INVESTIGATING REGULATORS OF THE ALTERNATIVE PATHWAY OF
COMPLEMENT AND THE MODULATORY ROLE OF FACTOR H AND
FACTOR HRELATED PROTEINS
by
ALEXANDRA HOPE ANTONIOLI
B.S., Yale University, 2007
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Molecular Biology Program
2016
This thesis for the Doctor of Philosophy degree by
Alexandra Hope Antonioli
has been approved for the
Molecular Biology Program
by
Arthur GutierrezHartmann, Chair
V. Michael Holers, Advisor
Joseph A. Brzezinski
Elan Z. Eisenmesser
Thomas E. Morrison
Date: 08/19/2016
ii
Antonioli, Alexandra Hope (Ph.D., Molecular Biology)
Investigating Regulators of the Alternative Pathway of Complement and the
Modulatory Role of Factor H and Factor HRelated Proteins
Thesis directed by Professor V. Michael Holers
ABSTRACT
Immunemediated diseases such as rheumatoid arthritis (RA), agerelated macular degeneration (AMD), systemic lupus erythematosus (SLE), and atypical hemolytic uremic syndrome (aHUS) are chronic and costly illnesses. Although the pathogenesis of each of these diseases is complex, it is known that dysregulation of the complement system plays a key contribution. Therefore, a better understanding of complement regulation and specific complement regulatory proteins is crucial for developing therapies that could improve the lives of many individuals.
The overall objective of this work was to explore the interrelationships between the complement regulatory protein Factor H (FH) and a group of closely related molecules called the Factor HRelated (FHR) proteins. FH regulates complement activation on selfsurfaces, thus allowing the innate immune response to discriminate between self and pathogens. FH and the FHR proteins consist in their entirety of compact repeating domains known as short consensus repeats
(SCRs). The FHRs share a subset of structural and functional traits with FH including the capacity to bind complement component C3b and glycosaminoglycans
(GAGs). However, few functional studies have been carried out on the FHR proteins, and they have not been studied in any in vivo models of inflammatory disease. Here we report the generation and characterization of recombinant murine FH and three
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FHR proteins. Results from hemolytic assays using erythrocytes indicate that two of the FHR proteins, mFHRA and mFHRB, antagonize the protective function of FH on some cell surfaces. For example, both mFHRA and mFHRB increase cell surface C3b deposition on a human retinal pigment epithelial cell line (ARPE19) and a murine kidney proximal tubular cell line (TEC). We also determined the apparent KD values of murine FH and the murine FHR proteins for a putative binding partner, murine C3d. To better understand the role of FHR proteins in vivo, preliminary experiments were performed to investigate the effects on complement activation on kidney cells in a murine model of complementmediated kidney injury.
The results from this work suggest that like their human counterparts, mFHR proteins appear to have an important role in complement regulation. Further work is warranted to define their in vivo contextdependent roles and determine whether the
FHR proteins are suitable therapeutic targets for the treatment of complementdriven diseases.
The form and content of this abstract are approved. I recommend its publication.
Approved: V. Michael Holers
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I dedicate this work to my family.
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ACKNOWLEDGEMENTS
I would like to thank my mentor, V. Michael Holers, for the opportunity to work with a world leader in the field of complement research. I am grateful for the many opportunities to attend extraordinary scientific conferences and for providing me with space to grow as a scientist. I also would like to acknowledge Liuda Kulik for sharing her knowledge of complement and antibodies with me. Thank you to Joshua
Thurman and members of his lab, Jennifer Laskowski and Brandon Renner, for their time and help with animal experiments. Thank you to Shaun Bevers and the
Biophysics Core for their help and Lori Sherman and Michelle Randolph in the
Cancer Core.
My training would not have been the same without Philippa Marrack, who gave me the opportunity to work in her laboratory and learn about a new area of science. I am thankful for her mentorship and guidance and for a truly wonderful experience in her lab. I would also like to thank John Kappler for our discussions about protein biochemistry. I am thankful to Janice White who provided help and expertise with the antibody development in this project and to Fran Crawford for her exceptional knowledge of protein biochemistry and her assistance with biacore experiments. Thank you also to other members of the KapplerMarrack (KM) lab including Gina Clayton, Haolin Liu, Alana Montoya and Ella Kushner. I would also like to acknowledge Jo Alamri for her support and encouragement. In addition to the
KM lab, I am very appreciative to the Clinical Complement Lab at National Jewish
Hospital and in particular, Ashley FrazerAbel and Patricia Giclas, for allowing me the opportunity to perform certain experiments and for providing me with reagents.
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I would like to acknowledge the members of my thesis committee including
Arthur GutierrezHartmann, Joseph Brzezinski, Elan Eisenmesser, Thomas
Morrison, and Rui Zhao, for their help and guidance throughout my training. I am also appreciative of the support of the Molecular Biology Program. In particular, I would like to express my gratitude to the Victor W. and Earleen D. Bolie Family for providing financial support for my graduate training and also for providing funding to attend an international conference. Thank you to both Arthur GutierrezHartmann and Angie Ribera for their mentorship and for always having time to talk with their students about science or life. I would like to thank our Molecular Biology and MSTP administrators, Sabrena Heilman, Jodi Cropper, and Emily Dailey. Additionally, I would like to also acknowledge Dr. Liron Caplan who served as my clinical preceptor for my Foundations of Doctoring course. Dr. Caplan has tremendous clinical skills and working with him for five years was inspiring and a true privilege.
On a more personal note, I would also like to thank my friends and numerous colleagues for their help and companionship during graduate school. The past five years have been an amazing journey, and I am blessed to have had their support and encouragement. Finally, I would like to thank my parents, Sandra and Peter as well as my sister, Gabrielle, and uncle Milt for all of their love, support, and endless encouragement. While my grandparents, Milt, Ruth, and Della, and my uncle Mark are no longer with us, I would like to acknowledge them for providing me with a magical childhood full of love and for encouraging me to dream big and stay strong.
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TABLE OF CONTENTS CHAPTER
I. INTRODUCTION ...... 1
Immune System Network...... 1
Complement Pathways ...... 2
The Classical Pathway ...... 4
The Lectin Pathway ...... 5
The Alternative Pathway ...... 6
Complement Regulation ...... 8
MembraneBound Complement Regulators ...... 8
Cell Surface Receptors for Complement Components ...... 10
Fluid Phase Regulators ...... 11
Regulation by Factor H ...... 13
CFH Genomic Position and Structure ...... 16
Mapping of the CFHR Genomic Region ...... 19
Human FHR Proteins ...... 22
FHR1 ...... 24
FHR2 ...... 24
FHR3 ...... 25
FHR4 ...... 26
FHR5 ...... 27
Human Disease Associations ...... 28
C3 Glomerulopathy ...... 29
Atypical Hemolytic Uremic Syndrome ...... 30
AgeRelated Macular Degeneration ...... 32
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FH and FHR Protein Involvement with Pathogens ...... 33
Murine Factor HRelated Gene Family ...... 36
Initial Characterization of Murine FHR Transcripts ...... 36
Initial Characterization of Two Murine Factor HRelated Proteins ...... 39
Regarding Current Knowledge of the Murine Factor HRelated Gene Family ...... 42
Scope of Research ...... 47
II. MATERIALS AND METHODS ...... 50
Protein Experiments ...... 50
Sequence Analysis ...... 50
MFH and MFHR Construct Design ...... 50
Mutant MFHR Construct Design ...... 51
MC3d Construct Design ...... 53
Glycerol Stocks of MFH and MFHR Constructs ...... 54
MFH and MFHR Protein Expression ...... 55
MFHRB Expression in Pichia pastoris ...... 57
MFH and MFHR Protein Purification ...... 57
Column Calibration ...... 58
Murine C3d Protein Purification ...... 59
SDSPAGE Analysis ...... 60
Deglycosylation of Recombinant Glycoproteins ...... 61
Removal of His6tag from Recombinant Proteins ...... 61
Western Blot Analysis ...... 62
Differential Scanning Fluorimetry ...... 62
Circular Dichroism (CD) Spectroscopy ...... 63
Ligand Binding Experiments ...... 64
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EnzymeLinked Immunosorbent Assay (ELISA) with MC3d ...... 64
Surface Plasmon Resonance ...... 64
Functional Assays ...... 65
Erythrocyte Hemolytic Assays ...... 65
CellSurface Assays ...... 65
C3b deposition on Renal Tubular Epithelial Cells (TECs) ...... 65
C3b deposition on Retinal Pigment Epithelial Cells (ARPE19) ...... 66
Measuring Transepithelial Resistance (TER) of ARPE19 Cells ...... 66
Antibody Studies ...... 67
MFH and MFHR Antibody Production ...... 67
B Cell Fusions ...... 68
Screening Hybridomas ...... 68
SPR Analysis of AmFH Antibodies ...... 69
Quantifying MFH by Sandwich ELISA ...... 70
Immunoprecipitation (IP) of MFHR Proteins from Serum ...... 71
Mass Spectrometry ...... 71
Sample Preparation for Mass Spectrometric Analysis ...... 71
Mass Spectrometry ...... 72
Database Searching and Protein Identification ...... 73
Animal Studies ...... 73
Renal Ischemia/Reperfusion Model ...... 73
Statistical Analysis ...... 74
III. RECOMBINANT MFH AND MFHR PROTEIN EXPRESSION AND ANALYSIS ...... 75
Expression and Purification of Complement Proteins ...... 75
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Selecting an Expression System for producing MFH and MFHR Proteins ...... 75
Expression of MFHR Proteins using 293F Cells ...... 79
Assessing Quality and Thermal Stability of MFH and MFHR Proteins ...... 83
Summary ...... 87
IV. ASSESSING THE FUNCTIONAL ROLES OF MFHR PROTEINS ...... 91
General Background ...... 91
MFHRA and MFHRB are Potent Antagonists of MFH on SRBCs ...... 95
V. CHARACTERIZING THE MFH AND MFHR PROTEIN INTERACTION WITH MC3D ...... 98
General Background ...... 98
MFH and MFHR Proteins bind MC3d and Mutant MFHR Proteins are Inactive ...... 100
Determination of MC3d Binding Affinity for MFH and MFHR Proteins ... 104
Mutant MFHRB Exhibits Loss of Function in Hemolytic Assays ...... 106
Summary ...... 107
VI. MFHR PROTEINS ACTIVATE COMPLEMENT ON DIFFERENT CELL SURFACES ...... 110
MFHRA and MFHRB Induce Complement Activation on Murine TECs110
MFHRA and MFHRB Enhance Complement Activation on Retinal Pigment Epithelial Cells ...... 111
Summary ...... 116
VII. CREATION OF MFH AND MFHR SPECIFIC ANTIBODIES ...... 117
Monoclonal Antibodies Specific for MFH ...... 118
Developing MFHR Specific Antibodies ...... 121
Summary ...... 129
VIII. IN VIVO STUDIES ...... 132
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IX. DISCUSSION ...... 135
X. FUTURE DIRECTIONS ...... 146
Characterizing Additional MFH and MFHR Interactions and Functions . 146
Identifying MFH and MFHR Interactions with Different Host Polyanions 148
Characterizing the Interaction of MFHRs and Sialic Acid ...... 148
Animal Models ...... 150
Antibody Development ...... 152
REFERENCES ...... 153
APPENDIX ...... 177
A. Mouse Complement FH Gene and Protein Information ...... 177
B. Mouse Complement FHRA Gene and Protein Information ...... 178
C. Mouse Complement FHRB Gene and Protein Information ...... 179
D. Mouse Complement FHRC Gene and Protein Information ...... 180
E. Mouse Complement FHRD Gene and Protein Information ...... 181
F. Mouse Complement FHRE Gene and Protein Information ...... 182
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LIST OF TABLES TABLE
1. SPR Analysis of the Interactions between MFH/MFHRs and MC3d ...... 106
2. Reactivity of 14 AntiMFH Hybridomas from Initial Screen ...... 119
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LIST OF FIGURES FIGURE 1. Complement Activation Pathways ...... 3
2. Alternative Pathway (AP) Amplification Loop ...... 7
3. MembraneBound and Fluid Phase Complement Regulators ...... 11
4. Regulators of Complement Activation (RCA) Gene Cluster ...... 17
5. Comparison of Human and Murine CFH and CFHR Gene Organization ...... 20
6. Human Factor H and Factor HRelated Proteins ...... 23
7. Initial Characterization of Murine Factor HRelated (mFHR) Transcripts ...... 40
8. Outline of Murine Factor HRelated Experiments and Conclusions by Hellwage et al. 2006 ...... 43
9. Murine FH and FHR Gene Family and Schematic Representation of MFH and MFHR Proteins ...... 46
10. MFH and MFHR Expression Construct Design...... 77
11. Expression Trials of MFHRB Protein in 293F cells and P. pastoris .... 79
12. MFHRA Purification using IMAC ...... 81
13. Purification of Recombinant MFH ...... 82
14. Alignment of Human and Mouse SCRs 7 ...... 83
15. SDSPAGE of Glycosylated and Deglycosylated MFH, MFHRA, and MFHRB ...... 84
16. Chromatographic Separation and Calibration Curve of Protein Standards ...... 85
17. Thermal Shift of MFH, MFHRA, MFHRB, MFHRC, and MFH 19 20...... 87
18. AH50 Assay with MFH and MFHR Proteins Influences Alternative Pathway Activation ...... 93
19. Hemolytic Assay Schematic with MFH and MFHR Proteins ...... 94
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20. MFHRA and MFHRB are Potent Antagonists of MFH on HostLike Surfaces ...... 96
21. Human FH SCRs 1920: C3d Interface ...... 99
22. Sequence Alignment of Human FH SCRs 1920 with MFH and MFHR proteins showing Putative C3d Binding Sites ...... 100
23. Recombinant MC3d Expression and Analysis ...... 101
24. MFH and MFHRs Bind MC3d and Mutant Proteins are Inactive...... 103
25. Binding of Murine C3d and MFH and MFHR Proteins ...... 105
26. Mutant MFHRB does not inhibit MFH Function on Cell Surfaces ...... 107
27. MFHRA and MFHRB Act as Potent MFH Antagonists on Murine Tubular Epithelial Cells (TECs) ...... 112
28. MFHRA and MFHRB Increase Complement Activation on ARPE19 cells ...... 114
29. Complement Activation Induced by MFHRA Results in Loss of TER of ARPE19 cells ...... 115
30. Affinities of AmFH55.7 and AmFH70.6 for Recombinant MFH...... 120
31. Quantitation of Murine Factor H in Serum Using AmFH55.7 and AmFH70.6 Monoclonal Antibodies ...... 122
32. Western Blot Analysis of Murine FHR Proteins in WT and FH/ Serum and Plasma ...... 125
33. Alignment of Exons 37 of Pseudogene Gm61332 and MFHRA and MFHRB sequences ...... 127
34. Mass Spectrometry Identifies a Peptide Sequence Specific to MFHR A ...... 128
35. Conserved Sialic Acid Binding Site Residues on MFH and MFHRs ... 150
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CHAPTER I
INTRODUCTION
Immune System Network
The immune system is a complex network of molecules, cells, tissues, and organs working together to protect the body from pathogenic organisms and guard against disease. Components of the immune system are organized under two branches known as the innate immune system and adaptive immune system. Each branch serves distinct roles, and together they act in a concerted manner to maintain and protect healthy host tissue.
Unlike the adaptive immune system, the innate immune system does not change through genetic modifications over the course of an individual’s lifetime. The innate immune system is the more ancient system, and components from this system are found in a range of multicellular organisms including vertebrates, invertebrates, and plants (1). A limited number of germline encoded receptors recognize foreign molecular structures or patterns and distinguish infectious agents from selftissues to provide a rapid first line of defense (13).
The adaptive immune response is based on antigenspecific responses of B and T cells providing a slower, yet more specific response to foreign invaders while creating immunological memory. Of central importance to both systems is the complement system, which acts as a complex innate immune surveillance system sensing danger signals and then translating those signals to activate the adaptive immune response (4). The complement system must maintain a careful balance between activation and inhibition, as having too much or too little regulation can
1
cause uncontrolled inflammatory responses and favor the development of autoimmunity.
Complement Pathways
The human complement system consists of over 30 proteins including serum glycoproteins and cell surface proteins which act to recognize pathogens and provide the host with a crucial first line of defense against infection. While most of these proteins are synthesized in the liver, local production of various proteins also occurs.
Complement activation initiates through direct engagement with pathogens or by indirect binding to pathogen bound antibodies. The three pathways of complement include the classical (CP), lectin (LP), and alternative (AP) pathway
(Figure 1). All three pathways are initiated by different mechanisms; however, they all generate homologous C3 convertase protease complexes (C3(H20)Bb and
C4b2a) which cleave complement component C3 to generate the active fragments
C3a (an anaphylatoxin) and C3b (an opsonin).
C3b tags antigens (and other surfaces) by means of a reactive thioester which preferentially forms covalent bonds with nearby amine and hydroxyl groups.
Deposition of C3b on target surfaces facilitates the phagocytosis of microorganisms by macrophages and the amplification of complement by formation of the AP C3 convertase, C3bBb. As the density of deposited C3b on an activating surface increases, additional C3b moieties also become incorporated into existing CP and
AP C3 convertases resulting in the assembly of C5 convertases (C3bBb3b and
C4b2a3b). These C5 convertases drive the release of the potent anaphylatoxin,
2
C5a, and initiate the formation of the membrane attack complex (MAC), the lytic pore forming terminal complement complex.
Figure 1 Complement Activation Pathways. Schematic showing activation of the complement cascade by three main pathways: the Classical, Lectin, and Alternative pathway. All pathways generate C3convertases which drive production of C3a (an anaphylatoxin) and C3b (an opsonin). C3b generated by the CP or LP can participate in the AP amplification loop (depicted by the green arrow).
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The Classical Pathway
Activation of the CP occurs mainly by antigenantibody complexes, but can also occur through antibodyindependent mechanisms. CP initiation can be triggered by exposure of components on damaged or apoptotic cells (5), Gramnegative bacteria (6), amyloid and prions (7), bacterial lipopolysaccharide (LPS), nucleic acids, and even retroviruses (8).
Once the CP is initiated, for example by the C1 complex binding to immune complex containing IgG or IgM antibody, the C1 complex undergoes a conformational change. One C1q molecule binds two molecules each of C1r and
C1s. Both are serine proteases and C1r cleaves C1s which acts on components C4 and C2 to produce the CP C3convertase (C4b2a). It is important to note that cleavage of C4 by C1s releases a small fragment, C4a, and yields a larger fragment,
C4b, which now has an exposed thioester group. This thioester group is important in the function of both C4 and another downstream molecule, C3. In native C4 or C3 molecules, the thioester is concealed within a hydrophobic pocket, and once C4b or
C3b are formed, the thioester becomes exposed and can readily form covalent amide or ester bonds with exposed amino or hydroxyl groups that may be found on the surfaces of pathogens. Once bound to membrane surfaces, C4b acts as a binding partner for the CP component C2, which is cleaved by C1s into components
C2a and C2b, resulting in formation of the C4b2a complex.
The next component in the pathway is C3, which is essential in both the CP and the AP. C3 binds noncovalently to C2a in the C3 convertase (C4b2a) complex and is cleaved by C2a, which releases C3a and exposes the C3b labile thioester
4
group. C3b can bind the activated C4b2a complex to form the CP C5 convertase
(C4b2a3b), or C3b can bind an adjacent membrane. A key point is that C3b generated at this step can crossover and go directly into the alternative pathway.
The next steps in the CP include activation of C5, which unlike C3 or C4 lacks a thioester group. C5 binds noncovalently to C3b in the C4b2a3b complex, and C2a cleaves and releases C5a and C5b while exposing a labile hydrophobic binding site on C5b for C6. The C5b6 formed at this step assists in MAC formation with components C7, C8, and C9 which assemble to form pores in the membrane of the cell being targeted for lysis.
The Lectin Pathway
Unlike the CP, the lectin pathway is an antibodyindependent route for activation of complement on the surfaces of pathogens such as yeast, bacteria, parasites, and mycobacteria. Each of these organisms possess conserved molecular patterns which can be recognized as foreign by the immune system. The
LP utilizes the binding of mannosebinding lectin (MBL), ficolin (FCN), and their
MBLassociated serine proteases (MASPs) to cell surface carbohydrates, such as
Nacetyl glucosamine residues present in bacterial cell walls.
Three MASPs have been characterized (MASP1, MASP2, and MASP3) with two other nonenzymatic fragments known as Map19 and Map44. MASPs serve a role that is homologous to C1r and C1s and upon recognizing a pattern on a foreign molecule, MASP1 and MASP2 are activated and cleave C4 and C2 resulting in the formation of the LP convertase, C4b2b. Both MASP1 and MASP2 act enzymatically, while the roles of MASP3, Map19, and Map44 are less defined
5
but are thought to play a role in regulating complement activation (911). Following activation of the LP convertase, the complement cascade proceeds through a central pathway with a series of reactions that are common to both the CP and LP.
The Alternative Pathway
The AP does not require any specific molecular recognition by its early molecule(s) for its initiation but is activated by hydrolysis of C3 to C3(H20) in the fluid phase which results in activation and production of C3b. This phenomenon, known as C3 tickover, occurs spontaneously allowing for the rapid initiation and amplification of complement (Figure 2). Important components of the AP include
Factor B (FB) which once bound to C3b is susceptible to cleavage by complement
Factor D (FD). FD cleaves C3bbound FB into Ba and Bb to generate C3bBb, the
AP C3 convertase. Studies have indicated that activation of the AP is dependent on formation of proteolytically active FD by cleavage of a five amino acid peptide from its inactive zymogen form (proFD). Other studies have shown that MASP1/3 cleavage of proFD in circulation is important for AP generation in mice and that serum from MASP1/3 knockout mice have defective AP function (12).
Once the C3 convertase complex is formed, another protein, properdin, acts to stabilize the complex on cell surfaces (13, 14). Properdin is a complement protein encoded by a gene on chromosome X (13, 15). Properdin is released by neutrophils in the presence of pathogens during cell activation and degranulation (13, 16).
Properdin acts in the alternative pathway to bind bacterial and apoptotic surfaces and stabilize the C3 convertases (17, 18). The ability of properdin to direct AP convertase formation provides another way to activate the AP (19).
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Figure 2 Alternative Pathway (AP) Amplification Loop. The amplification loop has the ability to greatly enhance C3 activation in fluidphase and on cell surfaces. The AP is responsible for 80% of the final downstream effect of initial specific activation of the CP and LP, therefore the AP and amplification loop must be well controlled. Factor H (FH) is an important AP regulator that serves three main roles in AP regulation. 1) FH acts as cofactor for FI inactivation of C3b. 2) FH acts to prevent formation of C3 preconvertase complex, C3bB. 3) FH accelerates the decay of the existing C3 convertase, which is responsible for cleaving C3 to further amplify C3b production. Given that the alternative pathway is constantly and slowly activated by spontaneous hydrolysis of the C3 thioester bond, and that this pathway is responsible for 80% of the final downstream effect of initial specific activation of the classical and lectin pathways, precise control of this pathway and the amplification loop is required (20).
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Complement Regulation
A number of membranebound and fluid phase regulators ensure that C3b deposition on selfsurfaces and tissues is controlled in all three pathways (Figure 3).
These molecules include DecayAccelerating Factor (DAF), Complement Receptor 1
(CR1), Membrane Cofactor Protein (MCP), and Factor H (FH) among others (2123).
Together these proteins work to accelerate the decay of C3 convertases and/or they act as cofactors for a serine protease known as Factor I (FI) (24, 25). FI is the molecule responsible for proteolytic degradation of C3b and C4b, and consists of four domains including a FImembrane attack complex (FIMAC) domain, a CD5 domain, two lowdensity lipoprotein receptor (LDLr) domains, and one serine protease (SP) domain, (26).
Understanding the regulatory mechanisms that control spontaneous activation of complement in the fluid phase and the amplification of complement on specific surfaces (known as activator surfaces) has important implications for understanding and treating complementdriven inflammatory disease. Outlined in the next section are the major membranebound and fluidphase regulators that control activation of the complement cascade.
MembraneBound Complement Regulators
Membrane cofactor protein or CD46 is a cell surface glycoprotein that inhibits complement activation on the host surface by acting as a cofactor for FI. The rodent counterpart to MCP is known as Crry (CR1related gene/protein Y) and is present in both mouse and rat (27). Similar to MCP, Crry is a cofactor for FI and also has
8
decay accelerating activity (DAA) for both the CP C3 convertase and AP C3 convertase.
CD35/CR1 serves as another membrane bound cofactor for FI and regulates both the classical and alternative pathways by binding C1q as well as C3b, iC3b, and MBL to aid in the clearance of opsonized particles (28). Yet another protein,
CD55/DAF protects cell surfaces by interacting with cell bound C4b (classical and lectin pathways) and C3b (alternative pathway) preventing the conversion of C2 and
FB to C2a and Bb and blocking the formation of the respective C3 convertases
(C4b2a and C3bBb) (29). Compared to human DAF which is expressed uniformly as a glycosylphosphatidylinositol (GPI)anchored molecule, two forms of DAF (DAF1 and DAF2) are encoded by genes in the mouse producing GPIanchored and transmembrane anchored proteins (30).
In humans, CD59, also known as protectin/MACinhibitory protein, attaches to host cells using a GPI anchor and blocks the interaction between C9 and C5bC8 to prevent polymerization of the complement membrane attack complex (MAC) (31).
Humans with deficiencies in CD55 and CD59 proteins cannot regulate complement activation on blood cells, especially erythrocytes, and the destruction of red blood cells can lead to a lifethreatening disease called paroxysmal nocturnal hemoglobinuria (PNH) (32). On murine erythrocytes, it has been shown that Crry rather than CD59 and DAF is necessary to protect erythrocytes from spontaneous complement attack (33).
Complement receptor of the immunoglobulin superfamily (CRIg), which is also known as Z39Ig and Vset and Ig domaincontaining 4 (VSIG4), binds C3b and
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iC3b and is selectively expressed on macrophages including Kupffer cells and dendritic cells (3436). This receptor is important for binding iC3b and aiding in the phagocytosis of C3opsonized particles; however, it may play additional roles in immune suppression (37).
Cell Surface Receptors for Complement Components
While CD35, CD46, CD55, CD59, and CRIg all serve important roles in complement regulation by acting as membranebound regulators, a number of other cell surface receptors are involved in the binding of complement components and possess unique functions that are involved in immune regulation. As described above, CR1 acts as a cofactor for FI and receptor for complement components C3b, iC3b, and C4b. Complement Receptor 2 (CR2/CD21) is found on the surface of human B lymphocytes, follicular dendritic cells and a subset of T cells (38, 39). CR2 binds complement C3 and fragments C3d, iC3b, and C3dg (40). CR1 and CR2 on B cells act as coreceptors for surface bound immunoglobulin regulating B cell differentiation and maturation. CR2 in complex with a C3 fragment acts as a molecular adjuvant to enhance the antigenic response of B cells and increase memory to an antigenthus bridging the innate and adaptive immune responses (41
44). Other cell surfaces receptors include CR3 (CD11bCD18, MAC1, M2 integrin) and CR4 (CD11cCD18, X2 integrin) which bind iC3b, C3dg, and C3d to enhance their interaction with opsonized targets and aid in their immune clearance
(45, 46).
