Functional analysis of a complement polymorphism (rs17611) associated with Rheumatoid Arthritis

Abstract

Complement is implicated in the pathogenesis of rheumatoid arthritis (RA); elevated levels of complement activation products have been measured in plasma, synovial fluid, and synovial tissues of patients. Complement polymorphisms are associated with RA in genome-wide association studies. Coding-region polymorphisms may directly impact protein activity; indeed, we have shown that complement polymorphisms affecting a single amino acid change cause subtle changes in individual component function that in combination have dramatic effects on complement activity and disease risk. In this study, we explore the functional consequences of a single nucleotide polymorphism (SNP) (rs17611) encoding a V802I polymorphism in C5 and propose a mechanism for its link to RA pathology. Plasma levels of C5, C5a, and terminal complement complex were measured in healthy and RA donors and correlated to rs17611 polymorphic status. Impact of the SNP on C5 functionality was assessed. Plasma C5a levels were significantly increased and C5 levels significantly lower with higher copy number of the RA risk allele for rs17611, suggesting increased turnover of C5 V802. Functional assays using purified C5 variants revealed no significant differences in lytic activity, suggesting that increased C5 V802 turnover was not mediated by complement convertase enzymes. C5 is also cleaved in vivo by proteases; the C5 V802 variant was more sensitive to cleavage with elastase and the "C5a" generated was biologically active. We hypothesize that this SNP in C5 alters the rate at which elastase generates active C5a in rheumatoid joints, hence recruiting neutrophils to the site thus maintaining a state of inflammation in arthritic joints.

Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis

“…Purification of C5 has been challenging; early methods were laborious and inefficient with a very low recovery of active C5 . Current affinity purification methods are faster but are complicated by low yield (> 50%) and poor functional activity of the protein, a consequence of its pH lability and tendency to associate with C6 if denatured . Here we took advantage of a recently described anti‐C5 therapeutic mAb, RO7112689, that was selected to recycle in vivo by shedding its cargo when exposed to acidic pH (~ pH5·5) in the endosome .…”

“…[6][7][8][9] Current affinity purification methods are faster but are complicated by low yield (> 50%) and poor functional activity of the protein, a consequence of its pH lability and tendency to associate with C6 if denatured. [10][11][12] Here we took advantage of a recently described anti-C5 therapeutic mAb, RO7112689, that was selected to recycle in vivo by shedding its cargo when exposed to acidic pH (~pH5Á5) in the endosome. 13 Such antibodies have been engineered to reduce therapeutic dose; 16,17 pH-dependence to release soluble antigen in the endosome is an essential property.…”

“…A particular problem was the propensity of C5 to bind C6 when denatured by precipitation or pH shift during the early stages of purification, resulting in low yields of active protein . Immunoaffinity methods using specific antibodies, commonplace for plasma proteins, are reported for C5; however, elution at extremes of pH significantly compromises C5 activity, in our hands resulting in > 50% activity loss despite rapid neutralization on elution.…”

Complement activation in polycystic ovary syndrome occurs in the postprandial and fasted state and is influenced by obesity and insulin sensitivity

“…C5a(desArg), C3a(desArg), factor D and properdin were quantified using their respective commercial assays from Hycult Biotech, as instructed by the manufacturer. Plasma C5, TCC and factor H were all measured using in‐house ELISA 24‐26 . All assays used purified protein (either C5, TCC or factor H) as a standard.…”

Genetic profiling of ocular inflammation: further evidence for a gender-specific association of C5 with uveitis

“…Meanwhile, study by Chai L et al, also reported that rs17611 was associated with periodontitis [30]. Furthermore, functional analysis on rs17611 showed that individuals homozygously expressing the risk allele G exhibit increased C5a and decreased C5 in plasma, which potentially explain the genetic association of C5 rs17611 with uveitis [31].…”