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Recombinant Expression and Analysis of Tetraspanin Extracellular-2 Domains a Thesis Submitted for the Degree of Doctor of Philosophy

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Recombinant Expression and Analysis of Tetraspanin Extracellular-2 Domains a Thesis Submitted for the Degree of Doctor of Philosophy Recombinant Expression and Analysis of Tetraspanin Extracellular-2 Domains A thesis submitted for the degree of Doctor of Philosophy John Samuel Palmer September 2015 Department of Molecular Biology and Biotechnology University of Sheffield Acknowledgments I would like to start by thanking my supervisors, Dr Lynda Partridge and Dr Pete Monk, for their guidance on all things tetraspanin and continuous support throughout my PhD. A great deal of help has also come from lab members both past and present, for which I am deeply grateful. Thanks to Rachel for keeping me chipper at work (for the most part), Marzieh and Kathryn for teaching me how to science, plus Fawwaz, Atiga, Ahmed, Ibrahim and Shamaa for helping with experiments (and my grasp of the Arabic language), and Dan “Coz-ed” Cozens, Jenny and Tom for their friendship and help with experiments. Special thanks go to Dr Mr Andrew O’Leary for going out of his way to help during challenging times and whose words of encouragement were invaluable. I’m grateful for the help from several members of other labs from whom I was able to sponge knowledge and lab equipment, with particular gratitude towards Shuo Jiang, Luke Johnson and Pete Davis for sharing their protein knowledge! The help and guidance offered by BioServe UK Ltd during the early years of my project was particularly helpful and I would like to express my gratitude towards Simon Smith and the other members of the BioServe team. This work could not have been completed without help outside of academia, from all those family and friends who kept my chin up when experiments were not behaving (more often than not). Thanks to Bin-ed, Kate, Charlotte, Sam, Simon and Jack for making my time in Sheffield as enjoyable as possible and taking care of me on visits back there! Thanks to the lovely Lowry family for keeping the larders stocked and the house as warm as it could be. More importantly, thank you for making the effort to come round for a catch-up and a cuppa and cooking an amazing Sunday dinner! I would especially like to thank Jude for the sagely advice during times of frustration (have you tried it slower, quicker, colder etc.) and Dad for the working lunches that kept things in perspective and organised my thoughts. I would also like to thank the siblings: Mike, Em and Soph, for making sure that home visits and holidays were always something to look forward to, I couldn’t have coped without the Palmer clan! Last but certainly not least I would like to thank Rebecca. Not only has she helped me (both academically and emotionally) through my PhD and put up with me during the write up, but without her motivation during countless late night/early morning revision session at undergrad I would not be where I am today. More importantly her love and support has kept me happy – Thank you! i Abstract Tetraspanins are a superfamily of membrane proteins which span the membrane four times; they are found predominantly at the cell surface but are also located on intracellular vesicles. Tetraspanins (with a few exceptions) do not have conventional receptor ligand functionality and instead form lateral associations with other molecules within the membrane. Binding partners include, but are not limited to, MHC proteins, integrins, signalling proteins and other members of the tetraspanin superfamily. This large network of interactions has led to the idea of tetraspanin enriched microdomains (TEMs) or the tetraspanin web, in which tetraspanins function by bringing together proteins to form functional clusters which allow processes (such as signal transduction or adhesion) to take place more efficiently. Due to the numerous diverse binding partners, tetraspanins have been implicated in a number of cellular and pathological processes. Despite tetraspanins being involved in fundamental physiological processes, relatively little is known about the function of individual members. Difficulties arise as only a few monoclonal antibodies are available to the native proteins and mouse knock outs often show only a mild phenotypic change. Our group and others have used recombinant human EC2 domains in the form of GST fusion proteins to assess tetraspanin involvement in several processes. This region is thought to attribute specificity to individual tetraspanin members and when recombinant versions are added to cells exogenously, they have been shown to modulate different cellular events including adhesion, migration and fusion. Due to a number of inherent drawbacks associated with bacterially expressed recombinant EC2 domains (such as LPS contamination and inferior folding), the initial aim of this work was to express the recombinant proteins in a mammalian host. Despite multiple attempts to express the proteins in mammalian or insect cells using different vector systems, this was not successful, although it was demonstrated that DNA was integrated into the host genome and that EC2 encoding mRNA was expressed. Following this, it was decided to focus on bacterial expression and use the EC2 domains generated to further our understanding of tetraspanin involvement in IgE mediated degranulation this is a critical first step in Type I Hypersensitivity and although several tetraspanin family members have been implicated in this pathway, their exact involvement is not yet clear. Here, recombinant EC2 domains of tetraspanins CD9, CD63, CD81 and CD151 were used for the first time in conjunction with RBL-2H3 cells (a cell line commonly used as a model for mast cell degranulation) to examine tetraspanin involvement in IgE mediated degranulation. These particular tetraspanins were selected because past studies ii have implicated these members in mast cell activation, but dispite an anti-CD63 antibody being able to down regulate degranulation, in this instance the EC2 domains did not exhibit any modulating effect on this form of degranulation in RBL-2H3 cells. The activity of these particular recombinant proteins was demonstrated in two other functional assays; bacterial adhesion to endothelial cells and bacterial induced giant cell formation. Later work sought to characterise the EC2 proteins in terms of their secondary structure, LPS content and their ability to bind to cells, with the hope of elucidating their mechanism of action. The binding of each EC2 domains to two cell lines was examined: RBL-2H3 where EC2 domains show no effect on degranulation, and HEC-1B cells, where EC2 domains were previously shown exhibit biological activity by reducing bacterial adhesion. At highest concentrations utilised, the EC2 domains were shown to bind to both cell lines significantly more than the GST control protein, but attempts to examine the specificity of the EC2 interaction with the cells by competitive inhibition gave inconclusive results. Whilst this may have been due to technical issues it is tempting to speculate that this indicates cellular interactions that do not follow conventional binding mechanisms. The LPS content of the EC2 domains was shown not to correlate with their ability to modulate bacterial-induced cell fusion. Finally, to facilitate structural and future functional studies, attempts were made to optimise the removal of the GST tag from the fusion proteins. CD spectroscopy was then performed and attributed the EC2 domains of CD9 and CD81 with 50% and 52% α-helical structure, respectively, as expected for these proteins. Although initial aims of producing mammalian EC2 domains were not fulfilled, they were successfully produced in bacteria and used in RBL degranulation assays. later work indicated that LPS contamination was not the causative component of EC2 preparations and confirmed some level of secondary structure. Furthermore, the apparent lack of effect of recombinant EC2 domains in RBL-2H3 activation may shed light on tetraspanin interaction with the high affinity IgE receptor. iii Table of Contents: Acknowledgments ......................................................................................................................i Abstract ...................................................................................................................................... ii Table of Contents: ..................................................................................................................... iv List of Tables .............................................................................................................................. x List of figures ............................................................................................................................ xii Table of abbreviations ............................................................................................................ xvi Chapter 1: Introduction ................................................................................................................ 1 1.1 The Tetraspanin superfamily .............................................................................................. 1 1.1.1 The history, evolution and distribution of tetraspanins .............................................. 1 1.1.2 Tetraspanin Structure .................................................................................................. 5 1.1.2.1 Intracellular regions .............................................................................................. 5 1.1.2.2 Transmembrane domains ................................................................................... 13 1.1.2.3 EC1 region ..........................................................................................................
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