The Journal of Immunology Modulation of the Alternative Pathway of Complement by Murine Factor H–Related Proteins Alexandra H. Antonioli,* Janice White,† Frances Crawford,† Brandon Renner,* Kevin J. Marchbank,‡ Jonathan P. Hannan,* Joshua M. Thurman,* Philippa Marrack,†,x,{ and V. Michael Holers* Factor H (FH) is a key alternative pathway regulator that controls complement activation both in the fluid phase and on specific cell surfaces, thus allowing the innate immune response to discriminate between self and foreign pathogens. However, the interrela- tionships between FH and a group of closely related molecules, designated the FH-related (FHR) proteins, are currently not well understood. Whereas some studies have suggested that human FHR proteins possess complement regulatory abilities, recent studies have shown that FHR proteins are potent deregulators. Furthermore, the roles of the FHR proteins have not been explored in any in vivo models of inflammatory disease. In this study, we report the cloning and expression of recombinant mouse FH and three FHR proteins (FHR proteins A–C). Results from functional assays show that FHR-A and FHR-B proteins antagonize the protective function of FH in sheep erythrocyte hemolytic assays and increase cell-surface C3b deposition on a mouse kidney proximal tubular cell line (TEC) and a human retinal pigment epithelial cell line (ARPE-19). We also report apparent KD values for the binding interaction of mouse C3d with mouse FH (3.85 mM), FHR-A (136 nM), FHR-B (546 nM), and FHR-C (1.04 mM), which directly correlate with results from functional assays. Collectively, our work suggests that similar to their human counterparts, a subset of mouse FHR proteins have an important modulatory role in complement activation. Further work is warranted to define the in vivo context-dependent roles of these proteins and determine whether FHR proteins are suitable therapeutic targets for the treatment of complement-driven diseases. The Journal of Immunology, 2018, 200: 316–326. nderstanding regulatory mechanisms by which the al- lysis of C3 to C3(H2O) in the fluid phase, which results in acti- ternative pathway (AP) controls spontaneous activation vation and production of C3b through the activities of factor D, U of complement in the fluid phase and the amplification factor B, and properdin. This phenomenon, known as C3 tick- of complement on specific surfaces has important implications over, occurs spontaneously and allows for the rapid initiation for treating complement-driven inflammatory disease. Unlike the and amplification of complement. Given that this pathway is re- classical or lectin pathways, the AP does not require any specific sponsible for $80% of the final downstream effect of initial molecular recognition for its initiation but is activated by hydro- specific activation of the classical and lectin pathways, precise control of the AP and its amplification loop is required (1). Factor H (FH) is a major soluble complement regulator that is *Department of Medicine, University of Colorado School of Medicine, Aurora, CO essential for controlling AP activation in the fluid phase and on cell 80045; †Howard Hughes Medical Institute, Denver, CO 80206; ‡Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom; surfaces. Several human diseases are associated with mutations and x Department of Biomedical Research, National Jewish Health, Denver, CO 80206; autoantibodies that alter either FH function, or the activities of the and {Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045 closely related FH-related (FHR) proteins. For example, mutations or polymorphisms in the CFH and CFHR gene family have been ORCIDs: 0000-0002-0809-5003 (A.H.A.); 0000-0002-6060-4119 (B.R.); 0000-0003- 1312-5411 (K.J.M.); 0000-0003-3318-2341 (J.P.H.); 0000-0002-3476-9956 (J.M.T.); linked to the renal diseases atypical hemolytic uremic syndrome 0000-0003-1883-3687 (P.M.). and IgA nephropathy as well as diseases that have glomerular Received for publication December 7, 2016. Accepted for publication October 27, pathologies, including dense deposit disease and FHR5 nephrop- 2017. athy, which are encompassed under the C3 glomerulopathy This work was supported in part by National Institutes of Health Grant R01 AR- umbrella (2–4). Other autoimmune diseases associated with 51749, an investigator grant from the Beckman Institute for Macular Research (to V.M.H.), University of Pennsylvania Health System Grant AI-18785 (to P.M.), the alterations within the CFH and CFHR gene family include sys- Victor W. Bolie and Earleen D. Bolie Graduate Scholarship Fund (to A.H.A.), and the temic lupus erythematosus and age-related macular degeneration University of Colorado Cancer