Cell surface signalling laboratory

The Cell surface signalling laboratory is interested in the molecular basis of cellular recognition. Using our technologies, we have shown that it is possible to identify extracellular protein interactions that are essential for cellular processes that are critical for disease pathogenesis. We are currently working on recognition events that are important in malaria, heart disease and fertilisation.

The team, headed by Gavin Wright, has developed a screening assay called AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes many of the technical problems associated with identifying interactions between receptor proteins. Using this method, the team takes a unique large-scale approach to systematically identify networks of extracellular protein interactions between cells.

The basic rationale is to compile recombinant protein libraries containing the full repertoire of receptor proteins displayed on the surface of known interacting cell types. These receptor proteins are then screened against each other to identify novel interactions. The cellular interactions that we select for study are important for fundamental biological processes or the pathogenesis of disease. Once we have identified the receptor-ligand interactions involved, we aim to determine their role using functional assays.

Taking this approach, we have identified critical receptor interactions that can then be targeted to develop novel therapeutics. Importantly, extracellular protein interactions are amenable to this approach because they are accessible to systemically delivered drugs such as therapeutic antibodies. Currently, the laboratory is focusing on three main cellular interactions:

  • P. falciparum:host interactions - Host pathogen interactions that are involved in the pathogenesis of malaria.
  • Platelet:Platelet - The cellular interaction responsible for heart disease and stroke, two of the leading causes of human mortality.
  • Sperm:Egg - The cellular interaction responsible for new life. Understanding this could lead to the development of novel contraceptives and fertility treatments.

[Anne Weston, LRI, CRUK, Wellcome Images]

Technology

Cells can interact and communicate with each other through specific protein interactions made by cell surface receptor proteins embedded within the membrane that surrounds each cell. Many of the instructive signals received by cells originate from their immediate environment, the cell surface. More specifically, signals are sent and received by membrane proteins: special proteins that bridge the plasma membrane and are able to convey extracellular binding events within the cell. It is these proteins, and especially the extracellular interactions that they make with other proteins, that are the main interest of the laboratory. We aim to discover entirely new signalling pathways by identifying novel cell surface receptor-ligand pairs. Despite the known importance of these interactions in biology, relatively few cell surface proteins have known binding partners because they are biochemically difficult to manipulate.

AVEXIS

How AVEXIS resolves extracellular protein interaction networks.

How AVEXIS resolves extracellular protein interaction networks. [Genome Research Limited]

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Extracellular protein interactions made by membrane-embedded receptors are challenging to study because the proteins are biochemically difficult to work with: they are relatively rare (103/105 copies per cell), hard to solubilise in their native conformation and often contain structurally-critical posttranslational modifications such as disulfide bonds and glycosylation. In addition, the interactions mediated by these receptor proteins are often extremely transient, having half-lives of less than a second.

To circumvent the biochemical difficulties of working with membrane-embedded receptors, we have developed an assay called AVEXIS (for AVidity-based EXtracellular Interaction Screen) which can detect low affinity extracellular protein interactions in a convenient and scalable format. The approach works by compiling protein libraries constituting the whole ectodomain of a cell's receptor repertoire expressed as soluble recombinant proteins in mammalian cells. This enables us to preserve the extracellular binding activity of the receptor but in a format that allows us to manipulate the proteins easily.

The ectodomains are expressed as a bait and/or prey: the baits are biotinylated and captured on streptavidin-coated solid phases (such as a glass slide or microtitre plate) and the preys are pentamerised and enzyme-tagged. By pentamerising the prey proteins, we increase the local concentration of the ectodomain which increases the overall binding strength (avidity) of the interaction enabling us to then detect even very transient (t1/2 <= 0.1sec) interactions with a low false positive rate. The assay is highly adaptable and can be used on microtitre plates and also protein microarrays. The approach depends upon the production of protein libraries that represent the repertoire of receptors present on the interacting cell types.

Research

We focus on three key areas of cell surface signalling:

A merozoite invading an erythrocyte.

A merozoite invading an erythrocyte. [Miller LH et al., 1979. Originally published in Journal of Experimental Medicine. doi:10.1084/jem.149.1.172]

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Malaria - P. falciparum interactions with host cells

Malaria remains a major global health burden. In 2012, there were 207 million reported cases and 627,000 deaths, the majority of these deaths were African children under the age of five.

It is an infectious disease caused by parasites belonging to the Plasmodium genus and infections by P. falciparum are responsible for the vast majority of mortality. All the clinical symptoms of malaria result from the blood stages of infection which are initiated when the merozoite - the invasive form of the parasite - recognizes and invades a host red blood cell.

We have compiled protein libraries that represent the cell surface and secreted protein repertoire of the P. falciparum merozoite and human erythrocyte and used them in our screening approach to identify novel receptor-ligand interactions involved in invasion. We have recently become interested in the other stages of the parasite lifecycle too and have expressed proteins that are displayed on the surface of the sporozoite. We will be using these proteins to identify host-pathogen interactions with both human host and insect vector receptors.

Coloured scanning electron micrograph (SEM) of a clump of activated platelets from a blood clot.

Coloured scanning electron micrograph (SEM) of a clump of activated platelets from a blood clot. [Steve Gschmeissner/Science Photo Library]

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Cardiovascular disease - Platelet:platelet interactions

Heart disease and stroke (together known as cardiovascular diseases) are the number one cause of human mortality, responsible for approximately 30 per cent of all deaths globally.

