cells for the envelope glycoprotein gp120 of the human immunodeficiency virus, and the site at which gp120 binds to CD4 has been delineated. It has therefore become possible to contrast the fine specificities of a natural ligand (gp120) and antibodies that interact with the receptor at the same site. Here we report that out of a panel of 225 anti-CD4 antibodies, only one showed fine binding specificity that was broadly like that of gp120, but the evidence was against this being an exact mimic. Thus the data indicate that the production of antibody mimics will occur very rarely or not at all and that the anti-idiotype approach is unlikely to be useful. This contention is supported by a review of the results of attempts to use this approach. Taking strict criteria for success, there is no example for which the anti-idiotype approach has led to the discovery of a previously undescribed receptor or other protein of interest.

Schockmel GA, Somoza C, Davis SJ, Williams AF, Healey D. 1992. Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4. J Exp Med, 175 (1), pp. 301-304. | Show Abstract | Read more

The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.

Beyers AD, Davis SJ, Cantrell DA, Izquierdo M, Williams AF. 1991. Autonomous roles for the cytoplasmic domains of the CD2 and CD4 T cell surface antigens. EMBO J, 10 (2), pp. 377-385. | Show Abstract

CD2 and CD4 are single chain transmembrane T cell surface molecules that are involved in signal transduction. Chimaeric constructs from rat CD2 and CD4 antigens were expressed in the Jurkat human T cell line to examine the role of extracellular, transmembrane and cytoplasmic domains in mediating functions controlled by CD2 and CD4. The results show that the large rise in concentration of cytoplasmic free Ca2+ mediated via CD2 crosslinking is controlled by the cytoplasmic domain and does not require the CD2 transmembrane and extracellular domains. Similarly the CD4 cytoplasmic domain alone was shown to encode the specificity for binding to the p56lck tyrosine kinase and to control down-modulation of CD4 after treatment with phorbol ester. Evidence was obtained that down-modulation of CD4 occurs when p56lck dissociates from the cytoplasmic domain due to phosphorylation of Ser 405.

Davis SJ, Ward HA, Puklavec MJ, Willis AC, Williams AF, Barclay AN. 1990. High level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J Biol Chem, 265 (18), pp. 10410-10418. | Show Abstract

The CD4 cell surface antigen is of interest as a marker of T lymphocytes that recognize foreign antigens in the context of MHC Class II antigen, as a receptor for the human immunodeficiency virus (HIV) and as a member of the immunoglobulin superfamily (IgSF) with four Ig-like domains present in the extracellular domain. In order to produce large amounts of soluble CD4 for x-ray crystallography and other molecular studies, a recently developed expression system based on selection via glutamine synthetase was used. Expression was attempted for rat CD4 corresponding to the full extracellular sequence (sCD4; domains 1-4), the NH2-terminal half (domains 1 and 2) and the first domain alone. Stable transfected Chinese hamster ovary cell lines were obtained that expressed sCD4 and sCD4 (half) at typical maximal levels in spent tissue culture supernatant of greater than 80 and 25 mg/liter, respectively. Domain 1 alone was not expressed and introduction of a N-linked glycosylation site did not facilitate expression. The role of glycosylation in the expression of sCD4 was investigated by mutagenesis of the constructs to remove each of the two N-linked glycosylation sites in turn and both together. All three forms were expressed at 60-120 mg/liter. The sCD4 (half) was not expressed after deletion of its N-linked site. The disulfide bonds of sCD4 were determined to be within domains 1, 2, and 4 and isolation of glycopeptides showed that both N-linked sites were glycosylated. Analysis of the hydrodynamic properties of sCD4 suggested that the molecule adopted an extended conformation in solution rather than folding to form a compact structure like an Fab. The possibility of dimerisation of CD4 was investigated but sCD4 dimers were not seen at an affinity cut-off of about 4 x 10(5) M-1.

Davis SJ, Brady RL, Barclay AN, Harlos K, Dodson GG, Williams AF. 1990. Crystallization of a soluble form of the rat T-cell surface glycoprotein CD4 complexed with Fab from the W3/25 monoclonal antibody. J Mol Biol, 213 (1), pp. 7-10. | Show Abstract | Read more

The structure of the T-lymphocyte cell surface glycoprotein CD4 is of considerable biological and medical interest. Recombinant rat CD4 expressed in soluble form in mammalian cells and complexed with W3/25 monoclonal Fab fragments formed crystals that diffract to 3.5 A and have the orthorhombic space group P2(1)2(1)2 or P2(1)2(1)2(1). The unit cell has dimensions a = 317 A, b = 161 A and c = 41.8 A and the asymmetric unit consists of two CD4:Fab complexes. These crystals are of suitable quality for X-ray diffraction analysis.

Williams AF, Davis SJ, He Q, Barclay AN. 1989. Structural diversity in domains of the immunoglobulin superfamily. Cold Spring Harb Symp Quant Biol, 54 Pt 2 pp. 637-647. | Read more

Davis SJ. 1988. Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum. J Cell Biochem, 38 (2), pp. 77-86. | Show Abstract | Read more

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.

