Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome
2009; Elsevier BV; Volume: 284; Issue: 23 Linguagem: Inglês
10.1074/jbc.m900814200
ISSN1083-351X
AutoresMarkus J. Lehtinen, Angelique L. Rops, David E. Isenman, Johan van der Vlag, T. Sakari Jokiranta,
Tópico(s)Adenosine and Purinergic Signaling
ResumoAtypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway. Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway. Atypical hemolytic uremic syndrome (aHUS) 2The abbreviations used are:aHUSatypical hemolytic uremic syndromeFHfactor HGAGglycosaminoglycanFH19–20domains 19 to 20 of FHPBSphosphate-buffered salinemGEnC-1mouse glomerular endothelial cell line 1. 2The abbreviations used are:aHUSatypical hemolytic uremic syndromeFHfactor HGAGglycosaminoglycanFH19–20domains 19 to 20 of FHPBSphosphate-buffered salinemGEnC-1mouse glomerular endothelial cell line 1. is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1.Ruggenenti P. Noris M. Remuzzi G. Kidney Int. 2001; 60: 831-846Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2.Kavanagh D. Goodship T.H. Richards A. Br. Med. Bull. 2006; 5: 5Crossref Scopus (107) Google Scholar). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3.Fremeaux-Bacchi V. Dragon-Durey M.A. Blouin J. Vigneau C. Kuypers D. Boudailliez B. Loirat C. Rondeau E. Fridman W.H. J. Med. Genet. 2004; 41: e84Crossref PubMed Scopus (284) Google Scholar, 4.Frémeaux-Bacchi V. Miller E.C. Liszewski M.K. Strain L Blouin J. Brown A.L. Moghal N. Kaplan B.S. Weiss R.A. Lhotta K. Kapur G. Mattoo T. Nivet H. Wong W. Gie S. Hurault de Ligny B. Fischbach M. Gupta R. Hauhart R. Meunier V. Loirat C. Dragon-Durey M.A. Fridman W.H. Janssen B.J. Goodship T.H. Atkinson J.P. Blood. 2008; 112: 4948-4952Crossref PubMed Scopus (312) Google Scholar, 5.Goicoechea de Jorge E. Harris C.L. Esparza-Gordillo J. Carreras L. Arranz E.A. Garrido C.A. López-Trascasa M. Sánchez-Corral P. Morgan B.P. Rodríguez de Córdoba S. Proc. Natl. Acad. Sci. U. S. 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More than half of the mutations have been found to originate in the HF1 gene that encodes FH and FH-like protein 1.The alternative pathway is initiated spontaneously by hydrolysis of C3 to C3H2O that forms the C3-convertase C3H2OBb (9.Lesavre P.H. Müller-Eberhard H.J. J. Exp. Med. 1978; 148: 1498-1509Crossref PubMed Scopus (124) Google Scholar, 10.Pangburn M.K. Schreiber R.D. Müller-Eberhard H.J. J. Exp. Med. 1981; 154: 856-867Crossref PubMed Scopus (313) Google Scholar). This enzyme complex converts numerous C3 molecules to C3b that are covalently bound onto practically any nearby surface (11.Law S.K. Levine R.P. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2701-2705Crossref PubMed Scopus (242) Google Scholar). On a so-called activator surface, such as a microbe, the surface-bound C3b molecules are not efficiently eliminated and therefore new C3bBb complexes are formed leading to more C3b depositions and eventually effective opsonization or damage of the target cell. On non-activator surfaces, such as viable self (host) cells, factor I cleaves C3b to inactive C3b (iC3b) in the presence of one of the cofactors (CD46, CD35, FH, and FHL-1) (12.Harrison R.A. Lachmann P.J. Mol. Immunol. 1980; 17: 219-228Crossref PubMed Scopus (24) Google Scholar, 13.Harrison R.A. Lachmann P.J. Mol. Immunol. 1980; 17: 9-20Crossref PubMed Scopus (132) Google Scholar, 14.Ruddy S. Austen K.F. J. Immunol. 1969; 102: 533-543PubMed Google Scholar, 15.Pangburn M.K. Schreiber R.D. Müller-Eberhard H.J. J. Exp. Med. 1977; 146: 257-270Crossref PubMed Scopus (521) Google Scholar, 16.Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (321) Google Scholar). FH is the only one of these cofactors that mediates recognition of self-surfaces making the alternative pathway capable of discriminating between activating and non-activating surfaces (17.Pangburn M.K. Müller-Eberhard H.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2416-2420Crossref PubMed Scopus (261) Google Scholar, 18.Weiler J.M. Daha M.R. Austen K.F. Fearon D.T. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3268-3272Crossref PubMed Scopus (440) Google Scholar, 19.Whaley K. Ruddy S. J. Exp. Med. 1976; 144: 1147-1163Crossref PubMed Scopus (423) Google Scholar).The two main functions of FH are to prevent the alternative pathway activation in plasma and on self-surfaces. This 150-kDa glycoprotein consists of 20 tandemly arranged short consensus repeat domains that are composed of ∼60 amino acids. Domains 1–4 are essential for the cofactor and decay accelerating activity (20.Gordon D.L. Kaufman R.M. Blackmore T.K. Kwong J. Lublin D.M. J. Immunol. 1995; 155: 348-356PubMed Google Scholar). In the middle region of FH (domains 5–15) there are two binding sites for C-reactive protein (21.Jarva H. Jokiranta T.S. Hellwage J. Zipfel P.F. Meri S. J. Immunol. 1999; 163: 3957-3962PubMed Google Scholar), one or two sites for glycosaminoglycans (GAGs) (22.Blackmore T.K. Sadlon T.A. Ward H.M. Lublin D.M. Gordon D.L. J. Immunol. 1996; 157: 5422-5427PubMed Google Scholar, 23.Ormsby R.J. Jokiranta T.S. Duthy T.G. Griggs K.M. Sadlon T.A. Giannakis E. Gordon D.L. Mol. Immunol. 2006; 43: 1624-1632Crossref PubMed Scopus (56) Google Scholar, 24.Pangburn M.K. Atkinson M.A. Meri S. J. Biol. Chem. 1991; 266: 16847-16853Abstract Full Text PDF PubMed Google Scholar, 25.Schmidt C.Q. Herbert A.P. Kavanagh D. Gandy C. Fenton C.J. Blaum B.S. Lyon M. Uhrín D. Barlow P.N. J. Immunol. 2008; 181: 2610-2619Crossref PubMed Scopus (157) Google Scholar), and one site for C3c part of C3b (C3b/C3c) (25.Schmidt C.Q. Herbert A.P. Kavanagh D. Gandy C. Fenton C.J. Blaum B.S. Lyon M. Uhrín D. Barlow P.N. J. Immunol. 2008; 181: 2610-2619Crossref PubMed Scopus (157) Google Scholar, 26.Jokiranta T.S. Hellwage J. Koistinen V. Zipfel P.F. Meri S. J. Biol. Chem. 2000; 275: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The C-terminal domains 19–20 (FH19–20) possess binding sites for the thiol ester domain of C3b (C3d or C3dg, TED domain) and GAGs (26.Jokiranta T.S. Hellwage J. Koistinen V. Zipfel P.F. Meri S. J. Biol. Chem. 2000; 275: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 27.Blackmore T.K. Hellwage J. Sadlon T.A. Higgs N. Zipfel P.F. Ward H.M. Gordon D.L. J. Immunol. 1998; 160: 3342-3348PubMed Google Scholar).The most common types of mutations found in aHUS are FH missense mutations located within FH19–20 that was recently solved as crystal and NMR structures (2.Kavanagh D. Goodship T.H. Richards A. Br. Med. Bull. 2006; 5: 5Crossref Scopus (107) Google Scholar, 28.Herbert A.P. Uhrín D. Lyon M. Pangburn M.K. Barlow P.N. J. Biol. Chem. 2006; 281: 16512-16520Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29.Jokiranta T.S. Jaakola V.P. Lehtinen M.J. Pärepalo M. Meri S. Goldman A. EMBO J. 2006; 25: 1784-1794Crossref PubMed Scopus (133) Google Scholar). The C terminus of FH is crucial in self-cell protection as demonstrated by the severity of the aHUS cases and also in a recent mouse model of aHUS where domains 16–20 had been deleted (30.Ferreira V.P. Herbert A.P. Hocking H.G. Barlow P.N. Pangburn M.K. J. Immunol. 