Insights into the effects of complement factor H on the assembly and decay of the alternative pathway C3 proconvertase and C3 convertase.

The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies ( Conrad, D. H., Carlo, J. R., and S. (1978) J. Exp. Med. 147, 1792–1805; K., J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2416–2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979) J. Immunol. 122, 75–81) indicated that the comple-mentalternativepathwayregulatorfactorH(FH)competeswith factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. More-over, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western

teolytic activation by the plasma serine protease factor D (FD), generating the Ba and Bb fragments. Ba dissociates from the complex, whereas the Bb fragment remains bound to C3b, generating the active C3 convertase C3bBb. Properdin (P) binding to C3 convertase stabilizes this otherwise labile complex (17,18).
To prevent complement activation on host tissues, AP C3 convertase activity is tightly controlled by the set of fluid phase and membrane-bound regulators (5,6,19), of which complement factor H (FH) is a prominent member (20 -22). Factor H is an abundant plasma glycoprotein that not only modulates fluid-phase complement but also has the ability to inhibit C3b amplification selectively on self-surfaces.
Very old studies (23)(24)(25) proposed that FH could prevent C3bB C3 proconvertase assembly by competing with FB for binding to C3b. In these experiments, binding assays performed on C3b-coated sheep erythrocytes showed that FB both displaces bound FH from the cell and inhibits the equilibrium binding of FH to C3b. Competition between FH and FB for C3b binding resulting in an inhibitory effect on C3 proconvertase assembly was supported by more recent surface plasmon resonance studies (26). The authors found that when FB, together with increasing amounts of FH, was flowed over a C3b-coated surface, C3bB C3 proconvertase formation decreased in an FH concentration-dependent manner. Notably, because following FB and FH injections the net mass change was the sum of both C3 proconvertase formation and FH binding to the C3b-coated surface, it was actually difficult to dissect between the binding and the inhibitory effects of FH on C3bB assembly. Tortajada et al. (26), in keeping with a previous report (27), found that FH does not accelerate decay of the pre-formed C3 proconvertase.
Whether FH affects the formation of C3bBb from C3bB has not been investigated yet. However, the role of FH in enhancing the dissociation of already formed C3bBb (decay-acceleration activity) is well documented (28 -30). Investigations into the formation and dissociation of the C3bB and the C3bBb are hampered by the instability of the two complexes and the paucity of sensitive and specific detection assays.
To overcome these shortcomings, in this study we set up user-friendly assays based on combined microplate and Western blotting techniques, which specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on the following: 1) the assembly and decay of the AP C3 proconvertase C3bB, and 2) the formation and decay of the AP C3 convertase C3bBb.
Selective C3bB C3 Proconvertase and C3bBb C3 Convertase Formation Assays-We set up a novel user-friendly method based on combined microplate and Western blotting (WB) techniques to selectively generate and detect C3bB and C3bBb complexes. ELISA plates were coated with C3b, blocked as above, and then washed with wash buffer supplemented with 2 mM MnCl 2 (33) or 10 mM MgCl 2 (34) for C3bB or C3bBb, respectively. C3bB(Mn 2ϩ ) complexes were assembled by incubating C3b-coated wells at 37°C for 1, 2, 4, and 8 h with FB (1000 ng/ml), diluted in assay buffer containing 2 mM MnCl 2 (Fig. 1A). C3bBb(Mg 2ϩ ) complexes were formed by incubating C3b-coated wells at 25°C for 5, 10, 30, and 45 min with FB (1000 ng/ml) and FD (5 ng/ml), in assay buffer with 10 mM MgCl 2 (Fig. 1B). After washes, the complexes were detached from microtiter wells by incubation with 10 mM EDTA and 1% SDS for 1 h at room temperature, subjected to 10% SDS-PAGE, and transferred by electroblotting to a PVDF membrane. Proteins were detected with polyclonal rabbit anti-human FB antibody (1:500) followed by HRP-conjugated anti-rabbit antibody (1:30,000) and the ECL chemiluminescence detection system. C3bB and C3bBb formation was evaluated by the visualization by WB of the B band (93 kDa) and the Bb band (60 kDa), respectively. The intensity of the band detected by WB was estimated by densitometry using ImageJ (National Institutes of Health).
In preliminary experiments, the binding efficiency of HS to the plate was verified by using an ELISA. Microtiter plates were coated with 3 or 30 g/ml HS in PBS, in the presence or in the absence of 3 g/ml C3b, by overnight incubation at 4°C, blocked with 1% BSA, 0.1% Tween 20 in PBS for 1 h at 37°C, and washed with 0.1% Tween 20 in PBS. HS immobilized on the surface were detected with a monoclonal mouse anti-HS antibody (1:100) followed by HRP-conjugated goat anti-mouse antibody (1:2000), both diluted in PBS. Color was developed using 3,3Ј,5,5Ј-tetramethylbenzidine substrate and stopped with 2 M H 2 SO 4 , and absorbance was measured at 450 nm. Each reaction was performed in duplicate, and the OD values were averaged.
