Complement Factor H C-terminus and its significance:
A structural narrative
Arnab Bhattacharjee
University of Helsinki
2014
Academic Dissertation
Complement Factor H C-terminus and its significance: A structural narrative
Arnab Bhattacharjee
Department of Bacteriology and Immunology Haartman Institute University of Helsinki Finland
and
Research Programs Unit, Immunobiology Faculty of Medicine University of Helsinki Finland
To be publicly discussed with the permission of the Medical Faculty, University of Helsinki, in auditorium 1 of Haartman Institute, on 11th April, 2014, at 12 noon.
Supervised by: Adj. Prof. T. Sakari Jokiranta, MD, PhD Department of Bacteriology and Immunology, Haartman Institute and Research Programs Unit, Immunobiology, University of Helsinki, Finland
Reviewed by: Adj. Prof. Arno Hänninen, MD, PhD Department of Medical Microbiology and Immunology, University of Turku, Finland
Adj. Prof. Veli-Pekka Jaakola, PhD Department of Biochemistry, University of Oulu, Finland
Opponent: Professor Péter Gál, PhD Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
Illustrations by the author ISBN 978-952-10-9854-3 (paperback) ISBN 978-952-10-9855-0 (PDF) Printed at Oasis media Finland Oy Finland 2014
‘Cogito ergo sum’,: I think, therefore I am
~René Descartes
Dedicated to
the honourable memory of my first biology teacher in school ,
‘Ajitda’ who seeded the interest in me to look through biological systems,
but couldn’t live to cherish the day
4" CONTENTS"""""!
CONTENTS
ORIGINAL PUBLICATIONS ...... 5
CONTRIBUTION TO PUBLIC DATABASES ...... 6
ABBREVIATIONS ...... 7
ABSTRACT ...... 8
1 INTRODUCTION ...... 11
2 REVIEW OF LITERATURE ...... 13 2.1 The complement system: an overview ...... 13 2.2 Activation of the complement system ...... 14 2.3 Regulation of the complement system ...... 16 2.4 The CFH family: focus on structure and functions ...... 19 2.5 CFH Interactions with Host Ligands ...... 25 2.6 Diseases associated with CFH family of proteins ...... 29 2.7 The microbial complement evasion: role of CFH ...... 32 2.8 Anti-FH autoantibodies and aHUS ...... 33
3 OBJECTIVES ...... 36
4 MATERIAL AND METHODS ...... 37 4.1 Materials ...... 37 4.2 Methods ...... 38
5 RESULTS ...... 48 5.1 Both domains (19 and 20) of CFH are involved in C3b binding ...... 48 5.2 A bigamic interaction of CFH revealed ...... 50
5.3 Borrelia burgdorferi binds CFH20 to evade complement ...... 52 5.4 Molecular basis for autoimmunity against CFH in aHUS ...... 55
6 DISCUSSION ...... 58 6.1 Molecular basis for the self non-self discrimination by AP ...... 58 6.2 The microbial complement evasion: a molecular perspective ...... 61 6.3 Link between the CFHR1 deficiency and the AI-aHUS puzzle ...... 62
6.4 Structural analyses of various structures of CFH19-20 ...... 68 6.5 Crystal artifacts, the pinch of salt ...... 69 6.6 Conclusions and future prospects ...... 70
7 ACKNOWLEDGEMENTS ...... 71
8 REFERENCES ...... 73
ORIGINAL"PUBLICATIONS"""""5"!
ORIGINAL PUBLICATIONS
This thesis is based on the following publications:
I) Bhattacharjee A., Lehtinen M. J., Kajander T., Goldman A. and Jokiranta T. S. (2010) Both domain 19 and domain 20 of factor H are involved in binding to complement C3b and C3d. Mol Immunol 47, 1686-91.
II) Kajander T., Lehtinen M. J.*, Hyvärinen S.*, Bhattacharjee A.*, Leung E., Isenman D. E., Meri S., Goldman A. and Jokiranta T. S. (2010) Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement. Proc Natl Acad Sci USA, 108(7), 2897- 902.
III) Bhattacharjee A., Oeemig J.S., Kolodziejczyk R., Meri T., Kajander T., Iwai H., Jokiranta T.S.*, Goldman A.* (2013) Structural basis for complement evasion by Lyme disease pathogen Borrelia burgdorferi. J Biol Chem 288,18685-695.
IV) Bhattacharjee A., Reuter S., Kolodziejczyk R., Seeberger H., Hyvarinen S., Prohászka Z., Szilágyi Á., Goldman A., Józsi M, Jokiranta T.S. (2013) The major AA epitope on CFH and the structure of its homologous site on CFHR1 explain why CFHR1 deficiency leads to aHUS. (Submitted)
*authors contributed equally to the article
The publications are referred to the text by their roman numerals. The articles have been reprinted with permission of the copyright holders. 6" CONTRIBUTION"TO"PUBLIC"DATABASES"""""!
CONTRIBUTION TO PUBLIC DATABASES
I) Bhattacharjee A., Lehtinen M. J., Kajander T., Goldman A. and Jokiranta T. S. (2010): 3KXV; Structure of complement Factor H variant R1203A, three-dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb). II) Bhattacharjee A., Lehtinen M. J., Kajander T., Goldman A. and Jokiranta T. S. (2010): 3KZJ; Structure of complement Factor H variant Q1139A, three-dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb). III) Kajander, T., Lehtinen, M. J., Hyvarinen, S., Bhattacharjee, A., Leung, E., Isenman, D. E., Meri, S., Jokiranta, T. S., Goldman, A. (2010): 2XQW; Structure of complement Factor H domains 19-20 in complex with complement C3d, three-dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb). IV) Bhattacharjee, A., Oeemig, J.S., Kolodziejczyk, R., Meri, T., Kajander, T., Iwai, H., Jokiranta, T., Goldman, A. (2013): 2M4F; Solution structure of Outer surface protein E, three-dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb). V) Bhattacharjee, A., Kolodziejczyk, R., Kajander, T., Goldman, A., Jokiranta, T.S. (2013): 4J38; Structure of Borrelia burgdorferi Outer surface protein E in complex with Factor H domains 19-20, three- dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb). VI) Bhattacharjee, A., Kolodziejczyk, R., Goldman, A., Jokiranta, T.S. (2013): 4MUC; Structure of the C-terminus of Factor H related protein I, three-dimensional structural data deposited in the RCSB protein data bank (www.rcsb.org/pdb).
ABBREVIATIONS"""""7"!
ABBREVIATIONS
AA Autoantibody AI-aHUS Autoimmune Atypical haemolytic uremic syndrome aHUS Atypical haemolytic uremic syndrome AMD Age related macular degeneration AP Alternative pathway CCP Complement control protein CFH Complement Factor H CFH19-20 Domain 19 and domain 20 of Complement Factor H CFHL-1 Complement Factor H like protein-1 CFHR Complement Factor H related protein CFHR1 Complement Factor H related protein 1 CFHR14-5 Domain 4 and domain 5 of Complement Factor H related protein 1 CFH-AA anti Complement Factor H autoantibody CP Classical pathway CR Complement receptor CRP C-reactive protein CS-A Chondroitin sulfate A DAF Decay accelerating factor (CD55) DDD Dense deposit disease FI Factor I GAG Glycosaminoglycan HS Heparan sulfate IC Immune complex iC3b Inactive C3b Ig Immunoglobulin LP Lectin pathway mAb Monoclonal antibody MAC Membrane attack complex MASP MBL-associated serine protease MBL Mannan binding lectin PBS Phosphate buffered saline RCA Regulators of complement activation RLA Radioligand assay RMSD Root mean square deviation SA Sialic acid SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis SPR Surface plasmon resonance Å Ångström (0.1 nm) 8" ABSTRACT"""""!
ABSTRACT
Complement is comprised of a cascade of proteins that recognizes and attacks the invading microbes and thus is the first line of defense for the human body against invading pathogens. It is initiated via different activation pathways that lead to C3b deposition on the target and sequentially to the formation of lytic membrane attack complexes (MAC). One of the complement activation pathways – the alternative pathway (AP) – can be activated on any surface, self or non-self, and is therefore tightly regulated. Complement Factor H (CFH) is the most important complement down-regulator and it mediates target discrimination between self and non-self cells. Several point mutations in CFH and/or autoantibodies (AA) against it are found to be directly associated with atypical haemolytic uremic syndrome (aHUS), a severe and often fatal disease triggered by the impaired regulation of AP on self surfaces leading to complement attack. CFH is composed of 20 homologous complement control protein domains
(CCP). The N-terminal domains 1 to 4 (CFH1-4) mediate inactivation of C3b on the self-surfaces and the C-terminal domains 19 and 20 (CFH19-20) are critical for the ability of CFH to discriminate between self and non-self structures. Self-surfaces are rich in anionic sialic acids (SA) and glycosaminoglycans (GAGs) that are not present on pathogenic microbes. CFH19-20 contains binding sites for both the C3d part of C3b and self-surface polyanions that enhance avidity of CFH to C3b on self surfaces and thus enhance C3b inactivation. CFH mutations that have been found in aHUS patients are mostly located in CFH19-20.
The previously solved X-ray crystal structure of CFH19-20 illuminated the location of aHUS related mutations. The aims of this thesis work were to study the functionality of CFH on a molecular level by studying the molecular structure of CFH C-terminus and its mutants along with structures of CFH in complex with its different interacting partners, as well as the structure of the domains of CFH-related protein-1 highly homologous to CFH19-20. The structures solved and the their relevance are described in the four articles attached to this thesis. In the first article, stability of the CFH C-terminus fold by aHUS mutation(s) was studied by analyzing the binding of the CFH19-20 mutant proteins to C3d/C3b ABSTRACT"""""9"! using radioligand assays and affinity chromatography. The X-ray crystal structures of
CFH19-20 with two different point mutations (of residues indicated to be involved in binding C3d/C3b) were solved. It was shown that these mutations did not result in the disruption of the basic fold of CFH19-20, but maintained the same fold with a prominent difference in the surface charge distribution in the zone of the residues. The results clearly indicated that the aHUS mutations on CFH do not disrupt the basic structural fold, but induce anomaly in the charge distribution of the molecule, explaining the effects of the mutations to its C3b/heparin binding abilities suggested to be critical for the pathogenesis of the disease. In the second article, we revealed the rationale of the molecular mechanism of
CFH19-20 mediated self–nonself discrimination and showed why point mutations in
CFH19-20 lead to aHUS. The CFH19-20 :C3d structure reported in this article revealed 19 two independent binding interfaces between CFH19-20 and C3d, namely the ‘CFH site’ and the ‘CFH20 site’. The results of deeper analysis of this structure also showed 19 20 that the simultaneous binding of the CFH19-20 via the CFH site to C3b and via CFH site to C3d was possible.
In the third article, the X-ray crystal structure of CFH19-20 was solved in complex with the outer surface protein E (OspE) from Borrelia burgdorferi in order to understand the molecular mechanism of sequestering CFH by microbes which results in complement evasion. The nucleomagnetic resonance (NMR) structure of the OspE protein from Borrelia burgdorferi reported in this paper was required for solving the structure of OspE in complex with CFH19-20. Chemical shift perturbations studies using NMR also confirmed the physiological viability of the complex structure. This complex structure was the first structure of CFH19-20 in complex with any microbial protein and thus answered the puzzle of the molecular mimicry used by microbes involving CFH C-terminus in order to evade complement.
In the fourth article the structure of the CFHR1 domains 4 and 5 (CFHR14-5) was solved and used to explain why AA bind to a common epitope on CFH domain
20, which is highly homologous to domain 5 of CFHR1. The CFHR14-5 structure revealed an important structural bigamy of CFH and its related proteins that can be used to understand why CFHR-1 deficiency and formation of AA against CFH (CFH- AA) lead to autoimmune aHUS (AI-aHUS). We extensively studied CFH-AA from more than a dozen of patients and their binding behavior to CFH and CFHR1. The results suggest a novel hypothesis on the pathogenesis of AI-aHUS. 10" ABSTRACT"""""!
In conclusion, the results have revealed the molecular mechanisms beneath different functionalities of the C-terminus of CFH. It is not only the most important molecule to facilitate the target discrimination by the AP, but is also a prominent tool used by the microbes in order to evade complement attack. Furthermore, the CFH C- terminus also houses the AA binding epitope and thus also plays a role in the pathogenesis of AI-aHUS. We were also quite surprised to find out that the molecular structure of CFH C-terminus is extremely stable, and hardly undergoes any changes in its conformation in the presence of other ligands. Cumulatively, the structures of the CFH in presence of its different partners of interactions contributed greatly to the knowledge pool of the understanding of the molecular mechanisms associated with CFH in complement activation and regulation in health and disease.
INTRODUCTION"""""11" !
1 INTRODUCTION
The human body needs to protect itself both from invading pathogens as well as from the side effects of the immune processes going on inside the body. The continuous challenge thrown towards our body is both internal and external in nature. The security system our body puts up in order to handle these challenges is called the immune (coming from the latin word immunitas) system, which with evolution has developed into an organized, sensitive and a robust system. Our immune system can be classified into two parts, namely the innate and adaptive immunity. The concept of innate immunity shaped through evolution to discriminate self from non-self cells has been expanded, during the last decades, owing to the description of additional functions of the innate defenses. This part of immunity not only recognizes non-self molecules or surfaces, but also utilizes more sophisticated mechanisms of recognition. It is the type of immunity that provides the early line of defense against invading microbes and is comprised of the cellular and biochemical defense. Innate immunity is pre-poised to respond to an external attack even before they take place, but responds to repeated infections in a non-specific manner. On the contrary, the adaptive immune responses come into action by an exposure to external invading agents and increases its defense capabilities with each successive exposure to the particular microbe. The most important components of the innate immune system are the epithelial barriers, phagocytic cells, dendritic cells, natural killer cells, cytokines and the complement system. The complement system was described in 1889 when Hans Büchner noticed a heat labile substance in blood serum which was able to kill bacteria, thus ‘complementing’ the action of adaptive humoral immunity. Paul Ehrlich first used the term “complement” by 1899 (1) for the phenomenon mentioned below in more detail. The mammalian complement proteins provide a first degree immune response against pathogens in blood and interstitial fluids (2). The primary aim of the complement system is to identify surfaces of bacteria, viruses and fungi, and also apoptotic host cells in order to be treated for cell lysis, clearance by phagocytosis and stimulation of the adaptive immune response. 12" INTRODUCTION"""""!
Since 1960s, there has been a remarkable transformation in our understanding of the immune system and its functions. Advances in cell culture techniques and monoclonal antibody (mAb) production, immunochemistry, recombinant DNA technology, studies of molecule structures at atomic resolution employing different methods, and the generation of transgenic animals have changed the nature of immunological studies. Most of the immunological phenomena are now explained in structural and biochemical terms silently leading to revolutionary advances in the field. This thesis deals with different aspects of complement system and its occasional failure in case of the onset of infections or autoimmunity using X-ray crystallography as a tool to study it at atomic (or close to atomic) resolution. REVIEW"OF"LITERATURE"""""13" !