These diseases are caused by the inappropriate aggregation of platelets to form a thrombus, occluding the blood supply to vital organs such as the heart and brain. Platelets aggregate through the adhesive interactions of the receptor proteins displayed on their cell surface but no systematic survey of these interactions has been performed.

By compiling a recombinant protein library containing whole ectodomain fragments of approximately 130 human platelet receptor proteins, we are aiming to use our AVEXIS and "protein chip" technology to identify novel receptor pairs that are important in platelet aggregation. By taking this systematic large-scale approach, we hope to gain an integrated, global view of platelet adhesion and provide new avenues for therapeutic intervention.

Fertilisation occurs when a sperm cell recognises and fuses with an egg.

Fertilisation occurs when a sperm cell recognises and fuses with an egg. [istockphoto.com]

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Fertilisation - Sperm:egg interactions

Most life begins when haploid gametes join to form a diploid zygote; in humans, this happens when a sperm recognizes and fuses with an egg. Fertilisation is arguably the most important cell recognition event in biology, yet surprisingly the molecular interactions that underpin the recognition and fusion of gametes are not characterised.

We have compiled both sperm and egg receptor protein libraries, and by recombinantly expressing the extracellular domains of these proteins it is possible to screen for low affinity interactions between cell surface receptors using AVEXIS, and thus discover unknown protein-protein interactions. By gaining a better insight to the molecular mechanisms of fertilisation, we hope to identify novel contraceptive targets and enhance our understanding of infertility.

Resources

Database

ARNIE - AVEXIS Receptor Network with Integrated Expression

Selected Publications

  • Juno is the egg Izumo receptor and is essential for mammalian fertilization.

    Bianchi E, Doe B, Goulding D and Wright GJ

    Nature 2014;508;7497;483-7

  • RH5-Basigin interaction plays a major role in the host tropism of Plasmodium falciparum.

    Wanaguru M, Liu W, Hahn BH, Rayner JC and Wright GJ

    Proceedings of the National Academy of Sciences of the United States of America 2013;110;51;20735-40

  • A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins.

    Crosnier C, Wanaguru M, McDade B, Osier FH, Marsh K, Rayner JC and Wright GJ

    Molecular & cellular proteomics : MCP 2013;12;12;3976-86

  • Biochemical analysis of the Plasmodium falciparum erythrocyte-binding antigen-175 (EBA175)-glycophorin-A interaction: implications for vaccine design.

    Wanaguru M, Crosnier C, Johnson S, Rayner JC and Wright GJ

    The Journal of biological chemistry 2013;288;45;32106-17

  • Semaphorin-7A is an erythrocyte receptor for P. falciparum merozoite-specific TRAP homolog, MTRAP.

    Bartholdson SJ, Bustamante LY, Crosnier C, Johnson S, Lea S, Rayner JC and Wright GJ

    PLoS pathogens 2012;8;11;e1003031

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Nature 2011;480;7378;534-7

  • Jamb and jamc are essential for vertebrate myocyte fusion.

    Powell GT and Wright GJ

    PLoS biology 2011;9;12;e1001216

  • DeltaC and DeltaD interact as Notch ligands in the zebrafish segmentation clock.

    Wright GJ, Giudicelli F, Soza-Ried C, Hanisch A, Ariza-McNaughton L and Lewis J

    Development (Cambridge, England) 2011;138;14;2947-56

  • Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling.

    Martin S, Söllner C, Charoensawan V, Adryan B, Thisse B, Thisse C, Teichmann S and Wright GJ

    Molecular & cellular proteomics : MCP 2010;9;12;2654-65

  • A cell surface interaction network of neural leucine-rich repeat receptors.

    Söllner C and Wright GJ

    Genome biology 2009;10;9;R99

  • Large-scale screening for novel low-affinity extracellular protein interactions.

    Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A and Wright GJ

    Genome research 2008;18;4;622-30

  • Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.

    Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH and Barclay AN

    Immunity 2000;13;2;233-42

Team

Team members

Josefin Bartholdson Scott
jb28@sanger.ac.ukPostdoctoral Fellow
Enrica Bianchi
Postdoctoral Fellow
Kirsten Dundas
PhD Student
Julia Knoeckel
Postdoctoral Fellow
Nicole Muller-Sienerth
nms@sanger.ac.ukLaboratory Manager
Abi Perrin
PhD Student
Cecile Wright-Crosnier
Senior Staff Scientist

Josefin Bartholdson Scott

jb28@sanger.ac.uk Postdoctoral Fellow

I graduated from the University of Edinburgh in 2004 with a BSc Honours degree in Microbiology and Infection. I continued my studies in Edinburgh with a PhD in Biological Chemistry working on virulence factors of the multi-resistant cystic fibrosis bacteria Burkholderia cenocepacia. I joined the Sanger Institute in 2009.

Research

I am currently investigating novel interactions between Plasmodium proteins involved in malaria infection and receptors on the erythrocyte surface.

References

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

    Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

    Funded by: Medical Research Council: G19/9; NCEZID CDC HHS: R36 CK000119-01; NIAID NIH HHS: 2T32 AI007535-12, R01 AI057919, R01 AI057919-05, R01AI057919; Wellcome Trust: 077108, 089084, 090532

    Nature 2011;480;7378;534-7

  • Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia.