Selective C3bB C3 Proconvertase and C3bBb C3 Convertase Decay Assays-To study spontaneous or FH-mediated decay of C3 proconvertase and C3 convertase over time, C3bB(Mn 2ϩ ) and C3bBb(Mg 2ϩ ) were allowed to form for 2 h at 37°C and 10 min at 25°C, respectively, then were washed and incubated with selective assay buffers in the presence or in the absence of FH (2640 ng/ml; molar ratio FB/FH ϭ 1:1.6) for the following time periods: C3bB(Mn 2ϩ ), 30, 60, 120, and 240 min; C3bBb(Mg 2ϩ ), 2, 4, 8, and 16 min) (Fig. 1, C and D). For C3bB(Ni 2ϩ ) and C3bBb(Ni 2ϩ ) formed together during a 30-min period at 37°C, both spontaneous and FH-mediated decay were monitored by incubating the complexes at 37°C with assay buffer alone or with FH (2640 ng/ml) for 5, 30, 60, and 120 min (Fig. 1I). Following washes, the remaining complexes were detached from microtiter wells with 10 mM EDTA and 1% SDS and subjected to WB analyses as described above. The amount of C3bB and C3bBb formed before decay was also evaluated as baseline. The percentage of residual Bb band was calculated as the ratio of the densities (in pixel 2 ) of the Bb bands after decay and the corresponding baseline Bb band density before decay ϫ 100.
C3bBb(Mg 2ϩ ) C3 Convertase Formation and Decay in the Presence of Properdin-To evaluate the ability of P to stabilize C3bBb(Mg 2ϩ ) formation and decay, the complexes were generated by incubating C3b-coated wells with FB (1000 ng/ml), FD (5 ng/ml), and P (114.5 ng/ml; molar ratio FB/P ϭ 5:1) (35) in the presence or in the absence of FH (2640 ng/ml; molar ratio FB/FH ϭ 1:1.6) for 10 min at 25°C (Fig. 1E). Following washes, spontaneous or FH-mediated decay was performed for a further 10 min at 25°C with assay buffer alone or FH (2640 ng/ml). The remaining complexes were detached from microtiter wells with 10 mM EDTA and 1% SDS and subjected to WB analyses as described above.
C3NeF-IgG Purification-Human plasma was obtained from three healthy volunteers and 11 patients presenting histological and clinical evidence of C3 glomerulopathies (C3G), and the IgG fraction was purified as described (36).
C3NeF-IgG hemolytic activity was quantified by determining the ability of the IgG fraction purified from plasma to stabilize the cell-bound C3bBb convertase in the hemolytic assay (HA) (36). Under these experimental conditions, the residual convertase activity (expressed in %) in control experiments performed in the presence of normal IgG (400 g) was below 20%. The presence of C3NeF activity in the assay resulted in increased residual convertase activity. The study was approved by the Bioethical Committee of the Azienda Sanitaria Locale in Bergamo (Number 00148858/III). Informed consent was obtained from patients and controls.
C3NeF-stabilized C3bBb(Mg 2ϩ ) C3 Convertase Formation and Decay-To evaluate the ability of IgG fractions to stabilize C3bBb(Mg 2ϩ ) complexes both in spontaneous or FH-mediated decay, C3NeF-stabilized C3bBb(Mg 2ϩ ) complexes were generated by incubating C3b-coated wells for 10 min at 25°C with purified IgG (50 and 100 g/ml), immediately followed by FB (1000 ng/ml) and FD (5 ng/ml) diluted in assay buffer with 10 mM MgCl 2 . The complexes formed were detached from wells and detected by WB as above. The amount of C3 convertase formation before decay was evaluated as baseline. Spontaneous or FH-mediated decay of the complexes was monitored by further incubation for 10 min at 25°C with buffer alone or FH (2640 ng/ml), respectively. Following washes, the remaining complexes were detached from microtiter wells and quantified as above (Fig. 1F). The % of residual Bb band was quantified as the ratio of the densities (in pixel 2 ) of Bb bands after decay and the corresponding baseline Bb band density before decay ϫ 100.
To evaluate the effect of FH on C3NeF-stabilized C3 convertase formation, C3bBb(Mg 2ϩ ) was generated in the presence of C3NeF-IgG or control IgG (50 or 100 g/ml) and P (114.5 ng/ml; molar ratio FB/P ϭ 5:1) for 10 min at 25°C with assay buffer alone or FH (2640 ng/ml; molar ratio FB/FH ϭ 1:1.6). Spontaneous or FH-mediated decay was performed for a further 10 min at 25°C, and % of residual Bb band was evaluated as described above (Fig. 1G).
Quantification of Complement Fragments Bb and Ba-Bb and Ba fragment levels were measured in the supernatant of the reactions of C3bB(Mn 2ϩ ) and C3bBb(Mn 2ϩ ) complexes in the presence or in the absence of FH (Fig. 1A), by commercial ELISA kit following the protocol procedure (Quidel, San Diego).
C3bBb(Mg 2ϩ ) C3 Convertase and C5b Formation in the Presence of Normal Human Serum-To obtain C3 convertase formation by using a physiological mixture of complement components, microtiter wells were coated with 3 g/ml C3b in PBS by overnight incubation at 4°C, blocked with 0.5% BSA in PBS for 1 h at 25°C, and washed with wash buffer supplemented with 5 mM MgCl 2 . C3bBb(Mg 2ϩ ) complexes were formed by incubating C3b-coated wells at 37°C for 30 min with 20% NHS diluted in PBS containing 5 mM MgCl 2 in the presence or in the absence of 1 mM EGTA (Fig. 1J). After washes, formed C3bBb(Mg 2ϩ ) complexes and bound FH were detached from wells and detected by WB as described previously. In addition, C5b formation was evaluated by WB using polyclonal rabbit anti-human C5/C5b (1:300) antibody, followed by HRP-conjugated anti-rabbit antibody (1:30,000). C5b was visualized as ␣Ј-chain C5b (104 kDa) band.