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Figure 3 MembraneBound and Fluid Phase Complement Regulators. Several proteins are responsible for complement regulation and are schematically displayed under the pathway in which they primarily function. Membrane bound regulators include CD35/Complement Receptor 1, CD46/Membrane Cofactor Protein, GPI anchored proteins: CD55/DecayAccelerating Factor and CD59, and the complement receptor of the immunoglobulin superfamily known as CRIg. Fluid phase complement inhibitors include Factor H, FHL1 protein, C1inhibitor (C1INH), and C4 Binding Protein (C4BP). Carboxypeptidase N regulates all three pathways by binding anaphylatoxins C3a, C4a, and C5a. Two important complement activators are also displayed. Properdin binds cell surfaces and stabilizes the AP C3 convertase while C1q forms a complex with C1r and C1s and activates the classical pathway through binding of IgG/IgM coated pathogens.
Fluid Phase Regulators
Soluble complement regulators include C1q and properdin (which serve as complement activators) and C1inh, C4BP, FH, and FHL1 (which are molecules that inhibit complement activation). Terminal pathway regulators include carboxypeptidase N, clusterin, and vitronectin among others.
C1inh or C1 esterase is a member of the serpin family and serves as a serine protease in the classical and lectin pathways (47). C1inh regulates complement activation in both pathways by preventing downstream cleavage and activation of C2 and C4. In the classical pathway, C1inh binds irreversibly to the
11
activated C1r and C1s proteases while in the lectin pathway it inhibits MASP1 and
MASP2 in MBL complexes in a similar manner (48). In addition to acting as a complement regulator, C1inh has an important role in the clotting cascade and inhibits kallikrein, factor XIa, and factor XIIa (49, 50). Mutations in the gene encoding
C1inh or deficiencies in the protein have been linked to a range of both autoimmune diseases, such as systemic lupus erythematosus (SLE) as well as vascular conditions, such as hereditary angioedema (5154).
The classical pathway regulator C1q associates with C1r and C1s to form the
C1 complex. This complex has an important role in host defense and is an activator of the classical pathway. C1q binds IgG and IgM coated targets and aids in their elimination (55, 56).
C4 binding protein or C4bp is another classical and lectin pathway inhibitor that serves as a cofactor for FI and aides in the decay acceleration of the CP C3 convertase (57). When C4bp is bound to C4, FI is able to inactivate the C4 molecule and prevent the formation of the C4b2a C3 convertase.
Several other regulators that act to regulate complement activation include carboxypeptidase N, clusterin, and vitronectin. Carboxypeptidase N regulates all three complement pathways by binding and inactivating the anaphylatoxins C3a,
C4a, and C5a (58, 59). Clusterin (SP40 or apolipoprotein), is a member of the heat shock protein family and molecular chaperone. Clusterin is associated with high density lipid complexes but also acts in the terminal complement pathway by binding
C7, C8, and C9 (60). Another protein, vitronectin (complement S protein), acts in a
12
manner similar to clusterin by binding near the C5b9 complex rendering it water soluble and preventing the MAC from inserting into cell membranes (61).
As described above, complement inhibitors work to insure that complement levels are maintained in the absence of a target and that complement activation on normal host cell surfaces does not occur. At the same time, complement activators work to sense foreign invaders and quickly amplify the signal. In the next section, a potent regulator of the AP will be discussed. Given that the AP amplification loop has the ability to greatly enhance C3 activation, regulation of this pathway by FH is crucial. Additionally, many human diseases are associated with mutations and autoantibodies that alter FH function. Therefore, understanding the regulatory mechanisms by which the AP controls spontaneous activation of complement in the fluid phase and the amplification of complement on specific surfaces (known as activator surfaces) has important implications for understanding and treating complementdriven inflammatory disease.
Regulation by Factor H
Complement factor H protein was initially isolated in 1965 from human serum and identified as 1H globulin (62). Human FH is a 155 kDa single polypeptide chain glycoprotein expressed by liver cells, monocytes, endothelial cells, fibroblasts and myoblasts. FH is an important soluble plasma protein found at concentrations varying between 115562 µg/ml (0.73.6 µM) (6366). The halflife of FH in serum has been estimated to be six days (67).
FH regulates the alternative pathway of complement both in fluid phase and on cell surfaces by binding C3 components and acting as a cofactor for FIdriven
13
C3b inactivation. FH also competes with FB to prevent formation of the C3b(H2O)B and C3bB preconvertases and accelerates the decay of existing C3bBb convertases (68). The molecular structure of FH consists of 20 short consensus repeating (SCR) units, each of which are ~60 amino acids in length (69, 70). The alternatively spliced isoform of this protein, FHL1, is comprised of 7 SCR domains and is expressed at lower concentrations. FHL1 has regulatory functions similar to
FH. The FI cofactor and decayaccelerating regions of FH are housed within the four
Nterminal SCR modules (SCRs 14). The Cterminal domain (SCRs 1920) is critical for FH binding of C3b and host polyanionic markers. These domains are essential for FHmediated complement regulation on host surfaces (7174).
Absence or mutation of the CFH gene results in unregulated control of the
C3 convertase and consumption of C3 in fluid phase. Hereditary deficiency of complement FH in pigs and humans results in membranoproliferative glomerulonephritis type II (MPGN II), which is characterized by massive complement deposition in the renal glomeruli (75). In humans, FH deficiency or mutation is also associated with hemolytic uremic syndrome (HUS) among other diseases (76, 77).
FH knockout mice have been shown to develop both fluid phase C3 deficiency and renal abnormalities that are analogous to human dense deposit disease (78).
Studies have demonstrated that replacement of FH (using purified human FH) reverses the C3 deficiency and reduces renal complement deposition in FH/ mice
(79).
The murine equivalent of factor H (mFH) was identified in 1986 and shares similarity to human FH in both structure and function. MFH is a glycosylated protein
14
with a molecular weight ~155 kDa. This glycoprotein is primarily synthesized in the liver and is estimated to have a similar serum concentration to human FH, although an exact concentration has not been determined (80). Both human and murine FH have several predicted glycosylation sites. These Nlinked sites are classified as glycosidic linkages to nitrogen on asparagine residues appearing in the sequence
AsnXSer/Thr where X is any amino acid except for proline. While nine sites are predicted on human FH, eight of those sites have been shown to be glycosylated in vivo (81). Confirmed Nlinked sites include residues: Asn511, Asn700, Asn784,
Asn804, Asn864, Asn893, Asn1011 and Asn1077 within SCR domains 9, 12, 13, 14,
15, 17, and 18. One putative site within SCR4, residue Asn199, does not appear to be glycosylated on fulllength FH but may be glycosylated on FHL1 (81, 82). Murine
FH contains seven predicted Nlinked glycosylation sites with five confirmed sites found in SCRs 13, 17, 18, and 20. Notably, all but a single glycosylation site occurs within SCRs 820 on both human and murine forms of FH.
While changes in glycosylation patterns have been associated with diseases including cancer, one study has shown that increased fucosylation (addition of fucose to an Nglycan) of FH occurs in the setting of increased liver damage, but not cancer progression (83, 84). Changes that may occur in glycosylation do not appear to alter the function of FH in terms of complement regulation. Recombinant production of human FH in the yeast strain Pichia pastoris produced a functionally active protein in biological assays. Mutation of the glycosylation sites in FH from Asn to Gln to generate a FH mutant protein devoid of glycans also proved to be biologically active (85).
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CFH Genomic Position and Structure
The CFH gene is found within the Regulators of Complement Activation
(RCA) gene cluster on chromosome 1 (Figure 4). The RCA gene cluster can be divided into two groups containing more than sixty genes including a 650 kb region encoding factor H and the five factor Hrelated proteins (86). Genes within the RCA are thought to have evolved from an ancestral locus following multiple gene or exon duplication events (87, 88).
A common feature of at least thirteen complement proteins (including DAF,
CR1, CR2, MCP, and C4BP) and many other noncomplement proteins (including clotting factor XIIIb, the interleukin2 receptor achain and leukocyte cell adhesion molecules), is that they share common repeating domain motifs. These domains are known as short consensus repeats (SCRs) or complement control protein (CCP) modules (89, 90). The finding of these domains in ancient metazoan marine sponges suggests that these modules are highly conserved and have important structural and functional roles in a number of diverse biological functions (91). SCRs are characterized as compact repeating βsheet domains comprised of approximately 60 amino acids. Each of these beadlike modules contains a number of highly conserved residues, including an almost invariant tryptophan residue, and a number of other hydrophobic residues which are found within the core of each SCR.
Four conserved cysteine residues are disulfidebonded to each other form a Cys(I)
Cys(III) and Cys(II)Cys(IV) motif.
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Figure 4 Regulators of Complement Activation (RCA) Gene Cluster. In humans, a number of complement regulatory genes are encoded on the long arm of chromosome 1q in a region called the RCA gene cluster. These plasma and cell membrane associated regulatory proteins work to control both fluid phase and cell surface complement activation. In addition to encoding a number of fluidphase regulators such as CFH and the CFHR gene family members and C4 binding protein (C4BP), the RCA cluster encodes a number of other important regulators. These proteins include CD46 known as membrane cofactor protein (MCP) along with complement receptor 1 (CR1), complement receptor 2 (CR2), and decay accelerating factor (DAF) which were mapped through extensive sequence analysis using yeast artificial chromosomes (YAC) (88, 92). The RCA members are not only physically close to one another on the chromosome but they share structural similarities and are composed of repeating domains called short consensus repeats (SCR) (89, 90). On a functional level, these proteins target the complement cascade at various points, working to dissociate C3 and C5 convertases and aid in FI cleavage and inactivation of C3b and C4b.
The FH and FHR proteins exhibit extensive homology to one another and are all encoded on separate genes located at position 1q32 towards the telomeric end of chromosome 1 (69, 93). The human CFH gene consists of 23 exons spanning 94 kb of genomic DNA. Each exon, with some exception, encodes a single SCR domain by undergoing type I splicing after the first base of the exonterminal codon (94).
Exon 1 encodes the signal peptide for FH as well as the FHR proteins. FH SCR 2 is
17
encoded by exons 3 and 4 while exon 10 encodes the last amino acid residues
(SFTL) and the 3 untranslated region of an alternativelyspliced variant called FHL
1, which contains SCR domains 17. Situated next to CFH are the CFHR genes which contain several repeating regions believed to have resulted from non homologous recombination events leading to the production of FHR proteins with similar domains to FH.
The murine homolog of FH is positioned on the reverse strand of chromosome 1F. The mFH gene was identified to have 22 exons over a similar span of ~100 kb and shares 63% homology with human CFH (95). Unlike its human counterpart, the mFH gene does not have a FHL1 variant although it does contain an unspliced exon (exon 9) that could encode a SCR domain with a stop codon. At least five murine FHR genes exist and two proteins have been previously characterized; however, direct comparison to the human CFHR gene family is not straightforward (96, 97).
Extensive analysis of the human CFH and CFHR gene loci using Alu/L1 repeat dating established that duplication events between and FH and FHR exons likely occurred after the separation of rodent and primate lineages (94). The data suggest that direct comparisons between the human and mouse CFHR loci will be not as informative as experiments looking at how the proteins may act as functional homologs. The CFHR gene family is not the only group to have undergone more recent gene duplication events. C4BPAL1 is another complement gene within the
RCA locus that has undergone gene duplication from C4BPA after the separation of the rodent and primate lineages (98). Gene and exon duplications within the RCA
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cluster appear to be species specific and perhaps serve an important role in generating novel complement components. Given that one exon generally encodes one SCR functional domain and that each SCR can fold independently, it is not difficult to speculate how certain duplications of exons could create novel functional proteins that have the ability to exert regulatory control around complement components C3 and C4.
Mapping of the CFHR Genomic Region
More recently there has been an expansion in research towards understanding the five FHR protein family members. Unlike fulllength FH, the functional and physiological properties of these five molecules have not been extensively explored. One of the earliest papers on any of the CFHRs was a paper describing the isolation of murine FHRs by Vik et al. 1990 (96). Due to the extensive number of large genomic duplications between the exons of CFH and the CFHR genes, determining the genomic positions of the human CFHR genes was challenging and was performed throughout the early to mid1990’s.
Up until this point, it was only known that the CFH gene was ~7cM (~7Mb) away and positioned closer to the centromere than the other group of RCA gene members containing MCP, CR1, CR2, DAF, and C4BP (Figure 5). Characterization of the physical linkage of the gene encoding human coagulation Factor XIII established that this protein was encoded in a region between CFH and the other
RCA members (99). Next, four human FHR proteins were characterized in serum and the nomenclature for these proteins, labeling them as FHR14, was established
(100103).
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Figure 5 Comparison of Human and Murine CFH and CFHR Gene Organization. Analysis of the CFH and CFHR gene loci in humans determined that duplication events between CFH and CFHR exons likely occurred after the separation of rodent and primate lineages. Therefore, direct comparisons between the two species are not readily apparent. To complicate matters, the human CFH/CFHR gene locus is on the positive strand while the murine locus is on the negative strand. The human CFHR genes are numbered 15 while the murine genes are labeled as AE. MFHR A and mFHRD (in boxes) are labeled as pseudogenes in some genome browsers such as Ensembl.org. The nomenclature for the murine FHR family is not well established, so alternative names for each gene position are labeled under the gene.
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The position of CFHR2 was the first of the CFHRs to be determined when it was identified with a YAC placing it within the region between CFH and Factor XIII
(104). During the next several years, radiation hybrid mapping placed the other three
CFHR genes within the RCA cluster between CFH and CFHR2, but the similarities between the genes prevented the determination of their exact positioning relative to one another (105). The last CFHR gene, CFHR5, was initially discovered at the protein level in studies of glomerular immune complex deposition rather than at the cDNA level (106, 107). The genomic location of CFHR5 was determined using fluorescence in situ hybridization (FISH), radiation hybrid (RH) mapping and BLAST alignment analysis to map the position of CFHR5 (108).
Therefore, it wasn’t until 2002 that the genomic segment containing the CFH and CFHR gene family was confirmed to have the gene positions from centromere to telomere: CFH, CFHR3, CFHR1, CFHR4, CFHR2, CFHR5 as well as non complement factor XIIIB. Additionally, variants of FHR1 exist and alternatively spliced forms of CFHR4 have been identified as CFHR4A and CFHR4B leading to a total of nine or more different proteins and variants that are produced by the human CFH and CFHR gene family (109111).
By comparison, our knowledge of the murine FHR gene family and their gene positions is much more limited in scope as there have been far fewer studies examining the mouse FHR gene sequences and encoded proteins. Outside of the initial paper which characterized four murine transcripts isolated from liver in 1990, and another paper providing an initial characterization of two proteins, little is known about the functional homology between the murine and human FHRs (96, 97).
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Human FHR Proteins
All FHR proteins and variants share a high degree of sequence identity to FH in their two Nterminal SCR domains (ranging from 3694%) and their two Cterminal domains (ranging from 36100%) (Figure 6). The Nterminal domains of the FHR proteins are most similar to FH SCR domains 69 while the Cterminal domains share homology with FH SCRs 1920. An exception to this is FHR5 which shares homology to FH SCRs 67 as well as SCRs 1014 and 1920. The similar sequence homology of FHRs to FH SCR domains 67 and 1920 enables these proteins to bind molecules including heparin (SCR 7) and complement components such as
C3b or C3d. Notably, all FHR proteins lack domains that resemble FH SCRs 14, which are the domains responsible for the cofactor and decay accelerating activity of
FH.
While it was originally suggested that the ability of the FHRs to bind C3b/C3d may support FH function, for example by enhancing the cofactor activity of FH for FI, recent work has provided newer insights about different FHR functions. Recent studies have demonstrated that FHRs serve as antagonists of FH and can homodimerize or form heterodimers to compete with FH for binding of C3b/C3d
(112). The human FHR proteins can be divided into two groups: one group contains a dimerization motif shared by FHR1, FHR2, and FHR5 while the other group contains FHR3 and FHR4, which do not have a dimerization motif but may possibly form dimers. Outlined in the following section is a description of each of the different human FHR proteins.
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Figure 6 Human Factor H and Factor HRelated Proteins. Proteins encoded by the CFH gene family. FH consists of 20 short consensus repeat (SCR) domains. The first four domains are involved in regulation and C3b binding (red box) while the next four domains are involved in glycosaminoglycan (GAG) binding (green box), and the last two domains are involved in C3b/C3d and GAG binding (blue box). Factor H Like1 (FHL1) protein is an alternativelyspliced version of FH containing the first 7 SCR domains of FH. FHL1 has regulatory abilities. The FHR proteins 15 are shown with their SCR domains aligned to the SCR domain in FH with which they share the highest sequence identity (percentage identity is indicated). There are two isoforms of FHR4, a 9 SCR form (FHR4A) and a 5 SCR form (FHR4B). All FHR proteins contain highly homologous FH GAG binding regions (SCRs 58) and C3d/C3b binding domains (SCRs 1920). The FHRs lack the four regulatory domains (SCRs 14) found in FH. Putative Nlinked glycosylation sites are marked in orange and the molecular weights for each protein are indicated.
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FHR1
FHR1 contains five SCR domains and encodes two isoforms called FHR1, a 37 kDa protein and FHR1, a 43 kDa protein (110, 113). This protein shares higher homology with the first two domains of other FHR proteins than with FH.
FHR1 SCRs 12 have greater than 95% sequence identity to SCRs 12 of FHR2 and over 90% sequence identity to SCRs 12 of FHR5. The terminal three SCRs share nearly 100% homology with FH domains 1820. The two variants produced are denoted as the acidic variant (FHR1) and basic variant (FHR1) as they encode different residues, HLE versus YVQ, within SCR 3 (110). Two putative N glycosylation sites are predicted for this protein.
On a functional level, FHR1 binds to C3b and C3d and one study has shown that FHR1 inhibits complement C5 convertase activity and formation of the MAC
(114). FHR1 has also been shown to compete with FH for binding to C3 by forming homodimers and/or heterodimers with FHR2 and FHR5 to compete with FH for ligand binding (112, 115). The concentration of FHR1 in serum has been measured between 70100 µg/ml (2.1µM). Both FHR1 and FH have equimolar concentrations in serum (114, 116, 117).
FHR2
FHR2 consists of four SCR domains and is reported to have both a single glycosylated form (24 kDa) and a doubly glycosylated form (29 kDa) (100). Similar to
FHR1, FHR2 contains a dimerization motif within SCR1 and can form heterodimers with FHR1 but not FHR5 (112). The FHR2 domains with the highest sequence homology to FH are SCRs 34 which share 89% and 61% sequence homology with
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FH SCRs 1920. The concentration of FHR2 in serum is estimated to be around 50
µg/ml (1.72µM). While measurements of serum concentrations for FH, FHR1 and
FHR5 have been determined, the concentration of FHR2 is only an estimated value as there are no published measurements (112).
On a functional level, FHR2 protein has been shown in one study to bind
C3b and heparin and also act as a regulator of the alternative pathway (118). Other studies report that FHR2 does not compete with FH for binding to C3b and that
FHR2 is a regulator of complement activation (118, 119). Other research indicates that a hybrid FHR2/FHR5 protein acts in a deregulatory manner in patients suffering from C3 glomerulopathy with dense deposit disease (C3GDDD). Gene deletion in these patients produces a hybrid deregulatory FHR2/FHR5 protein that can stabilize the C3 convertase and reduce FH mediated convertase decay (120).
Other roles for FHR2 show that it is found in high density lipoprotein complexes
(121) and in addition to FHR1, can bind surface proteins on Borrelia burgdorferi
(122).
FHR3
The CFHR3 gene encodes a protein that contains five SCR domains. At least four glycosylated variants (between 35 kDa and 56 kDa) have been measured in human serum. FHR3 concentration is reported to be only 19 nM, or nearly 132 fold lower than FH (101, 115, 123). The FHR3 protein lacks the dimerization motif found in FHR1, FHR2, and FHR5. Three SCRs present at the Nterminal portion of FHR3 share high homology with FH SCR 6 (91%), SCR 7 (85%), and SCR 8
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(62%) while the last two domains of FHR3 (SCR 4 and SCR 5) share less homology with FH SCR 19 (64%) and SCR 20 (37%).
On a functional level, FHR3 binds the C3d region of C3b as well as heparin.
Early studies reported FHR3 to have weak FIcofactor activity (124). Later studies have shown that FHR3 acts as an antagonist of FH and competes for binding to
C3b on cell surfaces (115). FHR3 also competes with FH for binding to Neisseria meningitides and promotes immune activation (125). Most recently it has been shown that FHR3 has a novel role in B cell regulation. Data indicate that FHR3, unlike FHR1, binds C3d and blocks the interaction between C3d and CD21 impairing the molecular adjuvant effect that the coreceptor complex has with the B cell receptor (126).
FHR4
The CFHR4 gene has two alternatively spliced transcripts, encoding FHR4A and FHR4B (109, 127). The longer variant, FHR4A, contains nine SCR domains and has a MW of ~86kDa. FHR4A SCRs 14 and SCRs 57 are highly homologous to one another suggesting an internal duplication event has occurred. FHR4B is the shorter variant (~42kDa) and contains five SCRs that correspond to SCR 1 and
SCRs 59 of variant FHR4A. This FHR protein does not have the conserved dimerization domain that is found in the other FHRs.
Unlike FHR3, FHR4 does not reportedly bind heparin. However, in the presence of FH/FHL1, FHR4 may help enhance cofactor activity and inactivation of
C3b (124). Recent data has indicated that FHR4 SCR 1 can recruit Creactive protein (CRP), an acute inflammatory protein (128, 129). In addition to recruiting
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CRP, FHR4 can bind fluid phase C3b and activate complement to enhance local opsonization. FHR4 has been shown to activate the alternative pathway by acting as a scaffold for assembly of the AP C3 convertase. FHR4 bound to C3b can bind
FB and properdin to activate the C3 convertase and release C3a and C3b. This
FHR4C3bBb convertase was less sensitive to the decay acceleration activity of FH suggesting that FHR4 is a deregulator of the alternative pathway (129131).
FHR5
The CFHR5 gene encodes the longest human FHR glycoprotein which is composed of nine SCR domains and has a molecular weight of ~65 kDa (108). This protein contains a dimerization motif that is also present in FHR1 and FHR2. FHR
5 has been shown to circulate in serum as a homodimer as well as heterodimer with
FHR1 (112). Unlike the other FHR proteins, FHR5 has high sequence homology with FH SCR domains 1014 sharing between 4774% sequence identity. The C terminal domains of FHR5 have lower homology to FH SCR 19 (66%) and FH SCR
20 (43%). The concentration of FHR5 in serum is 36 µg/ml (between 0.050.09
µM), which is much lower than FH or FHR1 serum levels (132).
Studies with recombinant FHR5 show that heparin and CRP binding sites on
FHR5 are localized to SCRs 57 and that FHR5 inhibits AP C3 convertase activity in the fluid phase but not solid phase. While FHR5 has FIdependent cofactor activity, it is substantially less than FH. FHR5, like other FHRs, was observed to be part of high density lipid lipoprotein complexes (132). Allelic variations within FH and
FHR5 are present in MPGN II patients (133), and internal duplication of the first two
SCR domains of FHR5 creates a novel fusion protein found in patients with C3
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glomerulopathy (C3G) (134). Subsequent analysis of FHR5 and the internal duplicated mutant show that FHR5 can serve as a pattern recognition protein and can bind endothelial surfaces and anchor properdin (a complement activator). The mutant FHR5 bound endothelial surfaces and increased properdin recruitment which resulted in an increase in local complement activation (135). Another article has recently highlighted the role of FHR5 as a potential biomarker for metastatic progression in soft tissue sarcomas. The study reported that FHR5 levels are associated with the risk for metastatic progression, suggesting that FHRs may have important roles outside of complement regulation (136).
Human Disease Associations
Given the important role that FH has in regulating the AP of complement, a number of studies have linked FH dysregulation with disease. Mutations or polymorphisms in the CFH and CFHR gene family have been linked to a range of autoimmune diseases including several renal diseases such as atypical hemolytic uremic syndrome (aHUS), IgA nephropathy and diseases that have glomerular pathologies including dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and FHR5 nephropathy (76, 137, 138). Other autoimmune diseases associated with alterations within the CFH and CFHR gene family include systemic lupus erythematosus (SLE) and agerelated macular degeneration (AMD) (139141).
Several scenarios exist in which rearrangements result in the formation of hybrid proteins containing both FH and FHR segments. Other types of rearrangements include loss of one or more FHR proteins and production of autoantibodies against
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FH. Still other situations exist where FHR proteins have internal duplications which result in hybrid proteins.
Highlighted below are descriptions of the various genomic rearrangements that are known to occur within the CFH and CFHR gene family and a description of the illnesses with which they are associated.
C3 Glomerulopathy
The general definition of a C3 glomerulopathy is a disease resulting from abnormal complement activation through uncontrolled alternative pathway regulation leading to the deposition of characteristic C3 fragments within the glomerulus (142).
Diseases that are classified as C3 glomerulopathies include Dense Deposit Disease
(MPGNII), C3GN, and FHR5 glomerulopathy. Electrondense deposits in the middle layer of the glomerular basement membrane are pathognomonic for MPGNII while the C3GN and FHR5 glomerulopathies have subendothelial and mesangial deposits.
A common finding among patients with glomerulopathies is that they have experienced an infectious event prior to the clinical manifestation of their renal disease (143).
The glomerulopathy termed “FHR5 nephropathy” was originally identified in a cohort of patients of Cypriot origin and is characterized by a heterozygous internal duplication of SCR12 (exons 12) in FHR5 with the resulting mutant protein termed
CFHR51219 (144, 145). This same hybrid protein has also been identified in C3G patients of nonCypriot origin despite the proteins having different genomic breakpoints (134). In an Irish family, deletion of segments of CFHR3 and CFHR1
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created a hybrid protein containing SCR domains 12 of FHR3 combined with the entire FHR1 protein (CFHR312CFHR1) (146).
Duplications of FHR1 SCRs 14 have been reported in a Spanish family with
C3GN (147). These duplications created a FHR1 protein that was 9 SCRs in length rather than 5 SCRs and the mutant protein was able to form large multimers that blocked the ability of FH to protect surfaces from complement activation. A separate study showed deletion of exons 45 of FHR2 combined with fulllength FHR5 created a hybrid protein with the same structure as CFHR51219 (120). In the case of
DDD, two polymorphisms within the promoter region of CFHR5 were identified along with a Pro to Ser mutation occurring in FHR5 SCR 2 (133). While the exact functions of the FHR proteins are not fully understood, the mutant proteins illustrate the importance of these proteins in human health and how gene alterations within the CFH and CFHR gene family can influence complement regulation.