2 REVIEW OF LITERATURE
2.1 The complement system: an overview
Innate immunity is the first-line of the immune defense system, essential for cellular integrity, tissue homeostasis and modifying the adaptive immune response. The innate immune system comprises of several components and uses several receptors for the recognition of microorganisms. A major humoral component of innate immunity is the complement system, which was established early in evolution and is the primary immune system in invertebrates. Complement was initially identified more than 100 years ago as a serum component that ‘complements’ (thus contributing to the nomenclature) the antibody response towards pathogens (1,3). Now, more than a hundred years after these initial reports, the central role of this system in immune defense is much better known. It is now established as a system of a cascade of proteins and many of the biological effects of complement are understood in molecular terms and a role for complement in tissue homeostasis is emerging (2,4,5). Complement activation activates pro-inflammatory mediators, generates anaphylactic peptides, cytolytic compounds and antimicrobial compounds, recruits effector cells and induces effector responses. These proteins form a complex network of various recognition, effector, regulatory and receptor molecules that act in a finely tuned fashion, allowing complement to safely exert its functions (6,7). The activated complement system is beneficial for the protection of the host against invading pathogens (8). Other than protecting the host from infections by destroying and eliminating pathogens, complement maintains the integrity of the body by discriminating between healthy and injured tissue, in participating in the disposal of immune complexes and other cellular debris (9,10), also in inflammatory processes, angiogenesis and tissue regeneration (11,12). The individual complement reactions develop in a sequential manner, allowing regulation that modulates the intensity of the response and adjusts the effector functions for the specific immune response. It has also been established that complement is involved in the induction and regulation of both innate and adaptive cellular immune responses (2,13). 14" REVIEW"OF"LITERATURE"""""!
2.2 Activation of the complement system
Complement can be activated via three primary pathways, the classical pathway (CP), the lectin pathway (LP), and the AP. All pathways converge at the central component C3 (Figure 1), which eventually initiates the terminal pathway forming the membrane attack complex (MAC) The CP is initiated by C1q binding to surface-bound immunoglobulins (Ig, mainly IgG1, IgG3 or IgM) or apoptotic cells (14-18). C1q is reported to bind to the Fc regions of the antibodies in order to initiate the CP (19). It has been suggested that binding of C1q to multiple, or even multimerized Ig, on a surface results in changes of the conformation of the C1-complex; this would prevent fluid phase antibodies from activating the cascade (20). Bacterial lipopolysaccharides, DNA, C-reactive protein (CRP), serum amyloid P component (SAP), and pentraxin 3 (PTX3) have been also found to activate the CP (21-25), but the physiological importance of these phenomena remains mainly elusive. The LP is activated by binding of mannose-binding lectin (MBL) to repetitive carbohydrates, or by binding of ficolins to carbohydrate or acetylated groups on target cell surfaces (26-28). MBL binds mannose and N-acetyl-glucosamine residues present in bacterial cell walls. The LP activation starts with association of two C1r- and C1s- like MBL-associated serine proteases (MASP-1 and MASP-2) with MBL. Complement components C4 and C2 are both cleaved by MASP-2 leading to complement activation (29-31). MBL and C1q are structurally related but MBL possesses carbohydrate-binding lectin domains which is not present in C1q (32). LP can be also be activated by M-, L-, and H-ficolins (also called 1-, 2- and 3-ficolins), respectively. All of these ficolins function as pattern recognition molecules that recognize carbohydrates on microbial surfaces (28). The AP is spontaneously activated by the hydrolysis of the internal thioester group of C3 eventually forming the C3 convertase (classical/lectin pathway C3 convertase C4b2aand the AP C3 convertase C3bBb). (17,33). This complex cleaves fluid phase C3 to C3a (a small 9 kDa inflammatory mediator) and C3b, an opsonin that deposits on the surface of target cells and particles and promotes phagocytosis. (34-38). This spontaneous complement activation in fluid phase, a unique feature of AP, is constantly targeting all surfaces that are devoid of sufficient down-regulators. REVIEW"OF"LITERATURE"""""15" !
The larger fragment C3b formed as a result of C3 cleavage can also form additional C3 convertases with Bb and amplify complement activation. The cleaved C3b exposes a thioester group that can bind covalently to a target surface similarly to C4b in CP. At this stage the interaction between C3b and either factor B or CFH forms the basis of the self vs. non-self discrimination of AP (39,40). The amplification of AP occurs at this stage when C3 convertase activates more fluid phase C3 by cleavage to C3b (41). This amplification step causes effective C3b opsonization of the target (42,43). C3b can also bind to an already existing C3 convertase, to form the C5 convertase which cleaves C5 and activates the terminal complement pathway. This activation of C5 potentially leads to inflammation (via C5a generation) and results in the lysis of the target cell through the formation of membrane pores by the MAC (44). Whereas these activation processes are needed on microbial surfaces, they are destructive to host (self) cells.
Figure 1. Complement activation pathways and the regulatory role of complement factor H.
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2.3 Regulation of the complement system
The complement activity is important in our body for the clearance of foreign and altered host cells, whereas on the other hand, this activity must be well controlled in order to avoid self tissue damage. The spontaneous activation of AP as well as its secondary activation via the ‘amplification loop’ of classical/lectin pathway makes its regulation extremely important. Consequently, the process of complement activation is strictly regulated by membrane-bound and blood-plasma regulatory molecules that protect against complement activation by down-regulating the central proteolytic activity of the amplification and opsonisation steps. Deficiencies or mutations in complement regulators may predispose individuals to infectious and immune-related diseases or may lead to excessive complement activation and tissue injury. The complement system with its casacade of regulatory proteins may act during activation, amplification and effector steps of the cascade that enables fine- tuning of the activation in different physiological circumstances. These proteins compete with other complement components for the binding sites. The regulators of complement activation (RCA) gene cluster in chromosome 1 encode several complement regulatory proteins (45-50). All the RCA proteins are composed of several homologous domains that have probably arisen by gene duplication events (51). These domains are approximately composed of 60 amino acids each and these protein domains are arranged in a bead-like structure. These short consensus repeat domains (presently known as CCP domains) are generally arranged in proteins in a head-to-tail fashion resembling beads-in-string (52). The binding sites in CCP-bearing proteins may span over its domains and therefore the orientation of the CCPs is critical for the proteins to act. These RCA proteins include C4b binding protein (C4bp), CFH, CFH-like protein-1 (CFHL-1), CFH-related (CFHR) proteins and membrane bound regulators complement receptor 1 (CR1/CD35), membrane cofactor protein (MCP/CD46), and decay accelerating factor (DAF/CD55)(46-48).
2.3.1 Soluble complement regulators
C1 inhibitor (C1inh): C1inh is a 105 kDa serine protease inhibitor (serpin) with a heavily glycosylated single chain protein with a two domain architecture (53,54) which regulates the complement activation (via CP) by binding to C1r and C1s REVIEW"OF"LITERATURE"""""17" ! causing their dissociation from C1q. It also influences the C activation in fluid phase by acting on C1 that otherwise could be spontaneously autoactivated by non-immune activators at low levels (55). Deficiency in C1inh results in hereditary angioedema (HAE), a disease characterized by recurrent acute edema of skin or mucosa which is associated with the lack of regulation of kallikrein by C1inh (56).
C4b-binding protein (C4bp). C4bp is a 540-590 kDa protein consisting of several disulphide bonded subunits (57, 58) which interferes with the assembly of surface bound CP C3-convertase C4b2a by accelerating its decay. It also functions as a cofactor for factor I in cleavage of both surface bound and fluid phase C4b to C4c and C4d (59).
CFH and its family. CFH is an elongated, approximately 155 kDa single-chain glycoprotein (60) consisting of 20 CCP domains (61)(Figure 2). When bound on the surface, CFH regulates AP indirectly by acting as a cofactor for the inactivation of C3b by factor I (62). It also directly inhibits the formation of C3-convertases by interfering with the binding of FB to C3b (39) and furthermore increases the pace of the natural decay of already formed AP convertases (63,64). Mutations in CFH have been associated with aHUS (65,66), dense deposit disease (DDD, also known as membranoproliferative glomerulonephritis type II or MPGN II ) (67). CFH has been found to have some of its polymorphisms (68) associated with aHUS as well (69). One of the CFH polymorphisms also has been associated with age related macular degeneration (AMD) (70,71). Several other proteins from the RCA gene cluster have been found to have strikingly similar structural and functional features like CFH. These are all categorized in the family of CFH that will be discussed later.
Properdin. Properdin exists in plasma as oligomers of two to four monomers linked together in a cyclic fashion (72). Each 52 kDa monomer consists of six thrombospondin repeat-domains (73). Properdin is the only positive regulator of complement activation that stabilises the nascent C3/C5 convertases of the AP and thus subsequently slowing down their rate of decay. (74).
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Vitronectin. Also known as S-protein, vitronectin exists in at least two forms in plasma, a single chain molecule (84 kDa) or a double chain molecule where the subunits (69 kDa and 15 kDa) are linked together with disulfide bonds. It is also expected to form dimers and higher molecular weight aggregates in plasma (75). Vitronectin binds fluid phase C5b-7 in order to prevent its membrane-binding and thus prevent MAC insertion (76).
Factor I. Factor I is a 90 kDa enzyme consisting of a 50 kDa and a 38 kDa chain linked together by a disulphide bond. (77,78). Factor I regulates the complement system by inactivating C4b and C3b molecules (with either CFH, C4bp, CR1 or MCP working as cofactors) (79-81).
2.3.2 Membrane bound regulators
CR1/CD35. This is a 205 kDa polymorphic protein and was first isolated from erythrocyte membranes. It regulates complement activation on cell surfaces and facilitates the decay of fluid phase immune-complexes (82,83). CR1 gene is located in the RCA cluster.
Complement receptor of the Immunoglobulin family (CRIg). CRIg is a complement receptor expressed on tissue macrophages. It mediates opsonophagocytosis by binding C3b and inactive C3b (iC3b) on opsonized particles (84). CRIg regulates AP activation by binding to C3b and inhibiting the interaction of C3 and C5 with AP convertases (85).
Decay accelerating factor (DAF or CD55). DAF regulates the AP and CP by accelerating the decay of C3-convertases (86). DAF is a 70 kDa membrane protein composed of four CCP domains and a serine/threonine/proline (STP)-rich linker (87).
Membrane cofactor protein (MCP/CD46). It is 50-85 kDa heavily glycosylated membrane protein widely expressed on cells other than erythrocytes. It acts as a cofactor for C4b and C3b cleavage by factor I as mentioned before (81). MCP has four CCP domains and a heavily glycosylated STP region along with transmembrane and cytoplasmic domains as well (46).
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CD59 (protectin). CD59 is a 18-20 kDa single chain protein expressed by RBC and other cells (88). This has five intra-chain disulphide bonds (89), heavily glycosylated and binds to the membrane associated C5b-8 complex preventing the unfolding of C9 (90) and thus the membrane spanning by C9.
2.4 The CFH family: focus on structure and functions
CFH belongs to a protein family that also includes CFHL-1 (an alternative splicing product of the CFH gene) and five CFH-related proteins (CFHR) (91) (Figure 2). These proteins are composed of four to nine CCP domains, and share various degrees of sequence identity with each other and with CFH (Figure 2).
Figure 2. The schematic structure of factor H and its family members. For sequence identities of 32-49% the domains are displayed in white, for 57-84% identity in light blue, and for 85-100% identity in dark blue. The identity between the domains 3-5 of CFHR1 and domains 18-20 of CFH is indicated with percentages; *, sequence identity of the domain 3 of the basic isoform of CFHR1 is 100%, whereas that of the acidic isoform is 95% (216). CFH- like molecule-1 (CFHL-1) is an alternatively spliced transcript from the CFH gene having four unique residues after domain 7.
2.4.1 Complement Factor H (CFH) and target discrimination
CFH is the main soluble regulator of the AP (92). The CFH coding gene (CFH) is located in chromosome 1q32 within the RCA gene cluster (as mentioned before), adjacent to the five genes that code for the CFHRs. CFH is constitutively expressed in 20" REVIEW"OF"LITERATURE"""""! the liver and is distributed systemically in body fluids. Reported CFH plasma concentrations vary, depending on the study population, age, genetic and environmental factors, as well as on the method used for quantification (93). Previous studies overestimated CFH concentration, which was a result of measuring both CFH and CFHRs. So, while early reports suggested that the plasma CFH concentration ranges 265–684 µg/ml (94) and 116–562 µg/ml (68), current studies using monoclonal antibodies have established mean CFH concentrations of 233 µg/ml (in young adults), 269 µg/ml (in elderly individuals) (95) and 263 µg/ml (93), in different healthy control populations with the age group between 25-66 (mean 42) years. Thus, normal CFH concentrations in human plasma correspond to approximately 1–2 µM. In addition, CFH is also produced outside the liver by different cell types such as monocytes (96), fibroblasts (97), endothelial cells (98), keratinocytes (99), thrombocytes (100) and retinal pigment epithelial cells (101). Locally released CFH in tissues may help to limit complement activation and maintain an anti-inflammatory environment. Each of the autonomously folding globular domains (see 2.3.1) is composed of about 60 amino acids and is stabilized by two internal disulfide-bonds (61). As mentioned earlier, CFH regulates complement activation by inhibiting the assembly of the AP C3 and C5 convertase enzymes via competition with FB for C3b binding; thus by facilitating the dissociation of the convertases by displacing bound factor Bb; and by acting as a cofactor for the serine protease factor I in the cleavage and inactivation of C3b (64,102). These regulatory activities are mediated by the four N- terminal domains 1–4 of CFH (103,104) (Figure 3), whereas the C-terminal domains 19-20 are responsible for target recognition (Figure 3) (105-107). One of the important targets for CFH binding in the vicinity of C3b on host cells are polyanionic surface molecules, such as GAGs and SA, which increase the affinity of CFH for C3b (40). Thus, CFH is also able to control complement activation on self-surfaces (Figure 2, also see section 2.5) (108,109). The target discrimination by AP is mediated by CFH and it has been established that the negatively charged polyanions or SA on the self cells contributes to higher CFH:C3b (surface) interaction and thus leads to high cofactor activity of CFH (39,40,64,110). In contrast, host-like polyanionic molecules are normally not present on the surface of pathogens, rendering them susceptible to complement attack. REVIEW"OF"LITERATURE"""""21" !
Figure 3. Complement activation pathways and the regulatory role of factor H. The primary function associated with FH domains are also indicated. Sparsely dotted regions indicate (domains 6-7, domains 19-20) heparin and microbial binding sites while the densely dotted region (Domains 1-4) is associated with cofactor and decay acceleration activities. In addition to C3b and polyanionic molecules, CFH interacts with several other endogenous ligands. These interactions allow CFH to regulate complement on certain host surfaces (such as the glomerular basement membrane, the extracellular matrix, and late apoptotic cells), which are otherwise not properly protected due to a reduced expression or the lack of membrane-anchored complement regulators (i.e., membrane cofactor protein, decay accelerating factor and CD59). Several of these ligands and structures activate complement via interactions with C1q, MBL, or ficolins. The simultaneous binding of both complement activating molecules (i.e., C1q, MBL) and molecules inhibiting the activation of complement (i.e., CFH, C4b- binding protein) on such ligands and cells may facilitate their opsonisation and safe removal, but at the same time prevents an exaggerated complement activation that could lead to inflammation, unnecessary cell lysis, and subsequent tissue damage (111). 22" REVIEW"OF"LITERATURE"""""!