2006; 177: 6308-6316Crossref PubMed Scopus (125) Google Scholar, 31.Pickering M.C. de Jorge E.G. Martinez-Barricarte R. Recalde S. Garcia-Layana A. Rose K.L. Moss J. Walport M.J. Cook H.T. de Córdoba S.R. Botto M. J. Exp. Med. 2007; 204: 1249-1256Crossref PubMed Scopus (217) Google Scholar). Histopathology of aHUS in these mice had all the characteristics of human aHUS being concordant with the similarity of binding sites for C3b, heparin, and human umbilical vein endothelial cells between human and mouse FH domains 18–20 (32.Cheng Z.Z. Hellwage J. Seeberger H. Zipfel P.F. Meri S. Jokiranta T.S. Mol. Immunol. 2006; 43: 972-979Crossref PubMed Scopus (27) Google Scholar). Binding of mouse or human FH to glomerular endothelial cells has not been characterized despite the fact that in aHUS damage occurs mainly in the small vessels, especially in the glomeruli.The molecular pathogenesis leading to the clinical aHUS in patients with FH mutations remains elusive. The suggested molecular mechanisms for some aHUS-associated mutations include defective binding of the mutated FH to GAGs, endothelial cells, or C3b/C3d (28.Herbert A.P. Uhrín D. Lyon M. Pangburn M.K. Barlow P.N. J. Biol. Chem. 2006; 281: 16512-16520Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29.Jokiranta T.S. Jaakola V.P. Lehtinen M.J. Pärepalo M. Meri S. Goldman A. EMBO J. 2006; 25: 1784-1794Crossref PubMed Scopus (133) Google Scholar, 33.Józsi M. Heinen S. Hartmann A. Ostrowicz C.W. Hälbich S. Richter H. Kunert A. Licht C. Saunders R.E. Perkins S.J. Zipfel P.F. Skerka C. J. Am. Soc. Nephrol. 2006; 17: 170-177Crossref PubMed Scopus (104) Google Scholar, 34.Manuelian T. Hellwage J. Meri S. Caprioli J. Noris M. Heinen S. Jozsi M. Neumann H.P. Remuzzi G. Zipfel P.F. J. Clin. Investig. 2003; 111: 1181-1190Crossref PubMed Scopus (300) Google Scholar). The aim of this study was to define the effects of nine aHUS-associated FH mutations and five other structurally closely located mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. We identified three primary defects of the mutants: impaired C3b/C3d binding, enhanced mGEnC-1/heparin binding, and impaired mGEnC-1/heparin binding that could lead via three mechanisms to incapability of FH to eliminate C3b on plasma-exposed self-cells. The results clarify the mechanism of target discrimination of the alternative pathway by the C terminus of FH.DISCUSSIONAtypical hemolytic uremic syndrome is a rare but severe disease that typically affects children and adolescents. Mutations in several complement proteins have been reported from the patients but more than half of these are accounted for by mutations in the HF1 gene that codes FH. Molecular mechanisms underlying the association of mutations in FH with aHUS have, however, largely remained elusive. In this study we have characterized the effects of nine aHUS-associated and five non-associated mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. The results show that the aHUS-associated FH mutations cause different combinations of three primary defects. We also show, for the first time, the binding site for mouse glomerular endothelial cells in FH domain 20 and demonstrate that the binding site for C3b/C3d is likely to extend from domain 20 to domain 19. Finally, the results indicate that FH19–20 needs to bind to C3b/C3d to enable full activity of FH.Three primary defects were identified in our set of nine aHUS mutations: impaired binding to C3b/C3d, enhanced binding to mouse glomerular endothelial cells, and impaired binding to glomerular endothelial cells (Table 1). Five of the aHUS-associated mutants had impaired binding to C3b (W1157LaHUS, R1182AaHUS, W1183LaHUS, T1184RaHUS, and R1210AaHUS) and two of these did not have either of the other primary defects. This indicates that defects in C3b binding of FH19–20 