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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Clinical and Experimental Sciences Recombinant Expression of Functional Trimeric Fragments of Human SP-A and SP-D by Alastair Samuel Watson Thesis for the degree of Doctor of Philosophy March 2016 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE Clinical and Experimental Sciences Doctor of Philosophy RECOMBINANT EXPRESSION OF FUNCTIONAL TRIMERIC FRAGMENTS OF HUMAN SP-A AND SP-D by Alastair Samuel Watson The lung collectins, surfactant proteins A and D (SP-A and SP-D) are important innate immune molecules, localised predominantly in the pulmonary surfactant of the lung. SP- A and SP-D bind to carbohydrates on the surface of pathogens and enhance their neutralisation, agglutination and clearance. Moreover, they are important modulators of the inflammatory immune response. Lung collectins form trimers comprised of an N- terminal domain, collagen-like domain, a neck region and a C-terminal carbohydrate recognition domain (CRD). A trimeric recombinant fragment of human SP-D (rfhSP-D) comprised of only the neck, CRD and a short collagen-like stalk, which lacks the N- terminal domain important for the collectin oligomeric structure, has previously been produced. This has provided insights into the importance of the N-terminus and oligomeric structure for functions of SP-D and its mode of calcium-dependent ligand binding. Here I report the first successful expression and purification of an equivalent functional trimeric fragment of human SP-A (rfhSP-A). Through use of a novel expression tag (NT), a functional trimeric rfhSP-A molecule was successfully expressed in E. coli and purified. rfhSP-A was shown to bind to various natural SP-A ligands in a calcium-dependent manner, including mannan, BBG2Na (containing respiratory syncytial virus (RSV) G protein core region), recombinant HIV gp120 IIIB protein and LPS from K. pneumoniae, albeit at low levels. rfhSP-A was found to neutralise RSV and reduce infection of bronchial epithelial cells, as assessed by both RT-qPCR and flow cytometry, by up to a mean (± standard deviation) of 96.4 (± 1.9) % (p < 0.0001) to similar levels as the uninfected control. This fragment was significantly more effective (p < 0.0001) at neutralising RSV than native human (nh)SP-A which reduced infection levels by up to 38.5 (± 28.4) % (p < 0.05). This increased efficacy of rfhSP-A in neutralising RSV could be due to the absence of the SP-A N-terminal domain which may provide a route of entry for RSV to infect host cells; putative host cell receptors now need to be identified. Although nhSP-A and rfhSP-A were shown to bind BBG2NA, the mechanism through which SP-A neutralises RSV needs to be elucidated with purified recombinant proteins from various RSV strains. Use of the NT tag with two point mutations (NTdm) allowed the successful production of a functional trimeric rfhSP-D molecule without the requirement for refolding. Importantly, this novel expression method allowed a substantial increase in yields of rfhSP-D produced with a yield equivalent to 31.3 mg/litre of bacteria upon scale up as compared with 3.3 mg/litre obtained through the traditional refolding method; optimisation may further increase yields. rfhSP-D produced using the novel solubility tag prevented infection of RSV by up to 32.2 (± 25.6) % (p < 0.05), to a similar level as rfhSP-D produced using the traditional refolding method (37.8 (± 21.7) % (p < 0.05)). nhSP-D also reduced infection levels similarly by up to 47.2 (± 25.6) % (p < 0.001), suggesting that the N-terminal region and oligomeric structure of SP-D may not provide a route of entry for the virus, unlike for SP-A. nhSP-D bound various known ligands including RSV proteins, recombinant HIV gp120 proteins, inactivated HIV particles, house dust mite extracts, pollen extracts and H. influenza, K. pneumoniae and E. coli LPS. rfhSP-A may allow the crystal and solution structures of the human SP-A neck and CRD domains to be resolved. Moreover, it could allow the importance of the SP-A N-terminus in its interactions with an array of different pathogens to be investigated. This study has demonstrated the use of NTdm as a novel solubility tag for heterologous protein expression and has potentially increased the commercial viability of rfhSP-D. rfhSP-A and rfhSP-D may have therapeutic potential, particularly as adjunct treatments to current lipid surfactant therapies, which currently lack SP-A and SP-D. rfhSP-A and rfhSP-D could replace the deficient SP-A and SP-D in the neonatal lung and may prevent the emphysematous phenotype associated with neonatal chronic lung disease. Publication List Papers - Heath C, Del Mar Cendra M, Watson A, Auger J, Pandey A, Tighe P, Christodoulides M. 2015. Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells. Plos One: 10 (11): p. e0142773. - Fakih D, Pilecki B, Schlosser A, Jepsen C, Thomsen L, Ormhøj M, Watson A, Madsen J, Clark H, Barfod K, Hansen S, Marcussen N, Jounblat R, Chamat S, Holmskov U, Sorensen G. 2015. Protective effects of surfactant protein D treatment in 1,3-β-glucan-modulated allergic inflammation. Am J Physiol Lung Cell Mol Physiol: 309(11):L1333-43. Immunology News - Watson A. 2015. Translational Immunology. 17th International Congress of Mucosal Immunology. Immunology news, British Society for Immunology, September 2015 p. 40. - Watson A, Whitwell H. 2013. Translational Immunology. BSI Wessex immunology group meeting. Immunology news, British Society for Immunology, May 2013 p. 16-17. Patents - Clark H, Watson A, Johansson J, Kronqvist N, Rising A, Madsen J. Novel Expression of functional fragments of Surfactant Proteins A and D. Filed December 2015 (New Great Britain Patent Application No. GB1522610.3). Conferences - American Thoracic Society, USA (13-18th May 2016). Poster presentation: ‘First successful expression of a trimeric human fragment of SP-A and demonstration of its increased efficacy in neutralisation of RSV compared with native human SP-A’ Authors: Watson A, Spalluto C.M, Kronqvist N, Staples K.J, Johansson J, Wilkinson T.M.A, Madsen J, Clark H. - 17th International Congress of Mucosal Immunology. Berlin, Germany (14th-18th July, 2015). Poster presentation: Investigating the Interaction of SP-A and SP-D with Respiratory Syncytial Virus in Human Bronchial Epithelial Cells. Authors: Watson A, Spalluto C.M, Whitwell H, Staples K.J, Wilkinson T.M.A, Madsen J, Clark H. - Neonatal meeting (23-24th June 2015). Oral Presentation: ‘Investigating the interaction of SP-A and SP-D with RSV for the Development of Surfactant Therapeutics’. Author Watson A. - Faculty Conference. University of Southampton, UK (17th June 2015). Poster presentation: ‘Investigating the Importance of the SP-A Oligomeric Structure in RSV Neutralisation’. Authors: Watson A, Spalluto C.M, Kronqvist N, Johansson J, Staples K.J, Wilkinson T.M.A, Madsen J, Clark H. - Annual Oxford Developmental Biology Symposium (9th December, 2014) Poster presentation: ‘Investigating the Structure/Function Relationship of Lung Collectins’. Authors: Watson A, Kronqvist N, Spalluto C. M, Griffiths M, Staples K.J, Wilkinson T.M.A, Johansson J, Madsen J and Clark H. - British Society for Immunology Congress 2014. Brighton, UK (1-4th December, 2014). Poster presentation: ‘Evaluating the Importance of a Common Surfactant Protein D Polymorphism in Influenza Virus Infection’. Authors: Watson A, Madsen J, Clark H. - South African Immunology Society Infectious Diseases in Africa symposium. Cape Town, South Africa (21-26th October, 2014). Oral presentation and poster: ‘Investigating the Importance of the Structure of Lung Collectins in their Interaction with HIV’. Authors: Watson A, Kronqvist N, Griffiths M, Staples K. J, Wilkinson T.M.A, Madsen J, Clark H. - Faculty Conference. University of Southampton, UK (19th June 2014). Oral presentation: ‘Investigating the Structure/Function Relationship of Lung Collectins’. Author: Watson A. Contents Contents ....................................................................................................................... i List of tables ............................................................................................................... ix List of figures ............................................................................................................. xi DECLARATION OF AUTHORSHIP ................................................................. xvii Acknowledgements .................................................................................................. xix 1. Chapter 1: Introduction ..................................................................................
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