Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Markus A Loeven; Angelique L Rops; Markus J Lehtinen; Toin H van Kuppevelt; Mohamed R Daha; Richard J Smith; Marinka Bakker; Jo H Berden; Ton J Rabelink; T Sakari Jokiranta; Johan van der Vlag

doi:10.1074/jbc.M115.702506

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Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

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Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Markus A Loeven, Angelique L Rops, Markus J Lehtinen, Toin H van Kuppevelt, Mohamed R Daha, Richard J Smith, Marinka Bakker, Jo H Berden, Ton J Rabelink, T Sakari Jokiranta, …

The Journal of biological chemistry, Vol.291(10), pp.4974-4981

03/04/2016

DOI: 10.1074/jbc.M115.702506

PMCID: PMC4777835

PMID: 26728463

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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.

Protein Structure, Tertiary

Cell Line

Complement Factor H - immunology

Complement Activation

Endothelial Cells - metabolism

Glycosaminoglycans - metabolism

Humans

Tumor Necrosis Factor-alpha - pharmacology

Animals

Complement Factor H - metabolism

Protein Binding

Mice

Mutation

Complement Factor H - genetics

Heparin - pharmacology

Kidney Glomerulus - cytology

Complement Factor H - chemistry

Endothelial Cells - drug effects

Details

Title: Subtitle: Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro
Creators: Markus A Loeven - From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Angelique L Rops - From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Markus J Lehtinen - Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, FIN-00290 Helsinki, Finland
Toin H van Kuppevelt - Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands
Mohamed R Daha - Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, and
Richard J Smith - Department of Internal Medicine and Otolaryngology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
Marinka Bakker - From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Jo H Berden - From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
Ton J Rabelink - Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, and
T Sakari Jokiranta - Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, FIN-00290 Helsinki, Finland
Johan van der Vlag - From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands, johan.vandervlag@radboudumc.nl
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.291(10), pp.4974-4981
DOI: 10.1074/jbc.M115.702506
PMID: 26728463
PMCID: PMC4777835
NLM abbreviation: J Biol Chem
ISSN: 1083-351X
eISSN: 1083-351X
Publisher: United States
Grant note: DOI: 10.13039/501100002997, name: Dutch Kidney Foundation, award: CP09.03 (GLYCOREN), KJPB 09.01; DOI: 10.13039/100010281, name: Kidneeds, award: grant 2009; DOI: 10.13039/501100006209, name: Radboud Universitair Medisch Centrum, award: 2012; DOI: 10.13039/501100002341, name: Suomen Akatemia, award: 128646, 259793; name: Sigrid Juselius Foundation
Language: English
Date published: 03/04/2016
Academic Unit: Roy J. Carver Department of Biomedical Engineering; Molecular Physiology and Biophysics; Anatomy and Cell Biology; Stead Family Department of Pediatrics; Iowa Neuroscience Institute; Otolaryngology; Internal Medicine
Record Identifier: 9984007162602771

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