PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 April 8; 291(15): 8214–8230.
Published online 2016 February 22. doi:  10.1074/jbc.M115.693119
PMCID: PMC4825022

This article has been retractedRetraction in: J Biol Chem. 2017 April 14; 292(15): 6094    See also: PMC Retraction Policy

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase*

Abstract

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 Ruddy, S. (1978) J. Exp. Med. 147, 1792–1805; Pangburn, M. K., and Müller-Eberhard, H. 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 complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, 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 blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.

Keywords: complement, convertase, magnesium, manganese, Western blot, alternative pathway complement system, C3 convertase, C3 nephritic factor, C3 proconvertase, factor H

Introduction

The complement system is a complex network of plasma proteins that cooperate in the defense against foreign micro-organisms and in the maintenance of tissue homeostasis (1,3). Interest in the complement system was rekindled in the past decade by accumulating evidence that genetically determined complement dysregulation is involved in two rare devastating disorders of the kidney, atypical hemolytic uremic syndrome (aHUS)3 and C3 glomerulopathy (C3G) (4,8), as well as in age-related macular degeneration, the leading cause of blindness for older people (9). Finding that treatment with eculizumab (10), a monoclonal antibody that blocks the formation of terminal complement complex C5b-9, efficiently cured aHUS (11, 12) and C3G (13,15) further corroborated the role of complement in human disease (16).

Complement can be triggered by three activation pathways, the classical, the lectin, and the alternative (AP) pathways, all leading to the formation of unstable protease complexes, named C3 convertases, that cleave the inactive central component C3 into C3a and C3b fragments. C3b molecules deposited on pathogens or damaged self-cells provide a molecular platform for the formation of an active AP C3 convertase, C3bBb, which enzymatically generates many more C3b molecules, resulting in a positive feedback loop. Notably, the AP C3 convertase is crucial within the complement cascade as it also amplifies complement activation after C3b is generated through the other pathways.

The formation of the AP C3 convertase requires a two-step assembly process, in which factor B (FB) first binds to C3b in a Mg2+-dependent manner to form the labile C3 proconvertase C3bB. Once bound to C3b, the FB becomes susceptible to proteolytic 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 (23,25) 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.

Experimental Procedures

Complement Proteins, Chemicals and Antibodies

Microplates 96-microwell, Nunc-Immuno Maxisorp, and HRP-conjugated goat anti-mouse antibody (catalog no. 626520) were obtained from Thermo Scientific (VWR International PBI srl, Milano, Italy). Purified native complement proteins C3b, FB, FD and P were from Complement Technology Inc. (Tyler, TX). FH was purchased from Merck (Nottingham, UK). Bovine serum albumin (BSA), rabbit polyclonal anti-human FB antibody (catalog no. HPA001817), peroxidase-labeled polyclonal anti-goat IgG (whole molecule, catalog no. A5420) antibody, heparan sulfate (HS, H7640) sodium salt from bovine kidney, N-acetylneuraminic acid (sialic acid, A0812), and EGTA (34596) were obtained from Sigma-Aldrich. Peroxidase-labeled polyclonal anti-rabbit IgG (H+L, catalog no. PI-1000) antibody was purchased by Vector Laboratories Inc. (Burlingame, CA). 3,3′,5,5′-Tetramethylbenzidine substrate was from Bethyl (Tema Ricerca srl, Bologna, Italy). Mouse anti-heparan sulfate monoclonal antibody (catalog no. MAB2040) was purchased from Millipore (Temecula, CA). Polyclonal goat anti-human FH was obtained from Calbiochem (catalog no. 341276, Billerica, MA). Polyclonal goat antiserum to human FB was obtained from Quidel (catalog no. A311, San Diego, CA). Polyclonal rabbit anti-human C5/C5b antibody (catalog no. ab46168) was purchased from Abcam (Cambridge, UK). Hybond-P hydrophobic polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences) and the ECL Select chemiluminescence detection reagent (Amersham Biosciences) were purchased from GE Healthcare (Euroclone Spa, Milano, Italy). Normal human serum (NHS) was a pool obtained from 15 healthy volunteers.