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CFH also binds to nonhost ligands, such as certain surface proteins of microbes, which hijack host CFH in order to protect themselves from complement attack (112). AP evasion by microbes is considered to the primary reason for the sequestering of CFH on their surface. Streptococcus pyogenes was the first microbe to be identified with this phenomenon to evade complement-mediated opsonophagocytosis (113-115). Most microbes acquire CFH via two regions of this control protein, domains 5-7 and 18-20 (Figure 4). Domains 6-7 of CFH have been evidently functional in binding with Streptococcus and Neisseria surface proteins(114-119). Domain 18-20 of CFH has been suggested to mediate Neisseria gonorrhoeae complement evasion via Por1A and Por1B (119-121). Borrelia burgdorferi also uses two different surface proteins for CFH binding, viz. OspE for binding domains 18-20 of CFH (122,123) and BbCRASPs for binding to domains 6-7 (124,125). Some microbes such as Yersinia enterocolitica (126,127) and Streptococcus pneumoniae (128,129) have been shown to bind to non-conventional microbial binding domains in CFH. Interestingly, there are also microbes which have been found to bind CFH via two different domains using the same ligand on their surface, such as Pseudomonous aeruginosa (130) and Candida albicans (131,132). Sbi protein of Staphylococcus aureus and shiga toxin (Stx2) of enterohemorrhagic Escherichia coli have been reported to bind CFH in a non- conventional manner. Sbi binds domains 19-20 of CFH by forming a tripartite complex with C3b (133), whereas Stx2 binds both CFH domains 6-7 and 18-20 in fluid phase (134). Several microbial ligands involved in CFH interactions have not been identified. For example, Haemophilus influenzae (135), Fusobacterium necrophorum (136), Aspergillus fumigatus (137), Bordetella pertussis, Bordetella parapertussis (138) and Onchocerca volvulus (139) have been reported to bind to CFH, but the bacterial ligands remain unknown. All of these microbes interact at least with CFH domains 6-7 and/or 18-20.
2.4.2 The CFH like protein-1
CFHL1 is a 43-kDa protein deriving from an alternative splice product of the CFH gene. It shares the seven domains (1-7) with CFH and has four additional C-terminal amino acids. As expected owing to high sequence similarity, CFHL1 possesses the 24" REVIEW"OF"LITERATURE"""""! same complement regulatory activities as mediated by the N-terminus of CFH. CFHL1 may also play a role in age-related macular degeneration as the domain7 harbors the site for Y402H polymorphism (see 2.7). Consequently, the CFHL1 402H variant has impaired ligand-binding capacity, similar to that exhibited by the CFH 402H variant (140,141). However, an expression pattern differing from CFH and a distinct role in mediating cell adhesion have been reported for CFHL1 as well (142,143). CFHL1 has been observed to bind microbes which do not bind to CFH as well (144).
2.4.3 The CFH related proteins
The five CFHR proteins (CFHR1-CFHR5, Figure. 2) are from separate genes located adjacent to the CFH gene. They have domains homologous to the different domains of CFH, which are responsible for directing the complement regulatory activity of CFH to cell and tissue surfaces (91). These proteins have both similar and non- redundant ligands and functions with CFH. CFHR1, CFHR3, CFHR4 and CFHR5 bind to C3b, CFHR1, CFHR3 and CFHR5 to heparin, and CFHR4 and CFHR5 to CRP (145-151). In spite of similar cell and ligand binding properties, however, CFHR1 was reported to regulate the terminal complement pathway (145). In addition, it has been shown that both CFH and CFHR1 enhance neutrophil adhesion and activation during host cell-pathogen contact (152). Similar to CFH, CFHR4 binds to late apoptotic and necrotic cells, but in contrast to CFH, CFHR4 binds to the native pentameric form of CRP (153).
2.4.4 Discrimination of self and non-self by CFH
Identification of cell surfaces by the complement system is achieved by covalent binding of C3b molecules to the target surface through an exposed thioester in C3b that reacts with hydroxyl or amine groups. After initial binding of C3b or its homolog C4b by the initiation of complement cascade, the response (other than C4b2a) is amplified by enzyme complexes, or convertases, that form on the surface-bound C3b or C4b molecules. Host cells require soluble complement inhibitors, particularly CFH, which provide effective protection from unwanted complement-mediated damage, especially under conditions with strong complement activation. It is critical to protect noncellular surfaces, which do not carry surface-bound regulators. Several mutations REVIEW"OF"LITERATURE"""""25" ! in the two C-terminal domains of CFH have been linked to aHUS (109). The same mutations also exhibited reduced functioning of CFH (154). Self-nonself discrimination by CFH is mediated by domains 19-20 that bind to surface-bound C3b/C3d and host surface GAGs or SA (155). Critical for target discrimination is the formation of a ternary complex of CFH domains 19–20, surface-bound C3b and GAGs or SA or C3d from the self-surface. Host cells, so-called non-activators of complement, possess cell surface polyanionic molecules that promote CFH binding (40). In contrast, complement activators such as microbes or rabbit erythrocytes that lack SA and host-like GAGs do not allow significant CFH binding and thus activates complement. Heparin is generally used in studies as a representative of host GAGs, and the major heparin binding sites are located in domains 7 and 20 (154-157). Polymorphisms or mutations in domains 7 and 19-20 may affect interactions of CFH with host cells and basement membranes, and are implicated in the diseases AMD and aHUS (106).
2.5 CFH interactions with host ligands
Other than its role as a complement regulator, CFH has been shown to mediate cellular interactions by binding to receptors on various cells as well.
2.5.1 CFH interaction with C3b
The central complement protein C3b is the main host ligand of CFH. The C3b-CFH interaction is of particular importance for the functions of CFH, namely complement regulation and host surface recognition. In CFH, four binding sites have been reported for C3b and its fragments, each with a different binding preference, affinity and functional relevance (155,158,159). These binding sites are located in the domains 1- 4, 6-8, 12-14 or elsewhere in domains 8-18, and 19-20. Solid evidence supports the two primary C3b binding sites in domains 1-4 and 19-20, whereas evidence for additional binding sites remains elusive. The N-terminal domains 1-4 mediate CFH binding to intact C3b, domains in the middle of CFH (domains 8-18) to the C3c part of C3b (i.e., binds both C3b and the C3c fragment), and domains 19-20 to the C3d part of C3b (i.e., binds both C3b and the C3d fragment) (159). Surface plasmon resonance (SPR) analyses indicate that the main binding sites are in domains 1-4 and domains 19-20, with the latter having 26" REVIEW"OF"LITERATURE"""""! higher affinity (155), whereas additional domains may contribute to C3b binding. The crystal structure of the complex of C3b and CFH domains 1-4 showed that all four N- terminal domains of CFH are involved in this interaction (160), and that these domains are necessary and sufficient for both cofactor and decay accelerating activities (160). Thus, the deposited C3b and the surface GAGs (the latter absent on microbes) together allow CFH domains 19-20 to recognize the host cells.
2.5.2 CFH attachment to host Cells
In addition to membrane-bound regulators, host cells require soluble complement inhibitors, particularly CFH, which provide effective protection from unwanted complement-mediated damage, especially under conditions with strong complement activation (161). This is exemplified by the attachment of CFH to endothelial cells via cell surface GAG and C3b, as discussed above, which is impaired in aHUS and is associated with endothelial damage and acute renal failure (109,162). Also, CFH can bind to cell surface polyanionic molecules in the absence of C3b, although this binding is weak and not readily detectable in physiological buffers (109). Notably, this interaction is distinct from the binding to specific cellular receptors. Nonactivators of complement possess cell surface polyanionic molecules that allow for CFH binding (40). In contrast, complement activators such as microbes that lack SA and host-like GAG do not allow significant CFH binding and thus complement activation can proceed unchecked. Heparin is the highly sulfated form of heparan sulfate (HS) which contributes primarily to the negative charge of the endothelial cell surface glycocalyx and extracellular matrices, like the glomerular basement membrane (163). Soluble heparin is synthesized only in mast cells, but HS in the glycocalyx of the cells has heparin- like domains where CFH is thought to bind. Chondroitin sulfate A (CS-A) is the main GAG on thrombocytes. Upon thrombocyte activation, the fully sulfated form of CS-A is exposed on the surface and is also released from α-granules (164). CFH also protects thrombocytes from complement attack (100). The prominent negative overall charge of self surfaces are mediated by the SA or neuraminic acids other than the GAGs. SA are a diverse group of nine-carbon saccharides with a negative charge that are present as terminal monosaccharide in REVIEW"OF"LITERATURE"""""27" ! glycans of lipids and proteins (165). Sialic acid biology and specificity of the interactions with complement proteins are still poorly understood. The major heparin binding sites of CFH are located in domains 7 and 19-20, (155,166-169). Polymorphisms or mutations in domains 7 and 19-20 which affect their interactions with host cells and basement membranes lead to the pathogenesis of diseases AMD and aHUS. However, target surfaces bearing SA, especially erythrocytes, are recognized by CFH, which subsequently restricts complement activation on these surfaces (39,110).
2.5.3 CFH binding to apoptotic and necrotic Cells
To maintain tissue homeostasis, old and damaged cells must be removed and replaced by new ones, which is facilitated by apoptosis. Apoptosis is a programmed mechanism of cell death, involving changes like nuclear and cellular fragmentation, chromatin condensation and cell shrinkage. Changes in the cell membranes facilitate an efficient recognition and safe clearance of apoptotic cells by phagocytes. Complement proteins and pentraxins can bind to apoptotic cells and thus enhance their uptake by phagocytes via specific receptors (170). It has been shown that complement activation does not proceed to the terminal pathway on apoptotic cells, which is partly due to the binding of CFH (171). The loss of membrane-bound regulators on apoptotic cells is in part compensated by the acquisition of the soluble complement regulators CFH and C4b-binding protein (C4bp), protecting against complement attack that would otherwise lead to the release of potential autoantigens from the cells (172). The CFH binding site for apoptotic/necrotic cells is located within domains 6-20, which is outside the complement regulatory region (173). Thus surface-bound CFH is able to regulate complement activation.
2.5.4 CFH interactions with Pentraxins
Pentraxins are recognition molecules of the innate immune system. The classical short pentraxins CRP and serum amyloid P component circulate as pentamers in human plasma. The long pentraxins are comprised of the PTX3, PTX4 and neuronal pentraxins, which display a more complex structure (21). Human CRP is an acute-phase protein, whose synthesis by hepatocytes is upregulated in response to inflammatory stimuli. The main effector functions of CRP 28" REVIEW"OF"LITERATURE"""""! are the activation of the complement system and the stimulation of phagocytosis (174). A direct binding of CFH to CRP has been described (175,176), suggesting down-regulation of CRP-mediated complement activation on self-surfaces by CFH. Later it has been shown that CFH mainly interacts with the monomeric or denatured form of CRP (153,177,178), although an interaction of CFH with native pentameric CRP at acute phase concentrations has recently also been demonstrated (179). The interaction of CFH with monomeric CRP leads to CFH recruitment, which limits complement activation but increases phagocytosis of apoptotic cells and reduces the release of inflammatory cytokines by macrophages (180). Aside from the short pentraxin CRP, CFH and C4bp were also shown to bind to the long pentraxin PTX3 (181,182). PTX3 has a pentraxin domain, homologous to that of the short pentraxins CRP and serum amyloid P, and has an additional unique N-terminal domain. In contrast to the short pentraxins that are mainly produced in the liver and thus act systemically in the body, PTX3 is produced locally by various cell types, such as vascular endothelial cells, fibroblasts, monocytes, macrophages, myeloid dendritic cells and neutrophil granulocytes (21). The binding of CFH to PTX3 requires the presence of calcium and is mediated by at least two binding sites in CFH. The primary binding site located within domains 19-20 of CFH interacts with the N-terminal domain of PTX3, whereas a secondary binding site on domain 7 binds to the C-terminal pentraxin domain (181). Cumulatively, the above-mentioned in vitro studies suggest that CFH binding to pentraxins is important to limit local complement activation and inflammation.
2.5.5 CFH binding to extracellular matrix (ECM)
It has been shown that the ECM proteins fibromodulin, chondroadherin, and osteoadherin can bind C1q and the regulators CFH and C4bp, which together maintain a balance between complement activation and inhibition (111). Exaggerated complement activation in turn may lead to inflammatory disease. Complement activation and the roles of CFH and C4b-binding protein on endothelial cell-derived ECM has been analysed in vitro. ECM-bound CFH and C4b-binding protein acted as cofactors for the inactivation of C3b and C4b, respectively. Furthermore, their binding and thus cofactor activity were enhanced by PTX3 (182).
REVIEW"OF"LITERATURE"""""29" !
2.6 Diseases associated with CFH family of proteins
Any mishap in this complex enzyme cascade of complement system caused by complement gene mutations, AA or exogenous triggers may affect the balance between complement activation and inhibition, resulting in an attack on self tissue (13). Complement deficiencies and malfunctions in the complement system are associated with various infectious, inflammatory and (auto)immune diseases. CFH gene mutations and polymorphisms, as well as CFH-AA, are associated with several diseases that are characterized by severe complement dysregulation, e.g., the eye disorder AMD, the rare systemic diseases aHUS and DDD (91,183). Several studies in recent years (discussed below) clarified the roles of CFH and complement in the pathological conditions mentioned above.
2.6.1 Age-related macular degeneration
AMD is a leading cause of visual impairment in elderly populations. In recent years, complement gene mutations and polymorphisms have been found to be associated with AMD, pointing to a role of the AP activation in the pathogenesis of the disease (184). Although the underlying pathomechanism is not yet fully known, a role of complement-mediated inflammation in the eye is postulated. Correspondingly, several therapeutic compounds targeting the complement system are currently being evaluated in clinical trials (184). The common CFH polymorphism 402H has been identified as a major genetic risk factor for developing AMD (70,71,185,186). Functional analyses of the CFH 402Y and 402H variants revealed a reduced binding of the AMD-associated 402H variant to murine CRP (157,187-190). Since residue 402 in domain7 is involved in the GAG-binding site of CFH, there are also subtle differences between the variants in their interaction with heparin and GAG-analogs (191,192). In contrast, the 402H variant has a higher affinity for DNA and necrotic cells compared to the 402Y variant (190). No difference in binding to retinal pigment epithelial cells was found (189), but the disease-associated variant binds less efficiently to both the extracellular matrix protein fibromodulin (190) and the Bruch’s membrane in the retina (191). In addition, malondialdehyde, a lipid peroxidation product, has been described as a novel ligand of CFH on apoptotic/necrotic cells, and shown to bind the 402H variant less strongly, thus adversely affecting the anti-inflammatory role of CFH (193,194). Very recently, 30" REVIEW"OF"LITERATURE"""""! the rare CFH variant R1210C, previously described in aHUS patients, has been linked to AMD as well (195). This variant was shown to affect CFH interactions with C3b and cell surfaces (196). Altogether, these data suggest a CFH-associated defect in the proper, non-inflammatory handling of cellular waste and in the control of complement activation leads to damage retinal pigment epithelial cells. It is still unknown how these CFH defective functions cause or contribute to the late-onset disease AMD in affected individuals, and which other factors (genetic, environmental, lifestyle) influence the role of CFH (197).