alone can lead to aHUS and that binding of FH19–20 to C3b/C3d is essential for full activity of FH (Fig. 7, aHUS mechanism i). A role of impaired C3b/C3d binding in aHUS has also been previously suggested and so far a total of 10 mutations have been described with this defect (W1157RaHUS, R1182AaHUS, W1183LaHUS, W1183RaHUS, V1197AaHUS, E1198AaHUS, R1210CaHUS, R1215GaHUS, P1226SaHUS, and S1191L/V1197A) (29.Jokiranta T.S. Jaakola V.P. Lehtinen M.J. Pärepalo M. Meri S. Goldman A. EMBO J. 2006; 25: 1784-1794Crossref PubMed Scopus (133) Google Scholar, 33.Józsi M. Heinen S. Hartmann A. Ostrowicz C.W. Hälbich S. Richter H. Kunert A. Licht C. Saunders R.E. Perkins S.J. Zipfel P.F. Skerka C. J. Am. Soc. Nephrol. 2006; 17: 170-177Crossref PubMed Scopus (104) Google Scholar, 34.Manuelian T. Hellwage J. Meri S. Caprioli J. Noris M. Heinen S. Jozsi M. Neumann H.P. Remuzzi G. Zipfel P.F. J. Clin. Investig. 2003; 111: 1181-1190Crossref PubMed Scopus (300) Google Scholar, 35.Sánchez-Corral P. Pérez-Caballero D. Huarte O. Simckes A.M. Goicoechea E. López-Trascasa M. de Córdoba S.R. Am. J. Hum. Genet. 2002; 71: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar)). Therefore all the available data indicates that the loss of C3b/C3d binding is typical in aHUS and may lead to the disease in the absence of other functional defects.In this study we describe a novel defect of FH in aHUS, where three aHUS-associated mutants (T1184RaHUS, L1189RaHUS, and E1198AaHUS) have enhanced binding to mouse glomerular endothelial cells and heparin with normal C3b or C3d binding (Table 1). This gain-of-function does not lead to sequestration of FH to inappropriate compartments of the body because the patients with these mutations have at least normal plasma levels of FH (41.Pérez-Caballero D. González-Rubio C. Gallardo M.E. Vera M. López-Trascasa M. Rodríguez de Córdoba S. Sánchez-Corral P. Am. J. Hum. Genet. 2001; 68: 478-484Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 42.Richards A. Buddles M.R. Donne R.L. Kaplan B.S. Kirk E. Venning M.C. Tielemans C.L. Goodship J.A. Goodship T.H. Am. J. Hum. Genet. 2001; 68: 485-490Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 43.Vaziri-Sani F. Holmberg L. Sjöholm A.G. Kristoffersson A.C. Manea M. Frémeaux-Bacchi V. Fehrman-Ekholm I. Raafat R. Karpman D. Kidney Int. 2006; 69: 981-988Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Therefore the findings with these mutants suggest novel concepts for both the target discrimination between the activator/nonactivator surfaces by FH and the pathogenesis of aHUS in these patients. In target discrimination the typical model is that on nonactivator surfaces the C terminus of FH binds to the cell surface molecules such as heparan sulfate leading to enhanced elimination of the surface-bound C3b molecules by the N-terminal effector functions of FH. The model clearly contains the right players but it fails to consider that the C terminus of FH might eventually need to bind to the C3d part of C3b for effective elimination of the surface-bound C3b molecules. On the basis of our data it can be suggested that in a physiological situation heparin forms a temporary encounter complex with FH19–20 and subsequently there is transfer of FH19–20 to the C3d portion of C3b, because of the higher affinity of the protein-protein interaction relative to that of the protein/GAG interaction (Fig. 7).The suggested model of the alternative pathway discrimination is supported by the results that binding of the C terminus of FH to heparin and C3b is competitive (44.Hellwage J. Jokiranta T.S. Friese M.A. Wolk T.U. Kampen E. Zipfel P.F. Meri S. J. Immunol. 