ELISAs for C3bB C3 Proconvertase Assembly and C3bBb C3 Convertase Formation

For the generation of Ni2+-dependent AP C3bB C3 proconvertase and C3bBb C3 convertase, we performed an ELISA as described previously by Hourcade et al. (31, 32). ELISA plates were coated with 3 μg/ml C3b in PBS 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 wash buffer (8.1 mm Na2HPO4, 1.8 mm NaH2PO4, 0.1% Tween 20, and 25 mm NaCl) supplemented with 2 mm NiCl2. C3bB and C3bBb complexes were formed by incubating C3b-coated wells at 37 °C for 2 h with different concentrations of FB (0–500 ng/ml) in the absence or in the presence of FD (25 ng/ml), respectively, both diluted in assay buffer (8.1 mm Na2HPO4, 1.8 mm NaH2PO4, 4% BSA, 0.1% Tween 20, and 75 mm NaCl) containing 2 mm NiCl2. After washes, the C3bB and C3bBb complexes were detected by ELISA using polyclonal goat anti-human FB antibody (1:10,000) followed by HRP-conjugated anti-goat antibody (1:40,000), both diluted in antibody buffer (8.1 mm Na2HPO4, 1.8 mm NaH2PO4, 4% BSA, 0.1% Tween 20, and 25 mm NaCl) supplemented with 2 mm NiCl2. Color was developed using 3,3′,5,5′-tetramethylbenzidine substrate and stopped with 2 m H2SO4, and absorbance was measured at 450 nm. Each reaction was performed in duplicate, and the OD values were averaged. For the generation of C3bB(Mn2+) or C3bBb(Mg2+) complexes, C3b-coated microtiter wells were treated as above except that the incubation was performed in the presence of 2 mm MnCl2 for 2 h at 37 °C or 10 mm MgCl2 and for 30 min at 25 °C, respectively.

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 MnCl2 (33) or 10 mm MgCl2 (34) for C3bB or C3bBb, respectively. C3bB(Mn2+) 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 MnCl2 (Fig. 1A). C3bBb(Mg2+) 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 MgCl2 (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).

FIGURE 1.
Experimental design of microplate/WB assays.

For the generation of C3bB(Ni2+) and C3bBb(Ni2+) complexes, C3b-coated wells were incubated at 37 °C with FB (1000 ng/ml) and FD (5 ng/ml) in assay buffer containing 2 mm NiCl2, for different times (5, 10, 20, 30, 60, and 120 min). The Ni2+-protein complexes were evaluated by WB as described above (Fig. 1H).

To determine the effect of FH on C3bB and C3bBb formation, FH (2640 ng/ml) was added together with FB and FD to C3b-coated wells by using the selective experimental conditions described above (Fig. 1, A, B, and H), and it was visualized by WB as FH band of 150 kDa by using polyclonal goat anti-human FH antibody (1:1000) followed by HRP-conjugated anti-goat antibody (1:15,000). Physiological molar ratios among complement proteins were used (molar ratios FB/FD = 50:1 and FB/FH = 1:1.6). In the reaction performed in the presence of 2 mm NiCl2, FH was added together with FB and FD to C3b-coated wells, at different molar ratios (FB/FH = 10:1; 5:1; 1.6:1; 1:1.6; 1:5; 1:10). In the reaction performed in the presence of HS or SA, C3bB(Ni2+) and C3bBb(Ni2+) complexes were obtained by incubating FB (1000 ng/ml) and FD (5 ng/ml), diluted in assay buffer with 2 mm NiCl2, on C3b-coated (3 μg/ml) and HS-coated (3 and 30 μg/ml) or SA-coated (3 and 30 μg/ml) wells at 37 °C for 30 min, in the presence or in the absence of FH (Fig. 1H). C3bB(Ni2+) and C3bBb(Ni2+) complexes were evaluated by WB as described above.

