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.
Highlights
The R53H-CFH/LA-CFH mixture was less able to prevent ES hemolysis than R53H-CFH alone (Fig. 4E). This is consistent with non-depletion of C3, an expected consequence of the fluid-phase regulatory action of LA-CFH
Preincubation of LA-CFH with PspCN in a 2-fold molar excess produced little if any gain in affinity for C3b (KD ϭ 0.70 M versus 0.83 M and KD ϭ 0.60 M versus 0.86 M, respectively; Table 1). No effect of these mutations on the DAA of full-length CFH, assayed on the SPR chip surface, could be detected (Fig. 8B). These results suggest that the N-terminal region of CFH dominates its properties on the foreign surface of a SPR chip such that C-terminal mutations have undetectable effects
We previously reported that full-length D1119G-CFH, despite having wild type–like C3b-binding affinity and DAA activity on a foreign SPR chip surface, did not protect ES from hemolysis [50]
Summary
We produced the two common allotypic CFH variants with Ile or Val at position 62 of the mature protein (I62-CFH or V62-CFH, respectively) in recombinant P. pastoris. We compared binding of these three versions of CFH to C3d (Complement Technology) amine-coupled to a different flow cell on the same C1-sensor chip as used for the C3b-binding experiments (Fig. 2, A and C) Both I62-CFH and V62-CFH bound to immobilized C3d with KD values of ϳ1.9 M (Table 1). Very similar PspCN-induced enhancements of both plasma CFH and recombinant CFH binding to C3b and C3d were observed in multiple experiments; KD values were estimated for I62-CFH alone versus I62-CFH–PspCN complex (see Table 1). A 2-fold ratio of PspCN to R53-CFH did improve its CA on ES (Fig. 6F and Table 2) These functional deficiencies of R53H-CFH were not immediately evident in hemolysis protection assays. CFH, with or without PspCN, did not protect foreign ER (Fig. 4D), it was able to protect the more “self-like” ES (Fig. 4C)
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