    Loutet SA, Bartholdson SJ, Govan JR, Campopiano DJ and Valvano MA

    Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.

    Microbiology (Reading, England) 2009;155;Pt 6;2029-39

  • Effective binding and sensing of lipopolysaccharide: combining complementary pattern recognition receptors.

    Ganesh V, Bodewits K, Bartholdson SJ, Natale D, Campopiano DJ and Mareque-Rivas JC

    School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.

    Angewandte Chemie (International ed. in English) 2009;48;2;356-60

  • Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

    Bartholdson SJ, Brown AR, Mewburn BR, Clarke DJ, Fry SC, Campopiano DJ and Govan JR

    School of Chemistry, University of Edinburgh, Edinburgh, UK.

    The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D00134X/1

    Microbiology (Reading, England) 2008;154;Pt 8;2513-21

  • Temperate bacteriophages DK4 and BcepMu from Burkholderia cenocepacia J2315 are identical.

    Langley RJ, Kenna D, Bartholdson J, Campopiano DJ and Govan JR

    Cystic Fibrosis Laboratory, Medical Microbiology, University of Edinburgh, Medical School, Teviot Place, EH8 9AG, United Kingdom.

    FEMS immunology and medical microbiology 2005;45;2;349-50

Enrica Bianchi

- Postdoctoral Fellow

I graduated from the University of Bologna (Italy) with a degree in Biological Science and earned my Ph.D. in Reproduction and Development at the University of Rome “Tor Vergata” in 2008. During the following two years as a postdoc I investigated the reproductive defects in mice that were knocked-out for a gene encoding for an RNA-binding protein. In 2011 I moved to the Wellcome Trust Sanger Institute where I joined the Wright's group to study the sperm-egg interaction.

Research

Surprisingly the detailed mechanism of fertilization is far to be elucidated. Which and how many proteins are involved in the recognition, binding and fusion of the sperm with the egg is largely a mystery. Applying the large-scale systematic approaches to identify novel receptor-ligand pairs that have been developed in Wright's lab, this project aims to identify the proteins involved in the sperm-egg interaction. Interestingly this research could help filling the gap in this field and have relevant applications considering that no novel contraceptive has been developed since 1970.

References

  • Izumo meets Juno: preventing polyspermy in fertilization.

    Bianchi E and Wright GJ

    Cell Surface Signalling Laboratory; Wellcome Trust Sanger Institute; Cambridge, UK.

    Cell cycle (Georgetown, Tex.) 2014;13;13;2019-20

  • Juno is the egg Izumo receptor and is essential for mammalian fertilization.

    Bianchi E, Doe B, Goulding D and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

    Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.

    Funded by: Wellcome Trust: 098051

    Nature 2014;508;7497;483-7

  • FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

    De Cola A, Bongiorno-Borbone L, Bianchi E, Barcaroli D, Carletti E, Knight RA, Di Ilio C, Melino G, Sette C and De Laurenzi V

    IDI-IRCCS Biochemistry Laboratory, c/o Department of Experimental Medicine, University of Rome Tor Vergata, Roma, Italy.

    Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

    Funded by: Medical Research Council: MC_U132670600; Telethon: GGP09133

    Oncogene 2012;31;5;573-82

  • Ablation of the Sam68 gene impairs female fertility and gonadotropin-dependent follicle development.

    Bianchi E, Barbagallo F, Valeri C, Geremia R, Salustri A, De Felici M and Sette C

    Department of Public Health and Cell Biology, University of Rome Tor Vergata, 00133 Rome, Italy.

    Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads, whose ablation causes male infertility. Herein, we have investigated Sam68 expression in the adult ovary and its function in female fertility. Immunohistochemistry showed that Sam68 was localized in the nucleus of oocytes and follicular cells at all stages of folliculogenesis. Sam68(-/-) females were severely subfertile, and they showed a delay in the age of first pregnancy, increased breeding time for successful pregnancy and yielded smaller litters. Morphological analyses indicated a significant reduction in the number of secondary and pre-antral follicles in the ovary. These defects were associated with alteration of oestrous cycles and a reduced number of ovulated oocytes, which were only partially restored by the administration of exogenous gonadotropins. Crosslinking/immunoprecipitation experiments showed that Sam68 directly binds the mRNAs for the follicle-stimulating hormone (FSH) and the luteinizing hormone receptors (Fshr and Lhcgr), which were downregulated in ovaries of adult knockout females. Stimulation of immature females with FSH-like pregnant mare serum gonadotropin (PMSG), or of follicular cells with the FSH second messenger analogue 8Br-cAMP, caused the upregulation of Sam68. The increase in Sam68 levels paralleled that of the Fshr and Lhcgr mRNAs in the pre-ovulatory follicle and was required to allow accumulation of these transcripts in follicular cells. These studies identify a new crucial function for Sam68 in the regulation of female fertility and indicate that this protein is required to insure proper expression of the gonadotropin receptor transcripts in pre-ovulatory follicles in adult ovary.

    Funded by: Telethon: GGP09154

    Human molecular genetics 2010;19;24;4886-94

  • Expression of stemness markers in mouse parthenogenetic-diploid blastocysts is influenced by slight variation of activation protocol adopted.

