Abstract
Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a Kd of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a Kd for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a Kd of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a Kd of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a Kd estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a Kd of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 (Kd 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.
Highlights
Complement Factor H plays an essential role in homeostasis of the complement system
Activation of the complement alternative pathway is spontaneous and continuous. It must be inhibited or severe tissue damage will result as illustrated in diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and membranoproliferative glomerulonephritis (MPGN)
Factor H is the primary regulator of this system and its interactions with host polyanions and with C3b and the breakdown products of C3b are key interactions controlling alternative pathway activation
Summary
Complement Factor H plays an essential role in homeostasis of the complement system. It controls the spontaneous fluid phase activation of the complement alternative pathway and it prevents activation of this pathway on host surfaces [1] through specific interactions with host markers [1,2,3,4,5,6]. Control of complement activation on host tissues involves interactions of Factor H with surface-bound C3b, iC3b, C3d, with polyanionic host markers [4, 6, 8] and with malondialdehyde (MDA)-modified lipids or proteins [9]. Agerelated macular degeneration (ARMD) has been strongly linked to a single amino acid allotype in domain 7 (Y402H in CCP 7) [10] This region has been shown to bind polyanions, CRP, and MDA and this allele affects these interactions [1]. The CCP 19-20 region of Factor H contains binding sites for C3b, iC3b, C3d, MDA, and for polyanionic markers prevalent on human cells and tissues [4, 5, 8, 13,14,15]. Evidence is presented here localizing both of these binding sites
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