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 μg/ml HS in PBS, in the presence or in the absence of 3 μg/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 3,3′,5,5′-tetramethylbenzidine substrate and stopped with 2 m H2SO4, 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(Mn2+) and C3bBb(Mg2+) 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 = 1:1.6) for the following time periods: C3bB(Mn2+), 30, 60, 120, and 240 min; C3bBb(Mg2+), 2, 4, 8, and 16 min) (Fig. 1, C and D). For C3bB(Ni2+) and C3bBb(Ni2+) 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 pixel2) of the Bb bands after decay and the corresponding baseline Bb band density before decay × 100.

C3bBb(Mg2+) C3 Convertase Formation and Decay in the Presence of Properdin

To evaluate the ability of P to stabilize C3bBb(Mg2+) 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 = 5:1) (35) in the presence or in the absence of FH (2640 ng/ml; molar ratio FB/FH = 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 μg) 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(Mg2+) C3 Convertase Formation and Decay

To evaluate the ability of IgG fractions to stabilize C3bBb(Mg2+) complexes both in spontaneous or FH-mediated decay, C3NeF-stabilized C3bBb(Mg2+) complexes were generated by incubating C3b-coated wells for 10 min at 25 °C with purified IgG (50 and 100 μg/ml), immediately followed by FB (1000 ng/ml) and FD (5 ng/ml) diluted in assay buffer with 10 mm MgCl2. 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 pixel2) of Bb bands after decay and the corresponding baseline Bb band density before decay × 100.

To evaluate the effect of FH on C3NeF-stabilized C3 convertase formation, C3bBb(Mg2+) was generated in the presence of C3NeF-IgG or control IgG (50 or 100 μg/ml) and P (114.5 ng/ml; molar ratio FB/P = 5:1) for 10 min at 25 °C with assay buffer alone or FH (2640 ng/ml; molar ratio FB/FH = 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(Mn2+) and C3bBb(Mn2+) complexes in the presence or in the absence of FH (Fig. 1A), by commercial ELISA kit following the protocol procedure (Quidel, San Diego).

C3bBb(Mg2+) 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 μg/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 MgCl2. C3bBb(Mg2+) complexes were formed by incubating C3b-coated wells at 37 °C for 30 min with 20% NHS diluted in PBS containing 5 mm MgCl2 in the presence or in the absence of 1 mm EGTA (Fig. 1J). After washes, formed C3bBb(Mg2+) 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 α′-chain C5b (104 kDa) band.

Results

ELISA with Ni2+ 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 Ni2+ cation prolongs the half-life of both C3bB and C3bBb (27, 32, 37), and for this reason Ni2+ has been widely used, instead of the physiological Mg2+, 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 Ni2+ to generate C3bB(Ni2+) or C3bBb(Ni2+), 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).

FIGURE 2.
Detection of C3bB C3 proconvertase and C3bBb C3 convertase complex formation by ELISA and microplate/WB assays. A, detection of Ni2+-dependent C3bB and C3bBb. C3b-coated microtiter wells were incubated with increasing amounts of FB (0–500 ng/ml) ...

New Assays to Specifically Detect C3bB(Mn2+) C3 Proconvertase and C3bBb(Mg2+) 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 Mn2+ stabilizes C3bB in a form susceptible to FD cleavage, but the C3bBb(Mn2+), once formed, is highly unstable and dissociates immediately, so that only the C3bB(Mn2+) complex could be detected. At variance, Mg2+ stabilizes C3bBb but not the pro-enzyme C3bB (34).

On the basis of this evidence, the ELISA was repeated replacing Ni2+ with Mn2+ or Mg2+ 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 MnCl2, 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 MgCl2 (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(Mn2+) and the C3bBb(Mg2+) 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(Mn2+) and C3bBb(Mg2+), 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 Mn2+, 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 Mg2+ and FD (Fig. 2C, right panel). Neither B nor Bb bands were observed in the reaction with Mg2+ in the absence of FD.

We then analyzed the time course of C3bB(Mn2+) and C3bBb(Mg2+) formation. As shown in Fig. 3A, the B band of C3bB(Mn2+) 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.