    Bianchi E, Geremia R and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

    The importance of obtaining stem cells through alternative methods has increased progressively in the recent years due to the potential role that embryonic stem (ES) cells play in the field of regenerative medicine. In this regard, generation of parthenogenetic blastocysts allows the production of ethic-free ES cells without the need to manipulate normal embryos. Our work was aimed at clarifying whether variations in the method adopted to generate diploid parthenogenetic blastocysts could determine differences in the quality of blastocysts produced. In vitro development of mouse oocytes activated with three protocols, using Sr2+ and cytochalasin for different time, was compared with that of in vivo fertilized embryos. We have evaluated the efficiency of blastocyst formation and analysed the expression pattern of the stemness markers OCT4, CDX2, and NANOG. Our results indicate that the yield of diploid parthenogenotes and the segregation of the stemness marker OCT4 in the developing blastocyst are influenced by the parthenogenetic protocol adopted. Particularly, even if all methods tested allowed the production of blastocysts in vitro, the correct segregation of OCT4 occurred only in blastocysts developed from oocytes concomitantly treated for 4 h with Sr2+ and cytochalasin D. Our results indicate that the protocol employed to develop parthenogenetic blastocysts in vitro affects the quality of cells in the inner cell mass.

    Funded by: Telethon: GGP09154

    In vitro cellular & developmental biology. Animal 2010;46;7;619-23

  • Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis.

    Paronetto MP, Messina V, Bianchi E, Barchi M, Vogel G, Moretti C, Palombi F, Stefanini M, Geremia R, Richard S and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, 00133 Rome, Italy.

    Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68(-/-) male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68(-/-) mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

    Funded by: Telethon: GGP04118

    The Journal of cell biology 2009;185;2;235-49

  • Dynamic expression of the RNA-binding protein Sam68 during mouse pre-implantation development.

    Paronetto MP, Bianchi E, Geremia R and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

    The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.

    Funded by: Telethon: GGP04118

    Gene expression patterns : GEP 2008;8;5;311-22

Kirsten Dundas

- PhD Student

I graduated from the University of Bath with a degree in Biochemistry (MBiochem, (Hons)) in 2012. During my degree I completed several research placements, including at Cancer Research UK in Southampton and in the Department of Pharmacology at the University of Oxford. I joined the PhD programme at the Sanger Institute in 2012 and, after completing three rotation projects in various labs across the institute, I decided to carry out my PhD in the Cell Surface Signalling Laboratory.

Research

I am working on the liver stage of human malaria, caused by the parasite Plasmodium falciparum. I am investigating extracellular interactions between Plasmodium falciparum proteins and human receptor proteins, with the aim of identifying novel interactions which are important in the liver stage of malaria.

Julia Knoeckel

- Postdoctoral Fellow

I graduated with a diploma (MSc) in biology from the University of Bielefeld (Germany) in 2006. During my PhD at the Bernhard Nocht Institute for Tropical Medicine in Hamburg (Germany) I studied the Vitamin B6 biosynthesis enzymes Pdx1 and Pdx2 in the human malaria parasite Plasmodium falciparum. I received my PhD in Zoology from the University of Hamburg in 2009. From 2009-2013 I worked as a postdoctoral fellow in the lab of Louis Miller at the National Institutes of Health, USA. I investigated the question why culicine mosquitoes (Culex/Aedes spp.) do not transmit human malaria parasites.

Research

I joined the labs of Gavin Wright and Julian Rayner in the Sanger malaria programme in 2013. In my project I am working with mosquito stages of the malaria parasite P. falciparum. Plasmodium ookinetes and sporozoites have to travel from the midgut of the mosquito into the salivary gland to be transmitted to a human host. However, our knowledge on how the parasite reaches the salivary gland is quite limited. The aim of my project is to identify novel and essential interactions between parasite and mosquito proteins, which could provide new targets to block transmission of malaria.

References

  • Could culicine mosquitoes transmit human malaria?

    Molina-Cruz A, Lehmann T and Knöckel J

    Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

    Human malaria is known to be transmitted strictly by anopheline mosquitoes. Culicine mosquitoes such as Aedes spp. and Culex spp. are important vectors of other human pathogens including viruses and filarial worms, but have never been observed to transmit mammalian malarias. Culicines do transmit avian malarias and, interestingly, allow partial development of mammalian-infectious Plasmodium parasites, implying that physiological barriers in the mosquitoes prevent parasite transmission. Although the mechanism(s) are not known, the mosquito immune system is probably involved in eliminating Plasmodium. However, Plasmodium has shown substantial capacity to adapt to new vectors, and current ecological changes caused by humans could promote adaptation of human-infectious Plasmodium parasites to culicines. Such an event could have widespread epidemiological implications and therefore merits attention.

    Trends in parasitology 2013;29;11;530-7

  • An impossible journey? The development of Plasmodium falciparum NF54 in Culex quinquefasciatus.

