86 research outputs found

    Structure and function of the central part of complement factor H

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    Role of complement genetic variants in inflammatory diseases by an interactive database and protein structure modelling

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    The rare diseases atypical haemolytic uraemic syndrome (aHUS) and C3 glomerulopathy (C3G) are associated with dysregulation of complement activation. It is unclear which genes most frequently predispose to aHUS or C3G. Accordingly, a six- centre analysis of 610 rare genetic variants in 13 mostly complement genes from >3500 patients with aHUS and C3G was performed. A new interactive Database of Complement Gene Variants was developed to extract allele frequencies for these 13 genes using the Exome Aggregation Consortium server as the reference genome. For aHUS, significantly more protein-altering rare variation was found in the five genes CFH, CFI, CD46, C3 and DGKE than in ExAC. For C3G, an association was only found for rare variants in C3 and the N-terminal C3b-binding or C-terminal non-surface-associated regions of factor H (FH). FH is the major regulator of C3b and its Tyr402His polymorphism is an age-related macular degeneration risk-factor. To better understand FH complement binding, the solution structures of both allotypes were studied. Starting from known FH short complement regulator domains and glycan structures, small angle X-ray scattering data were fitted using Monte Carlo methods to determine atomistic structures for monomeric FH. The analysis of 29,715 physically realistic but randomised FH conformations resulted in 100 similar best-fit FH structures for each allotype. Two distinct molecular structures resulted; an extended N-terminal domain arrangement with a folded-back C-terminus, or an extended C-terminus and folded-back N-terminus. To clarify FH functional roles in host protection, crystal structures for the FH complexes with C3b and C3dg revealed that the extended N-terminal conformation accounted for C3b fluid phase regulation, the extended C-terminal conformation accounted for C3d binding, and both conformations accounted for bivalent FH binding to the host cell-surface. Finally, statistical analyses indicated that the structural location of rare variants in complement may predict the occurrences of aHUS or C3G

    Role of the complement factor H-related protein 5 in renal disease by protein expression and molecular solution structural studies

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    Complement Factor H-Related 5 (CFHR5) belongs to the same complement family as the major regulator Factor H. CFHR5 comprises nine short complement regulator (SCR) domains. The duplication of the N-terminal SCR-1/2 domains causes CFHR5 nephropathy, a cause of kidney failure in Cypriots. To clarify the molecular basis of CFHR5 nephropathy, E. coli expression systems were developed for SCR-1 and SCR-1/2 of CFHR5, and recombinant CFHR5 SCR-1/9 was obtained from a commercial mammalian expression system. First, the domain arrangement of CFHR5 SCR-1/9 was studied by analytical ultracentrifugation and X-ray scattering. Sedimentation velocity reported a molecular mass of 134 kDa, indicating that CFHR5 is dimeric. The CFHR5 sedimentation coefficient of 5-6 S decreased with increased NaCl, showing that this became more extended. X-ray scattering also showed that CFHR5 was dimeric. The X-ray mean radius of gyration RG was 5.5 ± 0.2 nm, and its maximum length was 20 nm. This length is low compared to that of 32 nm for monomeric Factor H with 20 SCR domains, indicating that CFHR5 possessed a more compact SCR arrangement than that of Factor H. Atomistic scattering curve modelling of CFHR5 that involved Monte Carlo simulations to generate physically realistic atomistic SCR structures showed that CFHR5 possessed a folded-back compact domain structure. Second, sedimentation velocity showed that SCR-1 was monomeric, while SCR-1/2 was dimeric, thus locating a CFHR5 dimerization site to its N-terminus. In summary, the solution structure of CFHR5 is markedly more compact than previously thought, and its dimerization site was located to SCR-1/2. The perturbation of SCR-1/2 may have a major role in causing CFHR5 nephropathy