FIGURE 3.
Time course of selective C3bB(Mn2+) C3 proconvertase assembly (A) and C3bBb(Mg2+) C3 convertase formation (B) detected by microplate/WB assays. C3bB(Mn2+) and C3bBb(Mg2+) complexes were obtained by incubating C3b-coated wells at time points indicated ...

The kinetics of C3bBb(Mg2+) (Fig. 3B) formation was faster than that of C3bB(Mn2+), 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(Mn2+) and C3bBb(Mg2+) complexes.

Effect of FH on C3bB(Mn2+) 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(Mn2+) 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).

FIGURE 4.
Effect of FH on C3bB(Mn2+) C3 proconvertase assembly (A) and C3bB(Mn2+) C3 proconvertase decay (B) detected by microplate/WB assays. A, time course of C3bB(Mn2+) assembly. The complexes were obtained by incubating C3b-coated wells at 37 °C for ...

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(Mn2+) 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(Mn2+) 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(Mg2+) 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(Mg2+) formation by incubating C3b-coated wells with FB and FD in the presence or in the absence of FH (Fig. 1B). In the presence of FH, the amount of C3bBb(Mg2+) 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(Mg2+) decay. C3bBb(Mg2+) 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(Mg2+) dissociated in a time-dependent manner, but a faint band was still detectable at 16 min. The decay of C3bBb(Mg2+) 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(Mg2+) 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.

FIGURE 5.
Effect of FH on C3bBb(Mg2+) C3 convertase formation (A) and decay (B) detected by microplate/WB assays. A, time course of C3bBb(Mg2+) formation. The complexes were obtained by incubating C3b-coated wells at 25 °C for 5, 10, 30, and 45 min with ...
FIGURE 6.
Effect of two different concentrations of FH on C3bBb(Mg2+) C3 convertase formation (A) and decay (B) by microplate/WB assays. A, time course of C3bBb(Mg2+) formation. The complexes were obtained by the incubation at 25 °C for 5, 10, 30, and 45 ...

Effect of FH on C3bBb(Mg2+) 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(Mg2+) 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(Mg2+) formation or the FH-mediated C3bBb(Mg2+) decay acceleration (Fig. 7).

FIGURE 7.
Effect of FH on C3bBb(Mg2+) C3 convertase formation and decay in the presence of P analyzed by microplate/WB assays. C3bBb(Mg2+) 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 ...

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 FH-mediated 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 ≥100%). No hemolytic activity was found in the purified IgG sample from a plasma pool of three healthy subjects. C3bBb(Mg2+) complexes were formed for 10 min in the presence of two different concentrations (50 and 100 μg/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 FH-mediated C3bBb decay in a dose-dependent fashion, so that in the presence of 100 μg/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 μg/ml C3NeF-IgG from patient 5 (Fig. 1G), FH-mediated C3bBb decay was almost completely prevented (81% stabilization of the C3bBb(Mg2+) complexes) (Fig. 9). Even though FH-mediated decay was efficiently blocked in this condition, C3bBb(Mg2+) 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(Mg2+) formed over 10 min in the presence of P and 100 μg/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(Mg2+) formed by 97%. These results would suggest that FH may have a direct effect on C3bBb formation from C3bB proconvertase.

FIGURE 8.
Effect of FH on decay of C3NeF-stabilized C3bBb(Mg2+) C3 convertase detected by microplate/WB assays. C3bBb(Mg2+) 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 MgCl2 in the ...
FIGURE 9.
Effect of FH on formation and decay of C3bBb(Mg2+) C3 convertase stabilized by C3NeF-IgG and P detected by microplate/WB assays. C3bBb(Mg2+) complexes were originated by incubating at 25 °C for 10 min C3b-coated wells with 1000 ng/ml FB, 5 ng/ml ...

Effect of FH on C3bBb C3 Convertase Formation from C3bB C3 Proconvertase

Mn2+ stabilizes C3bB in a form susceptible to FD cleavage, but the C3bBb(Mn2+) complex, once formed, is highly unstable and rapidly dissociates (33). Consistently, as shown in Fig. 3A, the time course of C3bB(Mn2+) assembly revealed that independently of the presence of FD, no Bb band of C3bBb(Mn2+) could be detected. We wondered whether in this condition, in the presence of FD, the formation of C3bBb(Mn2+) (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(Mn2+) formed and rapidly dissociated. As shown in Fig. 11, although the formation of C3bB(Mn2+) 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.

