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Structural Insight on CFHR1 Deficiency in AI-Ahus the Major Autoantibody Epitope on Factor H in Atypical Hemolytic Uremic Syndro

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Structural Insight on CFHR1 Deficiency in AI-Ahus the Major Autoantibody Epitope on Factor H in Atypical Hemolytic Uremic Syndro This accepted author manuscript is copyrighted and published by American Society for Biochemistry and Molecular Biology. The definitive version of the text was subsequently published in The Journal of Biological Chemistry. 2015 Apr 10;290(15):9500-10. doi: 10.1074/jbc.M114.630871. The final copyedited version is available at the publishers website: Structural insight on CFHR1 deficiency in AI-aHUS http://www.jbc.org/content/290/15/9500.long The major autoantibody epitope on Factor H in atypical Hemolytic Uremic Syndrome is structurally different from its homologous site in Factor H related protein 1 Arnab Bhattacharjee1,2, Stefanie Reuter3, Eszter Trojnár4, Robert Kolodziejczyk2,5, Harald Seeberger3, Satu Hyvärinen1, Barbara Uzonyi6, Ágnes Szilágyi4, Zoltán Prohászka4, Adrian Goldman2,5, Mihály Józsi3,7, and T. Sakari Jokiranta1 1Department of Bacteriology and Immunology, Haartman Institute, and Research Programs Unit, Immunobiology, University of Helsinki, Finland 2Institute of Biotechnology, University of Helsinki, Finland 3Junior Research Group for Cellular Immunobiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena, Germany 4Research Laboratory, 3rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary 5Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland 6MTA-ELTE Immunology Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary 7MTA-ELTE “Lendület” Complement Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary *Running title: Structural insight on CFHR1 deficiency in AI-aHUS Correspondence should be addressed to: Dr. Arnab Bhattacharjee, Haartman Institute; P.O. Box 21 (Haartmaninkatu 3); FIN-00014 University of Helsinki; Finland tel. +358 294 1911 fax. +358 294 191 26382. Email A.B: [email protected] or M.J: [email protected] Key words: Thrombotic microangiopathy, complement regulation, autoimmunity, atypical hemolytic uremic syndrome, structure-function study ___________________________________________________________________________________ Background: The aHUS patients with to CFHR1 domains 4-5 (CFHR14-5). Here, autoantibodies against CFH lack CFHR1. binding site mapping of autoantibodies from Results: The autoantibody epitope was mapped seventeen patients using mutant CFH19-20 and the structure of the corresponding part of constructs revealed an autoantibody epitope CFHR1 was solved. cluster within a loop on domain 20, next to Conclusion: The structural difference between the buried two residues different in CFH19-20 the autoantigenic epitope of CFH and its and CFHR1 . Crystal structure of CFHR1 homologous site in CFHR1 holds the key to the 4-5 4-5 revealed difference in conformation of the formation of autoantibodies in CFHR1 deficient autoantigenic loop on the carboxyl- aHUS patients. terminal domains of CFH and CFHR1 Significance: A plausible explanation of the explaining variation in binding of CFHR1 deficiency in autoimmune aHUS autoantibodies from some aHUS patients obtained to CFH and CFHR1 . The 19-20 4-5 ABSTRACT autoantigenic loop on CFH seems to be Atypical hemolytic uremic syndrome generally flexible as its conformation in previously published structures of CFH (aHUS) is characterized by complement 19-20 attack against host cells due to mutations in bound to a microbial protein OspE and a complement proteins or autoantibodies sialic acid glycan is somewhat altered. against the complement regulator factor H Cumulatively our data suggest that (CFH). It is unknown why nearly all the association of CFHR1 deficiency with patients with autoimmune aHUS lack CFH- autoimmune aHUS could be owing to its related protein-1 (CFHR1). These patients structural difference to the autoantigenic have autoantibodies against CFH domains CFH epitope suggesting a novel explanation for CFHR1 deficiency in pathogenesis of 19-20 (CFH19-20) which are nearly identical Structural insight on CFHR1 deficiency in AI-aHUS autoimmune aHUS. terminus of CFH, and inhibit the physiological CFH-mediated protection of host cells from INTRODUCTION complement attack(10,11,13,22,23). aHUS is a rare and often fatal systemic More than 90% of the patients with CFH- disease, characterized by hemolytic anemia, AA lack CFHR1 and CFHR3, resulting from a thrombocytopenia, microvascular thrombosis, homozygous deletion of the genomic region and kidney failure (1). It is associated with containing both of them(10,12,13,15). Some dysregulation of complement activation via either patients have other rarer genetic alterations, mutations, polymorphisms, or rearrangements in including homozygous CFHR1/CFHR4A deletion genes coding for various complement proteins (12), a combination of heterozygous (2). The mutations are mainly found in the gene CFHR1/CFHR3 and CFHR1/CFHR4A deletions coding for the complement regulator CFH (3,4), (12,13), or a combined heterozygous which mediates elimination of the