Jansson A, Barnes E, Klenerman P, Harlén M, Sørensen P, Davis SJ, Nilsson P. 2005. A theoretical framework for quantitative analysis of the molecular basis of costimulation. J Immunol, 175 (3), pp. 1575-1585. | Show Abstract

We present a theoretical framework for simulating the synaptic accumulation of the costimulatory molecules CD28, CTLA-4, B7-1, and B7-2, based on a system of mean-field, ordinary differential equations, and rigorous biophysical and expression data. The simulations show that binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects all profoundly influence complex formation at cellular interfaces. B7-2 engages 33-fold more CD28 than CTLA-4 at the synapse in contrast to B7-1, which ligates approximately 7-fold more CTLA-4 than CD28. Although B7-1 completely dominates interactions with CTLA-4, forming linear arrays of 7-18 receptor-ligand pairs, CTLA-4 is fully engaged by B7-2 when B7-1 is absent. Additional simulations reveal the sensitivity of CD28 interactions to modeled transport processes. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be due to the differential binding properties of B7-1 and B7-2 only. We discuss the apparent redundancy of B7-1 in the context of a potentially dynamic synaptic microenvironment, and in light of functions other than the direct enhancement of T cell inhibition by CTLA-4.

Evans EJ, Hene L, Sparks LM, Dong T, Retiere C, Fennelly JA, Manso-Sancho R, Powell J, Braud VM, Rowland-Jones SL et al. 2003. The T cell surface--how well do we know it? Immunity, 19 (2), pp. 213-223. | Show Abstract | Read more

The overall degree of complexity of the T cell surface has been unclear, constraining our understanding of its biology. Using global gene expression analysis, we show that 111 of 374 genes encoding well-characterized leukocyte surface antigens are expressed by a resting cytotoxic T cell. Unexpectedly, of 97 stringently defined, T cell-specific transcripts with unknown functions that we identify, none encode proteins with the modular architecture characteristic of 80% of leukocyte surface antigens. Only two encode proteins with membrane topologies found exclusively in cell surface molecules. Our analysis indicates that the cell type-specific composition of the resting CD8+ T cell surface is now largely defined, providing an insight into the overall compositional complexity of the mammalian cell surface and a framework for formulating systematic models of T cell surface-dependent processes, such as T cell receptor triggering.

Davis SJ, Ikemizu S, Evans EJ, Fugger L, Bakker TR, van der Merwe PA. 2003. The nature of molecular recognition by T cells. Nat Immunol, 4 (3), pp. 217-224. | Show Abstract | Read more

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

van der Merwe PA, Davis SJ. 2003. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol, 21 (1), pp. 659-684. | Show Abstract | Read more

Over the past decade, key protein interactions contributing to T cell antigen recognition have been characterized in molecular detail. These have included interactions involving the T cell antigen receptor (TCR) itself, its coreceptors CD4 and CD8, the accessory molecule CD2, and the costimulatory receptors CD28 and CTLA-4. A clear view is emerging of how these molecules interact with their ligands at the cell-cell interface. Structural and binding studies have confirmed that the proteins span small but comparable distances and that, overall, they interact very weakly. However, there have been important surprises as well: that TCR interactions with peptide-MHC are topologically constrained and characterized by considerable conformational flexibility at the binding interface; that coreceptors engage peptide-MHC with extraordinarily fast kinetics and at angles apparently precluding direct interactions with the TCR bound to the same peptide-MHC; that the structural mechanisms allowing recognition by costimulatory and accessory molecules to be weak and yet specific are very heterogeneous; and that because of differences in both binding affinity and stoichiometry, there is enormous variation in the stability of the various costimulatory receptor/ligand complexes. These studies provide the necessary framework for exploring how these molecular interactions initiate T cell activation.

Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, Stuart DI, van der Merwe PA, Davis SJ. 2002. The interaction properties of costimulatory molecules revisited. Immunity, 17 (2), pp. 201-210. | Show Abstract | Read more

B7-1 and B7-2 are generally thought to have comparable structures and affinities for their receptors, CD28 and CTLA-4, each of which is assumed to be bivalent. We show instead (1) that B7-2 binds the two receptors more weakly than B7-1, (2) that, relative to its CTLA-4 binding affinity, B7-2 binds CD28 2- to 3-fold more effectively than B7-1, (3) that, unlike B7-1, B7-2 does not self-associate, and (4) that, in contrast to CTLA-4 homodimers, which are bivalent, CD28 homodimers are monovalent. Our results indicate that B7-1 markedly favors CTLA-4 over CD28 engagement, whereas B7-2 exhibits much less bias. We propose that the distinct structures and binding properties of B7-1 and B7-2 account for their overlapping but distinct effects on T cell responses.

Bromley SK, Iaboni A, Davis SJ, Whitty A, Green JM, Shaw AS, Weiss A, Dustin ML. 2001. The immunological synapse and CD28-CD80 interactions. Nat Immunol, 2 (12), pp. 1159-1166. | Show Abstract | Read more

According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28.

Stamper CC, Zhang Y, Tobin JF, Erbe DV, Ikemizu S, Davis SJ, Stahl ML, Seehra J, Somers WS, Mosyak L. 2001. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature, 410 (6828), pp. 608-611. | Show Abstract | Read more

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 A resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.

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