Atypical Hemolytic Uremic Syndrome
Clinical diseases manifestations of hemolytic uremic syndrome (HUS) include hemolytic anemia, thrombocytopenia, and renal failure attributed to platelet thrombi in the microcirculation of the kidney. Atypical hemolytic uremic syndrome (aHUS) is different from HUS in that the disease is not induced by exposure to a shiga toxin producing bacteria or a virus. Even the initial symptoms of aHUS, including diarrhea, are not as severe. Unfortunately, patients often experience recurring disease and suffer from numerous complications including endstage renal failure. Also unlike
HUS, 60% of aHUS cases are genetic and attributed to mutations within genes
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encoding C3, CD46 (MCP), FB, CFH, CFHR1, CFHR3,C FHR4, and factor I among other genes, some of which are not part of the complement system (148).
Disease resulting from deficiency of FHR plasma proteins and presence of
FH autoantibodies is referred to as the disease subgroup, DEAPHUS and is responsible for ~6% of all aHUS cases (149, 150). Initial studies showed that deletion of CFHR31 (hFHR31Δ) was strongly associated with development of aHUS (151), while subsequent research demonstrated a correlation between hFHR31Δ and the development of autoantibodies against FH (152). The FH autoantibodies were mapped to the Cterminus of FH and in functional assays autoantibodypositive patient plasma enhanced hemolysis of sheep erythrocytes by inhibiting the protective function of FH on these surfaces (153). Deletion of CFHR1 and CFHR4 genes has also been identified in patients with DEAPHUS suggesting that FHR1 may have an important role in influencing autoantibody formation (154).
Interestingly, the one genome wide association study has identified the hFHR31Δ as conferring protection against another kidney disease, IgA nephropathy (155), while other research has identified rare variants in FHR5 that appear to increase susceptibility to the same disease (156).
Genome rearrangements between CFH and CFHR also occur and have been identified in different aHUS patients. These include rearrangements between FH and
FHR1 resulting in hybrid genes that link either the first 18 or 19 SCR domains of FH with the last 1 or 2 SCR domains of FHR1 (86, 157). This hybrid was shown to have decreased C3b/C3d binding rendering it less effective at protecting cells from
31
complement activation (158, 159). While this hybrid does not impact FH binding to surfaces, it has been shown to be a competitive inhibitor of FH (160).
Another hybrid consisting of 24 SCR domains has been reported and is a fusion between FH SCRs 119 with FHR3 SCRs 15 (161). Unlike the FH/FHR1 hybrid which was created though nonallelic homologous recombination secondary to the presence of segmental duplications, this hybrid arose by deletion occurring through microhomologymediated end joining (162). This abnormally large hybrid does not appear to have cell surface regulatory activity, but has normal fluid phase
FH activity.
AgeRelated Macular Degeneration
Agerelated macular degeneration (AMD) is a heritable retinal disease that impacts photoreceptors and results in the loss of central vision, predominantly in elderly individuals. During the early stages of disease, complement deposits, known as drusen, are formed between the retinal pigment epithelium and Bruch’s membrane. A number of complement components, including activators and regulatory proteins, have been found in the drusen deposits leading to the suggestion that drusen biogenesis could be mediated by chronic local inflammatory events and local complement regulation (163).
AMD is classified as wet AMD (neovascular or exudative) or dry AMD
(atrophic) with the former condition resulting in more severe vision loss. Wet AMD occurs when vascular endothelial growth factor (VEGF) synthesis and secretion induces vessel growth through the bloodretinal barrier into the subretinal space in a process known as choroidal neovascularization. Dry AMD is more common and
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occurs more slowly with drusen forming on the retina below the macula.
Inflammation and complement activation have an important role in the pathogenesis of dry AMD.
Several studies have shown that polymorphisms in FH, particularly the mutation of tyrosine to histidine at amino acid position 402 in SCR7, result in a loss and reduction of FH binding to heparin and CRP (164). Interestingly, homozygous deletion of hFHR31Δ lowers the risk for AMD (165), but increases susceptibility to aHUS as well as systemic lupus erythematosus (139, 151). Furthermore, one other
FHR protein, the FHR1A acidic variant, has been strongly associated with AMD
(166).
Given the strong association between the CFH and CFHR gene family and risk for AMD, a recent study examined the transcript levels of CFH and CFHR in the central and peripheral retina and retinal pigment epithelium/choroid/sclera (RCS) compared to transcripts in the liver. This study found no expression of any of the
FHR genes in any eye tissue, however FH and FHL1 transcripts were present in the
RCS and retina (167). All genes were found to be expressed by the liver. This study conflicts with earlier results that reported high expression levels of FHR1 and the presence of other FHRs within the retina (168, 169).
FH and FHR Protein Involvement with Pathogens
In addition to their association with autoimmunity, FH and FHR proteins have been associated with several pathogens and pathogenrelated illnesses. FH recruitment is a strategy used by pathogens to bind FH to their surface and evade immune detection. FH recognizes and binds polyanionic residues such as
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glycosaminoglycans (GAGs), heparin sulfate, and sialic acid primarily through SCR domains 68 (SCR 7 in particular) and SCR 20 (170172). In general, pathogens do not have rich polyanionic surfaces, however they have several surface proteins that can recruit FH and FHR proteins.
The common childhood disease, otitis media with effusion (OME), has been linked to AP dysregulation with FH and FHR proteins 15 identified in the middle ear effusion fluid (173). The Grampositive bacterium, Streptococcus pneumoniae, a common cause of otitis media, sinusitis, pneumonia and other diseases, and can bind FH, FHL1, and FHR1 through elongation factor Tu (Tuf) (174). The Group A streptococci M protein binds FH through SCR 7 (175). The M protein and another streptococcal collagenlike protein 1 (Scl1) are reported to bind FHL1 as well as
FHR1 (176180). FHR1, unlike FHRs 24, was shown to bind streptococcal proteins Scl1.6 and Scl1.55 suggesting this protein has a diverse and unique role which is distinct from other FHRs (177).
Another pathogen, Borrelia burgdorferi, has five surface proteins
(complement regulatoracquiring surface proteins, CRASP 15) that bind FH. FHR1 can bind three of the CRASP proteins (CRASP35) (181, 182) while FHR2 and
FHR5 have also been shown to bind these proteins (122). Other organisms that bind FHR proteins include Candida albicans (FH, FHL1, FHR4) (183, 184), conidia from Aspergillus fumigatus (FH, FHL1, and FHR1) (185), Borrelia spielmanii and
Borrelia hermsii (FHR1, FH) (186188), and Leptospira and Leptospira interrogans
(FH, FHL1, FHR1) (189, 190).
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Sialylation of group B Neisseria meningitidis has been shown to regulate alternative pathway activation by binding of FH (191). Both FH and FHR proteins interact with sialic acid structures on N. meningitides and N. gonorrhoeae.
Additionally, Por1A protein also interacts with FH, FHR1, and other FHRs (192).
Recently it has been demonstrated that competition between FHR3 and FH for binding to N. meningitides influenced susceptibility to disease (125). The bacterium cannot distinguish between FHR3 and FH binding, and it has been proposed that the level of protection against complement attack is related to the ratio between serum FH and FHR3 levels (125).
In addition to bacterial pathogens, FH and FHR proteins have also been shown to interact with parasites and viruses. Recruitment of FH to the surface of the human malarial parasite, Plasmodium falciparum, through FH SCRs 5 and 20 has been described (193, 194). FHL1 and FHR1 are also reported to bind schizonts as a complement evasion strategy by the parasite (194). FH has been shown to interact with gp41 and the C1 domain of gp120 of the human immunodeficiency (HIV) virus
(195, 196). A very recent study has shown that hepatitis B virus protein X can upregulate a microRNA (miRNA146a) which in turn downregulates FH expression
(197).
Taken together, these studies highlight a range of organisms which include bacteria, fungi, parasites and viruses, that are able to bind FH and FHR proteins as a way to subvert the host immune system. Hence, better characterization of the FHR protein family will be beneficial as it will help advance our understanding of the
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contextdependent functions of these proteins and hopefully generate new strategies for treatment.
Murine Factor HRelated Gene Family
Initial Characterization of Murine FHR Transcripts
Murine factor Hrelated (mFHR) proteins were first characterized by Vik et al.
1990 (96). Using BALB/c liver poly(A)+ RNA and fulllength mouse H cDNA as a probe, four different transcripts were isolated, one of which was fulllength mFH.
They next isolated several additional clones from a murine cDNA library (built with
RNA isolated from livers of C57B10.WR mice), again using fulllength mFH cDNA as a probe. From this screen, twelve different clones (with restriction maps that differed from mFH) were isolated and divided into seven groups based on their respective nucleotide sequences and length. From these seven groups, the mFHRs were divided into classes, which are labeled AD.
Class A clones were defined as having three regions: a 5untranslated region and putative leader sequence (reported as having <50% homology with FH), seven SCR domains, and a 3untranslated region. They deduced that this class contained SCR domains which share high sequence identity with SCR domains 59 and SCRs 1920 of fulllength factor H.
Within the class A group, four different clones (listed as 3A4, 8D11, 5G4, and
4C10) were described. The 3A4 clone was described as being the most similar to
FH sharing homology with mFH regions encoding SCRs 59 and SCRs 1920 with the exception of 51 nucleotides. However, this same clone contained two regions adjacent to a homologous mFH SCR 9 domain that did not share homology with
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mFH. It was assumed that sequences around this domain contained stop codons and/or were the result of retained introns from alternative RNA splicing. The other three clones did not appear to have the retained intron fragments around the SCR 9 like domain as seen in 3A4. Instead, these clones had different 5 start and 3 and stop positions.
The class A clone 5G4 was characterized as starting 12bp into a mFH SCR
6like domain, while another clone, called 4C10, was reported to start in a region corresponding to a mFH SCR 8like domain. Yet another clone, 8D11, was unique from the others as it contained 116 bp homologous to a region enconding mFH SCR
4, likely representing a partial cDNA from alternatively spliced mRNA.
Based on the different variants reported within this group all sharing high homology to mFH, alternative splicing likely plays a major role in this class of murine
FHR proteins. Therefore, the authors decided to assemble a prototype mRNA to represent class A. Given that all four clones within class A have different start and stop sites within domains that have high homology to mFH domains encoding SCRs
49 and mFH SCRs 1920, the prototype mRNA called 3A4/5G4 was assembled to represent this class of mFHRs. This 3A4/5G4 prototype was built using the 5 portion of the 3A4 clone and the 3 portion of the 5G4 clone and is predicted to encode a protein with 7 SCR domains.
The class B clone (listed as 23L1) sequence was deduced to have 4 SCR domains sharing high sequence identity with SCRs 5, 6, 7, and 19 of fulllength mFH. The class B clone also was reported to contain a unique 3untranslated region not found within the other classes. The class A and B clones were highly
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homologous to one another containing regions that resemble SCRs 57 and 19, although class A clones were found to have additional 2 additional SCR domains while the class B did not have these domains.
The class C clone, known as 9C4, was listed as having a similar structure to the class A and B clones, but was predicted to have more SCR domains. This class was described as having 13 SCRs with similar sequence identity to fulllength mFH
SCRs 614, SCRs 1617, and SCRs 1920. Additionally, this class was described as having a 5untranslated region similar to the 3A4 clone (with the exception of three nucleotides) and 3untranslated region resembling the sequence found in fulllength mFH.
The last group, called class D, contained clone 13G1 which was deduced to have five SCR domains corresponding to fulllength mFH SCRs 6 and 7, with two
SCRs sharing high identity with FH SCR 19, and one SCR similar to SCR 20. The first two SCR regions of class D clones have only 35% and 51% sequence identity to
FH SCRs 6 and 7, while the next two domains share 90% identity with SCR 19, and the last domain has 69% identity with SCR 20.
From the original four classes of mFHR that were described, the prototype A class was reported to be the most similar to fulllength mFH (96%) while class B was the next most similar (~94%), followed by class C (~90%), and then class D (62%)
(96).
To better understand whether the mFHR clones were encoded by separate genes, or whether they were products of alternative splicing by the mFH gene, the group also analyzed a separate group of nine genomic mFHR cosmid clones. These
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clones were isolated in a prior study that characterized the murine mFH gene locus using cosmid clones from a BALB/c library. The clones were found to be distinct from cosmid clones which define the mFH gene. From this separate analysis, they determined that the class A sequence was located 5 to the class B sequence followed by the class C gene. They could not characterize the gene location for the class D clone using these hybridization techniques as the sequence for this class was much different than FH.
In summary, the authors presented data providing the first evidence of murine
FHR transcripts and the presumed genomic order for classes AC. DNA sequence analysis of different clones isolated from a liverderived cDNA library was used to create these four different classes of mFHRs. Protein sequences for each of the transcript classes were deduced and compared to SCR domains found in fulllength mFH. A prototype for the class A mFHR was predicted to have 7 SCR domains.
Class A shares extensive homology with the class B mFHR transcript which was predicted to encode a protein with 4 SCR domains (now known as having 5 SCRs).
Class C was deduced to have 13 SCR domains (now known as having 14 SCRs) and class D clones, which were the least similar to mFH in identity, were deduced to have 5 SCR domains (Figure 7).
Initial Characterization of Two Murine Factor HRelated Proteins
After the initial characterization of the four classes of mFHR transcripts in
1990, this gene family was not thoroughly examined until Hellwage et al. 2006 published data on mFHR protein expression (97). After detecting two putative mFHRs with molecular weights of ~100 kDa and 38 kDa in mouse plasma using FH
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Figure 7 Initial Characterization of Murine Factor HRelated (mFHR) Transcripts. A) Schematic showing initial experiments and characterization of four murine FHR transcript classes by Vik et al. 1990 (96). B) Fulllength protein structure of murine factor H (mFH) containing 20 short consensus repeats (SCRs) with the regulatory region (red box), glycosaminoglycan binding region (green box), and C3b/C3d binding region (blue box) indicated. C) Four classes (AD) of mFHR mRNAs encode regions highly similar to mFH. The regions in blue were deduced to encode SCRs corresponding to the mFH SCR indicated by the number inside the box. The Class A 3A4/5G4 transcript is a prototype mRNA and was assembled based on the sequence of two class A clones. The 3A4 clone had intronlike sequences (orange) in a region resembling mFH SCR 9 which were omitted in the prototype 3A4/5G4 transcript sequence.
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specific antiserum, this group used a liver cDNA library to identify three transcripts.
These transcripts corresponded to the class B and class C clones described by Vik et al. 1990 (96).
Thorough analysis of these transcripts showed that several differences from the originally characterized transcript classes existed. They named the transcript resembling the class B clone as “FHRB” and described it as having 5 SCR domains, compared to the class B (23L1) clone which was originally predicted to have 4 SCR domains. They indicated that 23L1 may represent a splice variant of the mFHRB gene. Hellwage et al. 2006 also identified two transcript variants that resembled the class C clone (9C4) (97). They named these variants FHRC and
FHRC_v1 and described them as having 14 and 13 domains respectively. The
FHRC and FHRC_v1 varied from 9C4 by 16 different amino acids in each domain.
Following their sequence analysis of the of mFHRB and mFHRC transcripts, this group examined the expression of mFHRB in various C57BL/6 murine organs and reported that mFHRB levels were ~140fold higher in liver than fulllength mFH transcript levels. They also successfully expressed recombinant mFHRB using
Pichia pastoris, adding that this protein appeared overglycosylated using this expression system. ELISA assays using immobilized human C3b showed binding of the recombinant mFHRB protein (not deglycosylated). The interaction between deglycosylated recombinant mFHRB and heparin was also investigated using heparin affinity chromatography followed by western blot.
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To examine native mFHRB and mFHRC interactions, wildtype mouse plasma was incubated in wells of a microtiter plate coated with either human C3b or heparin. Following incubation of the mouse plasma with the coated C3b wells, proteins were eluted and analyzed using western blotting with mouse FHspecific antiserum. From bands present on western blot analysis, they concluded that mFHRC bound to human C3b. They did not observe a band at molecular weight
~40 kDa, which would be representative of mFHRB. Therefore, they suggested that the mFHRB and C3b interaction was a weaker affinity interaction than the interaction between mFHRC and human C3b.
From this same analysis, the authors reported an apparent interaction between heparin and both mFHRC and mFHRB. Finally, they performed cell binding assays in which mouse plasma was incubated with human umbilical vein endothelial cells (HUVEC) and western blotting with mouse FHspecific antiserum was used to indicate binding of mFHRC protein. Taken together, this study confirmed transcripts from two previously defined classes (B and C) of murine FHR genes. They also established the nomenclature for labeling the mFHRs as AD based on their genomic positions on mouse chromosome 1. A summary of their experiments and conclusions is provided (Figure 8).
Regarding Current Knowledge of the Murine Factor HRelated Gene Family
With thousands of publications about human FH and over 100 publications concerning the human FHR family, it is surprising that there are less than a handful of publications regarding the murine FHR gene family. The two main publications by
Vik et al. 1990 and Hellwage et al. 2006, which were extensively described in the
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Figure 8 Outline of Murine Factor HRelated Experiments and Conclusions by Hellwage et al. 2006. A summary of the experiments and conclusions made by Hellwage et al. 2006 (97). Differences in sequence were noted for both mFHRB and mFHRC transcripts compared to the original description of these transcripts by Vik et al. 1990 (96).
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previous section, provide an introduction to the murine FHR family of transcripts and putative proteins (96, 97). However, little subsequent research has been done to characterize these genes and evaluate the functional roles of the proteins they encode.
A more recent publication by Mehta et al. 2014 reported the expression levels of mFHRB and mFHRC in different murine models of autoimmune disease (198).
They evaluated changes in mRNA levels of mFH, mFHRB, and mFHRC in mouse disease models of dense deposit disease (DDD), diabetes mellitus (DM), basal laminar deposits (BLD), collagen antibodyinduced arthritis (CAIA), and systemic lupus erythematosus (SLE). In their study, they noted expression of mFH, mFHRB, and mFHRC in liver tissue from diseasefree animals, however they observed a complete absence of mFHRC mRNA in two animal models (SLE and DM).
In addition to this study, a review outlining the human and murine gene families was published by Pouw et al. 2015 (199). The authors noted that while several studies have compared the structure and function of murine and human FH proteins, less is known about the similarities between murine and human FHR proteins. Additionally, the concentrations of murine FH and the FHRs in serum are unknown (199). The authors also outlined the various autoimmune diseases associated with the human CFH and CFHR gene family, and noted that in order to evaluate potential therapeutics, drug testing in murine models of disease is necessary. Given our incomplete knowledge of the murine factor Hrelated gene family, translating results from murine models to their corresponding human FHR counterparts is extremely limited.
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One of the initial limitations to studying the murine FHR gene family may be with understanding the nomenclature and how it contrasts with the human FHR gene family. When Hellwage et al. 2006 introduced the nomenclature for the murine FHR genes labeling them alphabetically as A, B, C and so forth, a current search of various genome browsers including the USCS genome browser or Ensembl, lists these same genes as CFHR4/2, CHFR2, FHR1 or FHR3 (97). For example, the murine gene referred to as “CFHR1” is found at the mFHRE gene position located furthest away from mFH. This same gene was originally classified as a “class D” mFHR transcript and is also listed as 13G1.
To avoid further confusion, a summary of the genomic positions of the murine
FH and FHR genes and their encoded proteins is provided and is the basis for the experiments described within the following chapters of this thesis (Figure 9). A table summary for each of the the different murine genes and encoded proteins is provided in Appendix A. Two online genome browsers were used to compile the information listed within these tables. The chromosomal locations for mFH and mFHRs are provided, in addition to number of exons, descriptions of possible splice variants, and other pertinent information such as alternate names, protein sequence information and Uniprot identification numbers.
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Figure 9 Murine FH and FHR Gene Family and Schematic Representation of mFH and mFHR Proteins. A) Representation of mFH and mFHR genes and their locations on murine chromosome 1. Boxed regions indicate genes listed by some databases as unprocessed pseudogenes. B) Similar to human FH, mFH has 20 SCR domains. The first four domains are involved in regulation and C3b binding (red box). The next four domains are involved in glycosaminoglycan (GAGs) binding (green box), and the last two domains are involved in C3b/C3d and GAG binding (blue box). Two of the murine FHR proteins, mFHRB and mFHRC, have been previously described. The mFHRA prototype is designed based on a sequence described by Vik et al. 1990 (96). mFHRD is listed as an unprocessed pseudogene, although it contains regions that are 94% homologous to mFH SCRs 1920 (grey circles). The mFHR proteins AE are shown with their SCR domains aligned with SCR domains of mFH (percentage identity shared is indicated). Splice variants have been reported for mFHRB and mFHRC but are not shown. Putative Nlinked glycosylation sites are marked in orange and the molecular weights for each protein are shown.
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Scope of Research
Complement factor H and FHR proteins have been reported in a wide range of species ranging from zebrafish to higher mammals such as humans, rats, and mice (200). Murine FH (mFH), like its human homolog, is heavily glycosylated, and is comprised of 20 SCRs. Both human and murine FH proteins share high sequence identity (~61%), suggesting functional similarity. Like their human FHR homologs, a total of five murine FHR (mFHR) genes have been identified and evidence for four mFHR proteins have been inferred from mRNA transcripts isolated from mouse liver.
These predicted proteins exhibit high sequence identity with important ligand binding and selfsurface recognition domains of FH. Given that the FHR proteins display a high degree of sequence similarity to the innate immune regulator FH, it is thought that these proteins play an important role in complement regulation and that imbalances or dysregulation of CFH gene family members associates with various diseases. One question that remains unanswered is whether the murine FHR proteins are functional homologs to their human counterparts.
Why study the murine FHR proteins at all? Mice provide an excellent model system for studying various diseases and genetically modified mice in which different complement components have been knocked out (such as FB/ and FH / mice) are available for use. Furthermore, mouse models have been used to investigate the different human FHR proteins. Recent studies have examined the interaction of monomeric and dimeric human FHR5 protein with mouse C3 in vivo.
Recombinant human FHR5 wildtype and mutant proteins intravenously injected in
FH/ mice showed that FHR5 interacts with mouse C3 along the glomerular
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basement membrane (GBM) in the kidney in a specific and dosedependent manner
(112). Therefore, having a better understanding of this system is critical in order to develop insight about both human and mouse proteins which may lead to therapeutic interventions in humans.
To date, only a single study has examined the in vitro function of mFHR proteins. Researchers demonstrated that mFHRB and a variant of mFHRC were detectable in mouse plasma and they showed that transcript levels of mFHRB were higher than mFH in liver tissue. Recombinant mFHRB (expressed using P. pastoris) was also shown to bind heparin (a GAGsurrogate) and human C3b. These data reveal interesting preliminary findings, yet provide only a starting point for characterizing the functions of murine FHR proteins.
To address my hypothesis that mFHRs, like their human counterparts, regulate complement activation by antagonizing FH function, a comprehensive characterization of the mFHR proteins was necessary. Therefore, expression systems for producing recombinant mFH and mFHR proteins were analyzed and techniques for analyzing protein quality were developed. Hemolytic assays were used to assess the function of recombinant proteins. To characterize the interaction between mFHRs and C3b/C3d fragments, a combination of ELISA and surface plasmon resonance experiments were performed. To elucidate the functional homology between a conserved C3d binding site on the human and murine FHR proteins, mutant mFHRs were generated and their activity was assessed. Using in vitro experiments with different epithelial cell lines, the role of mFHRs on complement regulation on different cell surfaces was evaluated. Additionally,
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hamster antimFH and antimFHR antibodies were generated to create tools to examine native mFH and mFHR proteins in order to better understand their functions. While trial in vivo studies were performed, the results were inconclusive and further research is needed to generate a more complete picture of how mFHRs regulate the AP of complement in different animal models. From these analyses should flow a better understanding of how to modulate FHR function in a way that can potentially ameliorate human diseases to which these proteins contribute in a pathophysiologically important manner.
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CHAPTER II
MATERIALS AND METHODS
Protein Experiments
Sequence Analysis
DNA and protein sequences for the human and murine FH and mFHR families and C3b/C3d proteins were analyzed using the Bioinformatics Resource
Portal (ExPASy) of the Swiss Institute of Bioinformatics (https://www.expasy.org), the Ensembl database which is a joint project between the European Bioinformatics
Institute and the Wellcome Trust Sanger Institute (http://www.ensembl.org), and the
UCSC Genome Browser hosted by the University of California, Santa Cruz
(https://genome.ucsc.edu).
MFH and MFHR Construct Design
DNA sequences of fulllength mFH and mFHR and truncated constructs including mFHRA (based on the sequence for clone 3A4/5G4), mFHRB, mFHRC, and mFH1920 were generated commercially by GeneArt. The unique Uniprot identifiers for each protein are as follows: mFH Uniprot ID: P06909; mFHRA Uniprot
ID: Q61407; mFHRB Uniprot ID: Q4LDF6; mFHRC Uniprot ID: Q0KHD3; and mFH1920 Uniprot ID: P06909 residues 11121234. Each DNA sequence was codonoptimized and was engineered into a Gateway pDONR221 entry vector
(Life Technologies, Carlsbad, CA).
The usage of a Gateway recombination cloning vector allows for the shuttling of DNA inserts from entry clones (pDONR 221) into expression vectors for all of the commonly employed systems utilized in recombinant protein production such as
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expression in E. coli, yeast, insect cells, or mammalian cell lines. Each of the engineered mFH and mFHR constructs was designed using the same format. This design included an IgG kappa chain leader sequence followed by a linker region
(GlyAlaGlyAlaGlyAla), a Nterminal hexahistidine tag (His6tag), followed by another linker region (AspTyrAspIleProThrThr), and a Tobacco Etch Virus (TEV)
Nuclear Inclusion a (Nia) protease cleavage site (GluAsnLeuTyrPheGlnGly) for removal of the His6tag which is positioned 5 to the gene of interest. The constructs were designed so that the endogenous signal peptide sequence was removed and replaced with the IgG kappa chain leader sequence to allow for secretion of the expressed proteins.
After receiving the synthetically generated constructs, an in vitro recombination reaction was performed between the pDONR221 entry vector and a pcDNA3.2/V5destination expression vector using Invitrogen™ Gateway™ LR
Clonase™ II enzyme (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. Constructs were sequenced using T7 and V5 forward and reverse primers.
Mutant MFHR Construct Design
To create a series of point mutations and deletion constructs, a Quick Change
Lightning Site Directed Mutagenesis Kit (catalog number: 210519, Agilent
Technologies, Santa Clara, CA) was used according to manufacturer’s directions.
Generation of a N340A/D342A mFHRA mutant was created with the following primers that were codonoptimized for mammalian cell expression: forward primer
5 cct atc gac gcc ggg gct atc acc agc ctg3 and reverse primer 3 gga tag ctg cgg
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ccc cga tag tgg tcg gac 5 to create the following sequence: PIDAGAITSL.
Generation of a Q362A/Y365A mutant mFHRA protein was performed with the following primers: forward 5 gac tac cag tgt gcg aag tac gcc ctg ctg aag ggc3 and reverse primer 3 ctg atg gtc aca cgc ttc atg cgg gac gac ttc ccg5 to create the following sequence: DYQCAKYALLKG.