2.6.2 Dense deposit disease
DDD, also termed membranoproliferative glomerulonephritis type II (MPGN II), is a rare renal disease that progresses to fatal renal failure in about 50% of patients. It is associated with uncontrolled AP activation in plasma that generates C3 activation fragments depositing in the glomeruli (198). In the majority of the DDD patients, C3- nephritic factor, i.e. an autoantibody against the C3 convertase, is detected in serum (199). This factor causes enhanced complement activation by stabilizing the convertase or limiting down regulation of the convertase. In some patients, CFH mutations have been identified in these patients (183,200). These mutations may lead to CFH deficiency and thus insufficient plasma complement control (201). Mutations in cysteine residues that are important for forming the disulfide bonds within the single CCP domains can result in a defective protein folding and a CFH secretion defect (202). Moreover, a mutation in CCP4 was described, where the mutant CFH protein was inefficient in its cofactor activity, while cell-binding functions remained unaffected (67). All these cases led to a defective C3 activation control in plasma, either due to a quantitative CFH deficiency or dysfunctional CFH. CFH-AA have been described in DDD (203). So far, only one case has been published where the AA was characterized in detail. The isolated CFH ‘mini-AA’ consisted of lambda light-chain dimers that bound to CCP3 of CFH, i.e., within the complement regulatory region of the molecule (204). Functional assays demonstrated that the AA inhibited the CFH-C3b interaction and caused an increased C3 turnover due to a blockade of the complement inhibitory activity of CFH (203). Due to the lack of systematic screening for such AA in DDD patients, at present the prevalence and the characteristics of DDD-associated CFH-AA are not known. REVIEW"OF"LITERATURE"""""31" !
2.6.3 Atypical haemolytic uremic syndrome (aHUS)
The rare systemic disease aHUS is characterized by haemolytic anemia, thrombocytopenia (low platelet count) and impaired renal function (205). Its pathology is related to dysregulation of the AP, caused by polymorphisms, mutations and deletions in complement genes, or due to CFH-AA (206). CFH mutations affect approximately 30% of aHUS patients. More than 100 CFH mutations have been described in aHUS patients and can be searched in an online database (http://www.fh-hus.org) (207). In most cases, these are heterozygous mutations affecting various domains of CFH. However, most of the mutations affect the C-terminal CCP domains 19 and 20. Functional analyses of several of these mutants showed an altered interaction with C3b, heparin and endothelial cells (196,208,209). Furthermore, gene conversion and gene deletions leading to hybrid CFH proteins with functionally affected C-terminal domains have been reported (210- 212). These data show a disturbance in the physiological interaction of CFH with host endothelial cells. Certain mutations in domain 20 were also found to reduce the binding of CFH to CRP (180). For many of these mutations, however, there is no functional effect known to date. CFH-AA of the IgG class are detected in approximately 10% of aHUS patients (213,214). This form of aHUS affects primarily juvenile patients. A further characteristic of this patient group is that >90% of the affected individuals lack the CFHR1 gene, indicating that this genetic defect predisposes to the development of CFH-AA (214-217). These AA can also occur together with mutations in the CFH, CFI, C3 or MCP genes (217). The antibody binding sites in several patients were determined using recombinant CFH fragments and it was observed that practically all patients have AA that bind to domains 19-20 of CFH, although in some cases reactivity with other domains was also seen, such as to domains 8-11 (214,217-219). In three patients anti-CFH IgA AA were found that similarly recognized domains 19- 20 (220). Functional studies indicated that the AA interfered with the recognition functions of CFH, namely, impairing its interaction with surface-bound C3b and inhibiting the CFH complement regulatory activity on host surfaces (218,220,221). This reduced protection from complement-mediated damage is likely to be involved in the endothelial injury associated with aHUS. Owing to the similarity in the C- terminal domains of CFH and CFHR1, most of the studied AA recognise both the proteins. CFHR1 in fact can hijack AA and rescue host cells when added to CFH-AA- 32" REVIEW"OF"LITERATURE"""""! positive plasma (220). CFH-AA from healthy people have not been studied for obvious reasons, so the presence of CFHR1 and CFHR3 in these patients are not known. Altogether, these genetic and acquired abnormalities affecting CFH allow a normal fluid-phase regulation, but result in an impaired cell binding and cell surface protection from complement attack, which apparently contribute to the endothelial damage and microvascular thrombus formation in aHUS. However, further studies are needed to understand the role and relevance of mutations affecting other domains of CFH in aHUS. A recent study showed that some of the mutations do not lead to any known functional effect on CFH, thus care should be taken when interpreting genetic data and advising patients (222).
2.7 The microbial complement evasion: role of CFH
Since complement plays an important role in protection against infections, numerous viruses, bacteria, fungi and parasites have acquired the ability to sequester host complement regulators like CFH (112). Other than bacterial complement evasion (which is discussed here), viral complement evasion has been reported as well in different families of viruses. Herpesviridae and Coronaviridae are reported to interfere with CP, poxviruses and herpesviruses encode and express proteins with functional similarities to complement regulators (223). Viruses belonging to the families Poxviridae, Herpesviridae, Retroviridae and Togaviridae can incorporate host complement control proteins in their viral envelope and/or upregulate expression of these proteins in infected cells (224). It must be mentioned here that CFH has not yet been reported to have a direct involvement in any viral complement evasion. One of the vaccinia virus complement control protein has a striking similarity with domain 16 of CFH (225). As a common theme, it appears that many of the microbial CFH binding proteins interact with the domains 6-7 and 19-20, the primary CFH domains relevant for host cell recognition (Figure 4) (226). It has been suggested that the CFH Y402H polymorphism may be associated with enhanced resistance to certain bacterial infections. The AMD-associated CFH 402H variant has a lower binding affinity to various group A streptococci compared to the 402Y variant, resulting in more efficient opsonization and phagocytosis (227,228). REVIEW"OF"LITERATURE"""""33" !
However, studies indicate that microbes may use CFH for purposes other than complement inhibition, such as mediating entry into host cells as well. The interaction of CFH with neutrophils in the context of host-pathogen interactions is being characterized (152). CFH, when bound on the human pathogenic yeast Candida albicans, served as a bridging molecule to enhance the adherence and antimicrobial activity of neutrophils. The CFH-CR3 interaction could enhance Streptococcus pneumoniae adherence and uptake by epithelial cells and neutrophils (229). Similarly, CFH facilitated the adherence of Neisseria gonorrhoeae to human CR3-transfected cells (230). These data indicate a role for CFH in cellular adhesion by interacting with CR3, even beyond pathogen-host cell interactions. Binding of CFH downregulates opsonization and prevents further amplification of the C cascade and formation of cytolytic MAC. While prevention of opsonization and subsequent phagocytosis is beneficial for practically all microbes, evasion of MAC formation is especially important for Gram-negative bacteria and spirochetes that have an outer membrane exposed to plasma and therefore vulnerable to MAC attack. The molecular mechanism of CFH sequestration by Neisseria meningitidis has been demonstrated by the FHBp:FH complex structure (118). It has been observed that some microbes utilize the other microbial binding site on domains 19-20 of CFH to evade complement attack. Some microbes have also been shown to use two microbial evasion sites of CFH (domains 6-7 and 19-20). For instance, B. burgdorferi sensu stricto, which causes Lyme disease, binds CFH via domain 7 using protein CRASP-1 and potentially other CRASPs (231) and via domains 19–20 using OspE and its paralogs (122). Borrelia hermsii is known to bind CFH using FhbA in order to evade complement attack (232). Leptospira interrogans is known to bind both CFH and CFHR1 via LfhA (233). Recent reports have confirmed the binding of Salmonella enterica to CFH via Rck protein (234).
2.8 Anti-FH autoantibodies and aHUS
AA to CFH have been reported in aHUS and DDD patients and are associated with the diseases (see 2.6.2 and 2.6.3). However, a clear understanding of the mechanisms of how AA contribute to endothelial cell damage along with the pathophysiology of aHUS is still elusive. AA to CFH in HUS patients were first reported in 2005 for three 34" REVIEW"OF"LITERATURE"""""! children of the French aHUS cohort. The AA-positive plasma inhibited binding of CFH to the C3bBb convertase, but did not affect the complement regulatory functions of CFH in fluid phase mediated by the N-terminal domains 1–4 of CFH (213). CFH-AA were later on reported in five additional patients, and their binding epitopes were localized to the C-terminal cell surface attachment region of CFH, to domains 19–20 (218). These AA do not inhibit the complement regulatory activity of CFH in fluid phase but block cell binding of CFH. Purified IgG AA of the patients reduced C3b binding of CFH, blocked binding of CFH to cell surfaces and inhibited haemolytic activity (218). The frequency of CFH-AA has been analyzed in different cohorts (213,214,216-218). This suggests a similar mechanism of AA and of C-terminal mutations in CFH. Consequently, it was hypothesized that these AA inhibit surface attachment and the complement regulatory functions of CFH on cellular surfaces (235).
Figure 5. Schematic diagram of the chromosome 1, region q32. The 20 domains of CFH, the five domains of CFHR3, the five domains of CFHR1, and the nine domains of CFHR4 are indicated by vertical bars. The position of the two duplicated homologous segments (B and B’) is shown in purple and orange boxes. Failure of DNA amplification downstream reveals a deletion of a genomic ~84kb fragment of CFH in 99% of the AA- HUS patients. Protein analyses of the CFH-AA-positive plasma samples demonstrated the complete absence of CFHR1 and CFHR3 (214). Further detailed analyses showed for 14 patients the complete absence of both plasma proteins and for two patients rather low, barely detectable protein levels (235). Genetic analyses showed for the 14 deficient patients the complete absence of CFHR1 and CFHR3 due to a homozygous deletion of a large 84 kb genomic fragment which includes the CFHR1 and CFHR3 genes (Figure 5). The AA of practically all patients with AI-aHUS recognize the C- terminus of CFH, and inhibit the protective activity of CFH against complement attack to host cells (214,218,221). The reason for the association between CFH-AA and the CFHR1 deficiency is not yet known. An infection often precedes emergence of CFH-AA (236), and it is known that a wide variety of microbes recruit host CFH onto their surface via domain REVIEW"OF"LITERATURE"""""35" !
20 of CFH, known as the common microbial binding site, to prevent complement attack (226). The association of CFHAA with any physiological or microbial ligand binding to CFH19-20 and the location of the binding sites of the AA within CFH19-20 have not, however, been shown. 36" OBJECTIVES"""""!
3 OBJECTIVES
To characterize the structure-function relationship of the C-teminus of CFH via three distinct objectives:
I. To identify the molecular mechanism of the self non-self discriminatory process by CFH, and its implications on aHUS mutations.
II. Structural basis of complement evasion by microbes using CFH20: Borrelia as a case study. III. Connecting CFHR1 deficiency and AI-aHUS using a structural approach. MATERIAL"AND"METHODS"""""37" !
4 MATERIAL AND METHODS
4.1 Materials
4.1.1 Proteins
Generation of the recombinant proteins generated specifically for the studies of this thesis by the author are described below in section 4.2.
Wild-type CFH19-20 was cloned without expression tags by Maria Pärepalo. The mutants R1203A and Q1139A used in study I were cloned and expressed by Del Markus J. Lehtinen. The CFH19 -20 (Q1137A/ Q1139A/ Y1142A) and CFH19-20Del (T1184G/ K1202A/ R1203A/Y1205A) used in study II were cloned, expressed and purified by Markus Lehtinen and Satu Hyvärinen. The C3d with its residue C17 (the thioester forming residue) mutated to alanine and the C3dg (along with the mutants D36A, E37A, E117A, D122A, E160A, D163A, and K291A) used in the study I and II respectively were cloned and expressed in E. coli and were procured from Prof. David Isenman’s lab (Toronto, Canada) (237,238).
Rabbit polyclonal antibodies against CFH19-20 and CFH, and monoclonal anti-
CFH19-20 antibody VIG8 (239) were received from Dr. Jens Hellwage (Jena, Germany). The mAb C18 (107) was purchased from Enzo Life Sciences (Lörrach, Germany). For NMR, OspE (residues 21-171) was cloned, expressed and purified with a His-tag in Dr. Hideo Iwaï’s lab. The GST-tagged OspE used for crystallization was cloned by Mia Eholuoto (122).
4.1.2 Microbial strains used
Cloning of the CFHR14-5 constructs was done in E. coli XL10-Gold® cells
(Stratagene) and the expression of CFH19-20 proteins in Pichia pastoris X-33 strain (Invitrogen). The construct of OspE used for NMR was cloned in XL10-Gold cells and was expressed in E. coli ER2566 strain grown in M9-medium.
38" MATERIAL"AND"METHODS"""""!
4.1.3 Bioinformatical resources used
The following web resources were used to obtain sequence and structure information: European Bioinformatics Institute (www.ebi.ac.uk), National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), Protein Data Bank (www.rcsb.org/pdb), and “Proteins, Interfaces, Structures and Assemblies (PISA) server” (http://www.ebi.ac.uk/msd-srv/prot_int/) of the European Bioinformatics Institute.
4.1.4 Patient material used
Sera were collected from aHUS patients with CFH-AA in Germany and Hungary. The studies have been approved by the Research Ethics Committee of the Medical Faculty of Friedrich Schiller University and were performed in accordance with the declaration of Helsinki.
4.2 Methods
4.2.1 General laboratory methods for proteins
The proteins were resolved based on their size using standard sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) techniques and visualized using staining with Coomassie Brilliant blue, silver staining, or by performing Western blotting where detection was done with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch). Protein concentrations were measured using absorbance at 280 nm wavelength, BCA assay (Thermo Scientific), and comparing intensity of the protein bands with a standard in the gel using Image J program (http://rsb.info.nih.gov/ij/). Buffer exchanges were performed using dialysis membranes or PD-10 buffer exchange columns (GE Healthcare).
4.2.2 Protein iodination
For radio immune assays (RIA) or radio ligand assays (RLA) proteins were iodinated using the Iodogen method in a hood of a class B radioactive work area (240). Iodogen tubes were prepared by mixing Iodogen to chloroform and dried. Iodination was performed by incubating 20-100 µg of protein with 0.2-1.0 mCi of 125-I for 10-20 minutes in PBS. After the reaction the mixture was transferred to a glass tube for MATERIAL"AND"METHODS"""""39" !
unreacted iodine ions to form molecular iodine I2. The free iodine was separated from proteins using PD-10 buffer exchange columns. The specific activity of the proteins was typically 1.0 – 4.0 x 106 cpm/µg (16.7 – 66.7 kBq/µg).
4.2.3 Expression and purification of GST-OspE used for X-ray crystallography
The recombinant GST-OspE fusion protein was expressed in E. coli BL21(DE3)RIL host cells. Briefly, a primary culture was started by inoculating a single colony from a fresh transformant plate to 50 ml of Luria-Bertani (LB) broth containing 100 µg/ml ampicillin. The culture was incubated at 37°C with shaking overnight. The culture was diluted 1:50 with 1500 ml of LB broth containing 100 µg/ml ampicillin and incubated at 37°C for 3 h, until the growth reached the mid-log phase with an optical density of 0.6 at 600 nm. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.7 mM. After an additional incubation of 3 h, the cells were harvested, washed, and sonicated. The GST-OspE fusion protein was purified by affinity chromatography on glutathione-sepharose matrix according to the instructions of the manufacturer (GE Healthcare). The recombinant OspE was cleaved from GST with bovine thrombin (Sigma) in thrombin cleavage buffer (150 mM NaCl, 100 mM KCl, 2.5 mM CaCl2, 1 mM dithiothreitol, 20 mM hepes, pH 7.6) for 3 h and then eluted with thrombin cleavage buffer. The cleaved OspE was dialyzed against 20 mM Tris-buffer containing 20 mM NaCl, pH 7. The purity of the OspE protein after the elution from the glutathione-sepharose beads was confirmed by SDS-PAGE analysis.