    Knöckel J, Molina-Cruz A, Fischer E, Muratova O, Haile A, Barillas-Mury C and Miller LH

    Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America. julia.knoeckel@gmail.com

    Although Anopheles mosquitoes are the vectors for human Plasmodium spp., there are also other mosquito species-among them culicines (Culex spp., Aedes spp.)-present in malaria-endemic areas. Culicine mosquitoes transmit arboviruses and filarial worms to humans and are vectors for avian Plasmodium spp., but have never been observed to transmit human Plasmodium spp. When ingested by a culicine mosquito, parasites could either face an environment that does not allow development due to biologic incompatibility or be actively killed by the mosquito's immune system. In the latter case, the molecular mechanism of killing must be sufficiently powerful that Plasmodium is not able to overcome it. To investigate how human malaria parasites develop in culicine mosquitoes, we infected Culex quinquefasciatus with Plasmodium falciparum NF54 and monitored development of parasites in the blood bolus and midgut epithelium at different time points. Our results reveal that ookinetes develop in the midgut lumen of C. quinquefasciatus in slightly lower numbers than in Anopheles gambiae G3. After 30 hours, parasites have invaded the midgut and can be observed on the basal side of the midgut epithelium by confocal and transmission electron microscopy. Very few of the parasites in C. quinquefasciatus are alive, most of them are lysed. Eight days after the mosquito's blood meal, no oocysts can be found in C. quinquefasciatus. Our results suggest that the mosquito immune system could be involved in parasite killing early in development after ookinetes have crossed the midgut epithelium and come in contact with the mosquito hemolymph.

    PloS one 2013;8;5;e63387

  • The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference.

    Knöckel J, Müller IB, Butzloff S, Bergmann B, Walter RD and Wrenger C

    Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

    The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5'-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6 de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

    The Biochemical journal 2012;443;2;397-405

  • Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer.

    Knöckel J, Jordanova R, Müller IB, Wrenger C and Groves MR

    Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Bernhard Nocht Strasse 74, D-20359 Hamburg, Germany.

    Background: Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

    Methods: Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

    Results: Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

    Conclusions: As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

    The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.

    Biochimica et biophysica acta 2009;1790;5;347-50

  • The assembly of the plasmodial PLP synthase complex follows a defined course.

    Müller IB, Knöckel J, Groves MR, Jordanova R, Ealick SE, Walter RD and Wrenger C

    Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

    Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers.

    Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.

    Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

    PloS one 2008;3;3;e1815

  • The apicomplexan parasite Toxoplasma gondii generates pyridoxal phosphate de novo.

    Knöckel J, Müller IB, Bergmann B, Walter RD and Wrenger C

    Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Bernhard-Nocht-Str. 74, D-20359 Hamburg, Germany.

    Molecular and biochemical parasitology 2007;152;1;108-11

Nicole Muller-Sienerth

nms@sanger.ac.uk Laboratory Manager

I studied Biology at the University of Hohenheim (Germany) and completed my Diploma (MSc) in Microbiology in 2004. I finished my PhD in 2009 and continued working as a PostDoc with Prof. Harald Wajant at the University Hospital of Wuerzburg (Germany) in the research field of Tumor Necrosis Factor (TNF) ligands and receptors. I joined Gavin Wright's Cell surface signalling laboratory as an Advanced Research Assistant in 2011. In 2013 I took over duties as the Lab Manager.

Research

As the Lab Manager I am responsible for the smooth running of the lab, including ordering consumables and equipment, repairs, maintenance and servicing, local co-ordinator duties regarding Health and Safety, Permit to Work, Risk Assessments, New Starter Induction into CL2 and supervision. My current project involves cloning of soluble recombinant Fusion proteins, expression in mammalian cells and purification. I am interested in finding yet unknown binding partners to different types of extracellular proteins expressed on human platelets, the Malaria parasite Plasmodium falciparum etc. Using Flow Cytometry I'm analysing their binding to a wide range of cancer cell lines.

References

  • Development of an antigen microarray for high throughput monoclonal antibody selection.

    Staudt N, Müller-Sienerth N and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom. Electronic address: ns8@sanger.ac.uk.

    Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host.

    Funded by: NINDS NIH HHS: R01 NS063400]; Wellcome Trust: 098051

    Biochemical and biophysical research communications 2014;445;4;785-90

  • Cloning of recombinant monoclonal antibodies from hybridomas in a single mammalian expression plasmid.

    Müller-Sienerth N, Crosnier C, Wright GJ and Staudt N

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    Antibodies are an integral part of biological and medical research. In addition, immunoglobulins are used in many diagnostic tests and are becoming increasingly important in the therapy of diseases. To express antibodies recombinantly, the immunoglobulin heavy and light chains are usually cloned into two different expression plasmids. Here, we describe a method for recombinant antibody expression from a single plasmid.

    Methods in molecular biology (Clifton, N.J.) 2014;1131;229-40

  • Genetic engineering of death ligands for improvement of therapeutic activity.

    Gerspach J, Schneider B, Müller N, Otz T, Wajant H and Pfizenmaier K

    Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

    Advances in experimental medicine and biology 2011;691;507-19

  • SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells.

    Müller-Sienerth N, Dietz L, Holtz P, Kapp M, Grigoleit GU, Schmuck C, Wajant H and Siegmund D

    Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.

    Background: Compounds mimicking the inhibitory effect of SMAC/DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells.

    BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12.

    Significance: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.

    PloS one 2011;6;6;e21556

  • Superior serum half life of albumin tagged TNF ligands.

    Müller N, Schneider B, Pfizenmaier K and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

    Biochemical and biophysical research communications 2010;396;4;793-9

  • Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2).

    Rauert H, Wicovsky A, Müller N, Siegmund D, Spindler V, Waschke J, Kneitz C and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFkappaB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFkappaB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFkappaB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFkappaB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFkappaB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.

    The Journal of biological chemistry 2010;285;10;7394-404

  • Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand.

    Wyzgol A, Müller N, Fick A, Munkel S, Grigoleit GU, Pfizenmaier K and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Wuerzburg, Germany.