Results
ELISA with Ni 2ϩ Stabilization Does Not Specifically Discriminate between C3bB C3 Proconvertase and C3bBb C3 Convertase Formation-To study the in vitro assembly of the AP C3bB C3 proconvertase and C3bBb C3 convertase, we first tried the ELISA previously reported by Hourcade et al. (31,32). Several studies have documented that Ni 2ϩ cation prolongs the half-life of both C3bB and C3bBb (27,32,37), and for this reason Ni 2ϩ has been widely used, instead of the physiological Mg 2ϩ , to generate complexes stable enough to allow satisfactory measurements by ELISA or to perform structural studies. In a typical assay, increasing amounts of FB or FB plus FD are added to C3b-coated wells in the presence of Ni 2ϩ to generate C3bB(Ni 2ϩ ) or C3bBb(Ni 2ϩ ), respectively, and the products are detected using an anti-FB antibody. As shown in Fig. 2A, the ELISA curves from the reaction of coated C3b with either FB alone or FB plus FD showed dose-dependent superimposable profiles. However, when the complexes that formed in the presence or in the absence of FD were detached from the wells and analyzed by WB, both B (93 kDa) and Bb (60 kDa) bands were found in both conditions ( Fig. 2A, right side) indicating that the ELISA cannot selectively discriminate between C3bB and C3bBb formation. Indeed, in the presence of FD only a portion of C3bB was converted to C3bBb. Conversely, in the absence of FD, some C3bBb also formed besides C3bB, possibly due to FD contamination in commercial FB plasma-purified protein (27,38).
New Assays to Specifically Detect C3bB(Mn 2ϩ ) C3 Proconvertase and C3bBb(Mg 2ϩ ) C3 Convertase Formation-In an effort to specifically generate either C3bB or C3bBb complexes, we exploited the selective stabilization ability of different divalent cations. Hourcade and Mitchell (33) documented that Mn 2ϩ stabilizes C3bB in a form susceptible to FD cleavage, but the C3bBb(Mn 2ϩ ), once formed, is highly unstable and dissociates immediately, so that only the C3bB(Mn 2ϩ ) complex could be detected. At variance, Mg 2ϩ stabilizes C3bBb but not the proenzyme C3bB (34).
On the basis of this evidence, the ELISA was repeated replacing Ni 2ϩ with Mn 2ϩ or Mg 2ϩ ions to stabilize either the C3bB or the C3bBb, respectively. For C3bB assembly, C3b-coated wells were incubated with FB at 37°C for 2 h in the presence of 2 mM MnCl 2 , whereas C3bBb formation was obtained by incubating C3b-coated wells with FB and FD at 25°C for 30 min in the presence of 10 mM MgCl 2 ( Fig. 1, A and B), based on published data (33,34). As shown in Fig. 2, B and C, in either condition the amount of the complexes formed was too small to be detected by ELISA (left panels), likely due to degradation of the C3bB(Mn 2ϩ ) and the C3bBb(Mg 2ϩ ) during post-reaction incubation times with primary and secondary antibodies.
At variance, when we detached the complexes immediately at the end of the reaction, and analyzed them on WB with an anti-FB antibody, the B and Bb bands of C3bB(Mn 2ϩ ) and C3bBb(Mg 2ϩ ), respectively, could be visualized well (Fig. 2, B and C, right panels).
It is relevant that only the B band of C3bB was visualized in the product of the reaction with Mn 2ϩ , even when FD was added to the incubation mixture (Fig. 2B, right panel). Similarly, only the Bb band of C3bBb was detected in the reaction with Mg 2ϩ and FD (Fig. 2C, right panel). Neither B nor Bb bands were observed in the reaction with Mg 2ϩ in the absence of FD.
We then analyzed the time course of C3bB(Mn 2ϩ ) and C3bBb(Mg 2ϩ ) formation. As shown in Fig. 3A, the B band of C3bB(Mn 2ϩ ) was clearly detected after 2 h of incubation and was still evident at 8 h independently of the presence of FD, further confirming the specificity of the test for C3bB assembly.
The kinetics of C3bBb(Mg 2ϩ ) (Fig. 3B) formation was faster than that of C3bB(Mn 2ϩ ), and indeed the Bb band was already well detected after 10 min of incubation and the intensity further increased at 45 min of incubation. For the subsequent experiments, we used the new combined microplate/WB assays for specifically studying C3bB(Mn 2ϩ ) and C3bBb(Mg 2ϩ ) complexes.
Effect of FH on C3bB(Mn 2ϩ ) C3 Proconvertase Assembly and Decay-To investigate whether FH prevents C3bB assembly by competing with FB binding to C3b (23-25), C3b-coated wells were incubated with FB in the presence or absence of FH added at a physiological molar ratio with FB (Fig. 1A). FH did not affect C3bB(Mn 2ϩ ) assembly after 1, 2, 4, and 8 h of incubation, as documented by no change in densitometry of the B bands on WB, compared with values of the reaction without FH (Fig. 4A).
As reported in Fig. 4A, top, FH band could already be visualized in the WB of the products detached from the wells after 1 h of incubation, before C3 proconvertase formation, and FH band increased at 2 and 4 h in parallel with C3bB(Mn 2ϩ ) assembly. These results would indicate that FH binds both C3b and C3bB.