The mutant N220A/D222A mFHRB construct was created with the following primers: forward 5ccc atc gac gct ggg gct atc acc agc ctg3 and reverse 3ggg tag ctg cga ccg cga tag tgg tcg gac5 to create the following sequence: PIDAGAITSL.
Generation of the Q242A/Y245A mutant mFHRB protein construct was performed using the following primers: forward 5 gac tac cag tgc gcg aag tac gcc ctg ctg aag ggc 3 and reverse 3 ctg atg gtc acg cgc ttc atg cgg gac gac ttc ccg 5 to create the following sequence: DYQCAKALLKG. Additionally, two truncated constructs containing SCRs 13 of mFHRA and mFHRB were generated using the following primer sets. For the mFHRA truncated construct, the following primers were used: forward 5 c atc cgg atc aag acc tag tga tgc agc gcc agc 3 and reverse 3 g tag gcc tag ttc tgg atc act acg tcg cgg tcg 5 to create the following sequence:
CIRIKT(Stop Stop)CSAS. For the truncated mFHRB construct, the following primers were used: forward 5 c aac agc acc agg acc tga tag tgt ggc ccc 3 and reverse 3 g ttg tcg tgg tcc tgg act atc aca ccg ggg 5 to create the following sequence:
NSTRT(Stop Stop) CGP.
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MC3d Construct Design
A construct encoding wildtype murine C3d contained in a pCR Topo vector was obtained from a previous laboratory stock. This construct was sequenced with
M13 forward 5 gta aaa cga cgg cca g3 and M13 reverse primers 5cag gaa aca gct atg ac3 to confirm that the construct sequence matched the following GenBank accession number: ACG50177.1 or Uniprot ID: P01027 (residues 10021303).
However, a point mutation was found in this sequence. To change the sequence from LYNVETTSY to the original GenBank sequence, LYNVEATSY, the following primers were used : forward: 5ctc tac aac gta gag gcc aca tcc tac 3 and reverse:
3gta gga tgt ggc ctc tac gtt gta gag5. Additionally, primers were used to mutate
C1010A to prevent dimerization and reactivity of C3d by mutating the sequence from
PAGCGEQN to PAGAGEQN. The thioestercontaining domain (TED) of C3b is contained within this sequence. Four residues in this sequence (CysGlyGluGln) make up a fifteen member thiolactone ring which facilitates the covalent interaction between C3b and cell surfaces through residue Q1013. These primers (forward: 5 ccc gca ggc gct ggg gaa cag aac 3 and reverse: 3gtt ctg ttc ccc agc gcc tgc ggg
5) and the Quick Change Lightning Site Directed Mutagenesis Kit were used
(catalog number: 210519, Agilent Technologies, Santa Clara, CA) followed by transformation of X10Gold Ultracompetent (Stratagene, USA) cells with the PCR reaction and selection of the corrected construct.
Subcloning of the corrected mC3D construct into a pGEX2T GST expression vector (GE Healthcare Life Sciences, Uppsala, Sweden), which contains a thrombin cleavage site to remove the GST tag, was performed. Dual digestion of both vectors
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was performed with BamHIHF and EcoR1HF restriction enzymes in Buffer 4 per manufacturer’s specifications (New England Biolabs, USA) followed by gel purification and fragment ligation using T4 DNA ligase. Both pGex 5 forward (5 ggg ctg gca agc cac gtt tgg tg 3) and 3 reverse (5cg gga gtg ca tgt gtc aga gg
3) primers were used to sequence and confirm the identity of the mC3dpGEX2T
GST expression construct prior to transformation of BL21 (DE3) cells.
An additional mC3d construct was created after expression trials with the mC3dpGEX2T construct produced lower protein yields and removal of the GST tag with thrombin was not successful. A pGex6P1 vector (GE Healthcare Life
Sciences) was chosen because this vector has a PreScission protease site for on column cleavage of the GST tag. Briefly, the mC3d sequence was subcloned into the new pGex6P1 expression vector from the pGEX2T construct using the same restriction enzymes and protocol as discussed above.
Glycerol Stocks of MFH and MFHR Constructs
Glycerol stocks of the mFH, mFHR, and mC3d constructs were produced in order to streamline production of plasmid DNA. DH5α™ competent cells (Invitrogen) were transformed with mFH and mFHR constructs. XL10Gold cells (Agilient
Technologies) were transformed with mC3d constructs. Single colonies were selected from a LuriaBertani (LB) plate containing ampicillin and starter cultures were grown overnight shaking at 37ºC in 5 ml LBamp. To 0.5 ml of the overnight culture, 0.5 ml of 80% sterile glycerol was added in a sterile screw cap microcentrifuge tube. The sample was vortexed and frozen at 80°C. To streak out a single colony from the glycerol stock, a sterile flamed metal inoculating loop or
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disposable loop was used to scrape cells from the frozen glycerol stock onto a plate without allowing the original stock to thaw.
MFH and MFHR Protein Expression
DH5α™ competent cells were transformed with each of the constructs or glycerol stocks were used as starter cultures. Approximately 500 ml cell cultures were grown in LB to a cell density between 34 x 109 cells/ ml. Either an Invitrogen maxiprep kit or alternatively a QIAGEN™ HiSpeed® maxi prep kit was used to isolate ultrapure plasmid DNA. The yield of DNA from 1x500 ml culture typically produced enough DNA for transfection of ~1L of human embryonic 293 kidney cells
(Freestyle™ 293F).
The 293F Expression system (Thermo Fischer Scientific) was used for transient expression of mFH and mFHR proteins in serum free media. This system was chosen because of the ability to grow highdensity suspension cultures with native human glycosylation patterns in a serum free system. Transfections were performed according to the manufacturer’s instructions. Freestyle™ 293F cells
(Invitrogen, Carlsbad, CA) were obtained and subcultured in Freestyle™ 293
Expression Medium every three days.
Cells were passaged a minimum of five times after thawing prior to transfection. For a 125 ml culture volume, a 500 ml sterile disposable polycarbonate
Erlenmeyer flask with vent cap was used (Corning, NY, USA). Total cell density was approximately 2.5 x108 cells per each 125 ml culture. On the day of transfection, the cell density was approximately 1 x106 cells/ml as determined by counting cells using a Beckman Coulter Counter. For 125 ml transfections, ~150 µg of purified DNA was
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diluted into 2.5 ml OptiPro™ SFM. To this reaction, ~150 µl of FreeStyle™ MAX
Reagent was diluted into 2.5 ml OptiPro™ SFM and slowly combined with the DNA to a final reaction volume of 5 ml. After a 5 minute (min) incubation at room temperature (RT) to allow the DNAlipid complexes to form, cells were transfected by adding the mixture in a dropwise manner while swirling the flask. Transfected cells were incubated on an orbital shaker (135 rpm) in a 8% CO2 humidified incubator at 37°C. Cells were harvested 57 days posttransfection using centrifugation at 8,000 rpm for 15 min in sterile 250 ml tubes. Supernatant was filtered with a 0.22 μm filter and stored in aliquots at 80°C for later purification.
Proteins were purified using immobilized metal affinity chromatography (IMAC).
Polyethylenimine (PEI, Polysciences, Inc.) is a costefficient transfection reagent compared to Freestyle™ 293F Max Reagent. A PEI transfection protocol was also used for expressing protein in these studies. Using this method, 293F cells were subcultured every three days or when the cell density was between 4 x105 and
3 x106 cells/ml. The total transfection volume was the volume of medium used on day 1 in addition to an equivalent volume that was added on day 2 (therefore 50 ml of transfected cells on day 1 and 50 ml day 2 for a total transfection volume of 100 ml).
One day prior to transfection (day 0), cells were split to a density of 1 x106 cells/ml. On day 1, cells were counted and resuspended in fresh Freestyle™ 293F
Expression Medium to a density between 2.53 x106 cells/ml and DNA was diluted to
0.5 µg/µl in fresh medium (using 300 µg DNA /100ml transfection volume). DNA was added to the cells and incubated for 5 min with gentle shaking. A PEI stock solution
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was diluted to 0.5 µg/µl (using 900 µg PEI/100ml transfection volume) and added to the cells before incubating the cells on an orbital shaker according to previously described conditions. On day 2, a 220 mM stock of valproic acid sodium salt (VPA) was freshly made in sterile water. Cells were diluted 1:1 in prewarmed medium and
VPA was added to obtain a final VPA concentration of 2.2 mM. Cells were cultured for an additional 57 days before harvesting the supernatant for further purification.
MFHRB Expression in Pichia pastoris
Trial expression of mFHRB was performed in Pichia pastoris. A mFHRB construct codonoptimized for expression in yeast was designed and the synthetic
DNA construct was subcloned from the carrier plasmid into a P. pastoris pPICZα expression vector with Zeocin resistance. This vector included a strong inducible alcohol oxidase (AOX1) promoter to allow for high level gene expression in a variety of Pichia strains including X33, SMD1168H, and KM71H (201). The X33 P. pastoris strain was transformed with the linearized mFHRB construct using an
EasyComp™ Transformation Kit (Thermo Fisher Scientific) and 200 ml cultures were grown in shaker flasks. Protein was purified using IMAC techniques described in the following section.
MFH and MFHR Protein Purification
Purification of the FH and FHR proteins was performed using IMAC chromatography. Harvested supernatant from transfections was diluted using 5X buffer (0.1 M sodium phosphate pH 7.8, 0.1 M imidazole, and 2.5 M NaCl). Samples were applied to either a 5ml HisTrap (GE Healthcare Life Sciences) or handpoured
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column containing the appropriate volume of HisPur™ NiNTA Resin (Thermo Fisher
Scientific) as per manufacturer’s instructions. After applying supernatant over the Ni
NTA column, bound Histagged proteins were eluted using a linear imidazole gradient from 20 mM and 0.5 M imidazole. Most proteins were completely eluted from the resin using 0.2 M imidazole; however, 12 ml fractions were collected and analyzed by monitoring the OD ratio at 260/280. Protein purity was assessed by
SDSPAGE. Fractions containing purified protein were pooled and concentrated at
RT using a Vivaspin 20 spin concentrator (Millipore Inc.). Proteins were buffer exchanged into Dulbecco’s Phosphate Buffered Saline (DPBS: 2.7 mM KCl, 1.5 mM
KH2PO4, 138 mM NaCl, 8.1 mM Na2PO4, pH 7.4). A protein polishing step was performed by applying protein to a S300 HiPrep 16/60 Sephacryl size exclusion column (GE Healthcare) on an ÄKTAPure high pressure liquid chromatography system (GE Healthcare) which had been equilibrated with DPBS. Peak fractions were eluted in 1 ml increments and analyzed by SDSPAGE. Protein concentrations were determined using a NanoDrop Spectrophotometer (Thermo Fisher Scientific) by entering molecular weight (MW) values and molar extinction coefficients for each protein. Purified proteins were flash frozen in 50100 µL aliquots using liquid nitrogen and stored at 80°C. The S300 column was cleaned using a protocol recommended by the manufacturer and the column was stored in a 20% ethanol solution.
Column Calibration
To determine molecular weights of mFH and mFHR proteins using a S300
HiPrep 16/60 Sephacryl size exclusion column (GE Healthcare Life Sciences), a column calibration experiment was performed. A protein mixture (GE Healthcare Life
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Sciences, catalog: 17044501) was reconstituted using 115 µl of PBSA. Next, 40 µl
(10 mg/ml) BSA, 30 µl (10 mg/ml) ovalbumin, 30 µl (10 mg/ml) myoglobin, and 100
µl PBS were added to the mixture. The mixture was filtered through a spin column and 500 µl was injected onto the S300 HiPrep 16/60 Sephacryl column at a flow rate of 0.5 ml/min in filtered PBSA. A standard curve was generated by plotting the volume of each protein peak (ml) versus the log (molecular weight) of the protein using GraphPad Prism software version 7 (GraphPad Software, San Diego, CA).
Murine C3d Protein Purification
BL21 (DE3) cells were transformed using either mC3dpGEX2T or mC3d pGex6P1 expression constructs. Overnight 5 ml starter cultures were used to inoculate 8 x 500 ml of 2xYT medium (1.6% bacto tryptone, 1.0% bacto yeast extract, 0.5% NaCl, pH 7.0) plus ampicillin. Cells were grown at 37°C at 170 rpm until a cell density (OD600) of 0.3 was reached. Cultures were induced using 0.3 mM
IPTG and grown overnight at 20°C.
The following day, cells were harvested by centrifugation at 6000 rpm for 25 min at 6°C and pellets were resuspended using GST column buffer (50 mM TrisHCl
(pH 8.0), 250 mM NaCl, 1 mM EDTA). cOmplete™ EDTA protease inhibitor cocktail
(Roche) was added prior to sonication. Cells were lysed on ice using a XL2000
Qsonica machine by pulsing samples (using an amplitude of 40) six times for 10 seconds with 30 seconds rest between each step. The soluble protein sample was clarified using ultracentrifugation at 10,000 x g for 20 min before applying the sample to a 5 ml GSTrap 4b column (GE Healthcare Lifesciences, Pittsburgh, PA) which was preequilibrated with 10 column volumes (CV) of buffer.
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To remove the GST tag from mC3dpGex2T, 50 Units of thrombin was added to the column overnight at 4°C. The following day, mC3d was eluted using
GST column buffer. Uncleaved GSTmC3d was removed by adding GST column buffer with reduced glutathione. To the eluted mC3d fraction, 20 mM 4(2
Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) serine protease inhibitor was added to a final concentration of 1 mM. Both mC3d and GSTmC3d fractions were concentrated and protein purity was analyzed by SDSPAGE. For a polishing step, mC3d was applied to a S100 HiPrep 16/60 Sephacryl size exclusion column (GE Healthcare) on an ÄKTADesign high pressure liquid chromatography system (GE Healthcare) and protein was eluted from the column in DPBS.
SDSPAGE Analysis
To analyze mFH, mFHR, and mC3d protein purity, dithiothreitol (DTT) was added to samples which were heated at 90°C for 5 min. Samples were loaded onto either 10% NuPAGE BisTris gels (Invitrogen) or PhastGel 1012% gradient gels, which were run according to manufactuer’s instructions. For the NuPAGE BisTris gels, MES SDS running buffer was used and gels were run at 150 V for 1 hour for proper separation. A PageRuler™ Prestained Ladder (Invitrogen) or Novex® Sharp protein standard was used as a protein molecular mass marker. Gels were stained for 1 hour using Coomassie Blue Stain (0.5 g/L Brilliant Blue R250 dye, 50% methanol, 10% acetic acid) and destained (50% water, 40% methanol, 10% acetic acid) until protein bands were visible.
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Deglycosylation of Recombinant Glycoproteins
Proteins were deglycosylated using Peptide: NGlycosidase F (PNGaseF,
New England Biolabs), which cleaves between the innermost GlcNAc and asparagine residues of high mannose and complex oligosaccharides from Nlinked glycoproteins. Briefly, between 1020 µg of glycoprotein was added to a reaction containing glycoprotein denaturing buffer. Reactions were heated to 100ºC for 10 min before adding G7 reaction buffer, 10% NP40, dH2O, and PNGaseF. Reactions were incubated at 37ºC for 1 hour before deglycosylated proteins were analyzed using SDSPAGE.
Removal of His6tag from Recombinant Proteins
Per manufacturer’s instructions, AcTEV™ Protease (Thermo Fisher) was used to remove the Histag from recombinant mFH. mFH was buffer exchanged into
TrisHCl pH 8.0 with 1 mM EDTA, TEV buffer, and 0.1 M DTT. AcTEV™ Protease is a more stable version of the Tobacco Etch Virus (TEV) protease which recognizes a seven amino acid sequence (GluAsnLeuTyrPheGlnGly, cleaving between Gln and Gly). Histags were removed by incubation with the protease overnight at 4ºC.
Trial cleavage reactions were also performed at 37ºC for 4 hours. Following cleavage of the Histag, samples were buffer exchanged into PBS and applied to a
S300 HiPrep 16/60 Sephacryl size exclusion column to separate the cleaved Histag from the protein fractions. Fractions were subsequently analyzed using SDSPAGE.
Because an internal TEV cleavage site is predicted in mFH and mFHR proteins, further attempts at removing the Histag were not performed.
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Western Blot Analysis
Western blot analysis was performed to examine the expression levels of His tagged FH and FHR proteins in 293F cell supernatant. To detect the polyhistidine tagged proteins, two different antihistidine antibodies were tested. The first commercial antibody that was evaluated was an antipolyhistidine Clone HIS1 antibody (SigmaAldrich, catalog number: H1029). A 1:4000 secondary antimouse
IgGAlkaline Phosphatase antibody was used (SigmaAldrich, catalog number:
A3562). Western blots were performed according to the manufacturer’s instructions using a nitrocellulose membrane. This antibody did not appear to give consistent results. Therefore, a second antibody was used. The second antihistidine antibody tested was a HisTag® antibody HRP conjugate (Novagen, catalog number: 71840
3). A Polyvinylidene difluoride (PVDF) transfer membranes was blocked using 5% milk in TBST for 1 hour at RT. A 1:1000 dilution of antibody in blocking buffer was added to the membrane and incubated for 1 hour at RT. After washing the membrane, Pierce™ ECL Western Blotting Substrate reagent was added in order to detect proteins. Western blots were developed either manually using CLXPosure film (Thermo Fisher Scientific) and Kodak 2000A processor, or by scanning the membrane using a Typhoon FLA 9500 with a variable mode laser scanner (GE
Healthcare Life Sciences).
Differential Scanning Fluorimetry
To assess the folding and thermal stability of recombinant proteins, differential scanning fluorimetry, also known as Thermofluor, was used. The thermal stability profiles of mFH, mFHR, and C3d recombinant proteins were investigated by
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adding 75 µg/ml of protein in PBS (Corning, catalog: 21040CV) and dye in a total reaction volume of 30 µl. A fresh 300X stock of SYPRO Orange (5,000X) was diluted in fresh DMSO (SigmaAldrich Sweden AB, Stockholm, Sweden) and a final concentration of 10X SYPRO Orange was used. This specific dye has excitation/emission wavelength profiles of 490 nm and 575 nm. Experiments were performed in a 96well PCR plate (BioRad Laboratories AB, Solna, Sweden) and plates were sealed using OpticalQuality Sealing Tape (BioRad). An iQ5 Realtime
PCR Detection instrument was used to heat samples at 1°C/min from 20°C to 95°C with a fluorescence reading recorded every 0.2°C. The melting point (Tm) for each of the proteins was determined by fitting data to a Boltzmann model and data were normalized in GraphPad in order to make comparisons between different proteins
(202).
Circular Dichroism (CD) Spectroscopy
Recombinant mC3d was analyzed using CD spectroscopy. A Jasco J815 spectropolarimeter (Jasco, Inc., Easton, MD) with a Jasco DP500/PS2 system was used in this experiment. A Lauda model RMS circulating water bath (LAUDA
Brinkman, Delran, NJ) was used to maintain temperature control of the optical cell.
Results are expressed as mean molar ellipticity [θ] (deg·cm2·dmol1) and were calculated using the following equation:
� = ���� ∙ ���/ 10�� where θobs is the observed ellipticity in millidegrees, MRW is the mean residue weight of the peptide (peptide molecular weight/number of residues), l is the optical path length of the cell in centimeters, and c is the peptide concentration in mg/ml. A
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solution of 1 mg/ml mC3d in PBS was used in this experiment. Variable wavelength measurements of peptide solutions were scanned at 5°C from 195 nm to 250 nm, in
0.5 nm increments at a scan rate of 50 nm per minute.
Ligand Binding Experiments
EnzymeLinked Immunosorbent Assay (ELISA) with MC3d
To examine the interaction between mC3d and mFH/mFHR proteins, ELISA assays were used. Clear flat bottom polystyrene 96well microplates (Corning®) were coated overnight at 4°C with 50 µl/well of either mC3d or human C3d at a concentration of 10 µg/ml in 50 mM sodium bicarbonate buffer pH 8.8. Plates were washed 3 times using wash buffer (PBS + 0.05% Tween 20) before blocking with
100 µl PBS+ 1% BSA. Plates were washed three times in wash buffer before adding mFH or mFHR proteins. Following incubation with mFH/mFHR proteins for 1 hour at
RT, plates were washed again before adding a 1:1000 dil of anti6X His tag® antibody (Abcam, catalog: ab1187) in 100 µl PBS + 1% BSA for 1 hour at RT.
Finally, plates were washed three times before addition of 1Step Ultra TMBELISA substrate solution. After a 30 min RT incubation, samples were quenched with
H2SO4. Plates were read at wavelength 450 nm using an automated microplate reader.
Surface Plasmon Resonance
A Biacore® 2000 instrument (GE Healthcare Life Sciences) was used to calcuate apparent KD values for mFH and mFHR proteins and mC3d. A Series S
Sensor CM5 chip (GE Healthcare Life Sciences) was chemically coupled with 2500 response units (RU) of mC3d in 10 mM Acetate buffer pH 4.0. A pH scouting
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experiment was performed to determine the optimal buffer pH for amine coupling of mC3d to the chip. To assess binding to mC3d, mFH and mFHR wildtype and mutant proteins were serially diluted in PBS. Binding data were fit using a twostate binding model as determined using the Biacore® evaluation software.
Functional Assays
Erythrocyte Hemolytic Assays
The functional activities of mFH and mFHR proteins were analyzed using erythrocyte hemolytic assays. Sheep or rabbit red blood cells (Complement
Technology Inc.) were obtained and resuspended in MgEGTA buffer (0.1 M MgCl2,
0.1 M EGTA pH 7.3). To each 24 µl reaction, 0.51x106 cells (in MgEGTA buffer) were added to the bottom of a PCR tube. Either mFH or mFHR proteins were added to the cells followed by normal mouse serum (NMS) from C57BL/6 mice (40% final concentration). Reactions were incubated at 37°C for 30 min and quenched using
200 µl gelatin veronal buffer (GVB) buffer containing EDTA. Following centrifugation,
180 µl was read at OD412. As a negative control, the same reaction mixture was prepared using cells and normal mouse serum in PBS without recombinant protein.
The percentage of hemolysis was determined by subtracting the OD412 of the background hemolysis control (PBS + NMS) and dividing by the maximum hemolysis of 0.51x106 cells in sterile water.
CellSurface Assays
C3b deposition on Renal Tubular Epithelial Cells (TECs)
Murine renal tubular epithelial cells (TECs) were cultured in DMEM media supplemented with 10% FBS, 0.1 g/ml penicillin/streptomycin, and 1% non
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essential amino acids (Thermo Fisher Scientific). Cells were grown to 80–90% confluence prior to FACS analysis. The cells were trypsinized and then washed with
PBS. Cells were incubated in 10% normal mouse serum (NMS) in the presence or absence of increasing amounts of recombinant mFHR proteins. Surfacebound C3b was determined by incubating cells with a 1:50 dilution of FITClabeled goat anti mouse C3 Ab (Cappel #55500) in the dark on ice for 30 min. Cells were then washed twice and resuspended in FACS buffer (1X PBS with 1% BSA and 5 mM
EDTA). Samples were run on a FACSCalibur machine (BD Biosciences), and the results were analyzed using FlowJo software.
C3b Deposition on Retinal Pigment Epithelial Cells (ARPE19)
Cell conditions and experiments were performed according to previously published methods (203). ARPE19 cells, which are an immortalized cell line derived from human retinal pigment epithelial cells, were cultured in Dulbecco’s modified
Eagle’s medium F12 (Invitrogen) with 10% FBS, and 1X penicillin/streptomycin. For flow cytometry experiments, cells were treated with Accutase (Innovative Cell
Technologies), washed, and resuspended either in PBS or H2O2, which was used to stress the cells. Next, cells were incubated with mFHR proteins for 15 min prior to addition of 10% NMS and further incubation at 37°C for 35 min. Cell surface C3b deposition was determined using the same methods described for TEC cells.
Measuring Transepithelial Resistance (TER) of ARPE19 cells
The experiment listed in this section was performed by our collaborator
(Rohrer lab, Medical University of South Carolina). ARPE19 cells were grown using the previously described cell culture conditions on Transwell filters (Costar) to form a
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polarized monolayer. At least 24 hours prior to experiments, FBS was removed completely and media was changed prior to taking measurements. TER of the cell monolayer was determined by measuring the resistance across the monolayer using an EVOM voltohmmeter (World Precision Instruments). TER of the monolayer is proportional to membrane permeability and provides a method for evaluating barrier function of the RPE monolayer. Complement activation on the monolayer results in loss of barrier function and decreased TER values. For these experiments, two standards with H2O2 and either 2% or 10% NMS were used. To determine the effect of mFHRs on TER, 100 ng/ml of mFHRA and 100 ng/ml mFHRB were used and the percent loss in TER was determined after four hours.
Antibody Studies
MFH and MFHR Antibody Production
Armenian hamsters were obtained from Cytogen Hamsters (West Roxbury,
MA). Hamsters were prebled to obtain a stock of serum prior to immunization. On
Day 0, hamsters were immunized by subcutaneous injection of 100 µg protein in
Complete Freund’s Adjuvant (CFA). On day 14, hamsters were boosted with 50 µg protein in Incomplete Freund’s Adjuvant. On day 21, hamsters were bled and serum was tested by ELISA. Once a reasonable antibody titer was determined or after day
35, hamsters were given an intraperitoneal injection with 50 µg protein in PBS. On day 38 or later, the spleen was harvested and fused with mouse myeloma SP2/0 cells (obtained from the KapplerMarrack lab).
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B Cell Fusions
To prepare for the B cell fusion, minimum essential media (MEM) without fetal bovine serum (FBS) was gassed using 10% CO2 and placed in a 37ºC bead bath.
Freshly prepared 40% polyethylene glycol 8000 in 60% MEM (PEG8000) was sterile filtered. Spleen cells were teased apart in balanced salt solution (BSS), and cells were spun at 1500 rpm for 5 min. Cells were washed in 1X BSS and counted.
SP2/0 cells were also spun and washed in BSS and both cells were combined at a ratio of 5:1 (spleen cells: SP2/0) and incubated in a water bath at 37ºC for 10 min.
Following incubation, cells were centrifuged at 1500 rpm and the supernatant was aspirated from the cell pellet. Recombinant IL6 (500 Units/ml) was added to 120 mls of Complete Tumor Media (CTM) and used to resuspend the fusion pellet. Cells were plated in 96well microtiter plates with 100 µl/well and incubated at 37ºC in a
10% humidified CO2 chamber. To feed cells, a 3X HAT stock (10 mM sodium hypoxanthine, 40 µM aminopterin, 1.6 mM thymidine) was prepared and diluted in
CTM. On day 1 postfusion, 50 µl/well of 3X HAT was added to each well. Media was changed on days 5, 10, and 15 by adding fresh media (CTM+HAT+IL6).
Screening of hybridomas was initiated after day 10. Antibodies were purified using
Protein A affinity chromatography (GE Healthcare Life Sciences).
