The life-threatening kidney disease aHUS has been discussed extensively with respect to mutations that prevent the C terminus of complement FH from mediating selective regulatory action on self (rather than foreign) surfaces. We set out to discover whether a causal link could also be established between aHUS and mutations in the N-terminal module of FH, which is not involved in binding directly to surfaces.
It was important to first consider the widespread V62I polymorphism of FH. Ile62
has been reported to be protective (versus
) for aHUS (46
) as well as AMD (4
) and DDD (4
), an effect ascribed to its superior cofactor activity (51
). Indeed our results, obtained using FH1–4V62
, agree with previous findings showing that twice the concentration of full-length FHV62
, relative to full-length FHI62
, was needed to achieve 50% inactivation of C3b by FI in fluid-phase and on cell surfaces (51
). These authors also found that FHI62
binds slightly more tightly to C3b (KD
= 1.0 μm
) than does FHV62
= 1.3 μm
), consistent with higher cofactor activity. We, however, observed no difference in C3b binding between FH1–4V62
. Thus, the effect of I62V on C3b binding by FH is not exaggerated in the FH1–4 setting despite FH1–4 having only one C3b-binding site whereas full-length FH contains an additional, stronger, C3b-binding site.
We additionally showed that both FH1–4V62
variants accelerate decay of the C3bBb complex with equivalent activity. This also agrees with data from Tortajada et al.
), demonstrating similar relative decay accelerating activities for both wild-type variants of full-length FH. In summary, the new data establish that the CFH1–4I62
variants function efficiently in complement regulation with the CFH1–4I62
variant showing slightly better cofactor activity. Subtle differences between the two versions are consistent with their minor structural differences (56
) and their relatively weak associations with disease propensity at least until old age. These studies provided a useful base line for functional analyses of the mutants.
The poor affinity we observed between (R78G)FH1–4V62
and C3b agrees with inferences based on a co-crystal structure of C3b and CFH1–4 (57
) in which Arg78
in the N-terminal CCP forms an H-bonded interaction with Glu732
in the α′-N-terminal domain of C3b (c
). In vivo
, loss of affinity of the (R78G)FH N terminus for C3b could impair its ability to control AP convertases both in fluid phase and on cell surfaces. Therefore, the complement-regulating function of (R78G)FH1–4V62
was assessed and found to be very poor both in terms of cofactor and decay-accelerating activities. Taken together, these data support a disease mechanism in which (R78G)FH is functionally compromised by poor affinity for C3b, potentially resulting in severely impaired complement regulatory function and explaining low C3 levels in this patient (50
). The physiological significance and consequences of these observations depend on the levels of the various proteins and whether or not they are locally present in sub-saturating concentrations. Very often an aHUS patient with a disease-linked CFH
gene also has a normal version of the CFH
gene; haploinsufficiency, caused by missense or nonsense mutations in just one allele, has previously been associated with aHUS in many individuals (14
), illustrating the delicate balance between complement activation and regulation required for maintenance of homeostasis and health.
The unperturbed C3b-binding ability of a second aHUS-linked mutant, (R53H)FH1–4V62
, is consistent with the structural integrity of this mutant as reported previously on the basis of an NMR study (56
), and with the location of Arg53
, within the crystallized C3b·CFH1–4 complex, on an exposed face of FH rather than adjacent to C3b (c
). Hence, the diminished cofactor activity of (R53H)FH1–4V62
cannot be explained by the lower affinity for C3b as was the case with the R78G mutant. An alternative explanation is that Arg53
lies within a putative interaction site for FI, consistent with a previous suggestion that FI binds to CCPs 1–3 within FH as well as to the C345C domain of C3b (57
). The decay accelerating activity of (R53H)FH1–4V62
is also very poor. Given the wild-type-like affinity of (R53H)FH1–4V62
for C3b, these data suggest that impaired ability to accelerate decay of the convertase components may be due to weakened association with Bb. Thus, the side chain of Arg53
may participate in interactions with both Bb and FI, implying overlap between binding sites for these ligands within CCP module 1 of FH. The functionally deficient (R53H)FH mutant would likely compete with wild-type FH for binding C3b in heterozygous individuals and could thereby further diminish complement regulatory capacity.
In summary, both N-terminal FH mutants examined in the current study are defective in their ability to control the AP C3 convertase in the fluid phase and on cell surfaces, but the molecular mechanisms underlying the dysregulation caused by these mutant proteins are distinct. Thus, despite differences in their C3b-binding properties, both mutations have similar outcomes with respect to complement regulation and are associated with similar disease symptoms. It follows that a causal link between mutations and disease is the simplest explanation of these data. Previous functional analyses of C-terminal FH mutants have suggested that multiple mechanisms are responsible for defective cell surface regulation in aHUS (59
); these include decreased binding to C3b/C3d and/or to glycosaminoglycans and altered oligomerization of FH on the surface. Thus, the current observations greatly strengthen the hypothesis that a wide range of defects in FH activity can contribute to a similar disease phenotype.