To evaluate whether binding of FH to C3bB affects C3 proconvertase dissociation, C3bB(Mn 2ϩ ) was allowed to form over 2 h, and the complexes were subsequently incubated for different time intervals with buffer alone or with buffer containing FH (Fig. 1C). The intensity of the B bands did not change over 240 min decay versus baseline, either in the absence or in the presence of FH, indicating that in our conditions FH did not displace FB from C3b (Fig. 4B). Altogether the above results reveal that FH, at the physiological FH/FB molar ratio, did not affect either C3bB proconvertase assembly or its decay.
Effect of FH on C3bBb(Mg 2ϩ ) C3 Convertase Formation and Decay-We then wondered whether the capability of FH to bind C3bB results in inhibition of C3bBb convertase formation. Thus, we investigated the effect of FH on C3bBb(Mg 2ϩ ) formation by incubating C3b-coated wells with FB and FD in the FIGURE 2. Detection of C3bB C3 proconvertase and C3bBb C3 convertase complex formation by ELISA and microplate/WB assays. A, detection of Ni 2ϩ -dependent C3bB and C3bBb. C3b-coated microtiter wells were incubated with increasing amounts of FB (0 -500 ng/ml) and 2 mM NiCl 2 in the absence or in the presence of 25 ng/ml FD and analyzed by ELISA by using an anti-FB antibody (left side). The product of the reaction with 500 ng/ml FB, once detached from the wells, was also analyzed by WB with an anti-FB antibody (right side). B and C, selective formation of C3bB and C3bBb with Mn 2ϩ and Mg 2ϩ , respectively. C3b-coated wells were incubated with 500 ng/ml FB in the presence or in the absence of 25 ng/ml FD, 2 mM MnCl 2 , or 10 mM MgCl 2 to obtain C3bB(Mn 2ϩ ) and C3bBb(Mg 2ϩ ), respectively. Complexes were detected both by ELISA (left side) and WB (right side). C3bB and C3bBb formation was evaluated by the visualization of the B band (93 kDa) and the Bb band (60 kDa), respectively. One representative experiment of three is shown.
presence or in the absence of FH (Fig. 1B). In the presence of FH, the amount of C3bBb(Mg 2ϩ ) complexes, detected as Bb band density at each time point, was greatly reduced compared with the reaction without FH (Fig. 5A). We also analyzed spontaneous and FH-mediated C3bBb(Mg 2ϩ ) decay. C3bBb(Mg 2ϩ ) was allowed to form for 10 min and then incubated with buffer alone or with buffer containing FH (Fig. 1D). As shown in Fig.  5B, in the absence of FH, C3bBb(Mg 2ϩ ) dissociated in a timedependent manner, but a faint band was still detectable at 16 min. The decay of C3bBb(Mg 2ϩ ) was strongly accelerated in the presence of FH at a physiological molar ratio with FB. Indeed, no Bb band could be detected after 4 min of decay with FH, confirming that FH is very efficient in Bb displacement from C3b. Notably, the effect of FH on C3bBb(Mg 2ϩ ) formation and decay was concentration-dependent (Fig. 6). Because the decay of C3bBb in the presence of FH was very rapid, on the basis of the above results we could not dissect whether the apparent inhibitory effect of FH on C3bBb formation was due  to FH-mediated dissociation of C3bBb molecules as soon as they formed or to FH-mediated blocking of C3bB conversion to C3bBb.

Effect of FH on C3bBb(Mg 2ϩ ) C3 Convertase Formation and
Decay in the Presence of Properdin or C3NeF-IgG-In the attempt to prevent FH-mediated C3bBb decay, the above  experiments were repeated in the presence of P, which enhances the stability of C3bBb (40). P (at a physiological molar ratio with FB) was added together with FB and FD at the beginning of the incubation in C3b-coated wells for 10 min. Thereafter, the C3bBb(Mg 2ϩ ) complexes were allowed to decay for 10 min in the presence or in the absence of FH (Fig. 1E). Results showed that P did not affect either the FH-mediated inhibitory effect on C3bBb(Mg 2ϩ ) formation or the FH-mediated C3bBb(Mg 2ϩ ) decay acceleration (Fig. 7).
As an alternative strategy, we took advantage from patients with C3G who develop C3NeF, circulating IgG autoantibodies targeting C3bBb, which stabilize the complex and inhibit FHmediated decay (41,42). We isolated IgGs from 11 C3G patients and selected two preparations that were strongly positive for C3NeF by hemolytic assay (C3NeF-IgG activity Ն100%). No hemolytic activity was found in the purified IgG sample from a plasma pool of three healthy subjects. C3bBb(Mg 2ϩ ) complexes were formed for 10 min in the presence of two different concentrations (50 and 100 g/ml) of patient or control IgG, and they were then allowed to dissociate for an additional 10 min in the presence or in the absence of FH (Fig. 1F). As shown in Fig. 8, A and B, C3NeF-IgG from patient 5 (Fig. 8A) was the most potent and substantially limited FHmediated C3bBb decay in a dose-dependent fashion, so that in the presence of 100 g/ml C3NeF-IgG a Bb band with 64% intensity compared with baseline (before decay) was recovered at the end of decay with FH, although no Bb band was detected at the end of FH-mediated decay of C3bBb formed in the presence of control IgG. When the above experiments were repeated in the presence of both P and 100 g/ml C3NeF-IgG from patient 5 (Fig. 1G), FH-mediated C3bBb decay was almost completely prevented (81% stabilization of the C3bBb(Mg 2ϩ ) complexes) (Fig. 9). Even though FH-mediated decay was efficiently blocked in this condition, C3bBb(Mg 2ϩ ) formation in the presence of FH was not fully restored; FH, added together with FB and FD to C3b-coated wells, caused a 56% reduction in the amount of C3bBb(Mg 2ϩ ) formed over 10 min in the presence of P and 100 g/ml C3NeF-IgG, compared with the same reaction without FH (Fig. 9). For comparison, in samples with control IgG and P (in which FH-mediated decay was not prevented), FH reduced the amount of C3bBb(Mg 2ϩ ) formed by 97%. These results would suggest that FH may have a direct effect on C3bBb formation from C3bB proconvertase.