Screening Hybridomas
To screen for positive B cell hybridomas, mFH or mFHRs (100 µg/ml) in PBS were coated on Immunlon1B (Thermo Fisher Scientific) 96well plates overnight at
4ºC. The following day, plates were blocked using 200 µl TNN blocking buffer (10 mM Tris+150 mM NaCl+NaN3+30% FCS) and incubated for 30 min at 37ºC. Plates
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were washed three times using wash buffer (PBS+0.05% Tween20) and 100 µl of hybridoma supernatant was added to each well. Samples were incubated for 1 hour at RT and plates were washed three times. To each well, 100 µl/well of 1:10,000 goat antimouse IgGalkaline phosphatase (AP) conjugated antibody (Sigma, A
3562) was added and plates were incubated for 1 hour at RT. Following another wash step, 200 µl per well of substrate was added (onitrophenylphosphate+glycine buffer, pH 10.4) and incubated for another 30 min at RT. Plates were read by measuring the absorbance at OD405. A similar ELISA assay was used to determine the different isotypes of positive hybridomas. Instead of using goat antimouse IgG antibody, different specific biotinylated antibodies were used. These antibodies were produced and conjugated to biotin in the KapplerMarrack laboratory and were available for use. They included HIG65 (antiIgG2b), HIG88.2 (antiIgG2), HIG632
(antiIgG1), HIG229 (antikappa), and HIG346 (antilambda). A 1:3,000 dilution of
ExtrAvidin Alkaline Phosphatase (Sigma) was used as a secondary antibody.
SPR Analysis of AmFH Antibodies
A Biacore® 2000 instrument (GE Healthcare Life Sciences) was used to examine apparent KD values between recombinant mFH and two novel hamster anti mouse mFH antibodies, AmFH55.7 and AmFH70.6. HIG65 and HIG632 were immobilized to different flow cells on a Series S Sensor CM5 chip (GE Healthcare
Life Sciences). Injection of AmFH55.7 or AmFH70 bound HIG632. Next, recombinant mFH was injected in this twostep SPR assay. Kinetic analysis of
AmFH antibodies with recombinant mFH was performed using this binding sequence for 57 cycles followed by regeneration of the surface using regeneration buffer (2 M
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NaCl in acetate buffer, pH 4.6). BIAevaluation software was used to determine apparent KD values for both antibodies using a 1:1 binding model.
Quantifying MFH by Sandwich ELISA
A sandwich ELISA was developed to determine the concentration of mFH in different mouse sera. Both AmFH55.7 and AmFH70.6 were biotinylated for use in this assay as detection antibodies. Briefly, antibody was dialyzed into 0.1 M NaHCO3 pH 8.5. Following dialysis, 150 µl of 1 mg/ml NHSLCbiotin (Pierce, #21335) was added per mg of IgG. The reaction was rotated at RT for 4 hours and biotinylated antibody was subsequently buffer exchanged into PBS.
For the sandwich ELISA, either AmFH55.7 or AmFH70.6 were coated at 10
µg/ml in coating buffer (1X TNN) at 4ºC overnight. Plates were blocked with TNN +
1.0% BSA overnight at 4ºC. Plates were washed according to prior ELISA descriptions. Sera from wildtype, FB/, FH+/, or FH/ mice was serially diluted in
PBS. Serial dilutions of mFH (positive control) and mFHRs (negative control) were used in this experiment. BiotinylatedAmFH55.7 antibody (4 μg/ml) was added to plates and incubated for 2 hours at RT. Another wash step was performed and
1:3,000 dilution of ExtrAvidin Alkaline Phosphatase (Sigma) was used to detect biotin. Plates were read at OD405 using an automated microplate reader (BioTek, model ELx808) with program setting MKelisa. Data were analyzed by 4parameter logistic regression. mFH concentrations in the different sera were plotted using
GraphPad Prism software. Further work is needed to determine the reliability of these measurements and whether the antibodies can be used in reverse to obtain the same mFH measurements.
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Immunoprecipitation (IP) of MFHR Proteins from Serum
In order to IP mFHR proteins from serum, preclearing of the serum to remove all IgG antibodies was performed by adding 100 μL of Protein A Sepharose
4B (GE Healthcare Life Sciences) bead slurry to 50 μL of wildtype mouse serum.
Samples were incubated at 4ºC for 4 hours. Following centrifugation of the samples, precleared serum was incubated with 50 μg of AmFHRA231 antibody (which detects both mFHRA and mFHRB by ELISA) for 2 hours rotating at 4ºC. Protein A beads (50 μL) were added to the mixture for 30 min at 37ºC. Reactions were washed three times in PBS before eluting protein with 0.2 M glycine buffer (pH 2.6).
Precipitated proteins were separated under nonreducing conditions using gel electrophoresis as described in the next section.
Mass Spectrometry
Sample Preparation for Mass Spectrometric Analysis
Samples were loaded onto a commercial NuPAGE BisTris 10% gel
(Invitrogen). Electrophoresis was performed using MES SDS running buffer in an X
Cell midi gel system (Invitrogen) at 150 V for 1 hour, and the gel was stained with freshly prepared Coomassie Brilliant Blue. Selected gel pieces were destained in
200 µL of 25 mM ammonium bicarbonate in 50% v/v acetonitrile for 15 min and washed with 200 µL of 50% (v/v) acetonitrile. Disulfide bonds in proteins were reduced by incubation in 10 mM dithiothreitol (DTT) at 60°C for 30 min and cysteine residues were alkylated with 20 mM iodoacetamide (IAA) in the dark at RT for 45 min. Gel pieces were subsequently washed with 100 µL of distilled water followed by addition of 100 µL of acetonitrile and dried on SpeedVac (Savant Thermo Fisher).
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Then 100 ng of trypsin was added to each sample and allowed to rehydrate the gel plugs at 4 °C for 45 min followed by incubation at 37°C overnight. The tryptic mixtures were acidified with formic acid up to a final concentration of 1%. Peptides were extracted two times from the gel plugs using 1% formic acid in 50% acetonitrile. The collected extractions were pooled with the initial digestion supernatant and dried on SpeedVac (Savant Thermo Fisher).
Mass Spectrometry
Samples were analyzed on an LTQ Orbitrap Velos mass spectrometer
(Thermo Fisher Scientific) coupled to an Eksigent nanoLC2D system through a nanoelectrospray LC − MS interface. The source was operated at 2.25−2.5 kV, with no sheath gas flow, with the ion transfer tube at 275°C. A volume of 8 μL of sample was injected into a 10 μL loop using the autosampler. To desalt the sample, material was flushed out of the loop and loaded onto a trapping column (ZORBAX 300SB
C18, dimensions 5 x0.3 mm, 5 μm) and washed with 0.1% FA at a flow rate of
5 μl/min for 5 min. The analytical column was then switched online at 600 nl/min over an in housemade 100 μm i.d. × 150 mm fused silica capillary packed with 4 μm
80 Å Synergi Hydro C18 resin (Phenomex; Torrance, CA). After 10 min of sample loading, the flow rate was adjusted to 350 nl/min, and each sample was run on a 90 min linear gradient of 2–32% ACN with 0.1% formic acid to separate the peptides.
LC mobile phase solvents and sample dilutions used 0.1% formic acid in water
(Buffer A) (Optima LC–MS grade; Fisher Scientific, Pittsburgh, PA) and 0.1% formic acid in acetonitrile (Buffer B) (Optima LC–MS grade; Fisher Scientific, Pittsburgh,
PA). Data acquisition was performed using the instrument supplied Xcalibur™
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(version 2.1) software. The mass spectrometer was operated in the positive ion mode. Full MS scans were acquired in the Orbitrap mass analyzer over the m/z 300–1800 range with resolution 60,000 (m/z 400). The target value was 5.00 x105. The twenty most intense peaks with charge state ≥ 2 were selected for sequencing and fragmented in the ion trap with normalized collision energy of 35%, activation q = 0.25, activation time of 30 ms, and one microscan.
Database Searching and Protein Identification
MS/MS spectra were extracted from raw data files and converted into mgf files using a PAVA script (UCSF, MSF, San Francisco, CA). These mgf files were then independently searched against user database using an inhouse Mascot™ server (Version 2.2.06, Matrix Science). Mass tolerances were +/ 15 ppm for MS peaks, and +/ 0.6 Da for MS/MS fragment ions. Trypsin specificity was used allowing for 1 missed cleavage. Met oxidation, protein Nterminal acetylation, peptide Nterminal pyroglutamic acid formation, were allowed for variable modifications while carbamidomethyl of Cys was set as a fixed modification.
Animal Studies
Renal Ischemia/Reperfusion Model
An ischemia/reperfusion (IR) mouse model was evaluated in collaboration with the Thurman lab (University of Colorado) to examine how mFHR proteins modulate complement activation within the kidney. This in vivo model has been extensively used by the Thurman lab and is well described (204, 205). Briefly, animals were anesthetized with ketamine and given a retroorbital injection of 100 μg of either mFHRA, mFHRB, mFHRC, or PBS. Five animals per treatment group
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were used. Following injection of mFHRs, animals were placed on a heating pad and a midline incision was made. Both left and right renal pedicles were clamped for 20 min. Following removal of the clamps and closure of the incision, animals were given a 0.5 ml injection of sterile saline. Mice were sacrificed 24 hours postsurgery and blood and tissue samples were collected for further analysis. Renal function and degree of injury was assessed by measurement of blood urea nitrogen (BUN) and creatinine. Baseline BUN values were determined to be between 2030 mg/dL while unilateral damage was indicated between 4060 mg/dL and bilateral injury at values
>120 mg/dL. Kidney samples were processed for immunohistochemistry studies and sections were stained for C3b deposition using FITClabeled goat antimouse C3 Ab
(Cappell).
Statistical Analysis
GraphPad Prism software version 7 (GraphPad Prism Software Inc., la Jolla,
CA) was used for statistical analysis. For erythrocyte assays, the data are expressed as mean values +/ SEM. Descriptions of the various statistical calculations are provided in each section.
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CHAPTER III
RECOMBINANT MFH AND MFHR PROTEIN EXPRESSION AND ANALYSIS
Expression and Purification of Complement Proteins
Selecting an Expression System for producing MFH and MFHR Proteins
Several systems have been utilized to express complement proteins including
E. coli, baculovirusinsect cell expression, yeast systems using Pichia pastoris and mammalian expression systems with Chinese Hamster Ovary (CHO) cells and
Human Embryonic Kidney (HEK) 293F cells. While other groups have successfully produced truncated mutant FH constructs using an E. coli expression and purification scheme, their procedure required a protein refolding step which was deemed suboptimal for functional studies and proposed in vivo experiments. Given that our lab had previous experience with expressing complement proteins using a yeast system and a fermenter was available for use, this system was initially chosen and trial expressions were performed.
Pichia pastoris has previously been successfully employed to express a number of FH constructs, including fulllength FH, and other members of the RCA gene family (85, 97). Recently this strain has been reclassified as Komagataella pastoris, however it is still routinely referred to as Pichia pastoris (206). Several advantages existed for pursuing expression using this system including that this yeast provide a faster, less expensive method for large scale protein expression than insect or mammalian cell lines. In terms of yeast species, higher cell densities have been achieved using Pichia pastoris versus other yeast such as
Saccharomyces cerevisiae (207, 208).
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The mFHR construct that was chosen for trial expression was mFHRB as this construct and protein had been previously expressed in P. pastoris by another group (97). The DNA sequence for mFHRB was codonoptimized for expression in
P. pastoris and generated commercially. The construct was engineered to include both 5' and 3' unique restriction enzyme sites in order to extract and purify it from the commercially prepared carrier plasmid. The construct was also designed with an alpha secretion signal to allow for expression of protein into medium, and it also included an aminoterminal hexahistidine (His6) tag, a flexible linker region
(DYDIPTT), and a TEV (Tobaccoetch virus Nuclear Inclusion a (NIa)) protease cleavage site located at the 5' end of the DNA sequence. Two stop codons were engineered at the 3' end of the mFHRB construct (Figure 10).
Along with trial expression of mFHRB using P. pastoris, another expression system was selected for investigation. The 293F cell mammalian expression system was being used at this point in our laboratory to express the human FH and FHR proteins. Utilizing the same expression system for the expression of murine FH and
FHR proteins was seen as optimal for making comparisons between the human and murine proteins. To investigate the possibility of producing FHRs using this system, a collaboration with Dr. Kevin Marchbank from the Institute of Cellular Medicine
Newcastle University was initiated. Dr. Marchbank’s group had trialed the expression of recombinant mFHRs in another mammalian expression system. His group cloned sequences of three mFHR cDNAs into a pDR2EF1αHis mammalian expression vector which was used to transfect Chinese Hamster Ovary (CHO) cells. His
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laboratory had successfully demonstrated limited soluble expression for at least one of the mFHR proteins.
Figure 10 MFH and MFHR Expression Construct Design. mFH and mFHR proteins were synthetically generated to have attB cloning sites for easy recombination between donor vectors and entry clones. Constructs were designed to have a Kozak sequence followed by an Ig Kappa leader signal (for export of the protein into media), a linker region, His6tag, another linker, and a TEV cleavage sequence for removal of the His6tag. Sequences for mFH and mFHR proteins and their Uniprot IDs: mFH Uniprot ID: P06909; mFHRA Uniprot ID: Q61407; mFHRB Uniprot ID: Q4LDF6; mFHRC Uniprot ID: Q0KHD3; and mFH1920 Uniprot ID: P06909 residues 11121234.
These constructs were obtained and trial expressions were performed using
293F suspension cells. Trial expression of mFHRB protein in P. pastoris was compared to expression of the same protein using 293F cells. The predicted molecular weight of nonglycosylated mFHRB is ~40 kDa. Different molecular weights were observed for the protein which are most likely attributed to different glycosylation patterns between the two expression systems. Following deglycosylation with PNGaseF, the proteins ran at a similar molecular weight
(Figure 11). One substantial drawback to using P. pastoris for expressing mFHR proteins was that this system produced highly glycosylated proteins that could possibly increase or enhance the protein’s ability to form homodimers or multimers.
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This same phenomenon was reported by Hellwage et al. 2006 who reported bands at ~150 kDa prior to deglycosylation when they produced mFHRB in P. pastoris
(97).
Recently it has been described that using P. pastoris as an expression system does not always result in high yields of protein lower yields of protein are reported for proteins that form heterooligomers, are prone to proteolytic degradation, or are membraneattached proteins (206). While purification of FH was successful using P. pastoris, the human FHR proteins are reported to form dimers and possibly other higher order structures which may make recombinant protein production and purification of these proteins using P. pastoris more challenging than
FH (85, 112).
Given that the goal was to produce high yields of mFHR proteins that did not require deglycosylation to produce a uniform protein population that could be used in functional studies and in vivo disease models, the 293F system was selected as the better expression system. Glycosylation patterns from expression using 293F cells are closer to native protein expression patterns which may have implications for proper protein folding and function. Finally, 293F cell expression provides a serum free method for expressing and purifying mFHR proteins, while preventing cross reactivity and/or contamination from complement components and other proteins that are present in serumbased expression media.
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Figure 11 Expression Trials of MFHRB Protein in 293F cells and P. pastoris. 10% BisTris SDSPAGE reducing gel showing mFHRB protein expression. Expression trials of mFHRB were performed by transfecting 293F cells and the X 33 strain of Pichia pastoris. Lane 1 and lane 3 show mFHRB produced either by 293F cells or Pichia pastoris. Glycosylation patterns appear different between the two expression systems. Lane 2 and lane 4 represent mFHRB following deglycosylation with PNGaseF (indicated by green box). The red arrows depict the deglycosylated mFHRB protein (estimated molecular weight: ~35 kDa).
Expression of MFHR Proteins using 293F Cells
After determining a strategy for making mFH and mFHR proteins in 293F cells, expression constructs for mFH, mFHRA (sequence 3A4/5G4), mFHRB, mFHRC, and mFH SCR 1920 were designed and synthetically generated. These constructs were designed with the same format as the original P. pastoris construct; however, they were codonoptimized for expression in mammalian cell lines. Each construct has a Nterminal His6 tag that in theory, could be cleaved using TEV protease leaving only a single additional glycine residue located at the Nterminus of
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the target protein (Figure 10). This design was chosen rather than designing a construct with a tag located at the Cterminus, which has known C3b/C3d and GAG binding sites for FH with putative C3b/C3d binding sites predicted based on the mFHR protein sequences.
The same purification strategy was employed for all recombinant proteins.
Each transfection, which ranged from 500 ml to 1L, was harvested and proteins were purified using IMAC (Figure 12). Following IMAC, a polishing step was performed using gel filtration chromatography as described in Chapter II (Figure 13).
Fractions containing protein with the highest purity were selected using SDSPAGE under reducing conditions and protein was concentrated in PBS. Protein yield generally ranged from 210mg per 1L transfection for mFH and was substantially lower for the mFHR proteins ranging from 0.10.5mg per 1L.
With regard to removal of the His6tag from the proteins, several attempts were made to remove the His6tag from mFH using AcTEV™ protease. While different protease conditions and incubation temperatures were tested, cleavage of the tag resulted in very low protein yields (data not shown). Subsequent analysis of the mFH and mFHR protein sequences revealed a weak internal TEV cleavage site that exists within SCR 7 of murine FH that was likely attributing to the lower protein yields (Figure 14).
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Figure 12 MFHRA Purification using IMAC. (A) Chromatogram of gradient elution profile of mFH protein from 21ml HisTrap columns. Equilibrium buffer: 20mM sodium phosphate, 20mM imidazole, 0.5M NaCl, 1mM EDTA. Elution buffer: 20mM sodium phosphate, 0.5M imidazole, 0.5M NaCl (B) Fractions from the peak marked “mFHRA” are shown on reduced SDSPAGE (inset figure).
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Figure 13 Purification of Recombinant MFH. A HiPrep Sephacryl S300 size exclusion column was used as a polishing step in protein purification. (A) mFH elutes with a profile consistent with that of a primarily monomeric state. (B) SDS PAGE of the size exclusion fractions containing Histagged mFH (inset).
While the canonical sequence for TEV cleavage is ENLYFQG, TEV recognizes the linear epitope EXXYXQG/S where X can be any amino acid and cleavage occurs between Q (Gln) and G (Gly). Unfortunately, the mFH and the mFHR proteins have a predicted TEV internal site EKVYVQG, making cleavage of the His6tag less reliable. Our lab has also generated recombinant human FH and
FHR proteins using the same construct layout as the murine FH and FHR proteins; however, the human FH and FHR family does not appear to have the putative internal TEV sites that are predicted with the murine family members. Therefore,
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further experiments to cleave the His6tag from the recombinant proteins were not explored.
Figure 14 Alignment of Human and Mouse SCRs 7. Alignment of human FH SCR 7 (Uniprot ID: P08603 residues 387444) with mouse FH SCR 7 (Uniprot ID: P06909 residues 387444). Both human and mouse recombinant protein constructs were designed to have a TEV site for cleavage of the His6tag. However, a putative internal TEV cleavage site is present in murine FH SCR 7 (as well as in the other mFHR proteins which share identity to this domain), while it is lacking in human FH and FHR proteins. TEV cleavage occurs when sequences are closest to the consensus sequence EXXYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic residue and φ is any small hydrophobic or polar residue, preferably G/S (shown in red). Highlighted as well are several other important interactions reported for human FH SCR 7. Blue highlighted residues denote sites implicated in heparin and CRP binding (and are conserved in the mouse). Green highlighted residues are reported to bind heparin, CRP, and M protein and are lacking in the mouse. The residue highlighted with orange is reported to interact with heparin.
Assessing Quality and Thermal Stability of MFH and MFHR Proteins
In order to assess the quality and stability of mFH and mFHR proteins that were produced between subsequent transfections reactions, three different techniques were used. The first method for assessing protein quality was by SDS
PAGE analysis of glycosylated and deglycosylated proteins (Figure 15). This technique allowed for the examination of the relative purity of the proteins and estimation of their molecular weights under reducing and denaturing conditions.
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Figure 15 SDSPAGE of Glycosylated and Deglycosylated MFH, MFHRA, and MFHRB. The purity of recombinant proteins was assessed between different transfections under reducing conditions on SDSPAGE. Proteins are also shown deglycosylated with PNGaseF (marked by red arrow). mFHRB contains four putative Nlinked glycosylation sites which may contribute to the 810kDa molecular weight difference between glycosylated and deglycosylated forms of this protein.
The second technique employed gel filtration chromatography to assess the native state of these proteins and their molecular weights (MW). A Sephacryl S300
HR column (GE Healthcare Life Sciences) was calibrated using known protein MW standards per the methods outlined in Chapter II (Figure 16). The logarithmic values of the MWs of the known protein standards were plotted against their peak elution volumes to create a standard curve, which was used to calculate MW values for mFH and mFHR proteins. Two of the proteins, mFHRA and mFHRC, interacted with the column matrix and could not be accurately assessed by this method.
However, mFH, mFHRB, and the recombinant inhibitor mFH1920 were calculated
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to have molecular weights of 217,000 Da for mFH, 160,000 Da for mFHRB, and
20,000 Da for mFH 1920.
Figure 16 Chromatographic Separation and Calibration Curve of Protein Standards. (A) Chromatogram showing separation of a welldefined protein standard mixture on a Sephacryl S300 column in PBS with molecular weights (MW) indicated above each peak. (B) Calibration curve created by plotting the relationship between the logarithm of the MW of each protein and corresponding elution volume (C) GraphPad Prism software was used to plot the calibration curve and determine the slope found in this table (D) Chromatogram with elution profiles of mFH, mFHR B, and mFH 1920 and a table with predicted and calculated MW values.
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Differential scanning fluorimetry was the third technique used to analyze the quality of the recombinant mFH and mFHR proteins. This technique, also known as thermofluor, measures the temperature at which a protein unfolds, going from a well defined state to a more disordered state. This costefficient technology uses a small amount of protein sample, a realtime PCR machine, and a dye (SYPRO Orange).
The dye binds hydrophobic surfaces and emits a fluorescence signal. As a protein unfolds over a temperature range from 2090°C, there is an increase in signal. The melting point (Tm) of a protein is the temperature where half of the protein population is folded while the other half remains unfolded. The Tm is established by creating a fluorescence signal plot with the average of three replicates relative fluorescence unit (RFU) value subtracted from the background value and plotted against temperature values. Data are shown both as raw RFU values (yaxis) plotted against temperature (xaxis) and also normalized RFU values versus temperature for better visual and comparative purposes between proteins (Figure 17).
One of the concerns with producing recombinant proteins is that they maintain their biological function and are folded correctly. Long term storage at different temperatures or other buffer conditions may cause the protein to aggregate or unfold. Aggregates that may form as a result of highly concentrated samples or in certain buffer conditions exhibit no change in fluorescence intensity. Unfolded proteins have a high initial fluorescence and no melting curve is observed. This method was employed as a highthroughput method for quickly screening different protein aliquots. Based on these assays, all mFH and mFHR proteins exhibit a Tm between 6064°C in phosphate buffered saline (PBS).
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Figure 17 Thermal Shift of MFH, MFHRA, MFHRB, MFHRC, and MFH 1920. (A) Raw data showing relative fluorescence unit (RFU) intensity changes as a function of temperature for mFH (black), mFHRA (orange), mFHRB (red), mFHRC (green), and mFH 1920 (Blue). (B) Normalization of the RFU values for each protein allows for better a better visual comparison on one plot between different proteins. All FH and FHR proteins have a Tm between 6065°C.
Summary
Expression systems which have been used to generate recombinant complement proteins include E. coli, baculovirus, yeast systems using Pichia pastoris, and mammalian expression systems using CHO and HEK 293F cells. In
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this chapter, protein expression using Pichia pastoris and the mammalian 293F cell line was explored. Results from these experiments suggest that extensive glycosylation of mFHRB occurs with expression in Pichia pastoris. Therefore, expression using 293F cells in a serumfree system was selected as this method likely produces protein that is most similar to its native form. Subsequently, synthetic constructs for mFH, mFHRA, mFHRB, mFHRC and the recombinant inhibitor, mFH 1920, were designed and recombinant proteins were expressed and purified.
Three different techniques were used to analyze the quality of the recombinant proteins. SDSPAGE analysis was used to analyze protein purity, although in this method, proteins are in a reduced and denatured state. Gel filtration chromatography was used as a polishing step during protein purification and was also used to calculate the molecular weights of the proteins in their native forms.
Finally, differential scanning fluorimetry was used to assess the quality of the folded proteins and analyze their stability in PBS.
While gel filtration chromatography was not a useful technique for calculating the molecular weights of mFHRA and mFHRC, as these proteins appeared to interact with the column matrix and elute from the column in large volumes (>40mls), it was a useful technique for analyzing mFH, mFHRB, and mFH 1920. Two main explanations exist for the differences between observed protein molecular weights and calculated molecular weights. The murine FH protein is predicted to have eight potential Nlinked glycosylation sites, which is comparable to the human FH molecule that has nine potential Nlinked glycosylation sites, eight of which have been confirmed (81). Human FH Nlinked glycosylation is thought to be primarily
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with diantennary disialylated glycans, each having a molecular mass of 2,204 Da.
Deglycosylated murine FH has a MW estimated at ~137,000 Da, although with glycosylation, the mass difference would be around ~18,000 Da for a MW of
~154,000 Da, which is close to the MW observed on SDSPAGE. Studies of glycosylated human FH show that the protein MW is 155,000 Da, but can have an apparent size of 330,000 Da using gel filtration analysis relative to globular standards due to the flexible and extended nature of the FH molecule (209). It is likely that mFH also has a flexible and extended nature which may explain why the molecular weight calculated using gel filtration chromatography is 217,000 Da.
The mFHRB protein is predicted to have four Nlinked glycosylation sites which could contribute to the 8,00010,000 Da difference in MW between the glycosylated (~55kDa) and deglycosylated (~45kDa) protein as observed on SDS
PAGE. The MW calculated by gel filtration (~160kDa) may be attributed to the mFHRB protein forming homodimers or higher order complexes, similar to what has been observed with the human FHR proteins (112). The predicted MW of the recombinant mFH1920 inhibitor is 20,000 Da which is close to the calculated MW of
22,000 Da using gel filtration. One potential Nlinked glycosylation site is present in
SCRs 1920 which may account for the 2,000 Da difference that is observed.
Finally, differential scanning fluorimetry was used to screen the stability and folding of all recombinant proteins. All proteins had a Tm between 6064°C. This technique allowed for quick assessment of batches of protein produced from different transfection reactions and allowed for a way to examine protein stability in
PBS.
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Overall, this chapter explains how to successfully express and purify recombinant mFH and mFHR proteins using 293F cells. The three techniques that are discussed can be used to qualitycheck the recombinant proteins before subsequent binding experiments, in vitro functional studies, and in vivo experiments are performed.
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CHAPTER IV
ASSESSING THE FUNCTIONAL ROLES OF MFHR PROTEINS
General Background
Assays to test the function of complement pathways have been in existence for over 100 years dating back to when Jules Bordet first described complement fixation (210). Different assays can primarily measure the overall activity of individual pathways, such as a CH50 assay (classical pathway) or AH50 (alternative pathway).
