BASIC RESEARCH www.jasn.org Complement Factor H-Related 5-Hybrid Proteins Anchor Properdin and Activate Complement at Self-Surfaces † † Qian Chen,* Melanie Manzke,* Andrea Hartmann,* Maike Büttner, Kerstin Amann, ‡ † Diana Pauly, Michael Wiesener, Christine Skerka,* and Peter F. Zipfel*§ *Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany; †Department of Nephrology and Hypertension, Friedrich-Alexander University of Erlangen-Nuremberg, Germany; ‡University Hospital Regensburg, Regensburg, Germany; and §Friedrich Schiller University, Jena, Germany ABSTRACT C3 glomerulopathy (C3G) is a severe kidney disease for which no specific therapy exists. The causes of C3G are heterogeneous, and defective complement regulation is often linked to C3G pathogenesis. Copy number variations in the complement factor H-related (CFHR) gene cluster on chromosome 1q32 and CFHR5 mutant proteins associate with this disease. Here, we identified CFHR5 as a pattern recognition protein that binds to damaged human endothelial cell surfaces and to properdin, the human complement activator. We found the two N-terminal short consensus repeat domains of CFHR5 contact properdin and mediate dimer formation. These properdin-binding segments are duplicated in two mutant CFHR5 proteins, CFHR2-CFHR5Hyb from German patients with C3G and CFHR5Dup from Cypriot patients with C3G. Each of these mutated proteins assembled into large multimeric complexes and, compared to CFHR5, bound damaged human cell surfaces and properdin with greater intensity and exacerbated local complement activation. This enhanced surface binding and properdin recruitment was further evidenced in the mesangia of a transplanted and explanted kidney from a German patient with a CFHR2-CFHR5Hyb protein. Enhanced properdin staining correlated with local complement activation with C3b and C5b-9 deposition on the mesangial cell surface in vitro. This gain of function in complement activation for two disease-associated CFHR5 mutants describes a new disease mechanism of C3G, which is relevant for defining appropriate treatment options for this disorder. J Am Soc Nephrol 27: 1413–1425, 2016. doi: 10.1681/ASN.2015020212 Factor H-related proteins (CFHRs) are important uremic syndrome,10–15 deficiency of CFHR plasma regulators of the human complement system.1–3 proteins and factor H autoantibody positive hemo- CFHR5 was initially identified within glomerular lyticuremicsyndrome,16–20 age-related macular immune deposits in kidney biopsies derived from degeneration,21–23 IgA nephropathy,24 as well as sys- patients with membranoproliferative glomerulo- temic lupus erythematosus.25 CNVs in the CFHR5 nephritis as well as other glomerular diseases.4 In gene are identified in patients with C3G,5–7 a diseased glomeruli where complement activation oc- curred on the glomerular surface, CFHR5 colocalized with both C3b and terminal complement complex Received February 25, 2015. Accepted August 4, 2015. 4 C5b-9, thus suggesting that CFHR5-directed com- Published online ahead of print. Publication date available at plement activation in the glomeruli is relevant for www.jasn.org. disease pathogenesis. Correspondence: Prof. Peter F. Zipfel, Department of Infection In addition, copy number variations (CNVs) in Biology, Leibniz Institute for Natural Products Research and In- the human gene cluster that includes the five CFHR fection Biology, Beutenbergstr. 11a, 07745 Jena, Germany. genes are defined in several human diseases including Email: [email protected] – C3 glomerulopathy (C3G),5 9 atypical hemolytic Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: 1413–1425, 2016 ISSN : 1046-6673/2705-1413 1413 BASIC RESEARCH www.jasn.org devastating kidney disease characterized by C3 deposition in Monocytic THP1 cells express properdin,28 and native, cell- glomeruli. We previously reported two related German C3G derived properdin (green fluorescence) was evenly distributed on patients, both with the same 25 kbp chromosomal segment the cell surface (Figure 1F, panels on the left). CFHR5 (red fluo- deleted, which spans from the CFHR2- to the CFHR5 gene. rescence) bound to the THP1 surface and showed a rather spotty Both patients express a CFHR2-CFHR5 hybrid protein, which distribution (Figure 1F, upper row). CFHR5 colocalized with has the first two domains (i.e., short consensus repeats 1–2 properdin at the surface, as revealed by the yellow signal upon [SCR1–2]) of CFHR2 linked to all nine SCR domains of merging the images (Figure 1F, upper row, right panel). CFHR2 CFHR5. This CFHR2-CFHR5 hybrid protein deregulates the did not bind to the THP1 surface (Figure 1F, middle row). Thus C3 convertase in the fluid phase.5 Patients of a large Cypriot CFHR5 binds the complement activator properdin and at the cell cohort express a CFHR5 mutant protein, which has the first surface CFHR5 colocalized with native, cell-derived properdin. two domains, i.e., SCR 1 and 2, duplicated.6,7 Thus both the CFHR2-CFHR5 hybrid and the CFHR5 mutant have the C3G-CFHR5 Mutant Proteins Efficiently Bind Properdin N-terminal interaction segment