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Title: Molecular interactions of complement factor H and C4 using multidisciplinary biophysical methods
Author: Fung, K. W.
ISNI:       0000 0004 8503 2705
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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The first line of defence in the innate immune system is the complement system which is comprised of at least 30 soluble proteins that help to fight against pathogen invasion. The structural behaviour of two complement proteins, Factor H (FH) and C4b, has been subjected to multidisciplinary studies in order to clarify the molecular mechanism of action. FH is a major regulator of the complement system in the innate immune system and binds to heparan sulphate (HS) on host cell surfaces. The binding orientation of FH to HS is poorly understood and knowledge of this binding orientation will help us to understand the pathogenesis of FH-related disease. There are at least two independent heparin binding sites located on FH where heparin is an analogue of HS. In addition to HS, FH also binds to another surface glycosaminoglycan dermatan sulphate (DS). The solution structure of DS was determined by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). Structural comparison between DS and HS was made and the result showed that DS was less flexible and longer than HS. The binding orientation of FH to heparin was studied using dual polarisation interferometry (DPI). For the binding of full length FH to heparin dp38 and dp18 (dp stands for degree of polymerisation), an increase in thickness of the sensor chip of approximately 13 nm and dissociation constants KD of 1-4 μM were observed. In addition, a lower surface density of heparin dp38 and a shorter heparin dp18 surface resulted in weaker binding KD for full length FH. A recombinant SCR-19/20 fragment of FH containing the heparin binding site also interacted with the surface and this confirmed the bivalent and co-operative binding mechanism. The DPI data were successfully fitted to layer models which were consistent with the 'side on' orientation. C4 is activated to C4b during the classical and lectin pathway in which the thioester (TED) and CUB domains are vital for its role in attaching to activator surface. By using DPI, SAXS and AUC, the TED domain was determined to be highly flexible. In low ionic strength environment, C4b was shown to form compact structures and increases in ionic strength separates the TED domain from the MG1 domains. This result validated the crystal structure and solution structure of C4b.
Supervisor: Perkins, S. P. J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available