Briefly, in a CH50 assay the complement activity of serum on antibody sensitized sheep red blood cells (SRBC) is determined by measuring the amount of hemolysis that occurs. Low CH50 values resulting from decreased hemolysis indicate deficiencies in the classical or terminal pathways. Only antibody sensitized sheep red blood cells are used because nonsensitized sheep red blood cells (considered to be noncomplement activating surfaces) do not lyse in the presence of normal human serum. Factor H is thought to bind sialic clusters on sheep red blood cells preventing complement activation. Removal of sialic acid using neuramindidase has been shown to allow activation of the alternative pathway on that surface (211, 212).
To measure activity of the alternative pathway (AH50 assay), different erythrocytes and buffer are used. Rabbit erythrocytes, which are considered to be complement activating surfaces, lyse in the presence of normal human or mouse serum. These cells are thought to lack factor Hmediated cell surface protection in part because they have lower host polyanionic markers such as sialic acid. Analysis of erythrocytes from different animals has shown a direct correlation between the amount of cell surface sialic acid and resistance of the cell to alternative pathway
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activation (213). This assay is performed in buffer containing ethylene glycol tetraacetic acid (EGTA) which is used to chelate calcium (preventing activation of the CP or LP) but not magnesium (required for CP/LP C3 convertase formation).
Therefore, this assay measures overall AP function including the functional activity of factors D, FH, FB, properdin, C3 and downstream molecules.
To investigate the activity of mFH and mFHR proteins in an AH50 assay, a single trial experiment was performed. Rabbit erythrocytes were incubated in the presence of 40% wildtype (WT) mouse serum and increasing amounts of mFH or mFHR proteins were added. A control sample containing rabbit erythrocytes with
40% mouse serum was used as a baseline for hemolysis. Increased or decreased cell lysis was determined for samples containing mFH or mFHR proteins. Addition of mFH reduced cell lysis by nearly 50%, as one might predict. Interestingly, addition of mFHRA also appeared to significantly reduce hemolysis while addition of mFHRB and mFHRC increased hemolysis compared to the control (Figure 18).
Given that rabbit erythrocytes are thought to have limited FHcell surface protection, the ability of FHR proteins to antagonize FH function at cellsurfaces could not be investigated using this assay. The overall utility of this assay is that it can be used to explore whether overall alternative pathway activity is perturbed by addition of mFH or mFHR proteins. Here we observe that addition of mFH or mFHR
A appears to reduce overall alternative pathway activity, while addition of mFHRB and mFHRC appears to increase AP activity. However, it is possibly that addition of mFHRA could result in rapid consumption of C3 in fluidphase. This would give the impression that AP activation was reduced when in fact, it was increased. Therefore,
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a more specific hemolytic assay was used and is described in the next section to more precisely assess the activity of mFHRs and their ability to antagonize mFH on cell surfaces.
Figure 18 AH50 Assay with MFH and MFHR Proteins Influences Alternative Pathway Activation. An AH50 assay using rabbit erythrocytes was performed using 5 x105 rabbit erythrocytes in a 24 µL reaction volume with 40% normal mouse serum. Hemolysis was determined after incubation of the reaction for 30 min at 37°C followed by quenching with GVBE and measurement of the absorbance at 412 nm. Addition of either mFH or mFHRA appears to decrease AP activation while addition of mFHRB or mFHRC appears to increase AP activation. It is possible that addition of mFHRA resulted in rapid fluidphase C3 turnover which may appear as a reduced AP activation.
Previous research has shown that the Cterminal end of human Factor H plays a critical role in regulating complement activation on cell surfaces. FH SCR domains 1920 were shown to discriminate between nonhost and host surfaces.
While fluid phase complement activation was unaffected, hemolysis of normal sheep red blood cells occurred in the presence of the recombinant FH SCR1920 inhibitor.
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Both FH and the FH1920 inhibitor have 7fold higher affinity for surfaces coated with C3b and high amounts of sialic acid (214). Additionally, competition assays showed that FH1920 inhibits FHmediated control of alternative pathway driven complement lysis on hostlike polyanionbearing cells. Results from this work help to explain how aHUS patients who have mutations in the Cterminal domains of FH can maintain fluid phase complement homeostasis yet have complement activation occurring on host cell surfaces and tissues.
Figure 19 Hemolytic Assay Schematic with MFH and MFHR Proteins. Outline of functional assays performed to evaluate mFHR proteins. Endogenous mFH present in wildtype mouse serum controls activation of the alternative pathway on sheep red blood cells which resemble hostlike polyanion bearing cells. Addition of mFHRA or to a lesser extent, mFHRB, inhibits the protective function of mFH on these cells leading to the hemolysis of erythrocytes which is measured by evaluating the amount of hemoglobin released (A414).
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MFHRA and MFHRB are Potent Antagonists of MFH on SRBCs
To explore the roles that murine FHR proteins have on FH protection of host like surfaces, hemolytic assays using normal sheep erythrocytes were performed.
The mFH1920 inhibitor was expressed using the same methods described in
Chapter III. Normal sheep erythrocytes were combined with WT mouse serum and increasing amounts of mFHRA, mFHRB, mFHRC, mFH, and mFH 1920 (Figure
19). Our data confirm results from previous studies that describe how mFH1920 activates complement by inhibiting FH protection of SRBCs (214, 215).
Interestingly, both mFHRA and mFHRB also appear to be potent antagonists of mFH in this assay (Figure 20A). It should be noted that mFHRA is a prototype for a protein encoded by the mFHRA gene. A mRNA sequence isolated from a normal 5month old mouse liver (BC026782) shares high homology to this prototype and can be found in the UCSC Genome Database, however evidence for a mFHRA protein does not currently exist. Most interestingly is that the mFHRA prototype, which shares >95% identity to mFHRB SCRs 57 and 1920, is a more potent inhibitor of FH than mFH1920. The mFHRA prototype contains two additional SCR domains corresponding to mFH SCRs 89 which may enable the protein to more efficiently compete with FH for binding to C3b on cell surfaces. The additional SCR domains within the mFHRA and mFHRB proteins may help them to engage GAG surfaces better than mFH1920, thus making them more potent antagonists. Interestingly, mFHRC does not appear to be an antagonist of mFH.
Even after high amounts of mFHRC are added (20 µM), no hemolysis is observed
(Figure 20B).
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Figure 20 MFHRA and MFHRB are Potent Antagonists of MFH on HostLike Surfaces. Hemolysis assays were performed using 1 x106 sheep erythrocytes in a 24 µL reaction volume with 40% normal mouse serum. Hemolysis was determined after incubation of the reaction for 30 min at 37°C followed by quenching with GVBE and measurement of the absorbance at 412 nm. A) mFHRA is a more potent inhibitor of mFH than mFHRB. Almost 100% lysis is observed with less than 4 µM mFHRA, while mFHRB requires two fold more protein to induce just over 50% lysis of erythrocytes. B) mFH1920 is a previously characterized inhibitor of murine FH. This inhibitor, which is comprised of mFH SCRs 1920, competes with the Cterminal end of FH for binding to C3b and polyanionic rich surfaces. mFHRC does not antagonize mFH function, even at high concentrations.
Also, we cannot rule out that the mFHRA and mFHRB proteins may form forming dimers or higher order structures that increase their avidity for C3b on cell surfaces making them better antagonists of FH. Human FHR proteins 1, 2, and 5 all share a common dimerization motif which is not conserved in mFHRA, mFHRB or
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mFHRC, but is found in mFHRE. Dimerization of the human proteins has been shown to increase their avidity for C3b enabling them to act as competitive antagonists of FH.
Therefore, to better explore the molecular mechanisms that may underlie the different hemolytic activities observed in these functional assays, the next chapter will explore the interaction of mFH and mFHRs with the complement component,
C3d which approximates to the thioester containing domain in C3b.
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CHAPTER V
CHARACTERIZING THE MFH AND MFHR PROTEIN INTERACTION WITH MC3D
General Background
Numerous proteins have been proposed to interact with FH, but only a few of these interactions have been studied with great molecular detail. Extensive structural studies have been carried out to analyze the tertiary structure of FH and to elucidate how this molecule is able to concurrently engage C3b and cell surface polyanions.
To date, 14 wildtype or mutant FH structures encompassing a total of 13 of the 20
SCR modules of fulllength FH have been determined by Xray crystallography or
NMR spectroscopy.
Concerning the complex that FH forms with C3b, structures of the four N terminal SCR domains of FH in complex with C3b (FH1 4:C3b) and also of the two
Cterminal SCR domains of FH in complex with C3d (FH1920:C3d) have been determined (216, 217). C3d is a 35 kDa C3 proteolytic fragment which approximates to the thioester containing domain (TED) of C3b and is produced as a result of the further proteolysis of iC3b to form C3dg and subsequently C3d.
In the structure of the FH14:C3b complex, the four SCR modules of FH adopt an extended conformation making extensive contacts with several different domains of C3b including αNT, MG1, MG2, MG6, MG7, CUB and TED57 (216).
With regard to two determined FH1920:C3d complex structures, almost identical complex interfaces were elucidated which could be extrapolated to the context of the physiologic FH: TED interaction. These data show a crucial interface is formed between the Cterminal domain of human FH and C3b. Hence, mutations within the
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Cterminal region, such as those that have been reported in aHUS, have potential to disrupt important contacts between FH and C3b (218).
Additionally, NMR and mutagenesis experiments have separately confirmed the C3dFH1920 binding sites, and NMR has been used to identify GAGbinding sites also within the C3dFH1920 complex. FH residues important for making contacts within this interface include Ile1120, Ser1122, Asp1119, Asn1140, Tyr1142,
Gln1139, Asn1117, Pro1166, Tyr1190 (Figure 21) (216, 217). From these studies, it may be concluded that the Cterminal regions of FHR proteins may have important functional roles for binding C3b/C3d, and that mutations within these regions accordingly may predispose towards various diseases. Alignment of human FH with mFH and the mFHRs shows that all five residues with side chains that form hydrogen bonds with C3d are conserved (Figure 22).
Figure 21 Human FH SCRs 1920: C3d Interface. Residues on FH SCRs 1920 with side chains that form intermolecular hydrogen bonds with C3d, which approximates to the C3b thioester containing domain (TED) (216). These residues include Asn1117, Asp1119, Gln1139, Tyr1142, and Tyr1190. All residues except for Tyr1190 are housed within FH SCR 19. PDB: 3OXU
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MFH and MFHR Proteins bind MC3d and Mutant MFHR Proteins are Inactive
A previous study with mFHRB that was produced in P. pastoris provided the first evidence that murine FHRs interact with human C3b. However, differences between human and mouse C3b exist, therefore, recombinant murine C3d, which corresponds to the TED of C3b, was produced. Human C3d previously produced in our laboratory was also available in these studies for comparison.
Figure 22 Sequence Alignment of Human FH SCRs 1920 with MFH and MFHR Proteins showing Putative C3d Binding Sites. Alignment of human FH (hFH) SCRs 19 and 20 with corresponding murine SCR domains of mFH (Uniprot: P06909), mFHRA (shown in grey as this is a prototype sequence for mFHRA based on Vik et al. clone 3A4/5G4 Uniprot: Q61407), mFHRB (Uniprot: Q4LDF6), mFHRC (Uniprot: Q0KHD3) and mFHRE (Uniprot: Q61406). Residues highlighted in red have side chains that form hydrogen bonds with C3d. Mutations were made at positions N1117 and D1119 to create mutant mFHRA and mFHRB constructs used in ELISA and surface plasmon resonance assays. Residues marked by a blue box are part of the interface between hFH1920:C3d, yet are not found in all of the murine FH and FHR proteins. The residues in the orange boxes have been indicated in an additional proposed FH: C3d interaction, yet are not entirely conserved within the murine FH and FHR family. mFHRE has two SCR domains (SCRs 3 and 4) which share high sequence identity to hFH and mFH SCR 19. The mFHRD gene is also listed in various databases as an unprocessed pseudogene, and is not depicted here although one exon from this pseudogene has 94% nucleotide homology with the region that encodes mFH SCR 19 and 96% homology with mFHRE SCRs 3 and 4.
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Figure 23 Recombinant MC3d Expression and Analysis. Recombinant murine C3d was expressed in E. coli and the GSTtag was cleaved using thrombin followed by purification using size exclusion. A) 10% SDSPAGE of mC3d under reducing conditions shows a single band ~35 kDa. B) Circular dichroism spectrum showing the change in the mean residue ellipticity of mC3d as a function of wavelength. An helical secondary structure for mC3d is indicated. C) Differential scanning fluorimetry analysis of murine and human recombinant C3d proteins. Relative fluorescence values (RFU) have been normalized for comparison of the two curves of the proteins. The apparent Tm of murine C3d is slightly higher (>40°C) than that of human C3d (<40°C).
Recombinant production of human and murine C3d using E.coli has been previously performed in our laboratory, and three techniques were used to examine the recombinant protein. Following initial purification of the protein using a GST column, thrombin was used to cleave the tag from the protein. The protein was applied to a Sephacryl S100 size exclusion column and SDSPAGE separation was performed to show a product at the predicted molecular weight for mC3d at ~35kDa.
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Analysis of the mC3d secondary structure was performed using circular dichroism
(CD). The CD spectrum revealed an alpha helical confirmation for mC3d, which is in agreement with the xray crystallography structure of human C3d (219). Additionally, thermal shift assays for mC3d and hC3d were performed, and the apparent Tm for mC3d (>40°C) was estimated to be slightly higher than that for hC3d (<40°C). When compared to the murine FH and FHR proteins (Figure 17), the apparent Tm of both human and mouse C3d is much lower.
To investigate the binding interaction between murine FH and mFHRs with murine C3d, ELISA assays were performed. Microtiter plates were coated with C3d and increasing amounts of mFH and mFHR proteins were added. Binding of mFH, mFHRA, and mFHRA proteins was observed (Figure 24). While not shown, cross species reactivity between human C3d and mFH and mFHR proteins was also observed.
To further explore this interaction, mutant constructs were made. Two separate sets of mutations were performed for both the mFHRA and mFHRB constructs. Four residues within hFH SCR 19 and one residue within SCR 20 have side chains that form intermolecular hydrogen bonds with C3d. All five of these residues are conserved within the murine FH and FHR family. Several other residues are also important for the hFH1920:C3d interface and are indicated
(Figure 22). To test this interaction, mutations were made to either N1117A and
D1119A residues or Q1139A and Y1142 residues to make two sets of mFHRA mutants and mFHRB mutants. Mutant construct expression and purification was performed under the same scrutiny as used for wildtype constructs. Preliminary
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testing showed that either set of mutations appeared to be sufficient to inactivate the proteins (data not shown), therefore only the N1117A and D1119A mutation pair are described.
Figure 24 MFH and MFHRs Bind MC3d and Mutant Proteins are Inactive. ELISA analysis of platebound mC3d with mFH, mFHRA, and mFHRB. Residues on human FH SCR19 which contribute to sidechain interactions with C3d are conserved in mFHRA and mFHRB. Mutation of two residues was performed (N1117A and D1119A) and no binding was observed for the mutant mFHRA and mFHRB proteins.
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Determination of MC3d Binding Affinity for MFH and MFHR Proteins
Earlier studies have analyzed the interaction between murine FH and C3b using surface plasmon resonance (SPR). The binding of fulllength mFH, and truncated versions of mFH including mFH SCRs15 and mFH SCRs 1820 to both murine and human C3b has been demonstrated (220). However apparent KD values for this interaction were not determined and the interaction between murine FH (and the murine FHRs) and mC3d has not been evaluated.
Using previously described conditions for SPR experiments with mFH and
C3b, binding of mFH and mFHR proteins to mC3d was investigated. First, a pH scouting experiment was performed to determine a buffer condition (pH 4.5) that was optimal for amine coupling of approximately ~2500 resonance units (RU) of mC3d onto a CM5 sensor chip. Serial dilutions of mFH, mFHRA, mFHRB, mFHR
C, and mFH 1920 proteins were made and sensorgrams generated from the different dilutions were analyzed using BIA evaluation software. Data were fit using a
2state binding model and apparent KD values for each of the proteins were calculated.
The apparent binding affinities varied between the different proteins. No binding interaction was observed for mutant mFHRA and mFHRB proteins
(containing the N1117A and D1119A mutation set). Higher affinity binding was observed with mFHRA (KD= 136 nM) and mFHRB (KD= 546 nM) than mFHRC
(KD= 1.04 µM), mFH (KD= 3.85 µM) or the recombinant mFH 1920 inhibitor (KD=
22µM).
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Figure 25 Binding of Murine C3d and MFH and MFHR Proteins. Surface plasmon resonance was used to analyze the binding between murine C3d and mFH and mFHR proteins. Serial dilutions of mFH (A), mFHRA (B), mFHRB (C), mFHRC (D), and mFH1920 (E) were flowed over immobilized mC3d. Affinity values (KD) were determined by fitting data to a twostate reaction model. Different binding kinetics were observed for mFHRA and mFHRB compared to mFH, mFHRC, and mFH1920. Mutant mFHRA and mFHRB (inset figures) did not bind mC3d.
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Table 1 SPR Analysis of the Interactions between MFH/MFHRs and MC3d
Mutant MFHRB Exhibits Loss of Function in Hemolytic Assays
To assess the function of the mutant mFHRB protein compared with wild type protein, sheep erythrocyte hemolytic assays were performed following the same methods described in the previous chapter. Results show that mutation of two out of five major putative binding residues (N1117A and D1119A) that form intermolecular hydrogen bonds with C3d sufficiently abrogates the ability of mFHRB to inhibit mFH resulting in diminished hemolysis of erythrocytes (Figure 26). Testing has not yet been performed with the other set of mFHRB mutations (Q1139A and Y1142), however preliminary experiments with mutant mFHRA which has homologous mutations (Q1139A and Y1142) greatly diminished the ability of mFHRA to antagonize mFH (data not shown).
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Figure 26 Mutant MFHRB does not inhibit MFH Function on Cell Surfaces. Mutation of two key mC3d binding residues (which correspond to hFH residues N1117A and D1119A) on wildtype mFHRB greatly diminishes the ability of this protein to antagonize mFH function.
Summary
Here we explored the interaction between murine FH and mFHR proteins and murine C3d, which approximates to the C3b TEDcontaining domain. Binding of mFH, mFHRA, mFHRB, mFHRC, and mFH 1920 to mC3d was confirmed using
ELISA analysis and SPR. Crossspecies reactivity was observed between human
C3d and the murine proteins which supports previously published data with mFHRB and human C3b.
Alignment of human SCRs 1920 with mFH and mFHR proteins indicated that five key residues responsible for the hFH1920:C3d interaction are conserved among mFH and mFHR proteins. Results from SPR demonstrate higher nanomolar affinity interactions between mC3d and mFHRA and mFHRB proteins, compared with lower micromolar affinity interactions with mFH, mFHRC, and mFH1920.
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Additionally, data from SPR analysis suggest that mFHRA and mFHRB have different kinetic profiles than mFHRC, mFH, and mFHR1920. The different kinetics of mFHRA and mFHRB could be explained by dimerization or oligomerization.
Formation of higher order complexes may contribute to their higher apparent affinities for C3d, and provide a molecular mechanism for the underlying differences observed in hemolytic assays that were described in the previous chapter.
Furthermore, we show that mutation of only two of five important residues is sufficient to prevent binding of both mFHRA and mFHRB proteins to mC3d. Mutant mFHRA and mutant mFHRB do not bind mC3d in both ELISA and SPR experiments. Subsequent analysis of mutant mFHRB function using sheep erythrocyte hemolytic assays showed that mutant mFHRB does not antagonize mFH on cell surfaces.
These results not only establish that mFHR proteins contain a Cterminal binding site for C3d that is identical to the site found on human FH, but that mutation of two key residues (N1117A and D1119A) can greatly attenuate the ability of mFHRB to function as an inhibitor of mFH. Interestingly, two patients with HUS have been found to have fulllength FH mutations at position D1119. One patient with familial HUS is reported to have a missense mutation (D1119G) resulting in a nonpolar glycine residue at this position, however the patient’s C3 and Factor H levels were normal (138). Another patient was reported to have a missense mutation resulting in the mutation D1119N (221). Given that mutation of fulllength FH at position D1119 impairs function, it may be possible that mutation of the analogous residue may be sufficient to disrupt mFHRB (D1119) binding to C3d.
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A recent study by Hyvärinen et al. (2016) has used the recombinant FH1920 inhibitor to examine how different aHUS mutations that disturb sialic acid binding impact the ability of FH1920 to act as an inhibitor of mFH in erythrocyte assays
(222). Based on the structure of FH1920 bound to sialic acid (172), three mutant
FH1920 constructs (T1184R, L1189R, and E1198A) were created and two of the
FH1920 mutants, L1189R and E1198A, lost their ability to antagonize FH on cell surfaces. The conclusion of their study was that FH must simultaneously engage sialic acid and C3b to regulate complement activation on not only erythrocytes and platelets, but also endothelial cells which are rich in sialic acid residues. The main pathology of HUS is thrombotic microangiopathy resulting from complement mediated endothelial injury. Understanding how mFH and mFHR proteins regulate complement activation on specific cell surfaces is crucial for developing therapeutics to treat this disease and other complement mediated illnesses such as AMD.
Therefore, the next chapter examines the relationship between murine FHR proteins and complement activation on different cell types, and addresses the potential for exploring mFHRs using in vivo murine models of disease.
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CHAPTER VI
MFHR PROTEINS ACTIVATE COMPLEMENT ON DIFFERENT CELL SURFACES
As described in the Chapter I, FH and FHR proteins have been associated with a range of different lifethreatening and disabling autoimmune diseases including aHUS, C3 glomerulopathies, and AMD among others. Review of the online
FH aHUS Mutation database currently lists 193 mutations for FH (223). Furthermore, mutations and deletions of FHR genes, which create hybrid proteins, are also implicated to cause disease. One of the current challenges with understanding these molecules is that for example, deletion of hFHR31Δ has been shown to increase risk for both aHUS and SLE while conferring protection against AMD. Therefore, determining how these proteins regulate complement, especially in the context of different cell and tissue types, is of great importance for understanding how they contribute to the pathogenesis of various diseases. A comprehensive examination of the murine FHR proteins in vitro and in vivo is necessary as it can potentially provide key insights into how the human proteins influence disease.
MFHRA and MFHRB Induce Complement Activation on Murine TECs
Every cell in the body that is exposed to plasma experiences a low level of complement activation which requires constant monitoring by complement regulators such as CD55 (DAF) and CD59, and factor H. In addition to recognizing heavily sialylated surfaces, FH uses SCR7 and to a lesser extent, SCRs1920, to recognize polyanions such as heparan sulfate in order to bind different host cell surfaces and deactivate C3b (170). For example, mutation of FH Y402H has been shown to impair host tissue recognition by FH in the human eye. To explore whether the
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mFHR proteins impair mFH function on different cell surfaces, two relevant cell types were selected.
Murine renal tubular epithelial cells (TEC) have been shown to express the membranebound regulator Crry on their basolateral surface while factor H regulates complement activation on their apical surface (224). TECs are targets of complementmediated damage in vivo due to dysregulation and excessive activation of the alternative pathway (225). Exposure to 10% wildtype mouse serum induced complement activation, which was evaluated by measuring C3b deposition on the surface of TEC cells using flow cytometry. In a separate study, a FH binding partner, annexin 2 (A2), impaired complement regulation by FH and increased complement activation on TEC cells. The effect of A2 on C3b deposition was reversed by the addition of recombinant FH, but not addition of the inhibitor FH1920 (215).
To determine the effects on control of complement on this relevant cell type, the function of mFHRA and mFHRB were evaluated by incubating these proteins with TECs and 10% normal mouse serum. As expected based on earlier results from hemolysis assays, both mFHRA and mFHRB are potent antagonists of mFH and increase complement activation on TEC surfaces as indicated by the increase in
C3b deposition that is observed (Figure 27).
MFHRA and MFHRB Enhance Complement Activation on Retinal Pigment
Epithelial Cells
To further evaluate the complement enhancing effect of the mFHRA and mFHRB proteins, another relevant cell type was evaluated. Given the association
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with FHR proteins and AMD, the ability of the murine FHRs to enhance complement activation on a human retinal pigment epithelial cell line (ARPE19) was explored.
Figure 27 MFHRA and MFHRB Act as Potent MFH Antagonists on Murine Tubular Epithelial Cells (TECs). Increased complement activation is observed, as indicated by C3b deposition, when mFHRA and mFHRB proteins are incubated with TEC cells in the presence of 10% normal mouse serum.
Research by Thurman et al. (2009) has described a mechanism by which immortalized ARPE19 cells regulate complement activation on their surfaces. Using similar assays as described in the TEC experiments, they report that treatment of
ARPE19 cells with H2O2 to induce oxidative cell stress resulted in decreased surface expression of CD55 and CD59 and impaired FH cellsurface control (203).
Measurements of the transepithelial resistance (TER) across cell monolayers also
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decreased when cells were subjected to oxidative stress in complement sufficient serum. Sublytic activation of the MAC on the surface of these cells (which are C7 dependent) resulted in vascular epidermal growth factor (VEGF) release from their apical sides. In turn, the secreted VEGF bound receptors to induce further disruption in the monolayer.
Flow cytometry was used to examine the degree of C3b deposition on both unstressed and stressed ARPE19 cells by incubation with mFHRA or mFHRB in the presence of 10% normal mouse serum. Addition of mFHRA or mFHRB increased complement activation on both types of cells. The ability of mFHRA and mFHRB to increase complement activation on healthy cells (unstressed) further supports that mFHRA and mFHRB are potent antagonists of mFH on different nucleated cell surfaces (Figure 28).
To further investigate this result, experiments were performed by our collaborators (Rohrer Laboratory, Medical University of South Carolina) to measure the change in TER with the addition of mFHRA or mFHRB. Briefly, ARPE19 cells were plated in transwell plates and grown in the presence of fetal bovine serum
(FBS). Following the formation of monolayers, the cells were starved prior to the experiment so that complement sufficient normal mouse serum (NMS) provided the only source of complement. Two standards were used and consisted of either 10%
NMS and H202 or 2% NMS and H2O2, which was used to induce oxidative stress
(Figure 29).
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Figure 28 MFHRA and MFHRB Increase Complement Activation on ARPE19 cells. Incubation of mFHRA and mFHRB with ARPE19 cells in the presence of 10% normal mouse serum induces C3b deposition on the surface of A) stressed ARPE19 cells and B) unstressed ARPE19 cells.
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Figure 29 Complement Activation Induced by MFHRA Results in Loss of TER of ARPE19 cells. In this experiment performed by the Rohrer lab, two standards containing H2O2 (to stress the cells) and either 10% or 2% normal mouse serum (NMS) are shown. A control sample containing cells that were not exposed to NMS or H2O2 was used to establish the TER baseline value. Incubation of cells with 100 ng/ml of mFHRA or mFHRB results in complement activation. Addition of mFHRA results in loss of TER that is comparable to incubation of stressed cells in 10% NMS. mFHRB results in loss of TER comparable to stressed cells exposed to 2% NMS.
