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FHR5 Binds to Laminins, Uses Separate C3b and Surface-Binding Sites, and Activates Complement on Malondialdehyde-Acetaldehyde Surfaces

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FHR5 Binds to Laminins, Uses Separate C3b and Surface-Binding Sites, and Activates Complement on Malondialdehyde-Acetaldehyde Surfaces Published February 26, 2018, doi:10.4049/jimmunol.1701641 The Journal of Immunology FHR5 Binds to Laminins, Uses Separate C3b and Surface-Binding Sites, and Activates Complement on Malondialdehyde-Acetaldehyde Surfaces Ramona B. Rudnick,* Qian Chen,*,1 Emma Diletta Stea,*,2 Andrea Hartmann,* Nikolina Papac-Milicevic,†,‡ Fermin Person,x Michael Wiesener,{ Christoph J. Binder,†,‡ Thorsten Wiech,x Christine Skerka,* and Peter F. Zipfel*,‖ Factor H related-protein 5 (CFHR5) is a surface-acting complement activator and variations in the CFHR5 gene are linked to CFHR glomerulonephritis. In this study, we show that FHR5 binds to laminin-521, the major constituent of the glomerular base- ment membrane, and to mesangial laminin-211. Furthermore, we identify malondialdehyde-acetaldehyde (MAA) epitopes, which are exposed on the surface of human necrotic cells (Homo sapiens), as new FHR5 ligands. Using a set of novel deletion fragments, we show that FHR5 binds to laminin-521, MAA epitopes, heparin, and human necrotic cells (HUVECs) via the middle region [short consensus repeats (SCRs) 5-7]. In contrast, surface-bound FHR5 contacts C3b via the C-terminal region (SCRs8-9). Thus, FHR5 uses separate domains for C3b binding and cell surface interaction. MAA epitopes serve as a complement-activating surface by recruiting FHR5. The complement activator FHR5 and the complement inhibitor factor H both bind to oxidation- specific MAA epitopes and FHR5 competes with factor H for binding. The C3 glomerulopathy–associated FHR21–2-FHR5 hybrid protein is more potent in MAA epitope binding and activation compared with wild-type FHR5. The implications of these results for pathology of CFHR glomerulonephritis are discussed. In conclusion, we identify laminins and oxidation-specific MAA epi- topes as novel FHR5 ligands and show that the surface-binding site of FHR5 (SCRs5-7) is separated from the C3b binding site (SCRs8-9). Furthermore, FHR5 competes with factor H for binding to MAA epitopes and activates complement on these modified structures. The Journal of Immunology, 2018, 200: 000–000. he complement system is the central part of the innate extracellular matrix (ECM) (7, 8). The N-terminal SCRs1-2 con- immune system and controls many physiological reac- tain a dimerization motif and bind properdin (7, 9). FHR5 shares T tions. The self-amplifying cascade is spontaneously ac- sequence homology with factor H, the central inhibitor of the al- tivated and the counterbalanced action of activators and inhibitors ternative pathway. Factor H binds C3b, dissociates preformed C3- by guest on September 30, 2021. Copyright 2018 Pageant Media Ltd. directs the newly formed effector components to target surfaces. convertases, and assists the serine protease Factor I in degradation Regulation is required to protect intact self-cells and tissue surfaces of C3b (4, 5, 10). The N-terminal SCRs1-4 of factor H contain the from toxic activation products. In line with this, activators such as regulatory region, and the C-terminal end contains the surface- factor H related-protein 5 (CFHR5) and properdin as well as in- binding region, and the heparin and C3b interaction sites (11–16). hibitors like factor H and C4 binding protein are essential to control The factor H/CFHR gene cluster is a hotspot for genetic varia- complement action in time and space (1–4). tions and is associated with diseases (17–23). Variations in the FHR5 is a 65 kDa human plasma protein with a serum con- CFHR5 gene are linked to kidney diseases, i.e., C3 glomerulopathy centration of 3–6 mg/ml and consists of nine short consensus re- (C3G), dense deposit disease (DDD), IgA nephropathy, and atyp- peats (SCRs) (5, 6). This complement activator binds to necrotic ical hemolytic uremic syndrome (24–27). In CFHR glomerulone- http://classic.jimmunol.org human endothelial cells, activated mesangial cells, and the phritis, disease-associated genomic rearrangements concerning *Department of Infection Biology, Leibniz Institute for Natural Product Research and and from the European Community’s Seventh Framework Programme under Grant Infection Biology, 07745 Jena, Germany; †Clinical Department of Medical and Chem- Agreement 2012–305608 (European Consortium for High-Throughput Research in ical Laboratory Diagnostics, Medical University of Vienna, 1090 Vienna, Austria; Rare Kidney Disease; EURenOmics). R.B.R. is associated with the International ‡Research Center for Molecular Medicine of the Austrian Academy of Sciences, Leibniz Research School for Microbial and Biomolecular Interactions. E.D.S. was sup- 1090 Vienna, Austria; xInstitute