2002; 169: 6935-6944Crossref PubMed Scopus (98) Google Scholar) making a ternary complex between FH19–20, heparin (or other GAGs), and C3b unlikely. The key question is if it matters for function of full FH whether its C terminus is bound to heparin or the C3d portion of C3b on a surface. Our results with the mutant W1183L, found from an aHUS patient, indicate that it matters. This mutant shows normal heparin binding and decreased C3b/C3d binding indicating that normal heparin binding cannot compensate for loss of the C3b/C3d binding because the patient suffered from clinical aHUS. Therefore it is clear that a heparin-bound FH is not itself functionally fully active if it is unable to bind to the C3d part of C3b. This means that C3b/C3d binding is essentially needed for the physiological function of FH19–20 on cell-bound C3b. With this clarified model the explanation of the molecular pathogenesis of mutants T1184R, L1189R, and E1198A is logical. Because the heparin and C3b binding sites on FH19–20 are overlapping the enhanced heparin binding can lead to decreased C3b/C3d binding. Because normal C3b/C3d binding is needed for the fully functional molecule the elimination of cell-bound C3b is impaired leading to clinical aHUS. In this way the gain-of-function in heparin or cell binding eventually leads to loss-of-function in elimination of C3b (Fig. 7, mechanism ii).The third primary defect was described only by one mutant, R1215QaHUS, that had wild-type-like binding to C3b/C3d and impaired binding to heparin and possibly also to mGEnC-1 (Figs. 4D and 5D). Although the result for the mGEnC-1 binding was not statistically significant in our experiments, the role of Arg-1215 in cell binding is likely because a different mutation of the same residue, R1215GaHUS, has previously been shown to impair binding of FH8–20 to C3b, heparin, and human umbilical vein endothelial cells (34.Manuelian T. Hellwage J. Meri S. Caprioli J. Noris M. Heinen S. Jozsi M. Neumann H.P. Remuzzi G. Zipfel P.F. J. Clin. Investig. 2003; 111: 1181-1190Crossref PubMed Scopus (300) Google Scholar). Thus it may be that impaired binding of heparin/cells alone without loss of C3b/C3d binding can also lead to aHUS, as has been suggested before (Fig. 7, mechanism iii) (34.Manuelian T. Hellwage J. Meri S. Caprioli J. Noris M. Heinen S. Jozsi M. Neumann H.P. Remuzzi G. Zipfel P.F. J. Clin. Investig. 2003; 111: 1181-1190Crossref PubMed Scopus (300) Google Scholar, 45.Jokiranta T.S. Cheng Z.Z. Seeberger H. Jòzsi M. Heinen S. Noris M. Remuzzi G. Ormsby R. Gordon D.L. Meri S. Hellwage J. Zipfel P.F. Am. J. Path. 2005; 167: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This mutant, R1215QaHUS, however, is the only mutant published to date where a heparin binding defect has been observed in the absence of defect in C3b/C3d binding.We detected at least one functional defect with eight of the nine aHUS-associated mutants (Table 1). The only mutant that appeared functionally normal in our analyses was the mutant D1119GaHUS. The mutation has been described from one individual whose sibling was also affected but his/her sequence analysis has not been published (42.Richards A. Buddles M.R. Donne R.L. Kaplan B.S. Kirk E. Venning M.C. Tielemans C.L. Goodship J.A. Goodship T.H. Am. J. Hum. Genet. 