Loss of ~50% baseline TER was observed for cells incubated with 10% NMS and H2O2 compared to the control cells without serum added. Around ~30% loss in
TER was observed for cells incubated with 2% NMS and H2O2. To test the effect of mFHRA and mFHRB on TER, 100 ng/ml of each protein was added to cells in the presence of 2% normal mouse serum. The data show that addition of mFHRB results in a loss in TER which is similar to stressed cells exposed to 2% NMS.
Addition of mFHRA results in a greater loss in TER that is comparable to the standard containing stressed cells with 10% NMS.
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Summary
Here we explored two relevant epithelial cell types, renal tubular epithelial cells and retinal pigment epithelial cells, and examined the role that mFHR proteins have on inducing complement activation. The results from experiments with TEC cells in the presence of 10% normal mouse serum show that both mFHRA and mFHRB likely antagonize FH and induce complement activation. Similar results were observed using an immortalized human retinal pigment epithelial cell line
(ARPE19). Addition of H2O2 is required to stress cells and activate complement in reactions with 10% NMS. Here we show that addition of mFHRA or mFHRB in 10% mouse serum is sufficient to activate complement on healthy, nonstressed cells.
Experiments measuring TER of ARPE19 monolayers also support this data.
Addition of mFHRA, and to a lesser extent mFHRB, activates complement resulting in destruction of the cell monolayer, as determined by loss in TER. Addition of mFHRA results in loss of TER that is equivalent to what is observed for stressed cells in 10% NMS. Together, these results provide the first evidence that mFHRs are able to block mFH function on different nucleated cell types and activate the alternative pathway. The next chapter will discuss how this knowledge and development of mFHR antibodies can be used to explore the function of mFH and mFHR proteins in vivo.
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CHAPTER VII
CREATION OF MFH AND MFHR SPECIFIC ANTIBODIES
The underlying goal of this research is to characterize the murine family of
FHR proteins. As discussed in earlier chapters, mFHRA and mFHRB proteins antagonize the protective function of mFH on different cell surfaces by competing with mFH for binding to C3d and other cell surface ligand(s). To better understand the function of these proteins and explore the biological role of the mFHR proteins in vivo, generation of monoclonal antibodies specific for each protein was undertaken.
While polyclonal antibodies against mFH are commercially available, these antibodies exhibit crossspecies reactivity and react with multiple mFHR proteins.
That is not unexpected given the sequence similarities between the different mFHR proteins and mFH (Figure 9). Production of FHR antibodies using Armenian hamsters was chosen over antibody development using in another species, such as rats or rabbits, for several reasons. These hamsters provide a unique system for producing mFHR antibodies because they are phylogenetically distinct from mice, yet respond well to murine (as well as human antigens) and fuse productively with murine myeloma cell lines (226). Furthermore, Armenian hamster antibodies are nonimmunogenic in mice and have been used in previous studies to develop neutralizing antibodies against a range of murine molecules including tumor necrosis factor (TNF) (227). Given the high homology between mFH and the mFHR protein sequences, this system was deemed to be the most optimal for generating specific antibodies that could be used as tools to quantitate the individual levels of circulating mFH and mFHR proteins and potentially inhibit only one molecule.
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Monoclonal Antibodies Specific for MFH
Armenian hamsters were immunized using recombinant mFH protein according to methods described in Chapter II to create monoclonal antibodies specific for mFH. Over 372 positive hybridomas were screened for reactivity against mFH using ELISA analysis, and 14 hybridomas (hybrids) were selected for further testing. The specificities of the hybrids were tested by ELISA using plates coated with commercially purified human FH (Comptech), recombinant mFHRA, mFHRB, recombinant human FH, or HisC3d (to eliminate hybrids which displayed reactivity to a Histag) (Table 2).
From these fourteen hybrids, two hybrids (55 and 70, highlighted in yellow in
Table 2) were chosen for cloning and to undergo further analysis. These clones,
AmFH55.7 and AmFH70.6, were purified and determined to have IgG1 kappa isotypes. Affinity for mFH was analyzed using surface plasmon resonance. Briefly, a capture antibody (antiIgG1 kappa isotype antibody HIG632, Kappler/Marrack
Laboratory) was coated on a CM5 chip, followed by capture of AmFH55.7 or AmFH
70.6, and then recombinant mFH was flowed over the chip. Apparent affinities for
AmFH55.7 and AmFH70.6 for recombinant mFH were determined to be 5 nM and
160 nM respectively (Figure 30).
The concentration of human FH in normal serum is reported to be between
115562 µg/ml. Here we evaluated the concentration of murine FH by developing a sandwich ELISA using murine FH monoclonal antibodies AmFH55.7 and AmFH
70.6. Data from recombinant murine FH at known concentrations was fit using a 4 parameter logistic curve fit in order derive the protein concentrations of mFH found
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in different mouse sera. For this assay, AmFH70.6 is used as a capture antibody and biotinylated AmFH55.7 is used as the detection antibody. The amount of FH measured in C57BL/6J mice is between 700800 µg/ml. The concentration of FH measured in FH+/ mice (~300 µg/ml) and FH/ (<30 µg/ml) is substantially less as anticipated (Figure 31).
Table 2 Reactivity of 14 AntiMFH Hybridomas from Initial Screen
Commercial Recombinant Hybrid # mFH Human FH mFHRA mFHRB HisC3d Human FH
40 + +
55 +
65 + (+) + (+)
70 +
80 +
144 (+)
160 + + + (+)
171 + (+)
175
183
218
229 + + + +
240 +
372 +
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Figure 30 Affinities of AmFH55.7 and AmFH70.6 for Recombinant MFH. Surface plasmon resonance was used to determine the apparent KD values for AmFH55.7 and AmFH70.6 binding to recombinant mFH. AmFH55.7 shows higher affinity for mFH (5 nM) than AmFH70.6 (160 nM).
Interestingly, mFH was detected at higher concentrations in mice deficient in complement Factor B (FB). This was an incidental observation and this experiment needs to be further validated. However, FB is an alternative pathway activator and
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FB/ mice have absent alternative pathway activation and decreased CP activation, which is thought to be the result of loss of the AP amplification loop (228). Another study has also examined backcrossing of FB/ onto the MRL/lpr model of systemic lupus erythematosus. This study demonstrated that FB deficiency decreased proteinuria, lessened renal pathology including glomerular IgG deposition and vasculitis, and decreased C3 consumption compared to FB+/+ and FB+/ MRL/lpr models (229). Therefore, we could speculate that the higher FH concentration in FB knockout mouse serum compared to WT serum may result from loss of AP activation. Given that FH is a major regulator of the AP, loss of this pathway would suggest a lower requirement for FH control on cell surfaces which could explain the higher fluid phase FH levels that are observed. Decreased AP activation in combination with normal or increased levels of FH could contribute to the the renal findings observed in the FB/ MRL/lpr study. However, further work is necessary to determine the accuracy of mFH concentration measurements in this assay.
Developing MFHR Specific Antibodies
A substantial amount of effort was devoted to developing antibodies that are specific for the different mFHRs. Three Armenian hamsters were immunized with recombinant mFHRA, mFHRB, or mFHRC. Positive hybridomas were screened against the various recombinant proteins in ELISA assays as described above. To date, nearly a dozen hybrids showing specificity for mFHRA, mFHRB, or mFHRC have been selected and are frozen for future analysis.
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Concentration of Factor H in Murine Serum 1500 C57BL/6J (FH+/+) FH +/- 1000 FH -/- Complement Factor B -/-
500 g/mL of Factor H g/mL µ
0
FH +/- FH -/-
C57BL/6J (FH+/+)
Complement Factor B -/-
Figure 31 Quantitation of Murine Factor H in Serum Using AmFH55.7 and AmFH70.6 Monoclonal Antibodies. A sandwich ELISA assay was developed to measure the concentration of mFH in serum. AmFH70.6 was used as the capture antibody and biotinylated AmFH55.7 was used as a detection antibody in this system. The concentration of mFH measured was between 700800µg/ml in wild type mouse serum. The concentration of mFH in a FH+/ mouse was approximately ~300µg/ml while detection of mFH in the FH/ mouse was insignificant.
While characterization of the biochemical functions and different features of these antibody candidates is required, preliminary results from ELISA and Western blotting show that one of the antibodies, AmFHRA231, reacts against serum proteins with molecular weights corresponding to mFHRA or mFHRB. During hybrid screening, this antibody was observed to have strong reactivity against mFHRA and mFHRB, and weak reactivity against mFH. AmFHRA231 does not detect mFHRC by ELISA.
When a 1:10 dilution of either wildtype or FH knockout (FH/) mouse serum was separated under nonreducing SDSPAGE conditions, two bands can be
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visualized (Figure 32). One band can be visualized around ~110130 kDa and another band around ~4050 kDa. Under reducing SDSPAGE conditions, a single band is visualized at ~60 kDa. Given that similar size bands are detected in FH/ mouse serum, it seems unlikely that the detected protein/s are mFH. Therefore,
AmFHRA231 most likely detects mFHR proteins. However, a question that remains is whether AmFHRA231 detects mFHRA protein, mFHRB, or a combination of different mFHR proteins and/or possible dimer combinations.
Currently, the only publication that has examined murine FHR proteins in mouse plasma is by Hellwage et al. 2006 (97). In their study, murine FHRs were detected in mouse plasma and liver tissue by separation using 10% SDSPAGE under nonreducing conditions and western blotting. Rabbit antisera specific to either mouse FH or rat FH was used to probe for the mFHRs. FH/ liver extracts were also analyzed. From their western blot analysis of plasma and liver samples, they concluded that several bands present between 4055 kDa were likely to be mFHRB proteins. Several other bands between 90100 kDa were presumed to be proteins that were transcribed from the mFHRC gene locus. To date, no further analysis has been performed to confirm the identities of these bands. For comparative purposes in our own study, mFHR proteins were analyzed using a 10% SDSPAGE gel under nonreducing conditions and the membrane was blotted using a polyclonal hamster antimFHRA antibody (AmFHRA106) which detects all mFH and mFHR proteins by
ELISA. Multiple bands can be observed at similar positions that were described by
Hellwage et al. 2006 (97).
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Given that the identities of the different bands found in mouse serum have not been confirmed with specific antibodies, AmFHRA231 antibody was used to immunoprecipitation mFHR proteins from wildtype mouse serum. Proteins represented by positions marked as Band 1 and Band 2 were sent for mass spectrometry analysis (Figure 32). Analysis of Band 1 by mass spectrometry identified sequences from mFH as well as mFHRA and mFHRB which are shared between all three proteins. Additionally, mFH has a MW of ~140 kDa so it is possible that this band is mFH. However, the presence of Band 1 in FH/ serum suggests it is a mFHR protein, possibly a dimerized version of mFHRB or mFHRC which also shares sequence identity to mFH and mFHRA and mFHRB. Therefore, our analysis focused on Band 2 at ~50 kDa which contained peptide sequences identifying both mFHRA and mFHRB (blue boxes, Figure 33). A single peptide sequence (red box, Figure 33) identifies only mFHRA and not mFHRB. This sequence is found in fulllength mFH, so it is possible that the fragment is from an alternatively spliced form of mFH or degraded product, but the peptide sequence is not present in other mFHR proteins such as mFHRC or mFHRE. It is specific for mFH or a protein encoded by the mFHRA gene (Figure 34).
A critical point which must be emphasized is that the protein sequence of mFHRA is a prototype sequence. During the original characterization of the four murine transcripts by Vik et al. 1990, four classes of transcripts were identified (96).
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Figure 32 Western Blot Analysis of Murine FHR Proteins in WT and FH/ Serum and Plasma. A) Recombinant mFH and mFHR proteins (5 ng/lane) and serum from wildtype (WT) and FH/ mice (1:10 dilution in PBS) were separated by 10% SDS PAGE under nonreducing conditions. AmFHRA231(hamster antimouse mFHRA antibody) detected both mFHRA and mFHRB recombinant proteins. In wildtype serum and FH/ serum, two bands are observed. These two bands were analyzed using mass spectrometry B) When reducing conditions are used, a single band in both WT and FH/ serum and plasma is detected. C) 10% SDSPAGE under non reducing conditions followed by western blot using a polyclonal hamster antimouse mFHRA antibody that reacts with all mFH and mFHR proteins by ELISA. Similar observations were made by Hellwage et al. 2006 using rabbit antimouse FH antiserum (97).
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Within the Class A family, there were four separate clones with sequences containing different start and stop sites, but all were predicted to contain 7 SCR domains, which would produce a protein with a molecular weight ~5055 kDa. Based on these sequences, a prototype was designed by combining sequences from two clones (3A4 and 5G4).
Subsequent advances in sequencing of the mouse genome and improvements in annotation have allowed for the positions of the various original mFHR classes to be analyzed. Analysis of both the nucleotide sequences and predicted proteins of the murine FHR gene family shows that the prototype mFHRA
(3A4/5G4) sequence was likely based on a region which is currently annotated as an unprocessed pseudogene in the Ensembl.org genome browser.
This region was labeled as an unprocessed pseudogene because it contains possible frameshift mutations and stop codons that are predicted to disrupt the open reading frame (ORF). Furthermore, it is defined as an “unprocessed” pseudogene because it is predicted to have unspliced intron sequences. The Ensembl database lists the sequence of a potential Gm16332 transcript as having 16 exons. Analysis of exons 37 shows that this region could plausibly encode a protein with domains that share high sequence identity with SCRs 15 of the mFHRA prototype and SCRs 13 of mFHRB. The remaining 2 SCRs of the mFHRA prototype share sequence identity with all but two residues found in SCRs 4 and 5 of the mFHRB gene
(Figure 33).
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Figure 33 Alignment of Exons 37 of Pseudogene Gm61332 and MFHRA and MFHRB sequences. The Gm61332 pseudogene sequence contains 16 exons according to Ensembl.org. Translation of sequences from Exons 37 shows high homology to SCR domains found in mFHRA and mFHRB. Immunoprecipitation of mFHR proteins from wildtype serum using AmFHRA231 antibody followed separation by 10% nonreduced SDSPAGE revealed a band at ~50kDa which was sent for analysis by mass spectrometry. Blue boxes contain residues determined by mass spectrometry that identify mFHRA and/or mFHRB. The red box highlights a peptide sequence specific for mFHRA. This sequence is not found in other mFHR proteins but is present in fulllength mFH.
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Figure 34 Mass Spectrometry Identifies a Peptide Sequence Specific to MFHR A. Immunoprecipitation of FHR proteins from wildtype serum using AmFHRA231 and separation by nonreducing SDSPAGE. Excision of a protein band at ~50kDa and subsequent mass spectrometry analysis revealed the sequence shown (SCDMPVFENSITK) with m/z 764.34 amu identifying protein Cfhr2 Variant (Uniprot Q8R0I8) or mFHRA (Uniprot: Q61407, sequence of prototype 3A4/5G4). Observed fragment ions are indicated and marked by a line placed above or below the corresponding amino acid. While it is possible that the identified fragment may be the result of a splice variant of fulllength mFH, the molecular weight at ~55kDa is consistent with the predicted molecular weights of the Cfhr2 variant or a protein encoded by the mFHRA gene.
One explanation is that the current annotation of the genome is misleading and Gm16332 is not an unprocessed pseudogene, but is a processed gene with a mFHRA transcript that differs by only a few residues from mFHRB. The high degree of sequence identity between the mFHRA and mFHRB gene loci likely contributes to this confusion. Additionally, review of the UCSC genome browser does not list the Gm16332 pseudogene, but the same chromosomal region is labeled as a processed transcript called the “Cfhr_2 variant” which has 9 exons.
Review using a third genome browser (vega.sanger.ac.uk) lists the gene again as an
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unprocessed pseudogene. The annotation remarks in this genome browser define it as an “overlapping locus” or a region where exons of one locus overlap exons of a readthrough transcript of another locus.
Summary
One of the obvious challenges with characterizing the murine FHR gene family is the high sequence identity that mFHRA, mFHRB, and mFHRC genes share with fulllength mFH and to one another. Compared to the human FHR family which encodes several proteins (FHR1, 2, and 5) with Nterminal domains bearing
<45% identity to FH, the murine mFHR proteins have much higher sequence homology with fulllength mFH. The three Nterminal SCR domains of mFHRB differ from mFH SCRs 57 by only five residues while the two Cterminal domains differ by
14 residues. Any protein encoded by the mFHRA gene is predicted to have N terminal SCR domains that differ by five residues from mFHRB. Therefore, distinguishing between mFH, mFHRA, and mFHRB nucleotide and protein sequences is more challenging than mFHRC or mFHRE.
To add to the confusion, the initial expression analysis of mFHRB and mFHRC proteins performed by Hellwage et al. 2006 only speculated about the identities of different size bands analyzed by nonreducing western blotting with rabbit antisera specific to mouse or rat FH (97). Identification of these proteins using mass spectrometry and/or other techniques was not performed. Given that the predicted molecular weights of mFHRA, mFHRB, and mFHRE are all between
~4055 kDa, identification of bands in this region is very challenging. Furthermore, dimerization of mFHRA, mFHRB, or mFHRE proteins by disulfide bonding could
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create complexes appearing at ~100110 kDa on a nonreduced SDSPAGE gel, which is the predicted molecular weight of mFHRC protein. Alternative splicing of the mFHRB and mFHRC genes also may explain why several bands can be visualized when performing a western blot with a polyclonal mFH/mFHR antibody or antiserum.
Therefore, the objective of experiments in this chapter was to generate monoclonal antibodies that have specificity for mFH and the different mFHR proteins. Armenian hamsters were chosen as a unique system for creating these different antibodies. Initial characterization of two monoclonal antibodies, AmFH
55.7 and AmFH70.6, was performed and binding affinities of the antibodies for recombinant mFH were determined using SPR. Furthermore, a sandwich ELISA is being developed to determine the concentration of mFH in mouse serum.
Finally, attempts were made to create antibodies specific for native mFHRA and/or mFHRB proteins. ELISA and western blot analysis indicated that one antibody candidate, AmFHRA231, recognized recombinant mFHRA, mFHRB, and to a lesser extent, mFH but not mFHRC. A trial IP experiment was performed to examine native mFHRA and/or mFHRB proteins in wildtype mouse serum. Mass spectrometry analysis of a band at ~50 kDa identified sequences present in mFHR
A, mFHRB and mFH. A peptide fragment specific for mFHRA but not mFHRB was also identified at this molecular weight suggesting that the mFHRA may not be a pseudogene, but may actually encode a protein that shares high sequence identity and a similar molecular weight to mFHRB. The results from this experiment were
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complicated by the fact that mFH and mFHR share high sequence identity. Further testing is needed to understand the specificity of these antibodies for native mFHRs.
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CHAPTER VIII
IN VIVO STUDIES
To examine the different mechanisms of mFHR proteins in vivo, two separate pilot studies in animal models were investigated. A collaboration with the Rohrer lab
(Medical University of South Carolina) was used to assess whether mFHRA and mFHRB promote or otherwise modulate pathogenic alternative pathway activation in a murine model of choroidal neovascularization (CNV). The CNV model is a rodent model of wet AMD where lesions in Bruch’s membrane are generated through argonlaser induced photocoagulation (230). Injury to Bruch’s membrane results in AP activation and release of complement activation fragments, which generates VEGFdependent CNV. Tissue damage is assessed using imaging techniques and immunohistochemistry (231). Complement activation through the AP plays a central role in disease pathogenesis in this model (203).
For initial experiments, 6 animals (2 animals/group) were given a tail vein injection with either 25 µg, 75 µg, or 200 µg of mFHRA on day 0, day 2, and day 4 postCNV. This protein was determined to be the most potent antagonist of mFH in sheep erythrocyte assays which was the rationale for investigating this protein in vivo.
Results from this study indicated no change in CNV lesion size in the animals injected with either 25 µg or 75 µg mFHRA, however a 3fold reduction in CNV lesion size was observed in both animals that received 200 µg doses of mFHRA
(data not shown; results were described on 11/24/14 during oral conversations with
B. Rohrer and G. Schnabolk). The molar equivalent concentration of 200 µg mFHR
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A protein in a mouse with a total blood volume ~1.5 ml would be ~2.67 µM, which is equivalent to an estimated serum concentration of FH between ~0.73.6 µM.
The results from this initial study were unexpected given the potent inhibitory effect that 2.0 µM of mFHRA had on mFH function in sheep erythrocyte lysis assays and on complement activation in cell surface assays. Therefore, we decided to test whether a lower dose of mFHRA could impact CNV lesion size using the rationale that a high dose may have induced supraphysiological concentrations and effects. In a subsequent followup experiment, 8 animals (2 animals/group) were injected with either 5 µg mFHRA, 5 µg mFHRB, 5 µg mFH, or PBS. A single control animal was used for comparative purposes and the mouse not receive CNV lesions or any protein treatment. No change in CNV lesion size was detected in any of the treatment groups compared to controls (data not shown; results described by email correspondence with B. Rohrer and G. Schnabolk). Therefore, further CNV experiments were paused until more data regarding mFHR function was obtained.
One caveat to this study may have been that proteins were concentrated to achieve small volumes required for tail vein animal injections. Concentration of the protein to a smaller volume may have reduced protein stability and/or resulted in aggregation or alteration in function. Thermal stability assays were later used to assess the stability of mFHRs at different concentrations. At concentrations >0.5 mg/ml in PBS, proteins have been shown to be less stable. The concentration of the aliquot of mFHRA used in this study was recorded as 0.45 mg/ml. Additional work, including a study of the different halflife values of these proteins and where they may localize after injection, may help with the interpretation of these results.
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Another relevant animal model that was investigated was a mouse model of acute kidney injury. Alternative pathway activation on the basolateral surface of tubular epithelial cells is found in ischemic kidney injury in both rodents and humans, and is known to be pathogenic in murine models (224, 225, 232). C3 deposition on the basement membrane of injured tubules suggests that FH is unable to regulate
AP activation on these types of cells (233). For this doubleblinded study, 20 animals were used (five animals per treatment group). Prior to IR induction, C57BL/6 mice were given a 100 µg retroorbital injection of either mFHRA, mFHRB, or mFHRC proteins or PBS (control). Animals were sacrificed 24 hours postIR and tissues and sera were analyzed.
Assessment of serum blood urea nitrogen (BUN), which directly correlates with kidney injury, indicated that only one animal in each of the mFHRA and mFHR
B treatment groups had bilateral injury (BUN >120 mg/dL). Other animals had baseline BUN values (BUN 2030 mg/dL) or unilateral damage (BUN 4060 mg/dL)
Out of all of the treatment groups, the mFHRC group had baseline BUN values except for 1 animal with moderate injury (74 mg/dL). Additional analysis of C3b staining of kidney sections was also inconclusive. This model is reported to require 5 or 6 pairs of mice in order to show a difference between treatment groups (205).
Therefore, given the small number of animals in each treatment group and the variability in outcomes following IR in this animal model, a larger study is necessary in order to make any statistically significant conclusions.
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CHAPTER IX
DISCUSSION
The complement system is a major global mediator of immune surveillance and homeostasis serving as a bridge between the innate and adaptive immune systems. Research of the complement system in our laboratory is focused on understanding regulation of the different pathways, especially the alternative pathway which is the primary pathway by which the system damages cells and tissues in human diseases. The spontaneous hydrolysis of C3 leading to generation of C3b is known as AP tickover and must be wellcontrolled in order to prevent inappropriate and potentially rapid AP complement activation from occurring both in the fluid phase and on cell surfaces. Therefore, the central goal of this thesis work has been to expand our understanding of molecules that regulate the alternative pathway and explore the different mechanisms by which they exert their function.
Factor H is a major soluble complement regulator that is essential for controlling alternative pathway activation in plasma and on cellsurfaces.
Phylogenetic studies of the complement system have shown that FH, similar to the activation pathway proteins, has ancient origins (87). Comparison of FH to a homologous classical pathway protein (the chain of the C4b binding protein) has demonstrated that FH shares a closer evolutionary relationship with complement proteins found in barred sand bass and nematodes. The ancient origin of FH also agrees with the theory that the alternative pathway is evolutionarily older than the classical pathway. FH controls complement activation on host cell surfaces through engagement of polyanionic surface markers, such as sialic acid, and C3 fragments,
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such as C3d. Mutations or deletions in FH, particularly within the Cterminal region, are associated with a number of different autoimmune and inflammatory illnesses.
Pathogens and alteredhost cells (such as cancer cells) exploit the protective function of FH by recruiting FH to their surfaces as part of an immune evasion/modulation strategy.
While a significant amount is known about the structure and function of FH, the functional roles of a group of structurally similar molecules, called the factor H related (FHR) proteins, are less understood. Unlike FH, the FHR gene family is thought to have undergone more recent duplication events after the separation of the rodent and primate lineages. A single exon typically encodes one SCR domain, and duplication of exons within the FH and FHR family is suggested to be part of an ongoing process. Gene conversion, duplications of FH exons, and exon shuffling are all thought to contribute to the evolution of the FHR gene cluster. Therefore, could
FHR genes serve as a way for an ancient system to update and expand its repertoire of biological functions and interact with bacteria and other pathogens in a speciesspecific manner?
The CFHR genes are positioned adjacent to CFH on human chromosome 1 within the RCA gene cluster. These genes encode a series of proteins that are structurally similar and share high sequence homology to FH. They also exhibit similar functions to FH, such as binding to heparin and C3b and variants and deletions within the FHR family are associated with diseases including aHUS, C3 glomerulopathy (including MPGNII), SLE, and AMD. Deletion of FHR genes resulting in the absence of FHR proteins, such as FHR3 and FHR1 (hFHR31Δ) or FHR1
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and FHR4 (hFHR14Δ), does not appear to alter normal development but does appear to confer risk or protection to various diseases. The deletion of hFHR31Δ has been shown to increase the risk for the development of aHUS and FH autoantibodies while the same mutation is protective for AMD. The association of this deletion mutation with two different diseases that impact distinct organ systems indicates that FHRs have complex contextspecific roles in complement regulation.
While previous research has shown that FHR proteins possess regulatory functions such as weak cofactor activity, recent structurefunction studies by
Goicoechea de Jorge et al. 2014 show that a group of FHR proteins comprised of
FHR1, FHR2, and FHR5, contains a dimerization motif within their Nterminal SCR domains (112). Serum analysis revealed homo and heterodimerization of these proteins and functional assays were used to demonstrate how formation of higher order structures increased their avidity for C3 fragments allowing them to antagonize
FH function as demonstrated in experiments both in vitro and in vivo. Therefore, understanding the mechanisms by which FHR proteins regulate complement is essential for explaining their different genetic associations and proving causality.
Furthermore, animal models are required in order to evaluate the biological roles of the FHR proteins in various diseases and for the development of therapeutic interventions.
The first study of the murine FHR family characterized four classes of mFHR transcripts. Class A was predicted to bear the highest sequence homology to mFH and encode a protein with 7 SCR domains. Class B transcripts were predicted to encode a protein with 4 SCR domains, while Class C and D were predicted to have
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13 SCRs and 5 SCRs each. Subsequent research of the class B and C transcripts, referred to as mFHRB and mFHRC, indicated that the mFHRB gene encoded a 5
SCR domain protein while mFHRC was 14 SCR domains. Furthermore, recombinant production of mFHRB and initial characterization studies of mFHRB were performed; however, many questions were unanswered about the roles of these proteins and their correlation with the human FHR counterparts.
