Nitration of tyrosines in complement factor H domains alters its immunological activity and mediates a pathogenic role in age related macular degeneration.

Matthew Krilis,Peng Zhang,Mahmoud Abdelatti,Steven A Krilis,Jason W H Wong,Peter Mccluskey,Jian Qi,Miao Qi,John Whitelock,Bill Giannakopoulos,Michele C Madigan,Robyn H Guymer,Alex P Hunyor

doi:10.18632/oncotarget.14940

Abstract

Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis.Sections of eyes from deceased individuals with AMD (n = 5) demonstrated the presence of immunoreactive nitrotyrosine CFH. We purified nitrated CFH from retinae from 2 AMD patients. Mass spectrometry of CFH isolated from AMD eyes revealed nitrated residues in domains critical for binding to heparan sulphate glycosaminoglycans (GAGs), lipid peroxidation by-products and complement (C) 3b.Functional studies revealed that nitrated CFH did not bind to lipid peroxidation products, nor to the GAG of perlecan nor to C3b. There was loss of cofactor activity for Factor I mediated cleavage of C3b with nitrated CFH compared to non-nitrated CFH. CFH inhibits, but nitrated CFH significantly potentiates, the secretion of the pro-inflammatory and angiogenic cytokine IL-8 from monocytes that have been stimulated with lipid peroxidation by-products. AMD patients (n = 30) and controls (n = 30) were used to measure plasma nitrated CFH using a novel ELISA. AMD patients had significantly elevated nitrated CFH levels compared to controls (p = 0.0117). These findings strongly suggest that nitrated CFH contributes to AMD progression, and is a target for therapeutic intervention.

Highlights

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in people aged over 50 years in developed countries [1].Multiple lines of evidence suggest that dysregulation of the alternate complement system is an important contributor to AMD pathogenesis [2]
Paraffin sections of AMD eyes (n = 5, age of patients 69, 78, 83, 84 and 93 with Grade 1 to 4 Age-Related Eye Disease Study (AREDS) staging were examined for expression of complement factor H (CFH) and nitrotyrosine using single and double immunolabelling and confocal microscopy
In this study we have made the major findings that CFH can exist in a nitrated form in vivo, and that nCFH can be detected in retina/choroid of patients with AMD with increased levels in their plasma compared to controls

Summary

Introduction

Multiple lines of evidence suggest that dysregulation of the alternate complement system is an important contributor to AMD pathogenesis [2]. 3050% of genetic susceptibility to AMD is conferred most strongly by the complement factor H (CFH) Y402H polymorphism, resulting in a tyrosine(Y)-to-histidine(H) substitution at amino acid position 402 within the CFH protein [3,4,5,6]. CFH is a major negative regulator of the alternate complement pathway [7]. C3b forms the C3 convertase complex that through a positive feedback loop amplifies C3b generation, which goes on to form part of the C5 convertase complex, leading to the formation of the complement membrane attack complex. C3b is promiscuous in its ability to opsonize pathogen surfaces and healthy cell surfaces, and the latter can lead to colateral damage of the host cells unless C3b is promptly inactivated by regulatory proteins such as CFH at these sites [7]

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Conclusion