    The C3b Receptor (CR1) on Human Blood Cells

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    The C3b receptor (CR1) was first isolated from human erythrocyte membranes in 1979 and shown to be a large single chain polypeptide glycoprotein with a molecular weight of 205,000 daltons. CR1 isolated from erythrocyte membranes has been shown in vitro to possess cofactor activity for the I mediated cleavage of C3b to iC3b and C4b to iC4b. It also plays a role in the prevention of lysis of bystander erythrocytes by its ability to cause the decay dissociation of C4b2a3b and C3bBb formed on these cells. In addition erythrocyte CR1 in vivo is thought to play a role in the transport of opsonised immune complexes from the circulation to the reticulo enclothrlial system where they can be removed. On unstimulated phagocytic cells the primary function of CR1 is the binding of complexes opsonised with C3 and C4 degradation while on stimulated phagocytes CR1 is able to directly mediate the phagocytosis of opsonised particles. CR1 may play a role in the regulation of B lymphocyte function and on kidney podocytes CR1 may serve to prevent complement activation on the basement membrane of the glomerulus

    Glycosaminoglycan-protein interactions and human complement factor H

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    Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association

    Towards Immuno-profiling of Complex Biological Fluids in Patients Recovering from Major Surgery

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    The conventional biochemical diagnosis of disease using isolated blood biomarkers must be revisited by clinicians, replacing it with a multi-biomarker, personalised profile of human health. A label-free nanoparticle array technology, Liscar, has been developed. It is capable of performing rapid, multi-biomarker assays from complex biological samples which, if employed to assay the Complement cascade of the innate immune system, has the potential for a novel systemic profile of patient health. An assay for IgG has been developed on the Liscar platform with a detection limit of 380 ± 100 ng/mL IgG in model sera. Furthermore, addition of a chaotropic agent to the complex sample is shown to improve the accuracy of the IgG assay. Competitive binding between nonspecific interfering proteins and specific target analytes (IgG) at the sensor surface is studied, and a quantitative mathematical model is developed to analyse the data, yielding evidence for the active displacement of albumin by IgG-antigen binding. With a sensitive, accurate multi-biomarker detection platform, a systemic profile of patient health may be possible by examination of the Complement cascade. The Complement system can be activated by a variety of immunological challenges, causing large numbers of activation biomarkers to be produced quickly. Assays for three activation markers, C3d, TCC and Bb are developed, with detection limits of 0.864 ACS Units, 2.32 ng/ml and 54.7 ng/ml respectively. Complement activation was tested in a prospective cohort study of 45 patients undergoing major abdominal surgery. Patient recovery was monitored from admission to ~60 hours postoperatively by Complement activation and consumption using C3d, TCC, Bb, C3 and C4 as biomarkers. A response profile was obtained for the entire cohort for C3 and C4 assays by normalising with respect to individual analyte levels on admission, against which individual responses are compared. 22% of patients in the study suffered postoperative complications, and 73% showed Complement activation by increased levels of C3d, as expected from the initial trauma of surgery. Expansion of the trial is needed to establish clinical significance and utility, especially in relation to the presymptomatic diagnosis of disease

    Complement autoantibodies in atypical haemolytic uraemic syndrome and IgA nephropathy

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    PhD ThesisAtypical haemolytic uraemic syndrome (aHUS) is renal disease associated with mutations and/or polymorphisms in genes encoding complement proteins, including complement factor H (CFH), factor I (CFI) and membrane cofactor protein (CD46). Recently, deficiency of CFH-related (CFHR) proteins 1 and 3 (via loss of the CFHR3/CFHR1 gene block) was linked to the generation of autoantibodies to CFH. Around 10% of aHUS patients develop CFH autoantibodies, adding aHUS to a growing list of kidney diseases with a defined autoimmune component. IgA nephropathy (IgAN) is another such renal disease, where autoantibodies target an aberrantly glycosylated IgA1. To investigate the role of CFH and CFHR copy number variation in the control of complement activation in aHUS and IgAN, I have first generated a full panel of recombinant CFHR proteins in mammalian cell culture. These were then used to generate unique monoclonal antibodies (mAbs) and ELISA protocols to screen for autoantibodies. Using carefully optimised immunisation protocols, I used my recombinant CFHR proteins to produce several highly-specific CFHR mAbs. One of which targets CFHR1 (R1/1037) and three target CFHR4 (R4/244, R4/277 and R4/123). The generation of these antibodies have allowed putative ELISA screens to be developed to measure the concentration of CFHR4 in healthy individuals and aHUS patients. My full panel of CFHR proteins also enabled screening of both aHUS and IgAN patients for the presence of autoantibodies to CFH and the CFHR proteins. Screening of aHUS plasma did not indicate the presence of any novel CFHR autoantibodies. However, IgA autoantibodies against CFHR5 (~9%) and CFH (~32%) were detected in IgAN patients. Interestingly, 64% of IgAN patients show reactivity with bovine CFH. During this PhD, I have generated a panel of unique reagents for the study of CFHR proteins in health and disease. These have allowed me to demonstrate for the first time, the presence of CFH and CFHR5 autoantibodies in a preliminary cohort of IgAN patients