2001; 68: 485-490Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). At the time of the publication it was not known that in aHUS there are mutations also in other complement proteins and therefore, it is possible that the disease was not caused by the D1119GaHUS mutation because other known aHUS-associated mutations have not been excluded.The results of this study indicate that the C3b/C3d binding site is located in FH domains 19 and 20, and glomerular endothelial cell, and heparin binding sites in domain 20 (Fig. 6). Due to the similarity of the used mouse glomerular endothelial cells to human glomerular endothelial cells (39.Rops A.L. van den Hoven M.J. Baselmans M.M. Lensen J.F. Wijnhoven T.J. van den Heuvel L.P. van Kuppevelt T.H. Berden J.H. van der Vlag J. Kidney Int. 2008; 73: 52-62Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) it is likely that the observed FH binding site is the same for human cells. The binding site for C3b/C3d in domain 20 has been analyzed previously, but on domain 19 the only amino acid studied so far has been Trp-1157 (33.Józsi M. Heinen S. Hartmann A. Ostrowicz C.W. Hälbich S. Richter H. Kunert A. Licht C. Saunders R.E. Perkins S.J. Zipfel P.F. Skerka C. J. Am. Soc. Nephrol. 2006; 17: 170-177Crossref PubMed Scopus (104) Google Scholar). In that study, mutation W1157RaHUS decreased C3b binding but introduction of a charged arginine in place of the partially buried hydrophobic tryptophan may cause more distant changes in the overall structure without being part of the interacting surface. We now show that in addition to the W1157LaHUS mutation, the Q1139A mutation causes impairment in C3b/C3d binding (Figs. 2C and 3C). The Gln-1139 residue is surface exposed and therefore it is not likely to cause any global changes to the structure. Thereby the current results, showing that mutations D1119G/Q1139A, Q1139A, and W1157LaHUS in domain 19 lead to impaired C3b/C3d binding, suggests that the C3b/C3d binding site extends from domain 20 to domain 19. This observation would also explain the six different missense mutations in domain 19 found in aHUS patients.A heparin binding site on FH19–20 has previously been characterized in a nuclear magnetic resonance study of FH19–20, where heparin tetrasaccharide perturbed NMR spectral peaks of eight residues in domain 20 (Arg-1182, Lys-1186, Lys-1188, Lys-1202, Arg-1203, Arg-1215, Lys-1230, and Arg-1231), whereas no perturbations were observed in domain 19 (28.Herbert A.P. Uhrín D. Lyon M. Pangburn M.K. Barlow P.N. J. Biol. Chem. 2006; 281: 16512-16520Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Our study corroborates these results as we show that mutations of positively charged residues R1182AaHUS, K1186A, K1188A, R1206A, R1210AaHUS, and R1215QaHUS in domain 20 decrease heparin binding, whereas mutations in domain 19 do not have an effect on heparin binding. Interestingly, aHUS-associated mutations that enhanced heparin binding (Fig. 5D) introduce positive (T1184RaHUS, L1189RaHUS) or remove negative charge (E1198AaHUS) in the proximity of the heparin binding cluster in the structure of FH19–20. Thus T1184RaHUS, and L1189RaHUS seem to extend the binding site for heparin in domain 20 and E1198AaHUS removes repulsive charge from the edge of the cluster.Based on the identified binding sites we conclude that the glomerular endothelial cell binding site on FH19–20 is on domain 20 where it overlaps with the C3b/C3d binding site, and that the cell binding site is nearly identical with the heparin binding site (Fig. 6 and Table 1). This data fits well with the previous suggestion that the C3d and heparin binding sites on the FH15–20 background were found to be distinct but overlapping (44.Hellwage J. Jokiranta T.S. Friese M.A. Wolk T.U. Kampen E. Zipfel P.F. Meri S. J. Immunol. 