Effect of FH on C3bBb C3 Convertase Formation from C3bB C3 Proconvertase-Mn 2ϩ stabilizes C3bB in a form susceptible to FD cleavage, but the C3bBb(Mn 2ϩ ) complex, once formed, is highly unstable and rapidly dissociates (33). Consistently, as shown in Fig. 3A, the time course of C3bB(Mn 2ϩ ) assembly revealed that independently of the presence of FD, no Bb band of C3bBb(Mn 2ϩ ) could be detected. We wondered whether in this condition, in the presence of FD, the formation of C3bBb(Mn 2ϩ ) (Fig. 1A) could be detected indirectly by measuring Bb and Ba fragments released in the supernatant of the reaction.
We found higher Bb and Ba levels in the supernatant of the reaction with FB and FD compared with the same reaction without FD (Bb, 200 -500 ng/ml versus 75-98 ng/ml; Ba, 53-165 ng/ml versus 19 -52 ng/ml) (Fig. 10). These results confirmed that in this condition C3bBb(Mn 2ϩ ) formed and rapidly dissociated. As shown in Fig. 11, although the formation of C3bB(Mn 2ϩ ) was completely unaffected by FH (Fig. 11A), Bb and Ba release in the supernatant in the presence of FH was inhibited compared with the same reaction in the absence of FH (with FH, Bb 88 -109 ng/ml and Ba 30 -51 ng/ml versus without FH: Bb 200 -500 ng/ml and Ba 53-165 ng/ml) (Fig. 11B). Altogether these results support the hypothesis that FH does prevent the conversion of C3bB to C3bBb.
Effect of FH on C3bB(Ni 2ϩ ) C3 Proconvertase and C3bBb(Ni 2ϩ ) C3 Convertase Formation and Decay-To evaluate whether the diverse effect of FH on C3bB(Mn 2ϩ ) compared with C3bBb(Mg 2ϩ ) formation and decay was attributable to the different ions used to stabilize either complex (Mn 2ϩ versus Mg 2ϩ ), we repeated the microplate/WB experiments by incubating surface-bound C3b with FB and FD in the presence of Ni 2ϩ , which stabilized both C3bB and C3bBb and yielded both B and Bb bands on WB (Figs. 1H and 12, A and B). The formation of C3bB and C3bBb complexes peaked at 30 min. To evaluate the kinetics of C3bB and C3bBb formation at shorter time scales, we repeated the experiments using 5, 10, 20, and 30 min of incubation. B and Bb bands were already well detected after 5 min. The intensity of B band remained stable thereafter, whereas Bb band intensity further increased at 10, 20, and 30 min of incubation (Fig. 12B).
As shown in Fig. 12, A and B, C3bB(Ni 2ϩ ) assembly was not affected by FH. Dose-response studies showed that FH decreased C3bB(Ni 2ϩ ) formation only when added at FH/FB molar ratios 3-6-fold higher than physiological levels (Fig.  12C). FH, at physiological levels, strongly reduced the amount C3bBb(Mg 2ϩ ) complexes were generated by incubating at 25°C for 10 min C3b-coated wells with 1000 ng/ml FB, 5 ng/ml FD, and 10 mM MgCl 2 in the presence or in the absence of 114.5 ng/ml P and 2640 ng/ml FH. Spontaneous or FH-mediated decay was evaluated by further incubation at 25°C for 10 min with buffer alone or added with 2640 ng/ml FH. The amount of C3bBb formed was calculated as the intensity of the Bb (60 kDa) band and reported in the bottom graphs as pixel 2 ⅐10 6 . Results of a representative microplate/WB experiment of n ϭ 3 are shown.
of recovered C3bBb(Ni 2ϩ ) at all time points (Fig. 12, A and B), confirming the results obtained in the ion-selective conditions with Mg 2ϩ .