    For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.

    Journal of immunology (Baltimore, Md. : 1950) 2009;183;3;1851-61

  • Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.

    Müller N, Wyzgol A, Münkel S, Pfizenmaier K and Wajant H

    Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Germany.

    OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.

    The FEBS journal 2008;275;9;2296-304

  • Superior activity of fusion protein scFvRit:sFasL over cotreatment with rituximab and Fas agonists.

    Bremer E, ten Cate B, Samplonius DF, Mueller N, Wajant H, Stel AJ, Chamuleau M, van de Loosdrecht AA, Stieglmaier J, Fey GH and Helfrich W

    Groningen University Institute for Drug Exploration, Department of Pathology and Laboratory Medicine, University Medical Center Groningen, Groningen, the Netherlands.

    The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity-dependent lysis but also induces apoptosis by cross-linking of its target antigen CD20. Recent reports indicate that this apoptotic activity of rituximab can be synergized by cotreatment with Fas agonists. Here, we report on a strategy designed to exploit and optimize the synergy between rituximab and Fas signaling by genetically fusing a rituximab-derived antibody fragment to soluble Fas ligand (sFasL). The resultant fusion protein, designated scFvRit:sFasL, potently induced CD20-restricted apoptosis in a panel of malignant B-cell lines (10 of 11) and primary patient-derived malignant B cells (two of two non-Hodgkin lymphoma and five of six B cell chronic lymphocytic leukemia). ScFvRit:sFasL efficiently activated CD20 and Fas apoptotic signaling, resulting in a far superior proapoptotic activity compared with cotreatment with rituximab and Fas agonists. ScFvRit:sFasL lacked activity toward normal human B cells and also lacked systemic toxicity in nude mice with no elevation of aspartate aminotransferase and alanine aminotransferase levels or liver caspase-3 activity. In conclusion, scFvRit:sFasL efficiently activates CD20 and Fas-apoptotic signaling and may be useful for the elimination of malignant B cells.

    Cancer research 2008;68;2;597-604

  • Sustained JNK activation in response to tumor necrosis factor is mediated by caspases in a cell type-specific manner.

    Wicovsky A, Müller N, Daryab N, Marienfeld R, Kneitz C, Kavuri S, Leverkus M, Baumann B and Wajant H

    Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    In most cell types, tumor necrosis factor (TNF) induces a transient activation of the JNK pathway. However, in NFkappaB-inhibited cells, TNF stimulates also a second sustained phase of JNK activation, which has been implicated in cell death induction. In the present study, we have analyzed the relationship of cell death induction, caspase activity, JNK, and NFkappaB stimulation in the context of TNF signaling in four different cellular systems. In all cases, NFkappaB inhibition enhanced TNF-induced cell death and primed most, but not all, cells for sustained JNK activation. The caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-fmk) and overexpression of the antiapoptotic proteins FLIP-L and Bcl2 differentially blocked transient and sustained JNK activation in NFkappaB-inhibited KB and HaCaT cells, indicating that the two phases of TNF-induced JNK activation occur at least in these cellular models by different pathways. Although the broad range caspase inhibitor Z-VAD-fmk and the antioxidant butylated hydroxyanisole interfered with TNF-induced cell death to a varying extent in a cell type-specific manner, inhibition of JNK signaling had no or only a very moderate effect. Notably, the JNK inhibitory effect of neither Z-VAD-fmk nor butylated hydroxyanisole was strictly correlated with the capability of these compounds to rescue cells from TNF-induced cell death. Thus, sustained JNK activation by TNF has no obligate role in TNF-induced cell death and is mediated by caspases and reactive oxygen species in a cell type-specific manner.

    The Journal of biological chemistry 2007;282;4;2174-83

Abi Perrin

- PhD Student

I studied Natural Sciences at the University of Cambridge and specialised in Pathology. During this time I worked in several diffrent laboratories in and around Cambridge. One project involved transient transformation of plant leaves, another (here at Sanger) was looking at the evolution of Salmonella bacteria. I also investigated amplification bias in PCR for my third year lab project. During my PhD rotations I have worked in the malaria group and also looked at bacterial genome evolution. Outside of campus I am actively involved in science teaching and outreach (www.chaosscience.org.uk).

Research

I have been screening for protein-protein interactions between human platelets and P. falciparum merozoites. These protein libraries have been used previously in screens to identify erythrocyte invasion pathways in malaria, and to discover novel platelet-platelet interactions. Novel interactions from this screen will be fully characterised, to see if they have a role in the pathogenesis of malaria.

Cecile Wright-Crosnier

- Senior Staff Scientist

Cécile Crosnier graduated in Human Genetics at the Paris 7 – Denis Diderot University (France) in 1997. She obtained her PhD in 2001, working on the role of the JAGGED1 gene in Alagille syndrome, a pathology of the Notch signalling pathway, in the group of Dr Michele Meunier-Rotival. She then performed her postdoctoral studies in Dr Julian Lewis’ laboratory, Cancer Research UK, London, where she studied the role of Notch signalling in intestinal differentiation using the zebrafish as a model organism. She joined the Wellcome Trust Sanger Institute and Gavin Wright’s group in 2007.

Research

Since joining Gavin Wright's group, Cecile has been working on host-pathogen interactions between surface-exposed proteins to find novel receptor-ligand pairs involved in erythrocyte invasion by Plasmodium falciparum.