4.2.4 Cloning of the CFHR1 C-terminus domains
Since the C-terminus (domains 4 and 5) of CFHR1 is different from CFH19-20 only by 2 residues, we decided to generate the CFHR1 C-terminal construct by mutating the two residues of the CFH19-20 construct, S1191L and V1197A. Mutagenesis was done using QuikChange Multi site-directed mutagenesis kit (Stratagene). The primers were designed based on the criteria presented by the manufacturer of the mutagenesis kit and were obtained from Sigma-Aldrich (sequences given in study IV). The pPICZαB vector (Invitrogen) with the inserted CFH19-20 sequence was used as a template in mutagenesis PCR reactions, and then expressed in E. coli. The clones were selected using Zeocin® (InvivoGen) antibiotic selection on Luria-Bertani medium and then sequencing from 3’ and 5’ ends over the CFH19-20 region in the Haartman Institute sequencing core facility. The plasmids harboring the mutation(s) were transferred to 40" MATERIAL"AND"METHODS"""""!
Pichia pastoris for expression using electroporation according to manufacturer’s instructions (BioRad). The transformed cells were selected using Zeocin® and small scale culture expression in 10 ml. The media was screened for the presence of the protein using Western blotting with polyclonal anti-CFH19-20 antibody cross-reacting with CFHR-1. The clones that were recognized by the antibody and had the highest expression level were selected for large-scale expression.
4.2.5 Expression and purification of CFHR14-5
The CFHR14-5 protein was expressed using the Pichia pastoris expression system as described by the manufacturer (Stratagene). All the incubation steps were performed at 28°C under continuous shaking (225 rpm). Initially 5 ml of Buffered Methanol, Glycerol, Yeast extract (BMGY) medium with 100 µg/ml of Zeocin® was inoculated and incubated for approximately 17 hours, which was used to inoculate 50 ml of pre- tempered BMGY medium that was incubated for 16 hours. The cells were harvested by 5 min centrifugation at 3000 g at 22 °C. The cells were suspended to 1 liter of Buffered Methanol Medium (BMM) medium for expression. Ten ml of methanol was added to the culture after 24 hrs and 48 hrs of expression, which was stopped after 72 hrs. The supernatant was collected after centrifuging the cells at 5000 g for 15 min. The supernatant was filtered using 0.8 µm/0.2 µm filters. The pH of the filtrate (pH <
5) was adjusted to 6.0-6.5 with Na2HPO4 and diluted 1:1 with H2O. The proteins were purified from the filtrate using 1 ml Hi-Trap heparin columns (GE Healthcare) and eluted with 1 M NaCl in phosphate buffered saline (PBS: 137 mM NaCl, 2.8 mM KCl, 9.7 mM phosphate, pH 7.4). The protein preparations were further purified with Sephadex 75 (10/300 GL) gel filtration column using the ÄKTA® purifier system (GE Healthcare).
4.2.6 Light scattering and size exclusion chromatography analysis
Size exclusion chromatography coupled with multi angle light scattering (SEC-
MALS) was used to find the optimal proportions of CFH19-20 and OspE to form the complex in solution (Supplemental Fig 1b in study III). The proteins alone and their mixtures in two different apparent molar ratios (1:1 and 1:1.25) were run over a Superdex200 column attached to HPLC equipment (Shimadzu). MiniDAWN TREOS light-scattering detector and Optilab rEX refractive index detector together with ASTRA 5.3.4.20 software (Wyatt Technology Corporation) were used to calculate MATERIAL"AND"METHODS"""""41" ! masses of proteins and complexes eluted from the column. A size exclusion analysis of OspE and CFH19-20 alone and the OspE:FH19-20 complex was done by running the preparates through a Superdex200 column attached to an ÄKTA purifier HPLC system (GE Healthcare) (Fig. 2a of study III). All samples were eluted in PBS at 0.5 mL/min flow rate at 22˚C.
4.2.7 Generation of C3b
The C3b protein (used in study I) was prepared from plasma purified C3 (kindly provided by Karita Haapasalo-Tuomainen) using trypsin cleavage. First, 1 mg/ml of C3 in PBS was warmed in a water bath to 37 °C. Trypsin (Sigma- Aldrich) was added to the solution to make it 1 % (w/v), mixed, and incubated for 3 min. Soy bean trypsin inhibitor SBTI) (Sigma-Aldrich) was added to the solution to make it 2 % (w/v) and mixed to stop the reaction. The result was monitored with SDS-PAGE gel analysis, where a shift in the alpha chain of C3 indicated cleavage of C3a from C3 and generation of alpha’-chain. Trypsin, SBTI, C3a, and C3b were separated with HiLoad 16/60 Sephadex 200 gel filtration column attached to an ÄKTA® purifier system.
4.2.8 Radioligand assays
The proteins were attached to Nunc Polysorp Break Apart plates at 5-20 µg/ml in 0.5X PBS (68.5 mM NaCl, 1.4 mM KCl, 4.85 mM phosphate, pH 7.4) by incubating for 17 hours at 4°C. The wells were blocked with 0.5 - 2.0 % bovine serum albumin (BSA, Sigma-Aldrich) in 0.5X or 1X PBS for 2 hours at 22°C and washed 1-2 times with 0.5X or 1X PBS. In inhibition experiments the radioligand and inhibitor were usually premixed. In direct binding experiments the radioligand was pipetted onto the wells bearing the ligand. The plates were incubated for 2 - 3 hours at 22 °C and washed with ice cold 0.5X or 1X PBS once or twice. The radioactivity of the separated wells was measured with a Wizard gamma counter (Perkin-Elmer).
4.2.9 Surface plasmon resonance
SPR assays were performed on a Biacore 2000 equipment using carboxymethylated dextran chips CM4 and CM5 (GE Healthcare) .
4.2.10 Heparin affinity chromatography
Heparin affinity chromatography was performed with the ÄKTA® purifier system 42" MATERIAL"AND"METHODS"""""!
(GE Healthcare) and Hitachi LaChrom HPLC (Merck). The proteins were injected into a 1 ml Hi-Trap Heparin column (GE Healthcare) in 0.5X PBS and eluted using a linear salt gradient up to 0.5 - 1.0 M NaCl in PBS. The elution was monitored using absorbance at 280 nm. The elution fractions were verified using anti-CFH19-20 antibodies by Western blotting. The ionic strength of the elution buffer was monitored in real time with a conductivity meter in ÄKTA® or measured from the elution fractions using a conductivity meter CDM210 (Radiometer Analytical SAS).
4.2.11 X-ray crystallography
The proteins to be crystallised were purified to 99 % purity and were handled using robotic protein dispensers in order to set up the initial crystal screens. All of the crystallographic experiments and structure determination were performed in Prof. Adrian Goldman’s laboratory in the Institute of Biotechnology, Helsinki.
Both the CFH19-20 constructs with a single point mutation, either CFH19-
20Q1139A or CFH19-20R1203A, were crystallised at 22°C using 2M ammonium sulphate as a precipitant in 0.1 M Bis-Tris buffer, pH 5.5 or pH 6.5, respectively. X-ray data for both mutants were collected at beam line ID14-3 at 0.931Å wave-length in the European Synchrotron Radiation Facility (ESRF), Grenoble, using a ADSC Quantum 4 CCD detector. The data for both of the mutant protein crystals were collected at 100 K. The crystals belonged to the tetragonal space group I4122 (Table 1) with one molecule per asymmetric unit and a solvent content of approximately 68–70%. The
CFH19-20Q1139A crystals diffracted to 1.9 Å resolution and the CFH19-20R1203A crystals to 1.52 Å resolution (Table 1). The structures were solved using molecular replacement with MOLREP (241) and refined with REFMAC5 (242,243) with manual rebuilding, as necessary, in COOT (244). The final structures were refined using PHENIX (245) program “phenix.refine” with the alternative conformations. The crystals contained also sulphate molecules, as expected, and six of those per CFH19-20Q1139A molecule and two per CFH19-20R1203A molecule were also built similarly. The final work and free
R-factors were 20.4% and 24.5% (CFH19-20Q1139A) and 20.3% and 22.2% (CFH19-
20R1203A) with acceptable geometry, and there were no chain breaks in the models (Table 1). MATERIAL"AND"METHODS"""""43" !
44" MATERIAL"AND"METHODS"""""!
Table 2. Crystal screen set up with seven CFH19-20 mutants in order to see their crystallising behaviour in different pH. CFH19-20D1119G/Q1139A did not crystallise in any of them. The proteins were in 20mM Tris and 20mM Nacl. All of the crystallasing solutions contained 2 M ammonium sulphate as a salt precipitant.
Despite extensive trials, we were unable to crystallize non-mutated CFH19–20 with C3d, because CFH19–20 always crystallized by itself as a tetramer (study I) (106) in various crystal forms. We performed a small crystallographic trial with 7 of the
CFH19–20 mutants in different pH to identify the mutant which did not crystallise very easily in presence of 2M ammonium sulphate (Table 2). We found the double mutant
CFH19–20D1119G/Q1139A did not yield any crystals in the offered conditions. From this experiment, we reasoned that inhibiting tetramer formation would help crystallization of the complex, and therefore, we used this CFH19–20 double mutant for the rest of our crystallographic experiments in order to break the tetramer interface. The mutant still bound C3d well but crystallized poorly by itself. This allowed us to crystallize CFH19–
20D1119G–Q1139A:C3d complex because the homotetrameric crystals did not form. Small- angle X-ray scattering (SAXS) data also showed that the CFH19–20D1119G–Q1139A mutant is clearly monomeric, whereas the WT protein has a tendency to form higher oligomers and aggregates. The CFH19–20D1119G–Q1139A:C3d complex was crystallized at 22 °C from 0.1 M Hepes, pH 7.5, containing 14% PEG 4000 (160 µM CFH19–20, 200 µM C3d). Crystals appeared in 3–4 d. All data were collected at the European Synchrotron Radiation Facility (ESRF). The initial dataset, to 3.8-Å resolution, was collected at ID14-2; optimization of cryo-cooling conditions with 10% glycerol enabled us to collect data to 2.3-Å resolution at ID23-1 (Table 1). The initial molecular replacement solution was found with the 3.8-Å data. PHASER (246) MATERIAL"AND"METHODS"""""45" !
readily found two molecules of C3d but not CFH19–20. However, closer inspection of Fo–Fc and σA-weighted maps revealed very clear features of continuous extra density consistent with a single CFH19–20, and therefore, we manually built CFH20 into the density between the two C3d molecules, giving an asymmetric unit with two C3d molecules and a single CFH19–20. Successive rounds of building with Coot (244) and refinement with REFMAC (242,243) or PHENIX (245) against the 2.3 Å dataset allowed us to build an essentially complete model of the full heterotrimeric complex. The R factors (Rwork/Rfree) were 20.4%/24.3% with good geometry (Table 1).
Since CFH19-20 tends to crystallize as a homotetramer as stated above, we used
CFH19–20D1119G–Q1139A mutant for the crystallization experiments to prevent this from happening, as before. The CFH19–20D1119G–Q1139A mutant protein has similar binding affinity for OspE as the wild type CFH19-20 (mean IC50 of 0.55 vs. 0.42 µM)
(Supplemental Fig 4 of study III). The CFH19–20D1119G–Q1139Amutant:OspE complex was crystallized at 293 K from sitting drops in the presence of 2 M ammonium sulfate, 0.1 M citric acid, and 0.2 M sodium chloride at pH 5.5. Crystals appeared within 4 days and were cryo-protected in the mother liquor supplemented with 25% glycerol. The diffraction data (to 2.83 Å) were collected at ESRF ID14-4 beam line at 100 K on a Q315r ADSC CCD detector at 0.97372 Å. The data were indexed and scaled using XDS (247). A molecular replacement solution was obtained using the wildtype CFH19-20 structure (PDB code 2G7I) (106) and a truncated NMR structure of OspE (without the loops) with an R-factor of 0.3. After successive rounds of building with Coot (244) and refinement with REFMAC (242,243) or PHENIX (245), we could identify one OspE molecule bound to a single CFH19-20. The loops were modeled during real-space refinement using “phenix.refine” software without any applied restraints. The final R-factors (Rwork/Rfree) of the refined complex structure were 19.3/25.5 % (shown in Table 1). The last refinement cycles were done using TLS parameters (9 TLS groups). In the Ramachandran plot 94.0% of the residues in the structure were in the most favored regions.
CFHR14-5 was crystallized at 293 K from hanging drops in the presence of 2 M ammonium sulphate, 0.1M sodium acetate at pH 4.6. The cube shaped crystals appeared within 5 days and were cryo-protected by 25% glycerol (supplemented by the mother liquor). The diffraction data (to 2.9Å) were collected at ESRF ID14-4 beamline (248) at 100 K on a CCD ADSC Q315r detector at 0.979520 Å. The data was indexed and scaled using XDS (247). Using the structure of CFH19-20R1203A (PDB 46" MATERIAL"AND"METHODS"""""! code 3KZJ, study I) as a search model in PHASER(249) two molecules of CFHR14- 5 were identified in the asymmetric unit. After successive rounds of model building with Coot software and refinement in real space using ‘phenix.refine’ software (245) we could refine the structure to a higher precision (Rwork/Rfree = 0.20/0.26, also shown in Table 1). The last refinement cycles were done using TLS parameters. In the Ramachandran plot, 95% of the amino acid structures were within the most favoured region. The superpositions of different structures and structural illustrations were prepared using Pymol software (Schrödinger, Portland, USA).
4.2.12 NMR Spectroscopy
The NMR experiments were done by Jesper Oeemig under the supervision of Dr. Hideo Iwaï at Institute of Biotechnology, University of Helsinki.
4.2.13 Microtiter plate assays with the patient AA
The experiments with patient samples were done in collaboration with Dr. Mihály Józsi at Hans Knöll Institute, Jena, Germany (current address at Eötvös Loránd University, Budapest, Hungary).
4.2.14 Calculation of the electrostatic potential of the structures
Electrostatic potentials of the CFH19–20 wild type and the CFH19–20Q1139A and CFH19–
20R1203A mutant structures were calculated with APBS (250). Before calculation, the missing side chain atoms of some of the residues were added using COOT (244) on the basis of the location of the visible side chain atoms and electron density. APBS was run from PyMOL version 1.2 graphical interface. The program PyMOL (DeLano Scientific LLC) itself was used to generate the PQR file used to run APBS and add hydrogens to the structure. The positive isosurface contour was investigated after being displayed at +3 kT/e potential at Pymol.
