Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface–selective regulation of complement activation

Abstract

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1–20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris. We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES. PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES. Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.

An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy

“…Next, we evaluated the homodimeric mini-FH constructs in hemolysis protection assays using human C3b-coated sheep erythrocytes, which also bear human-like sialic acid. 54 In agreement with previous data, 43 FH 1-5^18-20 demonstrated marginally, but consistently, more potent decay accelerating and CFA i.e., approximately two-fold when compared with FH ( Figure 4, A and B, Supplemental Table 2). Strikingly, when compared with FH, FH 1-5^18-20^R1-2 demonstrated 33-and 64-fold increases in decay accelerating and CFA on the basis of mean IC 50 , respectively (Supplemental Table 2), whereas FH R1-2^1-5^18-20 demonstrated approximately 20-fold increases in both decay accelerating and CFA, respectively (Supplemental Table 2).…”

“…Interestingly, similar to FH 1-5^18-20 , 40,43 the homodimeric constructs also readily bound iC3b and C3d, suggesting these molecules are in an "open" conformation as opposed to the "foldedback" or closed conformation of FH. 14,54,62 This may be of great value. The homing of a therapeutic to the site of chronic complement activation may rely on the binding to a surface which has a high quantity of accumulated iC3b and C3d(g).…”

“…11,51,70 Under less flexible conditions, we can speculate that there remains potential for the two "arms" or component parts of the homodimeric mini-FH constructs to alternately anchor the molecule to the target surface in a homing fashion, allowing the "free-arm" to "patrol" an area adjacent to the C3b/GAGs that the first "arm" binds as well as rapidly recycling to other C3b molecules; this ability would be enhanced or restricted as a factor of the overall binding strength to the surface. An examination of "model patient" sera again highlights how finely balanced this is, i.e., FH 1-5^18-20^R1-2 is less effective than FH R1-2^1-5^18-20 in counteracting the ability of anti-FH antibody and "hybrid protein" 13,54 to deregulate alternative pathway activation. This implies that OX-24 binding to SCR 5 in FH R1-2^1-5^18-20 was obstructed, potentially, due to the proximity of the dimerization domain.…”

“…10,47,53 Under these conditions, both homodimeric mini-FH constructs displayed avid binding kinetics as demonstrated by a prolonged dissociation phase ( Figure 3A, Supplemental Table 1). Indeed, the very tight binding of the homodimeric mini-FH constructs meant we needed to use 40-fold less homodimeric mini-FH (at maximal concentration) than that commonly used in FH kinetic studies (normally start at 10 mM, 40,45,54 ), hence the low RU values noted for FH. Additionally, and unfortunately, the heterogeneous binding profiles of the homodimeric mini-FH constructs prevent accurate K D determination and traditional kinetic analysis.…”

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

“…A hemolytic assay was modified from a previously published procedure [65] to measure Sbi induced consumption of C3. Briefly, rabbit erythrocytes (TCS Bioscience) were resuspended in GVB buffer (5 mM veronal, 145 mM NaCl, 10 μM EDTA, 0.1 % (w/v) gelatin) by washing three times via centrifugation at 600 g for 6 mins.…”