References

  • New antigens for a multicomponent blood-stage malaria vaccine.

    Osier FH, Mackinnon MJ, Crosnier C, Fegan G, Kamuyu G, Wanaguru M, Ogada E, McDade B, Rayner JC, Wright GJ and Marsh K

    Pathogen Vector and Human Biology Department, Kenya Medical Research Institute Centre for Geographical Medicine Research, Coast, P. O. Box 230, 80108 Kilifi, Kenya.

    An effective blood-stage vaccine against Plasmodium falciparum remains a research priority, but the number of antigens that have been translated into multicomponent vaccines for testing in clinical trials remains limited. Investigating the large number of potential targets found in the parasite proteome has been constrained by an inability to produce natively folded recombinant antigens for immunological studies. We overcame these constraints by generating a large library of biochemically active merozoite surface and secreted full-length ectodomain proteins. We then systematically examined the antibody reactivity against these proteins in a cohort of Kenyan children (n = 286) who were sampled at the start of a malaria transmission season and prospectively monitored for clinical episodes of malaria over the ensuing 6 months. We found that antibodies to previously untested or little-studied proteins had superior or equivalent potential protective efficacy to the handful of current leading malaria vaccine candidates. Moreover, cumulative responses to combinations comprising 5 of the 10 top-ranked antigens, including PF3D7_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7_0606800, AMA1, Pf113, and MSRP1, were associated with 100% protection against clinical episodes of malaria. These data suggest not only that there are many more potential antigen candidates for the malaria vaccine development pipeline but also that effective vaccination may be achieved by combining a selection of these antigens.

    Funded by: Medical Research Council: MR/J002283/1; Wellcome Trust: 077092, 077176, 088634, 089833, 092654, 092741, 098051

    Science translational medicine 2014;6;247;247ra102

  • A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins.

    Crosnier C, Wanaguru M, McDade B, Osier FH, Marsh K, Rayner JC and Wright GJ

    Cell Surface Signalling laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK;

    Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.

    Funded by: Medical Research Council: MR/J002283/1; Wellcome Trust: 098051

    Molecular & cellular proteomics : MCP 2013;12;12;3976-86

  • Biochemical and functional analysis of two Plasmodium falciparum blood-stage 6-cys proteins: P12 and P41.

    Taechalertpaisarn T, Crosnier C, Bartholdson SJ, Hodder AN, Thompson J, Bustamante LY, Wilson DW, Sanders PR, Wright GJ, Rayner JC, Cowman AF, Gilson PR and Crabb BS

    Burnet Institute, Melbourne, Victoria, Australia.

    The genomes of Plasmodium parasites that cause malaria in humans, other primates, birds, and rodents all encode multiple 6-cys proteins. Distinct 6-cys protein family members reside on the surface at each extracellular life cycle stage and those on the surface of liver infective and sexual stages have been shown to play important roles in hepatocyte growth and fertilization respectively. However, 6-cys proteins associated with the blood-stage forms of the parasite have no known function. Here we investigate the biochemical nature and function of two blood-stage 6-cys proteins in Plasmodium falciparum, the most pathogenic species to afflict humans. We show that native P12 and P41 form a stable heterodimer on the infective merozoite surface and are secreted following invasion, but could find no evidence that this complex mediates erythrocyte-receptor binding. That P12 and P41 do not appear to have a major role as adhesins to erythrocyte receptors was supported by the observation that antisera to these proteins did not substantially inhibit erythrocyte invasion. To investigate other functional roles for these proteins their genes were successfully disrupted in P. falciparum, however P12 and P41 knockout parasites grew at normal rates in vitro and displayed no other obvious phenotypic changes. It now appears likely that these blood-stage 6-cys proteins operate as a pair and play redundant roles either in erythrocyte invasion or in host-immune interactions.

    Funded by: Wellcome Trust: 077108/Z/05/Z

    PloS one 2012;7;7;e41937

  • Semaphorin-7A is an erythrocyte receptor for P. falciparum merozoite-specific TRAP homolog, MTRAP.

    Bartholdson SJ, Bustamante LY, Crosnier C, Johnson S, Lea S, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom ; Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    The motility and invasion of Plasmodium parasites is believed to require a cytoplasmic actin-myosin motor associated with a cell surface ligand belonging to the TRAP (thrombospondin-related anonymous protein) family. Current models of invasion usually invoke the existence of specific receptors for the TRAP-family ligands on the surface of the host cell; however, the identities of these receptors remain largely unknown. Here, we identify the GPI-linked protein Semaphorin-7A (CD108) as an erythrocyte receptor for the P. falciparum merozoite-specific TRAP homolog (MTRAP) by using a systematic screening approach designed to detect extracellular protein interactions. The specificity of the interaction was demonstrated by showing that binding was saturable and by quantifying the equilibrium and kinetic biophysical binding parameters using surface plasmon resonance. We found that two MTRAP monomers interact via their tandem TSR domains with the Sema domains of a Semaphorin-7A homodimer. Known naturally-occurring polymorphisms in Semaphorin-7A did not quantitatively affect MTRAP binding nor did the presence of glycans on the receptor. Attempts to block the interaction during in vitro erythrocyte invasion assays using recombinant proteins and antibodies showed no significant inhibitory effect, suggesting the inaccessibility of the complex to proteinaceous blocking agents. These findings now provide important experimental evidence to support the model that parasite TRAP-family ligands interact with specific host receptors during cellular invasion.