of Pathology, University Hospital Hamburg- ported by a scholarship from the Societa` Italiana di Nefrologia. { Downloaded from Eppendorf, 20246 Hamburg, Germany; Department of Nephrology and Hypertension, Address correspondence and reprint requests to Prof. Peter F. Zipfel, Department of Friedrich-Alexander University of Erlangen-Nuremberg, 91054 Erlangen, ‖ Infection Biology, Leibniz Institute for Natural Products Research and Infection Germany; and Department Microbiology, Friedrich-Schiller-University, 07745 Jena, Biology, Beutenbergstrasse 11a, 07745 Jena, Germany. E-mail address: peter. Germany [email protected] 1Current address: Laboratory of Hepatology, Department of Biomedicine, University The online version of this article contains supplemental material. Hospital of Basel, Basel, Switzerland. Abbreviations used in this article: CFHR5, factor H–related protein 5; C3G, C3 2Current address: Nephrology, Dialysis, and Transplantation Unit, Department of glomerulopathy; DDD, dense deposit disease; DPBS, Dulbecco’s PBS; ECM, extra- Emergency and Organ Transplantation, University Aldo Moro, Bari, Italy. cellular matrix; GBM, glomerular basement membrane; hiHS, heat-inactivated hu- ORCIDs: 0000-0002-2732-1854 (R.B.R.); 0000-0002-8187-7677 (N.P.-M.); 0000- man serum; IC, isotype control; MAA, malondialdehyde-acetaldehyde; MDA, 0002-7489-9667 (F.P.); 0000-0002-6149-2411 (P.F.Z.). malondialdehyde; MFI, mean fluorescence intensity; MPGN, membranoproliferative glomerular nephritis; NHS, normal human serum; SCR, short consensus repeat; sh- Received for publication November 28, 2017. Accepted for publication January 30, BSA, sham-treated BSA; TMB, tetramethylbenzidine; WGA, wheat germ agglutinin. 2018. This work was supported by funding from Sonderforschungsbereich Grant 1192 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 (Immune-Mediated Glomerular Diseases—Basic Concepts and Clinical Implications) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701641 2 FHR5 ACTIVATES COMPLEMENT ON MAA- AND LAMININ-521 SURFACES the CFHR5 gene result in the expression of FHR1::FHR5, FHR2:: histochemically stained for FHR5. Tissue sections (1 mm) were depar- FHR5, FHR5::FHR2 hybrid proteins, or FHR5 proteins with affinized, rehydrated through graded ethanol solutions, and pretreated with duplicated segments (17, 24, 28–30). Two related C3G patients protease at 37˚C for 30 min before blocking with normal horse serum (S- 2000; Vector Industries). Sections were incubated with FHR5 mAbs (#635 and #638) with a 25 kbp chromosomal deletion express an (1:1000; R&D, Wiesbaden, Germany) overnight at 4˚C. For detection oligomer-forming FHR21–2-FHR5 hybrid protein, which over- using ZytochemPlus/POLAP100 (Zytomed Systems, Berlin, Germany), activates complement (7, 24). the slides were incubated with polymer 1, rinsed in PBS, and incubated There is evidence that complement activation is linked to lipids, with polymer 2. After washing in PBS, slides were stained in new fuchsin naphthol As-Bi phosphate substrate mixture (30 min) and in hemalaun which can be targeted by oxidation (31, 32). Phospholipids in par- (Mayer) for nuclear counterstaining (1 min). ticular are susceptible to oxidation induced by cellular stress. The oxidation leads to the formation of highly reactive degradation FHR5 binding to heparin products, which can modify self-molecules. As a consequence, Heparin-binding microtiter plates (BD, Heidelberg, Germany) were coated structural neoepitopes referred to as oxidation-specific epitopes are with heparin-albumin (50 mg/ml in DPBS; Sigma-Aldrich). After blocking generated. Oxidation-specific epitopes have been characterized as a (2% BSA in DPBS), recombinant FHR5, FHR5 fragments, or BSA novel group of damage-associated molecular patterns and include (100 nM in DPBS) were added and incubated at 37˚C for 1 h. Afterwards, bound FHR5 and FHR5 fragments were quantitated by c-myc mAb (clone malondialdehyde (MDA)-modified amino groups. MAA epitopes can 9E10; R&D) in combination with the corresponding secondary antiserum be detected on apoptotic cells, microvesicles, or oxidized low-density (Dako). TMB Plus2 substrate (Kem-En-Tec Diagnostics) was used to vi- lipoproteins (33–38). Malondialdehyde-acetaldehyde (MAA) repre- sualize binding. OD values were measured at 450 nm (Tecan). sents an advanced MDA-lysine adduct that is stable and able to in- FHR5 binding to laminin isoforms duce a potent immune response (31, 39). Recently, it was shown that factor H binds to MDA/MAA epitopes and bound factor H inhibits Laminins (BioLamina AB, Sundbyberg, Sweden) or gelatin (10 mg/ml in DPBS; Merck) were immobilized onto the surface of a MaxiSorp micro- complement progression and decreases the proinflammatory effects titer plate (Nunc). After washing and blocking (2% BSA in PBS-T), of MDA/MAA epitopes (31). recombinant FHR5 and the FHR5 fragments (50 nM in DPBS) were In this study, we identify
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