4.2.15 Curve fitting and statistical analyses
Curve fitting was done using non-linear regression models implemented in GraphPad Prism®. For HPLC data Unicorn® program of ÄKTA® system was used to analyze the elution curves (GE Healthcare). The statistical analyzes were performed using Microsoft Excel® and GraphPad Prism®. The comparison of mean values was done using combination of F-tests and t-tests, and ANOVA (as per the availability of the MATERIAL"AND"METHODS"""""47" ! replicating data). The results were presented with mean using standard deviation to indicate the error. 48" RESULTS"""""!
5 RESULTS
5.1 Both domains (19 and 20) of CFH are involved in C3b binding
5.1.1 The interaction of the CFH mutants with C3d/C3b and Heparin
Surprising results from previous studies (106,154) revealed that single point mutations in either the domain 19 or 20 of CFH lead to impaired binding of CFH19-20 to C3b and C3d. We wanted to know whether both the domains 19 and 20 of CFH are involved in binding to C3d and C3b, since point mutations could cause loss-of- function through secondary structural effects. In order to answer this question, we chose two representative mutant proteins with impaired C3d/C3b binding for further studies, one with a mutation in domain 19 and another with a mutation in domain 20. Mutation of the surface-exposed Q1139 to alanine in domain 19 led to one of the clearest losses of C3d/C3b binding (154). Therefore, we chose for the structural studies the CFH19-20Q1139A mutant protein as a representative of mutations in domain
19 with impaired C3b binding. We selected another mutant protein to be CFH19-
20R1203A owing to its least proximity from domain 19. We also considered the fact that binding of the construct CFH19-20R1203A to C3d and C3b was highly impaired compared to that of the wild type CFH19–20 than the previously published set of mutant proteins (154). Our own binding studies confirmed that R1203A mutation developed impaired binding to C3d/C3b by the protein to a very prominent level (Fig 1 of study I) We also studied the heparin binding profile of the mutants (used for the study
I). It was already reported previously from our group that CFH19-20Q1139A had similar affinity towards heparin (154). However, CFH19-20R1203A gave a completely different heparin binding profile while compared to the WT CFH19-20 protein. The R1203A mutation introduced in CFH19-20 resulted in a loss of binding to the heparin column (Fig. 2 of study I)
RESULTS"""""49" !
5.1.2 The structures of the mutants CFH19-20Q1139A and CFH19-20R1203A
The structures of the mutant proteins mentioned were solved to 2.0 and 1.65 Å resolution, respectively. The overall structures of both the mutant proteins were identical with the earlier reported wild type CFH19–20 (106) as seen by superimposing the Cα-atoms (Figure 5). The root mean square deviations (RMSD) of the mutant proteins (both CFH19-20Q1139A and CFH19-20R1203A) were extremely low when compared to the wild type CFH19–20 (study I).
Figure. 6. The Crystal structures of CFH mutants 19-20
superposed with CFH19-20WT The X-ray crystal structures of CFH 19-20Q1139A (Pdb code: 3KXV) and CFH (Pdb code: 3KZJ) 19-20R1203A superposed on CFH structure (Pdb code: 2G7I). 19-20WT The CFH is in Grey, CFH in light violet, 19-20WT 19-20Q1139A and CFH in slate colour. 19-20R1203A
We analysed the structures to determine if the mutations had effects on the side chains of the neighbouring residues of the mutated residue. None were observed, as only the side chain of the mutated residue was different. Even the main chain atoms of the mutated residues were seen to be in the same orientation (Fig. 4 of study I) Finally, we studied the changes in the surface electrostatics caused by the mutations. As expected on the basis of the lack of changes in the neighbouring residues and the nature of the mutation from charged to noncharged residue, no clear effect on surface electrostatic potential distribution was seen with the Q1139A mutation. On the contrary, a clear local change in terms of surface electrostatic potential occurred at the site of the R1203A mutation (Fig. 5 study I). This change was restricted to the surface area around the side chain of the mutated residue. Taken together, the Q1139A and R1203A mutations caused no changes other than the 50" RESULTS"""""! mutations in the structure despite their clear effect on binding of CFH19–20 to C3d and C3b. This indicated that both domains 19 and 20 of CFH are involved in binding to C3d part of C3b.
5.2 A bigamic interaction of CFH revealed
5.2.1 The CFH19-20:C3d structure
Earlier attempts to crystallise CFH19-20WT coupled with its several ligands repeatedly yielded on only CFH19-20WT crystals. We noticed that CFH19-20 readily crystallises whenever it is present in high concentration with ammonium sulphate. Accordingly, it was challenging to obtain crystals of CFH19-20 in complex with any other ligands. We decided then to find out a mutant of CFH19-20 that did not form crystals readily by doing a small experiment of crystallising different CFH19-20 mutants in the presence of
2M ammonium sulphate (Table 2). The CFH19-20D1119G/Q1139A was the mutant that did not crystallise (Table 2). Based on these results, we used the CFH19-20D1119G/Q1139A mutant for our experiments attempting to solve structures of protein complexes of
CFH19-20.
Figure 7. The crystal structure of CFH19- 20D1119G/Q1139A:C3d complex. The CFH19-20 mutant is in gray colour and C3d is in light blue.
RESULTS"""""51" !
We solved the CFH19-20D1119G/Q1139A:C3d structure (study II), which was initially determined by molecular replacement with C3d at 3.8 Å but refined against a higher-resolution dataset at 2.3Å. The asymmetric unit of the structure revealed a surprising CFH19–20:(C3d)2 heterotrimer (Figure 7) where two distinct sites on a single
CFH19–20 molecule bind two separate C3ds in different ways and at different sites of C3d. The overall structures of both C3d molecules are unchanged (RMSD = 0.32Å, Figure 8). Both binding sites on C3d are far from the thioester site used for covalent attachment of C3b to targets (Fig. 1A of study II).
Figure 8. Structural alignment between the C3d molecule in CFH19-20:C3d structure (PDB code: 2XQW, light blue in colour) and the C3d structure solved alone (PDB code: 1C3D, olive in colour).
One of the interfaces (the CFH20 site) is formed by the arginine rich tip of the domain 20, which binds in a strongly electrostatic manner into a negatively charged pocket on the concave top of the helical bundle of C3d (Fig. 1B, Fig. S2 A,C of study
II). The side chain of R1203 on CFH20 forms an ion pair with C3d E1153 located in the electronegative pocket of C3d. The total buried surface area of 490 Å2 includes three ion pairs and an extensive ionic network (Fig. S2 B,D of study II). The local 20 changes in the loops around the CFH site, when compared to structure of CFH19-20 only, are as follows: Lys1188 turns 180° to point towards C3d, and T1184 and R1171 19 pack against C3d. Another interface (the CFH site) of CFH19-20:C3d interaction is larger (620 Å2) and less charged (Fig. 1C and Fig. S2B of study II). This interface is formed by residues from both CFH19 and CFH20 that bind to the side of C3d at helices 104–118 and 170–189 and the Pro121 loop (the C3dFH19 site) (Fig. 1C and Fig. S2D of study II). The C3dFH20 site was observed to be partially buried in the full C3b molecule (Fig. 1B of study II). 52" RESULTS"""""!
5.2.2 Confirmation of the physiological relevance of the structure
We also modeled back the two mutated side chains D1119 and Q1139 to the crystal structure (Fig. S5 of study II). This indicated that D1139 would hydrogen bond to the I115 carbonyl group and Ser171, and Asp1119 would form an intermolecular helix cap with C3d (Fig. S5 of study II). The mutant bound C3d more poorly than WT, which was observed earlier (154).
Functional effects of some of the point mutations in the “CFH19 C3b site”
(D1119G, Q1139A, and K1188A) or the “CFH20 C3b site” (R1182S/A, W1183L, T1184R, and R1203A) have been reported to lead to reduced C3d/C3b binding (106,154,156) (study I and Fig. 2A and Table 1 of study II). These results verified the two binding sites we found in the crystal structure. In order to further verify the two binding sites, we tested the binding of seven C3dg mutants to CFH19–20. Both the mutations in the C3dFH19 site (E117A and D122A) and all four mutations in the C3dFH20 site (D36A, E160A, D163A, and K291A) significantly reduced the affinity of the interaction (Fig. 2B–D of study II), whereas the control (E37A) had no effect.
5.3 Borrelia burgdorferi binds CFH20 to evade complement
5.3.1 The Borrelial OspE structure
The solution structure of OspE was solved by NMR spectroscopy (Table 1, Fig 1a of study III). Residues 42-171 of OspE form a globular single-domain with a backbone RMSD of approximately 0.38 Å between the different NMR models indicating a very well defined and stable structure. The OspE core domain has a rigid α+β fold of eight β-strands and two short α-helices arranged in a repeating topology of four β-strands followed by an α-helix (β1- β2- β3- β4-αI- β5- β6- β7- β8-αII). The β -strands form antiparallel β-sheets where β1- β4 and β5- β8 are orientated almost perpendicularly. β1 and β8 are held together by hydrogen bonds thus forming a disoriented asymmetric β-barrel. It can be concluded as a β-barrel with one uniform and another non-uniform side. The β2 and β6 strands are highly twisted and deviate from the formation of classic β-barrel strands. β1, β2, and β6 are fairly long strands (10, 12 and 11 residues respectively) and β5 along with β8 are the smallest β-strands (6 residues each). RESULTS"""""53" !
Figure 9. The NMR ensemble of 20 different energy-minimised NMR structures of OspE. The NMR structures are superposed with the backbone of its globular domain (res 42-171)
The N-terminal region (residues 20-41) of OspE is structurally flexible as shown by superimposition of residues 42-171 from 20 NMR structures (Figure 9). The 15N relaxation data of OspE (Supplemental Fig. 2 of study III) also shows that these residues are moving freely.
5.3.2 The CFH19-20:OspE complex structure
Optimization of heterodimer formation by gel filtration analysis of OspE and CFH19-20
(Fig. 2a of study III) enabled us to crystallize OspE in complex with CFH19-20. Using the optimized 1:1 molar ratio of the proteins (Supplemental Fig 1b of study III) we obtained rod shaped co-crystals (Figure 10) of OspE and CFH19-20. The crystal structure of the OspE:CFH19-20 complex was solved at 2.83 Å resolution (Table 2,
Figure 6) using our previously solved CFH19-20 structure (106) and the NMR structure of OspE (described before) as the initial models in molecular replacement. In the complex, OspE binds to domain 20 of CFH with a contact surface area of 691 Å. The complex is held together by an E68OspE-R1182CFH-D73OspE ion triple, which is also hold together by a sulfate group forming an R1182CFH-SO4 --R66OspE ion triple, while R1182 CFH also forms an ion-pair with E1198CFH (Fig. 2c and 2d of study III). These interactions form the top part of a pocket for W1183CFH, which makes hydrophobic contacts with the aliphatic side chain of R1182CFH and with the backbone atoms around G75OspE and A83OspE. 54" RESULTS"""""!
Figure 10. The X-ray crystal structure of CFH19-20:OspE complex. The OspE molecule is shown in lemon and CFH19- 20 in gray colour. Inset: Rod type crystal obtained for the complex
The OspE structure in solution (NMR) and in complex with CFH19-20 (X-ray) was very similar (backbone RMSD of 1.43 Å/Cα) (Supplemental Fig. 7a of study
III). The structure of CFH19-20 in complex with OspE was also practically identical to the published structures of CFH19-20 in homo-tetramers (106) (backbone RMSD 1.43
Å/Cα) or in the CFH19-20:C3d complex (study II) (backbone RMSD 1.15 Å/Cα)
(Supplemental Fig. 7b of study III). The sulfate ion found in the OspE:FH19-20 interface did not affect the orientation of the side chains, not even R1182CFH19-20 in its close vicinity (Supplemental Fig. 8a,b of study III). When the region of OspE close to the sulfate ion was compared between the NMR ensemble and the crystal complex structure it was observed that orientation of E68 was similar while that of R66 was OspE slightly different, most probably due to a hydrogen bond between the NH2 of R66 and O of R1182CFH19-20.
5.3.3 The NMR chemical shift assay confirms the interaction site to be physiological
To further verify the existence of the observed interface between OspE and CFH19-20 under physiological conditions, or at least in fluid phase, we analyzed chemical shift perturbations in the NMR spectrum of OspE upon addition of saturating concentrations of wild type CFH19-20 (Fig. 3a, Supplemental Fig. 3 study III). The RESULTS"""""55" ! residues of OspE that shifted most (>0.4 ppm) are clustered on β-strands 1-4 that form the core of the OspE:FH19-20 interface in the crystal structure (Fig. 3b). Of the nine OspE residues involved in hydrogen bonding, six could be assigned in the assay and five of those were among the residues that shifted most (>0.4 ppm) (Table 3 of study III). The other three residues involved in binding (R66OspE, G80OspE and T84OspE) could not be assigned in the spectra of the CFH19-20 bound OspE, probably due to large changes in the microenvironment caused by formation of the complex. We also confirmed the interface of CFH19-20 and OspE from the CFH19-20 side using mutants of
CFH19-20 results of which have been reported elsewhere (226).
5.4 Molecular basis for autoimmunity against CFH in aHUS
5.4.1 The autoantibody binding site on CFH19-20
Binding of IgG from sera of eleven aHUS patient with AA against CFH (CFH-AA) to
14 CFH19-20 mutants was tested (Fig 2A of study IV). Binding of IgG of all the tested patients was diminished in case of the L1189R mutant (less than 70% of binding to the WT CFH19-20). Binding of IgG from nine patients was diminished to the CFH19-
20E1198A mutant. IgG from seven to eight patients were found to have impaired binding to the CFH19-20 constructs with mutations T1184R, K1186A, and K1188A while binding of IgG of only one to two patients was impaired to the mutants CFH19-
20Q1139A, CFH19-20R1182A, or CFH19-20W1183L (Fig. 2B of study IV). The binding of the patient IgG to WT CFH19-20 and to the mutants CFH19-20D1119G, CFH19-20W1157L, CFH19-
20R1203A, CFH19-20R1206A, CFH19-20R1210A, and CFH19-20R1215Q was similar (Fig 2B of study IV). The monoclonal antibody C18, which had been shown previously to have an epitope overlapping with that of the CFH-AAs (218,221) bound to the exact same site on the CFH C-terminus (Fig. 2A of study IV)
Surprisingly, the two residues that are different in CFH19-20 and CFHR14-5 were found to be located beneath the CFH-AA site. We therefore studied whether the small difference in the amino acid sequence had an influence on binding of the AA.
IgG from ten out of the six tested patient samples bound similarly to CFH, CFH19-20, and CFHR14-5 (Fig. 3 of study IV) while binding of one of the samples (patient 9) was clearly impaired to CFHR14-5 when compared to CFH or CFH19-20 (Fig. 3 of study IV). These results, in agreement with previous data (217,220), indicated that 56" RESULTS"""""! the conformation of the CFH-AA site could be slightly different in CFH domain 20 and CFHR domain 5 despite of the difference in only two buried amino acids.
5.4.2 The global structure of C-terminus of CFHR1
Figure 11. The structures of wild type CFH19-20 and CFR14-5 superposed. Both CFH19-20 and CFR14-5 are represented by cartoon diagrams. CFH19-20 is in gray and CFR14-5 is in orange. The CFH-AA site is marked.