    Unravelling the structure of glycosylated and deglycosylated immunoglobulin G antibodies

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    Immunoglobulin G (IgG) is composed of four IgG subclasses, IgG1, IgG2, IgG3 and IgG4, which although differ in function and structure, owing to variability in hinge length, all have a conserved N-linked glycan attached in the Fc region. However, the role of this glycan on the structure, stability and function of these IgG molecules is not fully understood. The focus of this thesis is to investigate the role of the Fc-glycan in respect to IgG1, IgG3 and IgG4 by using a multidisciplinary approach to study both their glycosylated and deglycosylated forms. Primarily by probing the full-length solution structure using small angle X- ray and neutron scattering, as well as analytical ultracentrifugation. Following this extensive computational modelling methods and analysis were used to extract the theoretical models which best fit the solution structure data in order to unpick the role of this glycan. Several studies have investigated the role of the IgG1 Fc-glycan using different structural methods, however, most of these studies investigated the Fc region of IgG rather than the full-length antibody, composed of Fabs, hinge and Fc. In this thesis all experiments are conducted on full-length IgGs, presenting a complete understanding on the effect of the Fc-glycan on the entirety of the IgG structure. Studies of IgG1 and IgG4 indicate that the Fc-glycan plays a role in restricting the flexibility of the Fc. This restriction is less obvious in IgG3; this may be owing to the molecule’s elongated hinge. Of the three antibodies studied, IgG3 has the longest hinge region, composed of 62 amino acids. This long hinge has historically made it difficult to study using other structural techniques, such as X- ray crystallography and NMR, therefore the study of the IgG3 solution structure presented herein is the most complete to date and provides insight into the dynamics of the hinge region

    Site-directed spin-labelling of proteins for EPR spectroscopy : application to protein complexes and development of new methods for cysteine rich proteins

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    The work described in this thesis is an experimental study into the application of Electron Paramagnetic Resonance (EPR) Spectroscopy for the study of biological systems. Using a variety of methods of site-directed spin-labelling (SDSL), this thesis aims to explore long range structure in an assortment of recombinant and native proteins, and complexes thereof. The work described in this thesis covers all aspects of the work, from experimental design, molecular biology and cloning, protein expression and purification, as well as functional characterisation, and finally EPR distance measurements, data analysis and interpretation. Challenges and pitfalls will also be addressed. Chapters 1 and 2 introduce EPR spectroscopy, and its application in the study of long range structure in biological systems. The experimental techniques employed throughout this thesis are also introduced. Chapter 3 details an investigation into the complement C3b:factor H complex. This chapter addresses the challenges associated with the SDSL of cysteine rich proteins. Utilising hidden cysteine residues in native proteins for spin-labelling purposes will also be addressed. Chapter 4 looks at the interactions of the human myosin regulatory light chain (RLC) with cardiac myosin binding protein C (cMyBP-C). Optimisation of expression and purification protocols will be the focus, as well as addressing issues with protein solubility and spin labelling efficiencies. Chapter 5 explores the development of new methods of SDSL, for the specific labelling of cysteine rich proteins. The ability of Escherichia coli to read through the amber stop codon will be exploited for the incorporation of unnatural amino acids for labelling purposes, and novel spin labels, specific for labelling cysteine pairs tested in several model systems. Furthermore, native paramagnetic centres in recombinant proteins will be explored as potential labelling sites
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