    Funded by: Medical Research Council: MR/J002283/1; Wellcome Trust: 098051

    PLoS pathogens 2012;8;11;e1003031

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

    Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

    Funded by: Medical Research Council: G19/9; NCEZID CDC HHS: R36 CK000119-01; NIAID NIH HHS: 2T32 AI007535-12, R01 AI057919, R01 AI057919-05, R01AI057919; Wellcome Trust: 077108, 089084, 090532

    Nature 2011;480;7378;534-7

  • A rapid and scalable method for selecting recombinant mouse monoclonal antibodies.

    Crosnier C, Staudt N and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK.

    Background: Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.

    Results: Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.

    Conclusions: This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

    Funded by: NINDS NIH HHS: R01NS063400; Wellcome Trust: 077108/Z/05/Z

    BMC biology 2010;8;76

  • Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.

    Crosnier C, Stamataki D and Lewis J

    Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

    The lining of the intestine is renewed at an extraordinary rate, outpacing all other tissues in the vertebrate body. The renewal process is neatly organized in space, so that the whole production line, from the ever-youthful stem cells to their dying, terminally differentiated progeny, is laid out to view in histological sections. A flurry of recent papers has clarified the key regulatory signals and brought us to the point where we can begin to give a coherent account, for at least one tissue, of how these signals collaborate to organize the architecture and behaviour of a stem-cell system.

    Nature reviews. Genetics 2006;7;5;349-59

  • Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine.

    Crosnier C, Vargesson N, Gschmeissner S, Ariza-McNaughton L, Morrison A and Lewis J

    Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

    The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. We have generated monoclonal antibodies to identify the absorptive and secretory cells in the epithelium, and we have used these antibodies to examine the part that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an aei mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium.

    Development (Cambridge, England) 2005;132;5;1093-104

  • JAGGED1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome.

    Crosnier C, Attié-Bitach T, Encha-Razavi F, Audollent S, Soudy F, Hadchouel M, Meunier-Rotival M and Vekemans M

    Unité INSERM 347 affiliée au CNRS, Le Kremlin-Bicêtre Cedex, France.

    Mutations of the JAGGED1 gene, encoding a NOTCH receptor ligand, cause Alagille syndrome (AGS), a complex malformative disorder affecting mainly the liver, heart, vertebrae, eye, and face. Minor and occasional features involving kidney, pharynx, systemic arteries, skeleton, and ear are in some cases associated with the syndrome. To describe the expression of JAGGED1 during human embryogenesis and to study its relationship with all the features of AGS, we performed in situ hybridization studies on human embryos and fetal tissue sections. JAGGED1 was mainly expressed in the cardiovascular system. In the liver, JAGGED1 transcripts were only detected in blood vessels. JAGGED1 was also expressed in other structures of mesenchymal origin (distal mesenchyme of limb buds; mesonephric and metanephric tubules of the kidney) and in epithelial structures including the ciliary margin of the retina and the posterior part of the lens, the ventral epithelium of the otic vesicle, the neurosensory epithelium of the ear vestibule, the epithelium of pharyngeal arches, and the developing central nervous system. The strong JAGGED1 expression during human embryo- and feto-genesis both in the vascular system and in other mesenchymal and epithelial tissues implicates abnormal angiogenesis in the pathogenesis of Alagille syndrome and particularly the paucity of interlobular bile ducts. However, it is probably not the only mechanism of the disease. Except for the central nervous system, there is a strong correlation between JAGGED1 expression and all the features of AGS. This implies that the features occasionally associated with the syndrome are not coincidental.

    Hepatology (Baltimore, Md.) 2000;32;3;574-81

  • Mutations in JAGGED1 gene are predominantly sporadic in Alagille syndrome.

    Crosnier C, Driancourt C, Raynaud N, Dhorne-Pollet S, Pollet N, Bernard O, Hadchouel M and Meunier-Rotival M

    INSERM Unité 347 affiliée au Centre National de la Recherche Scientifique, Département de Pédiatrie, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.

    Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an autosomal dominant disorder characterized by neonatal jaundice, intrahepatic cholestasis, and developmental disorders affecting the liver, heart, vertebrae, eyes, and face. We screened a large group of patients for mutations in JAGGED1 and studied transmission of the mutations.

    Methods: The coding sequence of the JAGGED1 gene was searched by single-strand conformation polymorphism and sequence analysis for mutations in 109 unrelated patients with the Alagille syndrome and their family if available.

    Results: Sixty-nine patients (63%) had intragenic mutations, including 14 nonsense mutations, 31 frameshifts, 11 splice site mutations, and 13 missense mutations. We identified 59 different types of mutation of which 54 were previously undescribed; 8 were observed more than once. Mutations were de novo in 40 of 57 probands.

    Conclusions: Most of the observed mutations other than the missense mutations in JAGGED1 are expected to give rise to truncated and unanchored proteins. All mutations mapped to the extracellular domain of the protein, and there appeared to be regional hot spots, although no clustering was observed. Thus, the sequencing of 7 exons of JAGGED1 would detect 51% of the mutations. Transmission analysis showed a high frequency of sporadic cases (70%).

    Gastroenterology 1999;116;5;1141-8

* quick link - http://q.sanger.ac.uk/cellsurf