Loeven Markus A, Rops Angelique L, Lehtinen Markus J, van Kuppevelt Toin H, Daha Mohamed R, Smith Richard J, Bakker Marinka, Berden Jo H, Rabelink Ton J, Jokiranta T Sakari, van der Vlag Johan
From the Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, FIN-00290 Helsinki, Finland.
J Biol Chem. 2016 Mar 4;291(10):4974-81. doi: 10.1074/jbc.M115.702506. Epub 2016 Jan 4.
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.
补体因子H（FH）可抑制补体激活，并通过其补体控制蛋白结构域19和20与肾小球内皮相互作用，这两个结构域也可识别硫酸乙酰肝素（HS）。FH异常与肾脏疾病非典型溶血性尿毒症综合征和致密物沉积病以及眼部疾病年龄相关性黄斑变性有关。尽管FH可系统性地控制补体激活，但临床表型却选择性地在肾脏和眼睛中表现出来，这表明存在疾病发展的组织特异性决定因素。最近的研究结果表明，组织特异性表达的硫酸化糖胺聚糖（GAG），如HS，在决定FH与宿主组织的结合及活性方面具有重要作用。因此，我们在酶联免疫吸附测定（ELISA）中研究了哪些GAG介导人FH及重组人FH补体控制蛋白结构域19和20（FH19 - 20）与小鼠肾小球内皮细胞（mGEnCs）的结合。此外，我们通过流式细胞术测量mGEnCs上C3b的沉积，评估了FH19 - 20突变体在补体激活过程中的功能缺陷。FH和FH19 - 20与mGEnCs的结合呈剂量依赖性，肿瘤坏死因子-α（TNF-α）处理可增加这两种蛋白的结合，而肝素酶消化以及与肝素/HS的竞争可抑制结合。此外，肝素的2 - O - 和6 - O - 去硫酸化而非N - 去硫酸化，显著增强了对FH19 - 20与mGEnCs结合的抑制作用。与野生型FH19 - 20相比，非典型溶血性尿毒症综合征相关突变体在mGEnCs补体激活过程中，在正常人血清中与FH竞争的能力较弱，证实了它们潜在的肾小球致病性。总之，我们的研究表明，FH和FH19 - 20与肾小球内皮细胞的结合由HS而非其他GAG差异介导。此外，我们描述了一种新型的、不依赖患者血清的竞争测定法，用于FH19 - 20突变体的致病性筛查。