2002; 169: 6935-6944Crossref PubMed Scopus (98) Google Scholar). Our results on the glomerular endothelial cell binding site on domain 20 (Fig. 6B) are concordant with the previously published studies on human umbilical vein endothelial cells, where mutations in domain 20 on FH8–20 and FH15–20 backgrounds decreased binding to cells (33.Józsi M. Heinen S. Hartmann A. Ostrowicz C.W. Hälbich S. Richter H. Kunert A. Licht C. Saunders R.E. Perkins S.J. Zipfel P.F. Skerka C. J. Am. Soc. Nephrol. 2006; 17: 170-177Crossref PubMed Scopus (104) Google Scholar, 34.Manuelian T. Hellwage J. Meri S. Caprioli J. Noris M. Heinen S. Jozsi M. Neumann H.P. Remuzzi G. Zipfel P.F. J. Clin. Investig. 2003; 111: 1181-1190Crossref PubMed Scopus (300) Google Scholar, 45.Jokiranta T.S. Cheng Z.Z. Seeberger H. Jòzsi M. Heinen S. Noris M. Remuzzi G. Ormsby R. Gordon D.L. Meri S. Hellwage J. Zipfel P.F. Am. J. Path. 2005; 167: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The binding of the mutants to cells thus correlates well with their binding to heparin indicating that the binding site for cells and heparin is located in domain 20 only. The only exception was mutant K1186A, which had increased binding to mGEnC-1 and impaired binding to heparin (Table 1). This indicates two things. First, FH19–20 binding to heparin that has been used as a model for cell surface heparan sulfate proteoglycans seems to be a relatively good model for endothelial cell binding. Second, the use of porcine heparin preparations may not reflect in all cases the actual binding of FH to sulfated GAGs on cells. Currently, we are further exploring the exact role of GAGs at the glomerular endothelial surface in binding FH.The data in this study shows that all aHUS-associated FH19–20 mutations do not cause the same primary functional defect but one or two of the three observed abnormalities in C3b/C3d or glomerular endothelial cell/heparin binding. It is likely that all three primary functional defects caused by the FH mutations in aHUS lead eventually to the same process of inefficient clearance of surface-bound C3b via three mechanisms (Fig. 7). In this study we have demonstrated a novel defect of enhanced binding of aHUS-associated mutants to glomerular endothelial cells or heparin that adds to the previously suggested two defects of impaired C3b/C3d binding and impaired cell/heparin binding. In addition, we have also mapped the binding site of FH19–20 for the glomerular endothelial cells to domain 20 and show that cell binding correlates well, but not fully, with heparin binding. This site is distinct from the C3b/C3d binding site that, for the first time, is shown to extend to domain 19. Finally, we have proposed a model to explain how the target discrimination by the alternative pathway essentially needs binding of FH C terminus to the C3d part of C3b. Atypical hemolytic uremic syndrome (aHUS) 2The abbreviations used are:aHUSatypical hemolytic uremic syndromeFHfactor HGAGglycosaminoglycanFH19–20domains 19 to 20 of FHPBSphosphate-buffered salinemGEnC-1mouse glomerular endothelial cell line 1. 2The abbreviations used are:aHUSatypical hemolytic uremic syndromeFHfactor HGAGglycosaminoglycanFH19–20domains 19 to 20 of FHPBSphosphate-buffered salinemGEnC-1mouse glomerular endothelial cell line 1. is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1.Ruggenenti P. Noris M. Remuzzi G. Kidney Int. 2001; 60: 831-846Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2.Kavanagh D. Goodship T.H. Richards A. Br. Med. Bull. 2006; 5: 5Crossref Scopus (107) Google Scholar). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3.Fremeaux-Bacchi V. Dragon-Durey M.A. Blouin J. Vigneau C. Kuypers D. Boudailliez B. Loirat C. Rondeau E. Fridman W.H. J. Med. Genet. 2004; 41: e84Crossref PubMed Scopu
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