Next, to assess the effect of FH on C3bB(Ni 2ϩ ) and C3bBb(Ni 2ϩ ) decay, the complexes obtained after 30 min at 37°C were incubated with buffer alone or with FH (Fig. 1I). C3bB(Ni 2ϩ ) did not undergo either spontaneous or FH-mediated decay at each time point compared with baseline (Fig. 13). At variance, C3bBb(Ni 2ϩ ) spontaneously dissociated in a time- FIGURE 8. Effect of FH on decay of C3NeF-stabilized C3bBb(Mg 2؉ ) C3 convertase detected by microplate/WB assays. C3bBb(Mg 2ϩ ) complexes were originated by incubating at 25°C for 10 min C3b-coated wells with 1000 ng/ml FB, 5 ng/ml FD, and 10 mM MgCl 2 in the presence or in the absence of C3NeF-IgG (50 and 100 g/ml) purified from two patients with C3G (A, patient 5; B, patient 10) or control IgG (CTR-IgG, 100 g/ml) purified from three healthy volunteers. Spontaneous or FH-mediated decay of the complexes was monitored by further incubation for 10 min at 25°C with buffer alone or added with 2640 ng/ml FH, respectively. The amount of C3bBb formed was calculated as the intensity of the Bb band (60 kDa) and reported in the bottom graphs as pixel 2 ⅐10 6 . The percentage of residual Bb band was calculated as the ratio of the densities (in pixel 2 ) of each Bb band after decay and the corresponding baseline Bb band density before decay ϫ 100. Results of a representative microplate/WB experiment of n ϭ 3 are shown. C3NeF-IgG from patient 5 efficiently stabilized C3bBb against FH-accelerated decay (64% stabilization with 100 g/ml); C3NeF-IgGs from patient 10 were less efficient to prevent FH-accelerated C3bBb decay (35% stabilization with 100 g/ml). . Effect of FH on formation and decay of C3bBb(Mg 2؉ ) C3 convertase stabilized by C3NeF-IgG and P detected by microplate/WB assays. C3bBb(Mg 2ϩ ) complexes were originated by incubating at 25°C for 10 min C3b-coated wells with 1000 ng/ml FB, 5 ng/ml FD, 100 g/ml C3NeF-IgG (C3NeF#5) or control IgG (CTR-IgG) and 10 mM MgCl 2 in the presence of 114.5 ng/ml P and with or without 2640 ng/ml FH. Spontaneous or FH-mediated decay of the complexes was monitored by further incubation for 10 min at 25°C with buffer alone or 2460 ng/ml FH, respectively. The amount of C3bBb was calculated as the intensity of the Bb band (60 kDa) and reported in the bottom graphs as pixel 2 ⅐10 6 . The percentage of residual Bb band was calculated as the ratio of the intensity (in pixel 2 ) of each Bb band after decay and the corresponding baseline Bb band density before decay ϫ 100. Results of a representative microplate/WB experiment of n ϭ 3 are shown. dependent manner and FH accelerated the decay (Fig. 13). Therefore, independently from the ion used, FH had no effect on C3bB, although it reduced C3bBb formation and accelerated its decay.
Effect of FH on C3bB(Ni 2ϩ ) C3 Proconvertase and C3bBb(Ni 2ϩ ) C3 Convertase Formation in the Presence of Heparan Sulfate or Sialic Acid-The binding of FH to C3b has been shown to be favored at the cell and tissue level by FH interaction with polyanions such as glycosaminoglycans on mammalian cell surfaces (39,43). Thus, to study the effect of FH on C3 proconvertase and C3 convertase formation in conditions mimicking FH interaction with cell surfaces, we performed C3bB(Ni 2ϩ ) and C3bBb(Ni 2ϩ ) formation assays using wells coated with mixtures of C3b and HS (3 or 30 g/ml, Fig. 1H). We confirmed HS coating on the microtiter plates by ELISA (Fig. 14A). Also in the presence of HS, FH did not substantially affect C3 proconvertase assembly, although it had a strong inhibitory effect on C3 convertase formation (Fig. 14B).
Because SA glycans, along with glycosaminoglycans, also are thought to act as self-markers (44), we repeated the above experiment using wells coated with SA and C3b (Fig. 1H). As observed for HS, in the presence of two different concentrations (3 or 30 g/ml) of coated SA, FH did not affect C3bB(Ni 2ϩ ) assembly, although it completely prevented C3bBb(Ni 2ϩ ) formation (Fig. 14C).
C3bBb(Mg 2ϩ ) C3 Convertase and C5b Formation in the Presence of Normal Human Serum-A previous study reported that C3 adsorbed to a polystyrene surface can form C3 convertase and mediate the AP of complement activation in the presence of pig serum (45). Thus, we wondered whether immobilized C3b in our assay could originate a C3 convertase functionally active in the presence of human serum. To this purpose, we incubated C3b-coated wells with NHS as source of complement proteins for 30 min at 37°C (Fig. 1J). In this condition, we confirmed C3bBb(Mg 2ϩ ) formation (Bb 60-kDa bands; Fig. 15), which was not affected by EGTA, that inhibits the classical and lectin pathways without affecting the AP (46). Notably, we also found formation of C5b, as clearly visualized by the detection on WB of an ␣Ј-chain (104 kDa) band (Fig. 15A). These results support evidence that the microplate-based method presented here can mimic in vivo assembly and dissociation of a functional C3 convertase, which upon binding to additional C3b molecules forms an active C5 convertase (Fig. 15B).

Discussion
In this study, we set up new user-friendly assays to specifically investigate the in vitro assembly and dissociation of the AP C3 proconvertase, C3bB, and the formation and dissociation of the AP C3 convertase, C3bBb. We then exploited the new methods to investigate the mechanisms underlying the effect of FH on either C3bB or C3bBb.
Traditionally, to evaluate formation and/or decay of the AP C3 convertase, hemolytic assays have been widely used (46,47). These assays, performed on the surface of C3b-coated sheep erythrocytes, make it possible to determine the functional activity of the cell surface-bound C3bBb by measuring the degree of erythrocyte lysis, but they do not provide a direct estimation of C3bBb complex formation and decay. In addition, surface plasmon resonance has largely been used to study C3bB and C3bBb formation and dissociation (33,38,48). Nevertheless, surface plasmon resonance is not universally available, and the method is not the most suitable for exploring the impact of FH on C3bB and C3bBb assembly and decay, because following FH injection the net mass change on the chip is a balance between FH binding to C3b and B/Bb release (26,49). Alternatively, an ELISA has been used, in which purified C3b-coated microplate wells are incubated with FB or FB plus FD to selectively generate C3bB or C3bBb, respectively (27,50). In most studies the ELISA was done in the presence of Ni 2ϩ instead of the physiological Mg 2ϩ , because Ni 2ϩ is well known to prolong the half-life of both C3bB and C3bBb (27,37,51). However, the above method cannot discriminate between C3bB and C3bBb, because WB analysis of the ELISA products showed the formation of both B and Bb bands of the C3bB and C3bBb complexes, independently of the presence of FD in the reaction mixture. The simultaneous detection of C3bB and C3bBb in the presence of FD is consistent with evidence that Ni 2ϩ strongly stabilizes C3bB (37). Moreover, C3bBb formation in the absence of added FD could be attributable to protease contaminants in the commercial plasma-purified FB protein (27,38).