central CFHR1/CFHR3 deletion in the presence of a complement activation component C3b. We and missense mutation in CFHR1 (12). The common others have shown how mutations in CFH19-20 feature in all these genetic alterations is the cause impaired regulation of C3b on host cells deficiency of CFHR1 (24,25). However, CFH- leading to complement attack against red blood AAs have also been described, although rarely, in cells, platelets, and endothelial cells as seen patients having two normal copies clinically in aHUS (5-8). Some aHUS cases, of CFHR1 and CFHR3 but mutations however, are caused by autoantibodies against in CFH, CFI, CD46, or C3 (12,13). CFH-AAs CFH (CFH-AA). These antibodies have been often cross-react with CFHR1 (13,23,26) but the identified in 5–11% of aHUS patients in different exact location of the autoantibody site on CFHR1 cohorts (9-13) but even 56% of 246 HUS patients has not been shown. On the basis of inhibition of have been reported with CFH-AA in India(14). autoantibody binding to CFHR1 by the mAb Patients with the autoimmune form of aHUS C18(26) and the sequence homology to the nearly always lack certain CFH-related proteins, carboxyl-terminus of CFH it is, however, likely primarily CFHR1(10,15). that the autoantibody binding site is within the last two domains of CFHR1, i.e. far away from CFH and CFHRs are encoded by its N-terminal dimerization site(27). adjacent genes and form a protein family (supplemental Fig. S1)(16). CFHRs are The reason for the association between composed of four to nine complement control CFH-AA and the CFHR1 deficiency has not been protein (CCP) domains, also called short known till date. In this study we aimed at solving consensus repeats, and especially the two most why deficiency of one molecule (CFHR1) carboxyl-terminal domains have relatively high predisposes to autoimmunity against another, sequence homologies with the carboxyl-terminal highly homologous, molecule (CFH) in aHUS. domains of CFH (supplemental Fig. S1)(16). We mapped the binding sites of CFH-AA within There are only two residues different in the last CFH19-20 and compared the CFH-AA binding two carboxyl terminal domains of CFH and sites to the previously reported ligand binding CFHR1 (domains 19-20 and 4-5, respectively). sites on CFH19-20. Since the autoantibody epitopes The functional importance of these minor formed a cluster next to the residues that are differences is obvious since the hybrid different in the two carboxyl-terminal domains of CFH/CFHR1 genes producing fusion proteins CFH and CFHR1, we decided to solve and CFH1-18/CFHR14-5 or CFHR11-3/CFH19-20 have analyze the structure of CFHR14-5 and study the been found in aHUS patients in the absence of potential differences in antigenicity of those two other mutants or CFH-AA(4,17-19). The domains molecules. We found structural differences in the 19-20 of CFH are responsible for directing its autoantibody binding site of CFH domain 20 and complement regulatory activity to cell and the corresponding homologous site of CFHR1 extracellular matrix surfaces by binding domain 5. Based on these data a novel model is simultaneously to both C3b and negatively proposed hereby suggesting how immunization charged glycosaminoglycans or sialic acid against CFH domain 20 could be linked to glycans on the surfaces (6,20,21). The CFHR1 deficiency. autoantibodies of nearly all the patients with autoimmune aHUS recognize the carboxyl- Structural insight on CFHR1 deficiency in AI-aHUS EXPERIMENTAL PROCEDURES using peroxidase-conjugated swine anti-mouse IgG. Proteins: Cloning, expression and purification of the wild type (WT) and mutant CFH19-20 proteins Binding of the patient CFH-AA (sera with the 14 single point mutations have been diluted 1:100) to CFH, its recombinant described earlier(5,7,28). Proper folding of the fragments, CFHR1 and CFHR14-5 was compared constructs has been verified for three mutants as above using 250 nM immobilized recombinant (Q1139A, R1203A and the double mutant proteins, plasma purified CFH (65 nM), or D1119G/Q1139A) by solving the structures using recombinant CFHR4B and albumin as negative X-ray crystallography(6,28,29) and for five controls (Fig. 2). Relative binding was calculated mutants (R1182A, W1183L, K1188A, E1198A, from representative datasets performed in and R1206A) by circular dichroism(30). triplicates. CFHR1 was generated by site directed 4-5 The assay comparing binding avidity of mutagenesis of CFH encoding DNA in 19-20 CFH and CFHR1 with the purified IgG of pPICZαB vector (Invitrogen). The primer used to 19-20 4-5 the patient 11 was done after coating CFH19-20 (10 introduce the S1191L and V1197A mutations µg/ml) to Nunc MaxiSorp plates. After blocking was CAG AAG CTT TAT TTG AGA ACA TCA (0.05% Tween-20 in PBS) for 120 min and GGT GAA GAA GCT TTT GTG. The mutations washes (0.02% Tween-20 in PBS), 40 µl of were confirmed by sequencing before expression patient IgG (3.8 µg/ml) and CFH19-20, CFHR14-5, of CFHR14-5 in Pichia pastoris (strain X-33) or CFH5-7 was added in different dilutions and using 1% methanol induction as described incubated for 120 min at 37oC followed by previously(7).
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