Core Protein Production Shared Resource, which (AMD) (5–7). receives direct funding support from the National Cancer Institute through Cancer Center Support Grant P30CA046934. Given the recent expansion in research toward understanding the Address correspondence and reprint requests to Dr. V. Michael Holers, University of human FHR protein family member disease associations, one Colorado School of Medicine, Division of Rheumatology, B-115, 1775 Aurora Court, question that remains unanswered is whether the mouse FHR M20-3102-E, Aurora, CO 80045. E-mail address: [email protected] (mFHR) proteins are functional orthologs to their human coun- The online version of this article contains supplemental material. terparts. The mouse FH (mFH) gene consists of 22 exons that share Abbreviations used in this article: AMD, age-related macular degeneration; AP, 63% homology with human CFH and encode a protein composed alternative pathway; CD, circular dichroism; DPBS, Dulbecco’s PBS; DSF, differ- ential scanning fluorimetry; FH, factor H; FHR, FH-related; mFH, mouse FH; of 20 short consensus repeat (SCR) domains (8). Unlike its human mFHR, mouse FHR; NMS, normal mouse serum; SCR, short consensus repeat; counterpart, the mFH gene does not have a FHL-1 variant, al- SPR, surface plasmon resonance; TEC, tubular epithelial cell; TED, thioester- though it does contain an unspliced exon (exon 9) that could containing domain; T , melting temperature. m encode an SCR domain with a stop codon. Similar to their Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 human FHR counterparts, a total of five mFHR genes have been www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602017 The Journal of Immunology 317 identified, and evidence for four mFHR proteins have been European Bioinformatics Institute and the Wellcome Trust Sanger Institute inferred from mRNA transcripts isolated from a mouse liver (http://www.ensembl.org), and the UCSC Genome Browser hosted by the cDNA library; however, direct comparison with the human CFHR University of California, Santa Cruz (https://genome.ucsc.edu). gene family is not straightforward (9, 10). These predicted mouse mFH and FHR protein expression proteins also exhibit high sequence identity with important ligand- DNA sequences of full-length mFH and mFHR constructs, including binding and self-surface recognition domains of mFH. After the mFHR-A [based on the sequence for class A clone 3A4/5G4 by Vik et al. initial characterization of the four classes of mFHR transcripts by (9)], mFHR-B, mFHR-C, and mFH19–20 were generated commercially Vik et al. (9), this gene family was not thoroughly examined until (GeneArt by Life Technologies). The unique UniProt identifiers for each Hellwage et al. (10) published data on mFHR protein expression construct include: mFH (UniProt: P06909), mFHR-A (UniProt: Q61407), mFHR-B (UniProt: Q4LDF6), mFHR-C (UniProt: Q0KHD3), and and binding partners. However, little subsequent work has been mFH19–20 (UniProt: P06909 residues 1112–1234). Each DNA sequence published that characterizes these genes, evaluates the functional was codon-optimized (Homo sapiens) and engineered into a Gateway roles of the proteins they encode, or examines the concentrations pDONR221 entry vector (Life Technologies) following previously pub- and functions of these proteins in vivo. lished construct design and experimental conditions (12). For protein ex- One potential limitation to studying the mouse FHR gene family pression, FreeStyle 293-F cells (Invitrogen) were obtained and subcultured in FreeStyle 293 expression medium every 3 d. Transfections were lies with understanding the published nomenclature as it relates to performed using FreeStyle MAX reagent (Life Technologies) accord- that of the human FHR gene family. Although the nomenclature ing to the manufacturer’s instructions. Cell supernatant was harvested for the mouse FHR genes (labeling them alphabetically as A, B, C 5–7 d posttransfection using centrifugation at 8000 rpm for 15 min in based on their position from mFH) was proposed more than a sterile 250-ml tubes. decade ago, a current search of genome browsers lists these genes Mutant mFHR constructs under various aliases, including CFHR4/2, CHFR2, FHR-1,or To create mutant mFHR constructs, a QuickChange Lightning site-directed FHR-3 (10). For example, the mouse gene referred to as CFHR-1 mutagenesis kit (Agilent Technologies, Santa Clara, CA) was used is located at the mFHR-E gene position, which is farthest from the according to the manufacturer’s directions. Generation of an N340A/ mouse FH gene. This same gene was originally classified by Vik D342A mFHR-A mutant was created with the following primers that et al. (9) as clone 13G1 and