With the aim of specifically generating C3bB and C3bBb complexes, we combined microplate and WB techniques and exploited the selective stabilization properties of Mn 2ϩ and Mg 2ϩ on C3bB and C3bBb (33,34,52), respectively. A previous study (33) documented that Mn 2ϩ stabilizes C3bB in a form susceptible to FD cleavage, but C3bBb(Mn 2ϩ ), once formed, is highly unstable and dissociates immediately. FB binding to C3b depends on elements in fragment Ba and on the metal ion-de-  (53). It has been proposed that Mn 2ϩ induces low affinity conformation of the MIDAS in C3bBb that leads to the rapid dissociation of the C3 convertase complex (33). At variance, Mg 2ϩ promotes the transition of the MIDAS from a low ligand binding affinity in C3bB to a high affinity conformational state in C3bBb, thus accounting for the increase in complex stability that accompanies the transition from C3bB(Mg 2ϩ ) to C3bBb(Mg 2ϩ ) (38,54). By using the new microplate/WB methods with ion-based selective stabilization, we could observe specific formation over time of C3bB(Mn 2ϩ ) or C3bBb(Mg 2ϩ ) in the absence or in the presence of FD, respectively. Thus, the new methods are easy useful tools for investigating how the C3bB and C3bBb are formed and regulated.
Assembly and regulation of the AP C3 convertase C3bBb is strictly modulated, and alterations in C3bBb control lead to tissue damage (4). Consistently, genetic abnormalities of the two components of AP C3 convertase, FB and C3, or of the main AP regulator FH, which result in increased C3 convertase formation and resistance to dissociation, lead to complementmediated severe tissue and organ injury and dysfunction, as observed in patients with aHUS, a rare thrombotic microangiopathy that targets the microvasculature of the kidney and other organs (4 -6, 46), or in patients with C3G (7,8,55). Understanding how the AP C3 proconvertase and C3 convertase are assembled and modulated by FH is therefore of great relevance.
An intriguing but poorly investigated aspect regarding AP complement regulation is whether FH has any effect on C3bB assembly (23,24). Here, for the first time, we document that FH does not affect C3bB assembly. The finding here that the C3bB complexes formed in the same amounts in the presence or in the absence of FH would suggest that FH does not compete enough with FB for binding to C3b to prevent C3bB assembly. A plausible explanation of our results derives from biophysical studies in the literature, showing that the binding affinity between C3b and FH molecules is lower compared with the affinity between C3b and FB (K d C3b-FH, 1 M, versus K d C3b-FB, 73 nM) (29,33,56), which would indicate that C3b-FB interaction is favored toward that between C3b and FH. Finding that FH band was detected before C3bB(Mn 2ϩ ) complex formation would indicate that the interaction between C3b and FH might have faster kinetics than that between C3b and FB. However, C3b-FH interaction was likely weaker than C3b-FB interaction because FH did not interfere with C3 proconvertase formation in our experimental setting. Notably, lack of a FH effect on C3bB formation was observed here independently of the ions used (Mn 2ϩ and Ni 2ϩ ) in the reaction mixture, further validating the new observation. C3b coated on plastic wells, as in the assays presented here, is not oriented as in physiological cell context via its thioester moiety, which suggests caution in interpreting these data. However, the experiments performed in the presence of human serum indicate that in this condition C3b provides a suitable molecular platform for the formation of functionally active C3 and C5 convertases.
FH binds to the host cell surface through interaction with oligosaccharides, such as glycosaminoglycans and sialic acid, bearing clusters of negative charges, and FH binding to such polyanions shifts FH from a latent to an active conformation, which significantly increases the affinity for C3b (39). This interaction prevents inadvertent action of the AP of complement on host cells and mediates its ability to distinguish between host cells and pathogens that do not have such glycans (57). The relevance of FH-glycan interaction in protecting cells and tissues from complement-activating products is highlighted by data that gene mutations resulting in reduced capability of FH to bind cell surfaces result in uncontrolled complement activation and aHUS (58,59). Our present results of C3bB and C3bBb assembly in the presence of HS or SA show that even in this more physiological pattern, FH does not substantially affect C3 proconvertase assembly but has a strong inhibitory effect on C3 convertase formation. However, we found that FH at molar ratios with FB higher than those present in normal blood was able to partially prevent C3bB proconvertase assembly. In addition, coating of HS or SA on a plastic well may not be sufficient to emulate a cell surface. Thus, we cannot exclude the possibility that in a cell-based context, SA and glycosaminoglycans present in high amounts in the glycocalyx and the cell surface, could favor FH clustering and influence the FIGURE 13. Effect of FH on C3bB(Ni 2؉ ) C3 proconvertase and C3bBb(Ni 2؉ ) C3 convertase decay detected by microplate/WB assays. Time course of spontaneous and FH-mediated decay of C3bB(Ni 2ϩ ) and C3bBb(Ni 2ϩ ), both formed during 30 min at 37°C by incubation of C3b-coated wells with FB (1000 ng/ml), FD (5 ng/ml), and NiCl 2 (2 mM), was monitored by further incubation at 37°C for 5, 30, 60, and 120 min with buffer alone or added with 2640 ng/ml FH. The amount of C3bB or C3bBb formed was calculated as the intensity of the B (93 kDa) or Bb (60 kDa) bands, respectively, and reported in the bottom graphs as pixel 2 ⅐10 6 . The percentage of residual B or Bb bands was calculated as the ratio of the densities (in pixel 2 ) of each B or Bb band after decay and the corresponding baseline B or Bb band density before decay ϫ 100. Results of a representative microplate/WB experiment of n ϭ 3 are shown.
interactions of C3b with FB or FH in favor of the latter (25), resulting in efficient antagonistic effects of FH on C3bB formation.