To detect possible differences in the identified CFH-AA site on CFH19-20 and the corresponding site of CHFR1 we used X-ray crystallography to solve the structure of
CFHR14-5. The structure of CFHR14-5 was obtained as a homodimer at 2.9 Å resolution (Table 1) from a different space group (a hexagonal P622) than the previously published CFH19-20 structures (tetragonal I4122) (Table 1 of study I and (106), but the structures aligned very well with each other (Fig. 4A of study I) and the region containing the mutations S1191L and V1197A was only slightly different (Fig. 5C of study IV). RMSD of 113 aligned Cα atoms was only 0.5-1.1 Å upon alignment of the CFHR14-5 structure with the previously published structures of
CFH19-20 solved as homotetramers (106) (0.5 Å) or in complex with C3d (study II) (1 Å) or Borrelial OspE protein (study III) (1.1 Å). This indicates that the structures were practically identical (Figure 15). RESULTS"""""57" !
The structures of CFH19-20 and CFHR14-5 were seemingly identical. However, the conformation of the CFH-AA site on CFHR14-5 was somewhat different from that on CFH19-20 (Figure 11) since both the backbone and loops forming the site (R281- K287 or CFHR1 and R1182-K1188 of CFH, respectively) had a slightly different orientation (Fig. 5D of study IV). The main difference in the backbone is a short alpha-helix in the loop of CFHR14-5 while no prominent helix was seen in the aforementioned zone in any of the previously solved structures of CFH19-20. The orientation of some of the side chains was also clearly different as all the residues from R281 through K287 of CFHR14-5 were at least 5 Å apart from the location of side chains of the corresponding residues of CFH19-20 (from R1182 through K1188) (Fig 5D of study IV). In order to exclude potential misrepresentation of X-ray diffraction data of the CFH-AA site, we also compared the electron density maps
(2Fo-Fc) of R1182-K1188 of CFH19-20 (106) and R281-K287 of CFHR14-5 (Fig. 6 of study IV). Clearly the model of CFHR14-5, but not of CFH19-20 fits very well with the electron density map of CFHR14-5 for the mentioned region (Fig. 6A and B of study
IV) while the corresponding region in the model of CFH19-20, but not of CFHR14-5, fit well with the electron density map of CFH19-20 (Fig. 6C and D of study IV). 58" DISCUSSION"""""!
6 DISCUSSION
In this thesis, X-ray crystallography and accompanying structural studies were used to unravel the molecular mechanisms regarding the involvement of two C-terminal domains of CFH in complement activation. The molecular basis of the role of CFH C-terminus in the target discrimination of self or non-self structures by AP was revealed in the first two studies of this thesis (study I and study II). In these studies we solved the structures of two mutants of CFH19-20 (one of them being an aHUS associated mutation) and the structure of CFH19-20 in complex with C3d. The structural basis of the process of complement evasion by microbes binding to CFH C- terminus was explained by solving the crystal structure of CFH19-20 in complex with a borrelial surface protein OspE (study III). We also proposed a novel explanation for the formation of CFH-AA in aHUS patients lacking CFHR1 by mapping the AA binding site on CFH and solving the structure of CFHR1 C-terminus (study IV).
6.1 Molecular basis for the self non-self discrimination by AP
The primary function of the C-terminus of CFH has been established to be the discrimination between activators and non-activators, i.e. protection of host cells from complement mediated damage while allowing activation on foreign targets. The importance of this region is very well documented by the onset of the systemic disease aHUS, primarily associated with point mutations in the C-terminus of CFH. The mutations in CFH are primarily located in the last two domains (183). CFH is a pattern-recognition regulator that will act on any surface, including the extracellular matrix, which carries host-specific molecular signatures. It has been shown earlier that negatively charged surfaces bearing SA and heparin were critical for protection of erythrocytes from complement attack by CFH (39,110,251). Later, the self and non-self discrimination of C3b deposited surfaces was reported to be mediated by the polyanion-binding site on CFH (40). Eventually, C3d/C3b and heparin binding sites were mapped on the CFH19-20 region (103,158,166) by the 1990s. The interaction between C3b and CFH is important for the proper complement function as demonstrated by the dramatic clinical effects caused by mutations interfering with this interaction in aHUS. DISCUSSION"""""59" !
Earlier several mutations in domain 20 of CFH were known to impair binding of CFH to either C3d/C3b or heparin or both, which led to the conclusive evidence of
CFH20 being functional in CFH binding to both heparin and cell-bound C3d/C3b. Since none of the CFH19 mutations impaired heparin or endothelial cell binding, but
CFH20 mutations did (154,156), it was likely that the binding site for heparin and endothelial cells is limited to domain 20, which is also supported by other reports (252). In study I of this thesis impaired binding to heparin was seen with the R1203A mutation in domain 20 but not with Q1139A in domain 19, fitting well with earlier data. On the basis of earlier reports, the role of domain 19 in binding of the C- terminus of CFH to C3d/C3b was not evident, as only the domain 20 seemed to be important. A role for domain 19 was, however, hinted by a few CFH19 mutations leading to impaired binding of CFH19–20 to C3d and/or C3b (154,156). The study I in this thesis shows that the Q1139A mutation in domain 19 also leads to impairment in binding of CFH19–20 to C3d and especially C3b, similarly to the R1203A mutation in domain 20. The X-ray crystal structure of both the CFH19-20Q1139A and CFH19-20R1203A mutants showed that only minor and local changes are caused to the structure of
CFH19-20 by the mutations. This led us to conclude that any effect on binding must therefore be due to the local changes in the CFH structure. The documented loss-of- function by the mutations Q1139A and R1203A can thus be conclusively attributed to the absence of the side chains of these residues at the binding interface and we concluded that both residues Q1139 and R1203, and thus both domains 19 and domain 20 of CFH are involved in binding C3d and C3b. Though the involvement of
CFH20 in aHUS was understood, the study I in this thesis also explains why mutations in domain 19 could also lead to impaired regulation of AP on surfaces leading to aHUS. Our study II revealed that the domain 20 of CFH has a binding site to C3d/C3b (Figure 7) and it has previously been shown to have a site for heparin, in the same zone as well (Fig. 3C of study II). Both the sites on domain 20 could in principle be involved in target discrimination. The mutual importance of these two sites in target discrimination is not evident. However, there are three key findings to evaluate the role of heparin vs C3d/C3b binding by domain 20. First, based on our results while CFH19 is bound to C3b, CFH20 can simultaneously bind to either the cell surface polyanions or another C3d or C3dg molecule (Fig. 3 of study II). There 60" DISCUSSION"""""!
seems to be steric hindrances for CFH19-20 to bind two C3b molecules (one via domain 19 and another via domain 20) but it is surely possible to bind one C3b via domain 19 and C3d via domain 20 (Fig. S3 of study II). Second, there are aHUS- associated mutations in the thioester domain of C3 at exactly the binding site for domain 20 of CFH. Since these mutations (or the regions around those) are not known to have any other functions it seems that disruption of C3d binding by domain 20 of CFH could lead to aHUS. Third, we have noticed that heparin inhibits binding of C3d to the aHUS mutant CFH19–20T1184R (a mutant associated with aHUS) even more than to CFH19Del–20 with mutations of three residues involved in the binding of CFH19 to C3b (study II) suggesting that the abnormally strong heparin binding by this mutant could interfere with function of CFH, e.g. by interfering with C3d binding by the CFH20 site, thus leading to aHUS. This has not been directly shown but the result indicates that strong heparin binding without C3b/C3d binding is not enough for efficient target discrimination by CFH. In conclusion, it is possible and even likely that CFH20 binding to both heparin and C3d is important for target discrimination and that loss of either of the interactions could lead to aHUS. One question which surfaced after our study II concerned the explanation of the CFH20 binding to C3d and the presence of aHUS mutations in CFH20 and the corresponding sites on C3b/C3d. If CFH19 is considered to be the only C3b binding site within CFH19-20, as suggested by Morgan et al. (162), the explanation of the presence of aHUS mutations in C3dFH20 binding site on as well as its binding behavior with C3d could not be explained. In addition to the model presented in the study II it is also possible that a conformational change of C3b upon binding to a non-activator surface exposed the C3dFH20 binding site. There is, however, no structural evidence for this.
Intriguingly, in our model of the C3b:CFH19–CFH20:C3d complex, the two thioester sites on C3d and C3b both face to the putative cell surface (Fig. 5 of study III). This is consistent with C3d (or iC3b) recruiting CFH on non-activator surfaces as proposed in our study II. Binding of the CFH20 site to C3d or iC3b could thus replace binding of CFH20 to non-activator surface polyanions, further explaining why the C3d and polyanion sites overlap on CFH20. Thus, our cumulative results in study I and study II suggest a comprehensive molecular mechanism for target discrimination of AP and provide evidence for negative feedback control of activation on non-activator i.e. host cell surfaces and extracellular matrix. This model explains DISCUSSION"""""61" ! widely, if not even fully, the location of aHUS point mutations (http://www.fh- hus.org) in CFH19–20 and the mutations within the C3d part of C3b. Mutations in
CFH19 affect C3b binding (154), and mutations in CFH20 affect, variously, glycosaminoglycan binding and/or C3d binding. All aHUS mutations in the C3d domain affect either CFH19 or CFH20 binding to C3d/C3b (154), which explains how all these mutations in CFH reduce control of C3b on non-activator surfaces, leading to the onset of the disease aHUS.
6.2 The microbial complement evasion: a molecular perspective
In our study III we resolved the structural basis of complement evasion by Borrelia burgdorferi via OspE. This study also provided information on microbial complement evasion in general. An extensive study on microbial binding of CFH19-20 (226) elucidates a multifaceted microbial angle of the story. The study III of this thesis revealed the structure of borrelial OspE in complex with CFH19-20, identified the binding surfaces and residues used for the interaction between OspE and host CFH, and explained how CFH is recruited by OspE onto the borrelial surface. A molecular model (Fig. 5 of study III) also revealed that OspE-bound CFH is sterically able to bind the central complement component C3b on the same surface. Our structure, however, contrasts with the previously suggested coiled coil structure for OspE (253). It has an 8-stranded up-down β-barrel globular structure (residues 42-171) linked to the membrane by a very flexible amino acid tail (Fig. 1 of study III), a fold found in SALP family, and primarily in the families of ‘Nucleic acid binding proteins’ and ‘Fatty acid binding proteins’ (Figure S5 of study III). The fold of the globular domain seems to be stable and fairly rigid since RMSD of the globular domain of the 20 NMR structures was only 0.38±0.05 Å for the backbone atoms and the structures of the CFH19-20:OspE crystal structure and the NMR structure of OspE were very similar (RMSD of 1.43 Å for Cα atoms). The flexibility of the tail can be explained by the tethering phenomenon that would naturally be useful for a bacterial surface protein. But why does the globular part of OspE needs to be so rigid? One possibility is that a rigid domain remains stable in variable molecular environments, allowing binding of CFH in all microenvironments of human and tick body. Alternatively, it could be useful if the fold did not change upon binding to a 62" DISCUSSION"""""! ligand since such an opening would allow new antigenic epitopes to be revealed – a phenomenon that would be harmful for the bacterium.
In our OspE:CFH19-20 structure, we could see that four residues (R1182, W1183, E1198, and R1215) in the CFH C-terminus play a key role in the interaction. The central role of these residues is supported by the chemical shift perturbations in the NMR spectrum of OspE upon addition of saturating concentrations of wild type
CFH19-20 and by the competition-binding assay with 14 mutant CFH19-20 proteins (226). The study III found the previously proposed theory of multiple lysine residues in OspE being functional in CFH binding (123) to be contradictory since in our
OspE:CFH19-20 structure there were no lysines in the interface. The results in study III also explain the previous data with deletion mutants of P21, an OspE homolog, (123) since the deletions used by Alitalo et al eliminated the beta strands involved in the interaction. It has been earlier shown that binding of CFH is essential for B. burgdorferi to survive in human serum, and that OspE (i.e. BbCRASP3-5) and BbCRASP1 are needed for this survival (122,254). It is obvious that the microbe benefits from the recruitment of host CFH onto the microbial surface since CFH acts as a cofactor for factor I in the degradation process of the central complement component C3b needed for both opsonization and propagation of the complement cascade to form membrane attack complexes. Therefore it is easy to understand that acquisition of host CFH onto borrelia correlates strictly with survival of the spirochetes in non-immune serum or blood, as observed earlier (255). The importance of OspE vs BbCRASP-1 mediated binding of CFH is, however, not known. The importance of OspE in immune evasion is supported by expression of OspE in vivo as judged by the emergence of anti-OspE antibodies in convalescent sera of patients with borreliosis. In addition, all the known strains of B. burgdorferi s.s. and B. garinii have at least one gene of OspE and most strains have several OspE paralogs that are homologous enough to take a similar fold (Fig.4 of study III).
6.3 Link between the CFHR1 deficiency and the AI-aHUS puzzle
AI-aHUS is a unique autoimmune disease associated with the deficiency of CFHR1 (a protein homologous to parts of CFH, see section 2.4.3). The reason for the presence of anti- CFH-AA in the absence of CFHR-1 in AI-aHUS has been a mystery. The DISCUSSION"""""63" ! study IV shows that the autoantigenic epitope on CFH, i.e. where CFH-AA bind to, form a cluster on domain 20 of the protein. This cluster is adjacent to the heparin and common microbial binding site on domain 20 of CFH (Fig. 2 of study IV) and also in close proximity to the two buried residues that are different in the C-terminal domains of CFH and CFHR1. It has previously been observed that CFH-AA from an AI-aHUS patient binds to CFH19-20 and CFHR1 differently, which is a phenomenon observed for full-length CFH as well (217,220) (see Figure 3 of study IV). We found minor differences in the X-ray crystal structures of the loop forming the auto-antigenic epitope on CFH and CFHR1 (Figure 7). Keeping in mind that the CFHR1 deficiency is found in nearly all the AI-aHUS patients, it is possible to conclude that CFHR1 plays a role in initiating and/or maintaining tolerance to CFH. The findings in study IV suggest that the structural difference in the loop forming the auto-antigenic epitope on the C-terminus of CFH and CFHR1 is the key to the puzzle of CFHR1 association and CFH tolerance. The patients from whom the sera for the study IV were taken from were from Germany and Hungary. Ethnically, the population represents fairly well a Central European population and genetic background but not worldwide distribution of, for example, major histocompatibility complex (MHC) genes involved in cellular immunity. It remains to be seen if patients from other parts of the world also exhibit the same behavior in terms of CFH and CFHR1 binding. Could the clustering of the CFH-AA epitope result from structurally remote effects of the mutations introduced to CFH19-20? Prior studies (study I) (226,256) confirmed that CFH19-20 mutations do not contribute to any prominent change in the domain fold of CFH via X-ray crystallography and circular dichroism studies. This indicates that the reduced CFH-AA binding in the epitope mapping study (Figure 2 of study IV) using the CFH19-20 mutants is not caused by any folding anomaly of the mutants but by a real clustering of the epitopes to a single loop on domain 20 of CFH (Figure 11). However, the CFH-AA binding analyses were all performed in a way that the mutants were coated to a plastic surface. This results in a possibility that binding of various mutants to the surface could be different and thus cause systematic error in the binding site mapping. The main reason why the autoantigenic epitope is real and not an artifact is the functional difference between the two terminal domains of CFH and CFHR1. The exact similarity (sequence and structure) of the CFH19 and CFHR14 64" DISCUSSION"""""!
leaves only the option of CFH20 and CFHR15 to be different enough to establish the clinical disease. It has already been shown that molecular pathogenesis of aHUS involves the loss of CFH binding either to C3b or to host cell surface structures such as GAGs or heparin (105,106,154,196,209, and study II). It is also known that AA from some patients impair CFH binding to C3b or to the cell surface structures (218). The impaired binding of CFH to either the cell surface structures or C3b influences the protection of the plasma exposed endothelial cells, thrombocytes, and red cells in aHUS (205). The location of the CFH-AA binding site we identified on domain 20 (Figure 2 of study IV) indicates that the AA are likely to block binding of GAGs/heparin to CFH, owing to its partial overlap with the GAG binding site on
CFH20 (Figure 12). It is also possible that CFH-AAs interfere with C3b binding of CFH as well owing to the large size of IgG. This is supported by the binding studies with C18 mAb against domain 20 of CFH (218).