duplicated, indicating a func- Mutant CFHR5 proteins with a duplicated N-terminal dimer- tional role of these duplicated interaction segments in disease ization region, derived either from CFHR2 or CFHR5, are pathology. Therefore, it is of interest to address the function of associated with C3G, and this suggests an important role of CFHR5 on target surfaces and to define the pathogenic mech- CFHR5 in C3G pathology. Todefine how these mutant proteins anism of CFHR5 mutant-associated C3G. cause pathology, both C3G-associated CFHR5 mutants were generated and recombinantly expressed. CFHR212-CFHR5 RESULTS (termed CFHR2-CFHR5Hyb)hasCFHR212 fused to intact CFHR5 and was identified in two German C3G patients; sim- CFHR5 Binds to Modified Surfaces and Enhances C3b ilarly CFHR512-CFHR5 (CFHR5Dup), which has the interac- Deposition tion segment of CFHR5 (CFHR512) duplicated, was initially Given the sequence similarities of the C-terminal domains of described in Cypriot patients. In addition, CFHR212- CFHR proteins with the surface recognition region of factor H, CFHR5Δ12 (termed CFHR2-CFHR5Var) was generated, which fi we analyzed surface binding of both CFHR5 and CFHR2. as a variant of CFHR2-CFHR5Hyb harbors the rst interaction i.e., CFHR5 derived from normal human serum (NHS), and segment, CFHR212, but lacks the second interaction seg- i.e., CFHR2 to a lesser degree, bound to the surface of nonhuman ment, CFHR512 (Figure 2A). Chinese hamster ovary (CHO) cells and to necrotic cells, but To determine how these CFHR5 mutant proteins affect fi veryweakly to intact human umbilical veinendothelial (HUVE) complement-mediated C3b surface deposition, rst properdin cells (Figure 1A, lanes 2–4). Surface binding was confirmed by binding was tested. When assayed either by ELISA or by SPR, flow cytometry using recombinant proteins. CFHR5 bound both the German and the Cypriot CFHR5 mutants bound dose-dependently to CHO and to necrotic HUVECells (Figure properdin about twice as strongly, as compared with CFHR2- 1B, Supplemental Figure 1A). CFHR5 bound to the surface of CFHR5Var or CFHR5 (Figure 2, B and C). This stronger fi necrotic human cells with 2.7-fold higher intensity than to interaction was con rmed when binding was assessed to fl CHO cells. CFHR5 bound with higher intensity than CFHR2 properdin-expressing THP1 cells, either by ow cytometry or (Supplemental Tables 1 and 2). by confocal microscopy (Supplemental Figures 3 and 4, CFHR5 attached to necrotic HUVE cells enhanced C3 Supplemental Table 6). Taken together, both C3G mutants deposition when the cells were challenged with NHS. This with the duplicated interaction segment bound with high effect was observed on the surface of HUVE cells but not for and with similar intensity to properdin. Apparently two copies CFHR5 attached to CHO cells (Supplemental Figure 1B). Thus of the N-terminal interaction region enhance properdin binding. surface-bound CFHR5 allows C3 convertase formation and To localize the region relevant for properdin binding, i.e., complement activation. In this set-up CFHR2 did not enhance additional mutants were generated, CFHR212-CFHR512, C3b deposition (Figure 1, B and C, Supplemental Tables 3 and 4). which represents exclusively the duplicated interaction seg- ments of CFHR2-CFHR5Hyb;CFHR512, as well as CFHR212, CFHR5 Binds Properdin representing exclusively the interaction segments of either Given this activating effect, we hypothesized that CFHR5 interacts CFHR5 or CFHR2. Recombinant CFHR2-CFHR5Hyb, when with properdin, the only known human complement activa- separated on a calibrated size exclusion column, eluted in tor.26,27 Indeed, when analyzed by ELISA, properdin bound to fractions with masses ranging from .700 to ca. 440 kDa immobilized CFHR5, and binding was dose-dependent (Figure (Figure 3, A and B). As monomer CFHR2-CFHR5Hyb has a 1D). CFHR2 bound with much lower intensity. In a reverse mass of 70 kDa, the large complexes are composed of more setting, CFHR5 bound to immobilized properdin (Supplemen- than ten, and the ca. 440 kDa complexes of six protein units. tal Figure 2). CFHR5::properdin interaction was also confirmed The recombinant CFHR212-CFHR512 fragment, which as a mono- by surface plasmon resonance (SPR) and CFHR5 formed strong mer has a mass of 30 kDa, eluted in fractions corresponding and stable complexes with properdin (Figure 1E). CFHR2 in the to molecular masses ranging from .700 to ca. 120 kDa, again same settings did not interact with properdin. demonstrating formation of higher-ordered complexes that 1414 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1413–1425, 2016 www.jasn.org BASIC RESEARCH Figure 1. CFHR5 binds to modified cell surfaces and interacts with properdin. (A) Intact CHO cells (lane 2), necrotic (lane 3) and living HUVE cells (lane 4) were incubated with NHS. Bound CFHR5, CFHR2, CFHR1 and factor H (arrows) were visualized by Western blotting. (B) Cells were incubated with recombinant CFHR5 or CFHR2 (50 and 200 nM each). Bound CFHR5 (left panel) or CFHR2 (right panel) was detected with CFHR5 or CFHR2 mAb by flow cytometry.