The few available data in the literature indicated that FH does not induce decay of assembled C3bB proconvertase (26,27), and this was confirmed by our finding here that C3bB was not dissociated by FH.
In contrast and in harmony with consolidated evidence (27,32), FH was very efficient at dissociating the C3bBb C3 convertase at a physiological molar ratio with FB, and P did not prevent FH-mediated C3bBb decay.
Co-crystal structural comparison of C3bBb with the complex between C3b and the regulatory N-terminal four complement control protein domains of FH (C3b/FH1-4) (56) indicated that FH-induced C3bBb dissociation is mediated by the first two complement control proteins of FH, which bind the ␣ЈNT, MG2, and MG6 -MG7 domains of C3b (56). Because the Ba fragment binds the ␣ЈNT domain of C3b (37,52,60) and Bb binds the MG domains, we speculate that FH cannot displace FB from C3b in C3bB because FB limits the accessibility of FH-binding sites on C3b. Once Ba is released from C3bB to form C3bBb, the ␣ЈNT region of C3b would become available to the interaction with FH, which in turn dislocates Bb from the C3bBb complex through steric hindrance (51,56). In addition, because the complementary surfaces of FH and Bb are both negatively charged, they could contribute to the destabilization of the C3bBb complex through electrostatic repulsion (56,61). More structural studies by NMR or x-ray crystallography are needed to better understand the underlying mechanisms.
It is relevant that we found an apparent strong inhibitory effect of FH on C3bBb C3 convertase formation, as documented by a failure to detect significant Bb band on WB from FIGURE 14. HS-coated ELISA (A) and effect of FH on C3bB(Ni 2؉ ) C3 proconvertase and C3bBb(Ni 2؉ ) C3 convertase formation in the presence of HS (B) or SA (C) detected by microplate/WB assays. A, microtiter plates were coated with 3 or 30 g/ml HS in PBS, in the presence or in the absence of 3 g/ml C3b. Immobilized HS was detected with a monoclonal mouse anti-HS antibody (1:100) followed by HRP-conjugated goat anti-mouse antibody (1:2000). Values are given as the OD averages with standard deviation (n ϭ 3 each). B and C, C3bB(Ni 2ϩ ) and C3bBb(Ni 2ϩ ) complexes were obtained by incubating C3b and HS-coated (B) or SA-coated (C) (3 or 30 g/ml) wells with FB (1000 ng/ml), FD (5 ng/ml), and NiCl 2 (2 mM) at 37°C for 30 min, in the presence or in the absence of FH (2640 ng/ml). The amount of C3bB or C3bBb formed was calculated as the intensity of the B (93 kDa) or Bb (60 kDa) bands, respectively, and reported in the bottom graphs as pixel 2 ⅐10 6 . FH band (150 kDa) could be visualized in the WB. Results of a representative microplate/WB experiment of n ϭ 3 are shown.
the reaction of C3b with FB and FD in the presence of FH. Based on the strong decay accelerating activity of FH on C3bBb, we first argued that this FH effect could be attributable to a rapid FH-accelerated dissociation of newly formed complexes. Finding that the addition of C3NeF and P, which almost completely prevented C3 convertase FH-mediated decay, did not fully restore C3bBb(Mg 2ϩ ) formation suggested that FH could also affect C3bBb generation. Data that in the reaction of coated C3b with FB and FD in the presence of Mn 2ϩ ions, FH almost completely prevented the release of Bb and Ba fragments in the supernatant, without modifying the amount of C3bB complexes, would indeed support the hypothesis that FH inhibits the conversion of C3bB to C3bBb. Together, these findings would suggest that the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration.
In summary, taking advantage of newly developed userfriendly assays based on combined microplate and WB techniques that specifically detect either C3bB or C3bBb, we shed new light on mechanisms underlying the regulation of the AP C3 proconvertase and C3 convertase by FH as follows. 1) We document that at the physiological molar ratio with FB, FH does not affect C3bB formation and dissociation even in the presence of HS or SA that favors FH-C3b interaction. 2) We confirm that FH blocks C3bBb formation and accelerates its decay. 3) We found that FH inhibits the conversion of C3bB to C3bBb. However, additional structural studies are needed to better understand the interaction between FH and the C3bB and C3bBb complexes, and cell-based studies are warranted to confirm such mechanisms in a more physiological context in which C3b is attached via its thioester to a surface nucleophile.
The assays presented here could be easy tools for studying the effect of other complement regulators on C3bB and C3bBb assembly and decay, as well as for a rapid screening of the effect of C3NeFs isolated from patients with C3G on C3bBb stabilization. A further application of the assays could be to analyze the functional consequences of FB, C3, or FH mutants/variants from patients with aHUS or C3G on C3bB and C3bBb formation and decay.