If a mutation were to impair the ability of fH to regulate complement at self-surfaces it would have particularly significant consequences for tissue and cell surfaces thought to rely on fH for protection from complement-mediated damage. An example is the glomerular basement membrane that lacks membrane-bound complement regulators, but is exposed to plasma (and therefore the alternative pathway of complement), by fenestrations between the endothelial cells of the glomerular microvasculature. Thus, fH mutations clustered within the surface-anchoring C-terminal region, which have been detected in a significant proportion of aHUS patients, may be expected to directly trigger or exacerbate pathogenesis. Understanding the precise properties of disease-linked mutants of fH will therefore help to clarify the molecular basis of aHUS. For example, disease-associated mutations in the C-terminal region may disrupt its three-dimensional protein structure. Alternatively, they could interfere with the recognition site in CCP 20 of fH for polyanionic self markers, or they may perturb the C-terminal binding site for surface-bound C3b, or they could affect both polyanion and C3b-binding sites simultaneously. In the present study, we sought to distinguish between these various possibilities.
Our NMR analyses show that none of the ten disease-linked mutations in the current study result in anything other than highly localized effects on the structures of CCPs 19 or 20. Consequently, any functional effects of these mutations may reasonably be ascribed to their location within, or near to, critical binding sites on the wildtype protein. C3b- and heparin-binding data collected on these and five further, structurally intact, “designer” mutations in the context of rH19-20 allow the most comprehensive delineation of binding sites for these ligands to date. This exercise (summarized in Figs. and ) suggests that positively charged binding sites for heparin and C3b overlap on one face of rH19-20. The binding surface for heparin (and by extension, for other glycosaminoglycans) appears to extend towards the C-terminus of fH while the C3b-binding surface extends into module 19. Changes in the electrostatic nature of the surface had the largest influences (gain of positive charge or loss of negative charge appeared to correlate with increases of both heparin and C3b-binding), thus it is possible that some of the residues concerned are involved in electrostatic steering effects rather than direct contacts with ligands. These findings are in partial agreement with previous reports (40
) but do not support a suggestion of simultaneously occupied binding sites on opposite faces of CCP 20 (41
Functional analyses of some aHUS-associated mutants reveal effects consistent with an intuitively feasible model of aHUS pathogenesis; i.e.
their reduced ability to bind to a polyanion-rich C3b-coated host-like surface correlates with their diminished performance in assays that measure directly affinity for C3b or for heparin. For example, although D1119G (involving loss of a negative charge) is unaffected in its ability to bind heparin, it has significantly diminished affinity for C3b (Fig. -) and this mutant was also the weakest of all the mutants in the cell surface assays. Moreover, analysis of some of the “designer” mutants (i.e.
those engineered for this study to help delineate C3b- and heparin- binding sites, but not identified in any aHUS patients to date) also produced results that appear to link an affinity for C3b and/or heparin to a capacity for blocking fH binding to a C3b-coated erythrocyte surface. Thus, the designer mutant R1210S binds C3b with wildtype affinity and is only slightly diminished in affinity for heparin, while maintaining near-wildtype activity in the cell surface assay. The case of the aHUS-linked R1210C is complicated by the likelihood that it forms disulfide bonds with itself or other proteins (54
) (recombinant R1210C was found to be glutathionylated in our study). In all, seven mutations (aHUS-linked mutants R1215G and R1182S, and designer mutants K1188Q, R1203S, R1210S, K1230A) display both weaker heparin and C3b binding (Figs. -) and, as expected, their performance is significantly impaired in the cell-surface assays (). It should be noted that the reduced heparin binding obtained with R1182S contrasts with the unaffected heparin binding previously observed with a mutant, R1182A, which has not been found in aHUS patients (40
Indeed all the aHUS-associated mutants exhibit a weaker than wildtype ability to compete with fH for erythrocyte surface binding (). For example, L1189R and W1183R perform very poorly in cell-surface assays (Fig. -), while T1184R and R1210C are the only aHUS-mutants that are nearly as good at inhibiting the protective effects of fH as the wildtype rH19-20. What is striking is that several of these mutations have a higher affinity both for heparin (Figs. and ) and for C3b (Fig. -). Such is the case for aHUS mutants W1183R, T1184R, L1189R, and the designer mutation E1172R, all of which involve an introduction of one or more positive charges. Note that in the case of the W1183R mutant, the observed increased affinity for C3b and heparin differed from what had been observed previously with another aHUS-linked mutant, W1183L (40
) that had reduced binding to C3b and heparin; unlike W1183L (40
), the W1183R mutant ran as a single band by SDS-PAGE (not shown). The L1189F mutation also shows increased C3b- and heparin-binding, albeit to a much lesser extent than L1189R (Figs. -). Another mutant (S1191L) with weaker than wildtype activity in the cell surface assays (Figs. -), has enhanced affinity for C3b (), contrary to previously reported results (54
). This same mutant had no effect on heparin binding (), in agreement with a previous study (55
). Interestingly, the presence of V1197A in the S1191L;V1197A double mutant has no additional effect in any of the assays tested versus
the S1191L mutant.
An overall summary of the heparin and C3b binding results (where a wide range of affinities for C3b and heparin were observed), and the cell-surface complement functional assays (where all aHUS-linked mutants were impaired), is illustrated in . Taken together these results unexpectedly show that, measured separately, affinities for C3b and heparin do not correlate consistently with the strength of binding to a polyanion-rich erythrocyte surface (on which C3b has been deposited); nor do they correlate with the disease-risk phenotype.
In the case of heparin it is conceivable that this material is not in fact representative of a putative host tissue-specific polyanionic ligand. The presence in such a ligand of particular patterns or densities of sulfation, for example, might result in differential binding amongst the mutants that correlates better with cell-surface assays. Studies aimed at identifying physiological polyanion species, as well as elucidating their chemical nature, are warranted.
The lack of correlation between the separate ligand binding assays and the cell based assays also suggests that a mutation may affect a third functional site in CCPs 19-20, perhaps different from those involved in C3b or polyanion binding. For example, a mutation could affect the region involved in the formation of putative dimers or tetramers (40
), which may be important for fH binding/function on the cell surface. In this regard, we have recently determined that polyanions greatly promote formation of fH dimers and tetramers, and that this dimerization/tetramerization leads to an increase in fH binding and complement control activity at the cell surface (58
). In addition, we have determined that this polyanion-induced self-assembly is mediated, at least in part, by C-terminus to C-terminus complex formation, specifically through CCP 18-20 (58
A further possibility derives from consideration of the relative affinities of fH for each ligand in the setting of the cell surface, and the order of binding events. An increased affinity between fH and polyanions, due to CCP 19-20 mutations, may result in a decrease in the ability of fH to diffuse normally over the cell surface, impairing its ability to efficiently encounter and bind C3b. Thus, such mutants would be less effective than normal CCP 19-20 that binds with the correct affinity to the combination of both ligands (polyanions and C3b/C3d). Likewise, the mutants that were found to have increased affinity for C3b may get retained/delayed in complexes with the products of decay acceleration and cofactor activity (i.e. C3b/iC3b).
Thus, we propose that aHUS-linked mutations disturb a complex relationship between affinities of fH for its individual ligands (C3b and physiological polyanions) or possibly for itself, in the context of the cell surface.