The research presented in this thesis provides a more extensive characterization of the mFHRB and mFHRC proteins in addition to evaluating a third protein, mFHRA, whose sequence is derived from the original characterization of class A mFHR transcripts. Following expression and purification of proteins using a 293F mammalian expression system, functional assays were performed to understand how mFHR proteins modulate complement activation by competing with mFH for C3b/C3d on selfsurfaces.
To assess the functional activity of recombinant mFH and mFHR proteins, different hemolytic assays were performed. An AH50 assay using rabbit erythrocytes was used to assess the overall impact of mFH and mFHR proteins on AP function.
In this experiment, mFHRA appeared to reduce overall AP activation while mFHRB and mFHRC appeared to increase AP activation. One explanation may be that addition of mFHRA in this assay may have resulted in the rapid consumption of C3 leading to decreased hemolysis of cells which could give the impression of decreased AP activity. Therefore, to better explore AP complement activation on specific cell surfaces, a different type of hemolytic assay was performed. Previous work has demonstrated that the last two SCR domains of FH (SCRs 19 and 20) are
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critical for FHmediated protection of cell surfaces. To address whether the mFHR proteins deregulate mFH cellsurface protection, functional assays using murine
FH1920 inhibitor, mFHRA, mFHRB, and mFHRC were performed. In these hemolytic assays, normal sheep erythrocytes are added to complement sufficient wildtype mouse serum, but hemolysis of the cells is prevented by mFH that is present in the serum. Reduction of mFH from the surface of these cells results in AP activation and subsequent hemolysis. Here we were able to demonstrate that addition of mFHRA and mFHRB in the presence of normal mouse serum induced hemolysis of sheep erythrocytes. Both mFHRA and mFHRB act as potent antagonists of mFH on sheep erythrocyte cell surfaces. While the mFH1920 inhibitor and mFHRB induced ~50% hemolysis at 7 µM concentration, mFHRA is more potent and induced 100% hemolysis at ~2 µM. Interestingly, mFHRC does not appear to increase hemolysis of sheep erythrocytes (and possibly is protective on specific cell surfaces). Taken together, the results from rabbit and sheep hemolytic assays show that mFHR proteins are able to perturb the overall function of the alternative pathway. The results from the different hemolytic assays suggest that mFHRs may have different biological functions and/or recognize different surface ligands.
Unlike mFHRC, our results with both mFHRA and mFHRB agree with functional studies of the human FHR proteins. Hemolytic assays with guinea pig erythrocytes show that both FHR1 and FHR5 inhibit FH function by competitively blocking the interaction between FH and C3b (112). Structural studies have established that this interaction occurs through FH SCRs1920 and the reactive
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thioester containing domain (TED) located on the C3d part of C3b. Five residues
(Asn1117, Asp1119, Gln1139, Tyr1142, and Tyr1190) within SCRs 1920 form intermolecular hydrogen bonds with C3d. Sequence alignment of human FH and the mFH family shows that these residues are conserved between the two species.
Therefore, we predicted that mFH and mFHRs would bind C3d and mutation of conserved residues within the binding interface would inactivate our proteins.
Using ELISA analysis and SPR, binding of mFH and mFHR proteins to C3d was assessed. Apparent dissociation (KD) values for mFHRA and mFHRB proteins were determined as 136 nM and 546 nM. These values are within the same range as apparent KD values calculated for the interaction between human C3d and human
FHR3 and FHR4. Using a similar method, C3d was immobilized on a chip using amine coupling and FHR3 and FHR4 proteins were analyzed in fluid phase.
Apparent dissociation values for FHR3 and FHR4 were calculated as 87 nM and
260 nM respectively (124). They also demonstrated that FHR3 and FHR4 have similar binding affinities with C3b and that the reverse analysis, with FHRs immobilized on a chip and C3d/C3b in fluid phase, produced similar results. One caveat is that the kinetics of the human proteins were analyzed using a 1:1 binding model while the murine proteins were fit using a 2state binding model. Taking this into consideration, the binding kinetics of mFHRA and mFHRB could also be compared to SPR results from the binding interaction observed between C3b and mutant human FHR512129 protein (112).
This mutant, found in patients with C3 glomerulopathy, has a duplicated dimerization domain (from duplication of SCRs 12). Binding affinities for wildtype
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mFHR5 and mFH to amine couple C3b were determined to be 1.7 µM and 6.6 µM.
While a binding affinity for the FHR512129 mutant was not determined, functional analysis of this mutant using hemolytic assays also showed greater hemolysis than wildtype controls. This result was attributed to the mutant’s increased ability to form higher order complexes and compete with FH for binding to C3b. Gel filtration chromatography was used to analyze the native mFHRB and results suggest that mFHRB may be capable of forming higher order complexes. Given that the fit of both the mFHRA and mFHRB curves resembles the curve for FHR512129, additional analysis of these proteins for the formation of higherorder complexes than dimers is necessary.
Comparison of binding affinities of mFHRC, mFH, and mFH1920 shows that these proteins exhibit weaker binding to C3d. Apparent dissociation constants were determined to be 1.04 µM for mFHRC, 3.85 µM for mFH, and 22 µM for mFH1920.
While binding of murine FH to murine C3b has been previously analyzed (220), affinity values were not calculated. However, two different dissociation constants have been calculated using SPR for the interaction between human C3d and FH19
20. Kajander et al. 2011 calculated a KD value of 0.18 µM for the C3d:FH1920 interaction while Morgan et al. 2011 determined a KD value between 6.28.2 µM
(216, 217).
To further analyze the functional homology between the human and murine
C3d binding site, mutation of the putative C3d binding site on mFHRA and mFHRB was performed. Results show that mutation of two residues (corresponding to human FH residues N1117 and Asp1119) was sufficient to disrupt mC3d binding of
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mFHRA and mFHRB in both ELISA and SPR assays. Additionally, mutant mFHR
B exhibited significant loss of function in a sheep hemolytic assay further suggesting that mFHRs compete with mFH for the same target (C3b/C3d) on cell surfaces. The direct correlation between mFHR binding affinity for C3d and functional activity in hemolytic assays further establishes how these proteins modulate complement through competition with mFH for C3b/C3d on cell surfaces.
Additionally, the important role of other cell surface markers must also be addressed. The sheep hemolytic assay is dependent on FH recognition of C3b/C3d as well as sialic acid residues that are present on the cell surface. The ability of FH to engage C3b increases 10fold in the presence of sialic acid residues and the structural basis for FH recognition of sialic acid on cell surfaces has been established (172). A recent study has proposed that FH simultaneously engages sialic acid and C3b in order to regulate complement activation (222). Mutation of sialic acid binding residues on FH1920 resulted in the loss of its ability to antagonize FH. This suggests that the behavior of FH, and perhaps the ability of
FHRs to antagonize FH function, is dependent on both C3b/C3d and surface sialic acid cell surface content. Implicit in this theory is that cells that possess low amounts of surface sialic acids (such as rabbit erythrocytes) are not subject to this same mechanism.
Understanding the behavior of FHRs on different cellsurfaces is critical for clarifying their associations with disease and explaining the different disease phenotypes. Complement activation by mFHRs on two relevant cell lines was examined. Our results show that increased C3b deposition on both murine tubular
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epithelial cells as well as normal human retinal pigment epithelial cells occurs when cells are incubated with normal mouse serum with addition of mFHRA or mFHRB.
With both cell types, mFHRA appears to generate greater complement activation than mFHRB which supports our other binding and functional data. Additionally, complement activation by mFHRs on a human cell line and demonstration that mFHRs can bind human C3d, provides evidence that the murine proteins can serve as orthologs of human FHRs in animal studies. Together, these data provide important evidence for further evaluation of the roles of mFHRs in vivo.
While it is fairly straightforward to compare the sequence homology of
C3b/C3d and GAGbinding sites between the different mFH and hFH protein families, it is much more challenging to make direct comparisons i.e. does mFHRB function more like human FHR3 and FHR4 or does it behave like FHR5? For direct comparisons such as this, better tools are required. Development of antibodies that recognize specific mFHR proteins is important for elucidating the different biological roles that these proteins may have. A description of two novel mFH antibodies was presented here and a sandwich ELISA is currently being tested to evaluate the concentration of mFH in serum. Additionally, another antibody that reacted with mFHRA and mFHRB proteins by ELISA was used to immunoprecipitate native FHR proteins from wildtype mouse serum. Separation of proteins by SDSPAGE followed by mass spectrometry analysis identified a band at
~50 kDa to be mFHRB and/or mFHRA protein. This experiment needs to be redone using FH/ serum in order to IP mFHRs in serum that does not contain mFH so that mass spectrometry analysis will be more informative.
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Finally, our results demonstrated that mFHRC does not inhibit FH function in a sheep erythrocyte assay, however it appeared to modulate AP activation in an
AH50 assay. These results suggest the possibility that mFHRC has other important contextdependent biological functions. Compared to the mFHRA and mFHRB proteins, mFHRC contains twice as many SCR domains (14 SCRs), but is 6 SCR domains shorter than mFH. Notably, mFHRC (as well as all other FHR proteins) lacks sequence homology to FH SCRs14, which are regulatory domains. Perhaps mFHRC has a different role in cellrecognition and complement regulation?
Although the in vivo IR study was not large enough to make any statistically significant conclusions, animals injected with mFHRC had noticeably lower BUN values than animals in any of the other treatment groups. One could speculate that mFHRC may have contextdependent functional roles in the kidney. Perhaps mFHRC can destabilize C3 convertases on TEC surfaces? Or maybe mFHRC has fluid phase regulatory abilities and acts as a cofactor for FI? These results suggest that we must evaluate the mFHR proteins in a variety of different contexts in order to fully understand how they modulate complement. We must examine how FHRs regulate complement both in fluid phase versus on different cell surfaces taking into consideration that these proteins may function differently in the context of an autoimmune disease model versus a cancer or infectious disease animal model.
From an evolutionary perspective, how would an ancient complement system keep up with a modern world with different environmental agents, viruses and other pathogens, and the species specific development of cancer? The evolution of the
FHR gene family may serve as a way for the complement system to diversify its
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biological roles. Understanding the differences between mFHRA/mFHRB and mFHRC may offer insight into how FHR proteins exert influence over complement regulation which could allow for strategic treatment against different diseases.
In summary, these results demonstrate that murine FHR proteins modulate complement activation by antagonizing FH function on cellsurfaces. Interestingly, we also show that neighboring genes, mFHRB and mFHRC, encode FHR proteins that have unique functions. While mFHRB is a potent inhibitor of mFH, mFHRC does not appear to have inhibitory functions which may be context dependent.
Similar to human FHR1 and FHR5, mFHRA or mFHRB compete with mFH for binding to C3b/C3d on cell surfaces and have similar C3d binding affinities to FHR3 and FHR4. We identified that the binding interaction between C3d and mFHR proteins is conserved between human and murine systems. Furthermore, we demonstrate that mFHR binding affinities for C3d directly correlate with their functional activity in hemolytic assays. This research will be helpful for guiding future work in different animal models of disease, such as aHUS and AMD, which could potentially lead to the development of novel therapeutics.
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CHAPTER X
FUTURE DIRECTIONS
Characterizing Additional MFH and MFHR Interactions and Functions
Do mFHRs bind C3b? To further evaluate the roles of mFH and the mFHR proteins in complement regulation, other binding assays could be performed. Using the same methods described to characterize the mC3d binding interaction, binding of mFHRs to murine C3b could be evaluated. Currently murine C3b must be purified from mouse serum; however methods are wellestablished for purifying C3 from plasma (234). C3b is a larger complement fragment than C3d and the main target for FH. Given the results from binding assays with C3d, we would anticipate similar affinities of the mFHRs for C3b.
Do mFHRs interact with other terminal complement components? To generate the terminal complement complex (TCC), C5 must be cleaved by either the
AP C5 convertase (C3bBb3b) or CP C5 convertase (C4a2a3b). Cleavage of C5 generates C5a and C5b. Binding of C5b and C6 (C5b6) initiates terminal complex formation and recruits C7C9. A study has demonstrated that FHR1 can regulate terminal complex formation by blocking C5 convertase activity (114). In order to test the ability of mFH and mFHR proteins to bind C5 and C5b6, purified human C5 and
C5b6 could be purchased (Complement Technology Inc.).
Do mFHRs have FIcofactor activities? Several studies have suggested that human FHRs exhibit different functional abilities. Early studies of FHR3 and FHR4 show weak FI cofactor activity for these proteins (124). Another study has shown that FHR5 also has weak FI dependent cofactor activity. This same study also
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reported that FHR5 inhibited AP C3 convertase activity in fluid phase but not on solid phase (107, 132).
A fluid phase cofactor assay could be used for determining whether mFHR proteins impact FI cleavage of human C3b. Incubation of C3b and FI with each of the FHR proteins could be performed followed by analysis of the C3b degradation products using SDSPAGE. The C3b αchain and βchains are separated based on size. In this assay, mFH is a positive control and cleavage of the C3b αchain by FI will result in the generation of fragments between 40kDa and 68kDa. A lane with
C3b and FI only is the negative control.
Do mFHRs influence C3/C5 convertase activity? To measure whether mFHRs impact C3 and C5 convertase decay, generation of the C3bBb complex is required. This complex is formed by incubating C3b and C3 with FD and FB.
Increasing amounts of the recombinant FHRs would be added to the reactions and the generation of C3a can be assessed by commercially available ELISA assays
(Quidel). For evaluating the role of mFHRs in C5 convertase regulation, sheep erythrocytes should be incubated with C3b prior to adding C3, FB, FD, and properdin. After formation of stable C5 convertases on the erythrocytes, mFHRs should be added in addition to C5. Quantification of C5a production can be used to assess the role of the mFHRs in convertase breakdown.
While these assays provide only a few examples of different experiments that can be performed, an important future goal should be to contrast the function of a
FHR protein in the fluid phase versus its behavior on solid phase. FHR5 antagonizes FH function on cell surfaces acting as a deregulator; however, the
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ability of the protein to regulate complement by inhibiting AP C3 convertase formation in fluid phase (but not on surfaces) suggests the function of these proteins may be context dependent.
Identifying MFH and MFHR Interactions with Different Host Polyanions
One way FH is able to discriminate and inhibit complement activation on host cell surfaces versus pathogen surfaces is through recognition of host polyanions including sialic acid, glycosaminoglycans, heparin sulfate (HS), and dermatan sulfate
(DS) (235, 236). To analyze the interactions of mFHRs with a range of GAGs, different techniques could be used. Commercially available bovine lung heparin, which is used as an analogue of HS, dermatan sulfate, and chondroitin sulfate C can be digested using proteases heparinase I, chondroitinase, or chondroitinase AC.
Digestion of these GAGs will produce hexasaccharide fractions which can be purified and biotinylated before being applied to a streptavidinconjugated matrix
(HiTrap Streptavidin HP, GE Healthcare). To test the interaction between mFHRs and different GAGs, proteins can be applied to the column and eluted by using a
NaCl gradient while monitoring OD280 absorbance.
Another alternative method to identify interactions between mFHRs and
GAGs could be using SPR. Biotinylated GAGs could be immobilized on a streptavidincoated flow cell and the recombinant mFHRs will be injected to investigate binding capabilities of the different proteins.
Characterizing the Interaction of MFHRs and Sialic Acid
While the previously described assays could provide a general way to identify different host polyanions that interact with mFH and mFHRs, a more directed
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approach would be to evaluate the interaction between mFHRs and sialic acid. The interaction between FH and sialic acid has been established and residues that contribute to the sialic bindingsite on FH1920 have been identified (172).
Several residues determined to be important for the interaction between FH and Nacetylneuraminic acid (Neu5Ac) are conserved in the mFH and mFHR family. These include L1181, W1183, E1198, and R1215. The backbone amide and carbonyl groups of E1198 and the backbone of W1183 form critical hydrogen bonds with the Neu5Ac ring. Furthermore, recent work has shown that mutations to FH19
20 (E1198A or L1189R), result in loss of FH1920 ability to antagonize FH function in a sheep erythrocyte hemolytic assay (222). While the L1189 residue is not conserved in the murine FH and FHR proteins, several other key residues are present. To test whether mFHR proteins compete with FH recognition for sialic acid, mutant proteins could be created. Two of the most interesting mutations would be
W1183R and R1215Q; these mutations have been associated with aHUS patients and are conserved among the different murine proteins.
Given the conservation between C3d and sialic acid binding sites between the human and murine FH and FHR proteins, a combination of hemolytic functional assays and binding experiments could be used to test whether mFHR proteins compete with mFH for binding of C3b/C3d and/or sialic acid. The advantage to testing both C3d and sialic acid mutants is that the murine system could offer an excellent model for examining the different types of aHUS mutations. Additionally, this same region contains residues that are important for binding of FH to the outer surface protein E (OspE) of the Lyme disease causing pathogen, Borrelia
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burgdorferi (172). Another possible direction could be to investigate whether mFHR compete with mFH for binding to OspE using different sets of wildtype and mutant proteins.
Figure 35 Conserved Sialic Acid Binding Site Residues on MFH and MFHRs. Several residues that are FH recognition of host sialic acid residues are conserved between humans and mice. These residues include L1181, W1183, E1198, R1215. Other residues are not conserved in the entire family are are shown as well in red. Additionally, the outer surface protein E (OspE) of Borrelia burgdorferi has been shown to bind FH through W1183, S1196, E1198, and R1215 through side chain interactions. Three out of four of these key residues are conserved in the murine family.
Animal Models
The data in this study provide excellent in vitro preliminary findings for evaluating the role of mFHRs in ophthalmologic, renal, and/or arthritic animal disease models. Trial experiments have been performed to examine whether mFHR proteins block mFH and promote alternative pathway activation in the eye in a murine model of choroidal neovascularization (CNV) in collaboration with the Rohrer lab (Medical University of South Carolina). Other preliminary experiments in collaboration with the Thurman lab (University of Colorado) have been performed to evaluate the role of mFHRs and alternative pathway activation in a renal ischemia/reperfusion (IR) model in C57BL/6 mice. In both trials, small animal sizes were used (<5 per treatment group) and while some animals appeared to have
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evidence of increased complement activation (such as elevated blood urea nitrogen and creatinine levels in the renal model indicating renal damage) other animals within the same test group appeared to have reduced complement activation and no statistically significant conclusions could be made from these early studies.
A third animal model, in which the roles of mFHRs have not been evaluated, is an animal model of rheumatoid arthritis (RA). The Holers lab has successfully examined the role of FH SCRs1920 (FH1920) using this model. Notably, the AP of complement is both necessary and sufficient for mice to develop passive transfer collagen antibodyinduced arthritis (CAIA) (237). Induction of CAIA, an immune complexinduced model of the effector phase of human RA, is produced by administration of a cocktail of four monoclonal antibodies to bovine type II collagen
(II). Administration of recombinant FH1920, which competes with fulllength FH, significantly worsened clinical disease activity, histopathologic injury, and increase deposition of C3 in both synovium and cartilage (238). These observations suggest that mFHR proteins may also regulate complement in this model and should be evaluated.
An alternative method, and perhaps more efficient way for evaluating the roles of mFHRs in different animal models, would be using silencing experiments with siRNA rather than in vivo injection of recombinant proteins. Experiments with siRNAs targeted specifically for the liver (where the majority of FH and likely FHR proteins synthesis occurs) in order to downregulate specific mFHRs may provide helpful insights into the functions of these proteins in vivo.
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Antibody Development
Chapter VII discussed development of mFH and mFHR specific antibodies.
Several antibody candidates specific for mFH or the different mFHR proteins have been tested and awaiting development. Further work to assess their utility in ELISA assays, Western blot analysis, immunohistochemistry, and any inhibitory capabilities is required. Having specific mFHR antibodies that recognize native proteins would provide several advantages from measuring endogenous levels of protein to testing the effectiveness of silencing experiments using siRNAs. Further development of these antibodies may help us better understand the role of FHR proteins in complement regulation.
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APPENDIX A
Mouse Complement FH Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011) mm10 UCSC Version
Mouse Complement Factor H (mFH) Mouse Complement Factor H (mFH) Cfh001 Cfh (uc007.cws.1) ENSMUST00000066859
Description Description Complement component factor h Mus musculus complement component factor h (Cfh), mRNA. MGI:88385 Synonyms Hf1, Sas1, NOM, Sas1, Mud1
Location Location Chromosome 1: 140,085,859140,183,411 mm10 chr1:140,085,855140,183,411 GRCm38:CM000994.2 Size: 97,557 Strand: Coding Region Position: mm10 chr1:140,086,326140,183,276 Size: 96,951 Strand:
Total Exon Count: 22 Total Exon Count: 22 8 Splice variants reported Transcript Length: 4361 Transcript Length: 4365
NCBI accession: NM_009888 NCBI accession: NM_009888
Protein: 1234aa Protein: 1252aa (Uniprot: P06909) (Uniprot: E9Q8I0 leader sequence included)
Protein accession: NP_001020746 Protein accession: NP_001020746
177
APPENDIX B
Mouse Complement FHRA Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011) mm10 UCSC Version
mFHRA presumed gene position mFHRA presumed gene position Gm16332001 pseudogene Mouse Gene Cfhr2 (uc007cwr.1) ENSMUST00000134229
Description Description Predicted gene 16332 Mus musculus complement factor Hrelated 2 (Cfhr2) MGI:3840155
Location Location Chromosome 1: 139,862,957139,936,477 mm10chr1:139,810,292140,018,921 Strand: Size: 208,630 Coding Region Position: mm10 chr1:139,810,823140,018,848 Size: 47,805 Strand:
Total Exon Count: 16 Total Exon Count: 9
Protein: None described Protein: 506 aa Uniprot: Q8R0I8 NM_001025575.2 Protein: 452 aa Uniprot: Q61407 (3A4/5G4)
Exon 7 position: Exon 7 position: Chr1: 139,890,400139,890,222 Chr1: 139,890,400139,890,225 Exon 7:179bp Exon 7: 179bp Translated Exon 7 protein sequence: Translated Exon 7 protein sequence: SCDMPVFENSITKNTRTWFKLNDKLDYE SCDMPVFENSITKNTRTWFKLNDKLDYE CLVGFENEYKHTKGSITCTYYGWSDTPS CLVGFENEYKHTKGSITCTYYGWSDTPS CYG CYG
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APPENDIX C
Mouse Complement FHRB Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011) mm10 UCSC Version
mFHRB Gene Position mFHRB Gene Position Cfhr2 Mouse Gene Cfhr2 (uc007cwp.2) ENSMUST00000094489
Description Description complement factor Hrelated 2 Mus musculus complement factor Hrelated 2 (Cfhr2), mRNA. MGI:3611575
Synonyms BC026782, Cfhrb_4/2, CfhGN, Cfhrb_4/2GN
Location Location Chromosome 1: 139,810,288139,858,702 mm10 chr1:139,810,292139,858,699 Strand: Size: 48,408 Coding Region Position: mm10chr1:139,810,823139,858,627 Size: 47,805 Strand:
Total Exon Count: 6 or 7 Total Exon Count: 6 2 Splice variants reported Cfhr2001:1619bp, 6 exons Cfhr2002:1189bp, 7 exons
NCBI accession: NM_001025575 NCBI accession: NM_001025575 NP_001020746
Protein: Protein: 332 aa Cfhr2001: 332 aa (Uniprot: Q4LDF6) Cfhr2002: 303 aa (Uniprot: Q61405)
179
APPENDIX D
Mouse Complement FHRC Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011)
mFHRC Gene Position mFHRC Gene Position Predicted Gene 4788 Gene Gm4788 (uc011wte.1)
Description Description Predicted Gene 4788 Mus musculus predicted gene 4788 (Gm4788), transcript variant 2, mRNA. MGI:3646434
Synonyms Cfhrc, EG214403
Location Location Chromosome 1: 139,697,623139,781,243 mm10 chr1:139,697,919139,781,239 GRCm38:CM000994.2 Size: 83,321 Strand: Coding Region Position: mm10 chr1:139,698,095139,781,168 Size: 83,074
Total Exon Count: 14 or 15 Total Exon Count: 14 or 15 3 Splice variants reported Same as reported by Ensembl.org Transcript Length: Gm4788001: 2958bp, 14 exons Gm4788002: 2935bp, 14 exons Gm4788003: 3187bp, 15 exons
NCBI accession: NCBI accession: NM_001029977 NM_001160303.1 NM_001160303 NM_001160304
Protein: Protein: Gm4788001: 808aa (Uniprot: E9Q8B5) Gm4788001: 808aa (Uniprot: E9Q8B5) Gm4788002: 820aa (Uniprot: E9Q8B6) Gm4788002: 820aa (Uniprot: E9Q8B6) Gm4788003: 879aa (Uniprot: E9PUM5/ Gm4788003: 879aa (Uniprot: E9PUM5 or Q0KHD3) Q0KHD3)
180
APPENDIX E
Mouse Complement FHRD Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011)
mFHRD Gene Position mFHRD Gene Position Unprocessed pseudogene
Description Description Complement factor Hrelated 3 MGI:3647418 AK015277 (uc007cwm.1) Synonyms Mus musculus adult male testis cDNA, RIKEN fulllength enriched library, clone: EG624286 4930431N10 productweakly similar to COMPLEMENT FACTOR HRELATED PROTEIN 1 PRECURSOR (FHR1) (H FACTOR LIKE 1) (H36) [Homo sapiens], full insert sequence.
Location Location Chromosome 1: 139,584,783139,660,899 mm10 chr1:139,574,841139,631,911 Strand: Size: 57,071 Strand:
Total Exon Count: 6 Total Exon Count: 7 Transcript Length: 1022bp Transcript Length: 1072bp Reported as unprocessed pseudogene Reported as unprocessed pseudogene
Protein: Protein: Q8BMW5 None reported
181
APPENDIX F
Mouse Complement FHRE Gene and Protein Information
Ensembl.org Genome.ucsc.edu GRCm38.p4 (Patched April 1, 2015) GRCm38 (December 2011) mm10 UCSC Version
mFHRE Gene Position mFHRE Gene Position Complement factor Hrelated 1 MGI:2138169 Cfhr1 (uc007cwl.1)
Description Description Complement factor Hrelated 1 Mus musculus complement factor Hrelated 1 (Cfhr1), mRNA. Synonyms
Cfhl1, CFHRB, AI194696
Location Location Chromosome 1: 139,547,053139,560,272 mm10 chr1:139,547,064139,560,222 GRCm38:CM000994.2 Size: 13,159 Strand: Coding Region Position: mm10 chr1:139,547,700139,560,158 Size: 12,459 Strand:
Total Exon Count: 6 Total Exon Count: 6 2 Splice variants reported Transcript length: 1732bp Transcript Length: Cfhr1001: 1792bp, 6 exons Cfhr1002: 408bp, 2 exons
NCBI accession: NM_015780, NP_056595 NCBI accession: NM_015780.2
Protein: Protein: Cfhr1001: 343 aa (Uniprot: Q61406) 343 aa (Uniprot: Q61406) Cfhr1002: 111 aa
182