CFH-AA Heparin/endothelial Common microbial site cell binding site binding site Figure 12. Surface representation of the domains 19 and 20 of CFH showing the CFH-AA site (light blue), heparin/endothelial cell binding site (purple) and the common microbial binding site (dark blue).
The risk for CFH-AA in the absence of CFHR1 is very high (Odds ratio over 400) (215). This motivated us to study the structural differences in the CFH-AA site in CFH domain 20 and the corresponding site on CFHR1 domain 5 (which are different by just 2 residues). Since only two residues are different in CFHR14-5 and
CFH19-20, it is not surprising that the crystal structures of CFHR14-5 and CFH19-20 are so similar. However, a minor difference was identified specifically at the CFH-AA DISCUSSION"""""65" ! site (Fig. 5 and Fig. 6 of study IV). The results obtained in study IV provided a structural basis to the phenomenon of CFHR1 deficiency in AI-aHUS. The hemolysis-inducing aHUS related mutation W1183L (220,257) of CFH being located in the centre of the CFH-AA binding site confirms the importance of the site in host cell protection. The issue of the crystal structure being an artifact has been discussed extensively in the study IV. Structurally, the most important fact that there were no direct contacts between the residues of the CFH-AA site (R281-L288) and the molecule in the neighboring crystal cell (Figure 13) made it unlikely to be an artifact. Functionally, the most important argument against the structure being an artifact is that the fusion proteins CFH1-18/CFHR14-5 or CFHR11-3/CFH19-20 associated with aHUS have different functions from normal CFH and CFHR1 (210,211,258). Together with other arguments given in the discussion of study IV, the functional difference between CFH20 and CFHR15 is likely to be in their ability to bind to heparin/GAGs on self cells (154,252) and the autoantibodies would naturally disrupt this interaction.
Figure 13. The CFHR14-5 structure shown along with its symmetry (crystal) mates and the corresponding crystal cell. CFHR14-5 with the CFH-AA site is shown in orange and its symmetry mates in olive colour.
Inset: close-up showing the CFH-AA site (also highlighted by line representation). The closest distance from its neighbour is shown by a dotted line (4.0 Å)
66" DISCUSSION"""""!
A novel hypothesis has been proposed which explains the association between formation of CFH-AA and deficiency of CFHR1 (Fig. 7 of study IV). Briefly, the model is based on the subtle conformational difference in the CFH-AA binding region of CFH and CFHR1. We think that the CFH-AA region of CFH could adopt an alternate conformation upon binding a microbial molecule to the adjacent microbial binding site on CFH domain 20, resulting in formation of a neo-epitope, identical to the natural CFHR15. For a CFHR1 non-deficient healthy individual, CFHR1 antigen would result in central tolerance to CFHR1 (and CFH domains 18-20). But in case of a homozygous CFHR1 deficient human, the alternative conformation naturally occurring in CFHR1 would not be introduced to the immature B-cells resulting in emergence of CFH-AA upon e.g. a microbial ligand binding to CFH20. However, as far as we know, all the patients with AI-aHUS do not have a history of a recent infection and there are even some cases without CFHR1 deficiency. Our model explains most issues in the autoimmunization but not all of them. This is likely to be caused by variability in the mechanism that could also explain some variety in the binding sites of antibodies against other parts of CFH than the C-terminus (219). Though the formation of a co-neoepitope in presence of a ligand of CFH and shared by it (Fig. 7 of study IV) leading to the formation of AA against CFH can be an explanation of the AI-aHUS pathogenesis as well, the theory does not explain the CFHR1 deficiency in AI-aHUS patients. If we consider that an altered self-molecule or an exogenous molecule forms the neoepitope in CFH20, the concept still fails to explain the issue of CFHR1 deficiency. Additionally, there have been reported cases of microbial infection to the patients preceding AI-aHUS, the explanation of which remains elusive as well. In conclusion, we found that in case of AI-aHUS, the AA bind to a common epitope that is adjacent to the polyanionic and microbial binding site on CFH20. The conformation of this epitope is slightly different in CFH and CFHR1, introducing a structural explanation why nearly all AI-aHUS patients are deficient of CFHR1. The results also do indicate that a just a subtle conformational change of the antigen has the potential to lead to an autoimmune disease. DISCUSSION"""""67" !
68" DISCUSSION"""""!
6.4 Structural analyses of various structures of CFH19-20
In this thesis there are all together five solved structures of the C-terminus of CFH (or CFHR1, red in Figure 14). Superimposition and RMSD analyses point very clearly to the fact that the distal end of CFH is structurally very coherent even in very different conditions. The rigidity of the structure can be assessed from the fact that almost 8 different structures (Figure 14) has an RMSD of around 1Å only while superposed on each other (Figure 15). Domain 19 and domain 20 of CFH has been always seen constantly with a ~32° angle in relative to each other’s axis, even in the CFH18-20 structure (259). aHUS mutations (eg CFH19-20R1203A) (study I), binding to C3d/C3b (study I), binding to microbial proteins (study I) could not induce any prominent structural change in the structure of CFH19-20. Not surprisingly, rigidity of CFH19-20 structure has also been recognized by at least one other group (259).
Figure 15. The CFHR14-5 (orange) structure shown along with its 8 other CFH19-20 homologues. The WT CFH19-20 structure is shown in gray colour.
DISCUSSION"""""69" !
Although the structures of CFH19-20 have been observed to be rigid in nature, an unexpected kink is seen between the domain 18 and 19 of CFH (259). The domain 18 of CFH has been noticed to be tilted by ~122° with respect to the long axis of CFH19, and by ~151° along the axis CFH19-20.
To visually compare the surface charges of WT CFH19-20, CFHR14-5, and
CFH19-20R1203A, the charge distribution was first calculated using APBS and plotted on the WT CFH19-20 structure (Fig. 5 of study I and Fig. 6 of study IV). As expected, we noticed distinguishable difference in the distribution of the contour of the positive isosurface in case of CFH19-20 and CFH19-20R1203A as displayed in the figure. Though not so prominent, we also noticed deficiency of an electronegative pocket in case of
CFHR14-5 as well.
6.5 Crystal artifacts, the pinch of salt
With the advent of the high throughput system of solving molecular structures, the rate of structure (protein) solving has gone up by several fold, which have revolutionised the way of understanding molecular interactions which happen in our body continuously. However, it is worth mentioning here that very often the crystallising conditions of the proteins (or any other molecules) are not very physiological, thus bringing potential misguidance to the very basic reason of structure solving. All of our structures of CFH and CFHR1 were solved in the presence of ~2M ammonium sulphate, which is not close to physiological conditions. Therefore, it is important to emphasize that the structures shown in this thesis have been verified by biochemical and immunological methods as well as has been possible.
We had to disregard an interface of CFH:C3d indicated in the CFH19-20:C3d structure solved by us (Pdb id: 2XQW, study II). Our previous binding studies clearly showed that it could not be the binding interface between C3d/C3b and CFH19-20 (106,154). 70" DISCUSSION"""""!
6.6 Conclusions and future prospects
In conclusion, the C-terminal domains 19 and 20 of CFH actively work in order to differentiate between the self and non-self molecules in order to prevent complement attack against self-structures while defending successfully against microbes. The mechanism that controls this target discrimination was revealed at a molecular level, which paved the way to understanding the molecular implications of the aHUS mutations in the C-terminus of CFH. Molecular mimicry by microbes which imitates the polyanionic host structures, in order to evade the complement attack, was also demonstrated in atomic resolution using B. burgdorferi as a model. The results also shed new light on the pathogenesis of AI-aHUS by the epitope mapping of the CFH- AA on the C-terminus of CFH and the molecular structure of CFHR1. All of these three separate molecular mechanisms involving the domains 19 and 20 of CFH were resolved to a high resolution in this thesis, thus increasing the understanding of aHUS disease established by complement malfunction, complement evasion by bacteria, and potentially also the process of autoimmunization in general.
The study of several newly solved structures (specifically different CFH19-20, Figure 14) structures have illustrated the process of target discrimination in detail. However, microbial exploitation of CFH as a complement evasion tool is still not understood extensively. We have made an attempt to understand it from the CFH19-20 point of view in our recent publication (226). The successful vaccine (Bexsero) against Neisseiria meningitidis serogroup B with the factor H binding protein of the microbe has made the field of CFH binding by microbes extremely important in next generation vaccinology. ACKNOWLEDGEMENTS"""""71" !
7 ACKNOWLEDGEMENTS
The research work presented in this thesis is done primarily in the Haartman Institute and the Institute of Biotechnology, University of Helsinki. I am indebted to Academy of Finland, the Sigrid Jusélius foundation, the Biomedicum Helsinki foundation and the University of Helsinki for financial and infrastructure support in order to carry out the research work. I am extremely honored to have Prof. Péter Gál as my esteemed opponent. My sincere thanks goes to Docent Arno Hänninen and Docent Veli-Pekka Jaakola for reviewing my thesis and improving it by their expert advice and criticism. It gives me immense pleasure to thank my mentor Sakari Jokiranta, for his confidence in me as a researcher, guiding me through my good as well as bad days, teaching me every nooks and corners and the basic dynamics of research. I could never be where I am today without his constant encouragement. My warm thanks is also extended to Adrian Goldman, who introduced me to Scandinavian research world and gave me the unique opportunity of learning X-ray crystallography (the basic craft which forms the skeleton of my thesis) in his lab under his skillful guidance. I take this opportunity to thank Seppo Meri and Mikael Skurnik for always being there for me whenever I needed their help. Their comments and discussions on various articles have enriched my understanding of science to a great extent. I am deeply indebted to the senior crystallographers Robert and Tommi for helping me to understand and solve the protein structures. I also appreciate their friendship and the ‘beer-time’ we spent together. I hereby thank all of my collaborators who contributed extensively to augment my thesis work. I deeply thank Jesper Oeemig and Hideo Iwaï from NMR group of Institute of Biotechnology, University of Helsinki for their contribution in solving the OspE structure. I also thank David Isenman along with Elisa Leung for providing C3d/C3dg used for the experiments, and the discussions we had. I had the great opportunity of having not only a fruitful collaboration but also deep scientific interaction with Mihály Józsi and his group including Stefanie Reuter, Harald Seeberger and Barbara Uzonyi. I thank all of them. I also thank Zoltán Prohászka along with his coworkers Ágnes Szilágyi and Eszter Trojnár for their contribution in the assays with the AI-aHUS patient samples. I profusely thank Derek for proofreading my thesis, and helping me to make it a better one, along with his personal friendly company. It was extremely beneficial for me to get critical comments on my research work from Neeta. Her non-complement, yet immunological perspective to a problem pertaining microbial immune evasion has helped me interpret my research data in a very fruitful manner, needless to mention of her personal helpful and supportive friendship offered to me in times of need. It was a sheer joy to work with Anna J, the hard working lady she is. Interactions with her have taught me to be critical of my own work. My heartiest thanks to Arun for all the help I got in using the infrastructure of the virology lab along with his selfless friendship. Words will always fail to express my deep gratitude to the ex and present members of the Jokiranta group, with whom the moments spent in and outside the lab turned out to be extreme fun yet learning. Have learnt a lot on practical immunological experiments and making statistical sense out of them from Markus and Taru, aspects of microbial experiments from Hanne and Karita. I will always 72" ACKNOWLEDGEMENTS"""""! remember Satu for all kinds of scientific and non-scientific discussions that enriched my life inside and outside the lab. It was an extreme delight to be the ‘taster’ of her baking delicacies on a regular basis. I will always remember the moments of sharing lab space and conference trips with Aino and Hannah with smile. Several other members of the Jokiranta-group like Anna H, Maarit, Sonja, Tanja, Tiira, and Valentina made the lab environment a very cosy one. It was a great enriching experience to be a part of the macromolecular crystallography group in the Institute of Biotechnology, Viikki as well. I consider myself extremely lucky to have made great friends like Juho, Wesley and Esko with whom the scientific discussions were very fruitful, not to mention the bar escapades together did bring the very necessary extra cheer to deal with the dark winters. I will always remember Seija and Sirpa for their motherly affection towards me. Seija’s excellent technical assistance in handling the crystallographic robots was extremely crucial to form the core of my thesis. The cakes baked by Ansko were very refreshing. Would also like to thank the other ex and present lab members of crystallography groups for the moments spent in the lab and symposiums: Andrzej, Anja, George, Heidi, Jaana, Jack, Katja, Kornelia, Kostya, Lari, Maria, Pirkko, Sanjay, Subhanjan, Tuulia, Veli-Matti and Vimal. I would also like to thank members (present and past) of the Meri-group for their co-operation and association in the lab. Special reference must be made for Hanna Jarva, for being the voluntary de facto guide to everything in the lab for every newcomer. Tobias, Nathalie, Ayman, Rauna, Anna-Helena, Kirsi V, Antti, Jorma, Sami, Inki, Marta B, Laura B, Hanna J, Hanna D, Ville, Martin: you all have made the SERO group a vibrant research group. My hearty thanks to all other members of the RPU, Immunobiology for having nice coffee room discussions together. Nelli, Laura R, Anni, Pirkka, Tamás, Eliisa and Tuisku from Arstila’s group, Julia, Kati, Laura, Mabruka, Anu, Juha, Marta BS, and Elif, from Skurnik’s group, Anne, Mercel, Judith and Eija, from de Vos’s group created a very positive atmosphere in the lab. I heartily thank Päivi for the scientific discussions we had in the RPU meetings. I also take this opportunity to thank my other collaborators for their scientific knowledge sharing. Sanjay Ram, Peter Rice, Olli Ritvos and Jutamas Shaughnessy deserve a special mention here. None of my research works would have been possible without the skillful assistantship from Marjatta, Pirkko, Marjo and Kirsti. Marko did a wonderful job in keeping the machines up and running. Kirsi and Taija with their jovial and helpful nature are always a boon for us foreigners with negligible knowledge of Finnish language. I thank all of them from the innermost recess of my heart. I would like to thank all of my friend members of the Helsinki Photography Club: ‘Kameraseura’, for their company in different photographic trips and meetings, helping me to co-host my photography exhibition, and finally maintain a vibrant photography club. Life without them would have been very dull in winters of Helsinki. Last but not the least, I would like to acknowledge the support of my family for being with me through my thick and thin, which made me achieve whatever I have achieved in life.
______Arnab Bhattacharjee / Nappi Helsinki, April, 2014 REFERENCES"""""73" !
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