title: Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro.
abstract: Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.
authors.affiliation: From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Loeven
authors.affiliation: From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
authors.forename: Angelique L
authors.initials: AL
authors.lastname: Rops
authors.affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, FIN-00290 Helsinki, Finland.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Lehtinen
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands.
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.affiliation: Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, and.
authors.forename: Mohamed R
authors.initials: MR
authors.lastname: Daha
authors.affiliation: Department of Internal Medicine and Otolaryngology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Smith
authors.affiliation: From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
authors.forename: Marinka
authors.initials: M
authors.lastname: Bakker
authors.affiliation: From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
authors.forename: Jo H
authors.initials: JH
authors.lastname: Berden
authors.affiliation: Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, and.
authors.forename: Ton J
authors.initials: TJ
authors.lastname: Rabelink
authors.affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, FIN-00290 Helsinki, Finland.
authors.forename: T Sakari
authors.initials: TS
authors.lastname: Jokiranta
authors.affiliation: From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands, johan.vandervlag@radboudumc.nl.
authors.forename: Johan
authors.initials: J
authors.lastname: van der Vlag
mesh_terms: D000818:Animals; D002460:Cell Line; D003167:Complement Activation; D017242:Complement Factor H / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D006025:Glycosaminoglycans; D014409:Tumor Necrosis Factor-alpha; D017242:Complement Factor H; D006493:Heparin
keywords: atypical hemolytic uremic syndrome; autoimmune disease; complement Factor H; complement system; endothelial glycocalyx; endothelium; heparan sulfate; innate immunity