FIGURE 10.
Bb and Ba fragments ELISA in the supernatant of the reactions of C3bB(Mn2+) and C3bBb(Mn2+). Bb and Ba fragment levels (ng/ml) were measured, by ELISA, in the supernatant of the reactions of C3bB(Mn2+) and C3bBb(Mn2+) complexes formed in the absence or ...
FIGURE 11.
Time course of selective C3bBb(Mn2+) C3 convertase formation in the absence or in the presence of FH, by microplate/WB assay (A) and results of Bb and Ba fragments ELISA in the supernatant (B). A, C3bB(Mn2+) and C3bBb(Mn2+) were obtained by incubating ...

Effect of FH on C3bB(Ni2+) C3 Proconvertase and C3bBb(Ni2+) C3 Convertase Formation and Decay

To evaluate whether the diverse effect of FH on C3bB(Mn2+) compared with C3bBb(Mg2+) formation and decay was attributable to the different ions used to stabilize either complex (Mn2+ versus Mg2+), we repeated the microplate/WB experiments by incubating surface-bound C3b with FB and FD in the presence of Ni2+, which stabilized both C3bB and C3bBb and yielded both B and Bb bands on WB (Figs. 1H and and12,12, 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).

FIGURE 12.
Effect of FH on C3bB(Ni2+) C3 proconvertase and C3bBb(Ni2+) C3 convertase formation detected by microplate/WB assays. A and B, time course of C3bB(Ni2+) and C3bBb(Ni2+) formation was obtained by incubation at 37 °C for 5, 30, 60, and 120 min ( ...

As shown in Fig. 12, A and B, C3bB(Ni2+) assembly was not affected by FH. Dose-response studies showed that FH decreased C3bB(Ni2+) 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 of recovered C3bBb(Ni2+) at all time points (Fig. 12, A and B), confirming the results obtained in the ion-selective conditions with Mg2+.

Next, to assess the effect of FH on C3bB(Ni2+) and C3bBb(Ni2+) decay, the complexes obtained after 30 min at 37 °C were incubated with buffer alone or with FH (Fig. 1I). C3bB(Ni2+) did not undergo either spontaneous or FH-mediated decay at each time point compared with baseline (Fig. 13). At variance, C3bBb(Ni2+) spontaneously dissociated in a time-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.

FIGURE 13.
Effect of FH on C3bB(Ni2+) C3 proconvertase and C3bBb(Ni2+) C3 convertase decay detected by microplate/WB assays. Time course of spontaneous and FH-mediated decay of C3bB(Ni2+) and C3bBb(Ni2+), both formed during 30 min at 37 °C by incubation ...

Effect of FH on C3bB(Ni2+) C3 Proconvertase and C3bBb(Ni2+) 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(Ni2+) and C3bBb(Ni2+) formation assays using wells coated with mixtures of C3b and HS (3 or 30 μg/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).

FIGURE 14.
HS-coated ELISA (A) and effect of FH on C3bB(Ni2+) C3 proconvertase and C3bBb(Ni2+) 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 μg/ml HS in PBS, in ...

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 μg/ml) of coated SA, FH did not affect C3bB(Ni2+) assembly, although it completely prevented C3bBb(Ni2+) formation (Fig. 14C).

C3bBb(Mg2+) 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(Mg2+) 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 α′-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).

FIGURE 15.
C3bBb(Mg2+) C3 convertase and C5b formation by using normal human serum detected by microplate/WB assays. A, C3bBb(Mg2+) complexes were obtained by incubating C3b-coated wells with 20% NHS at 37 °C for 30 min, in the presence 5 mm MgCl2. C3bBb ...

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 Ni2+ instead of the physiological Mg2+, because Ni2+ 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 Ni2+ 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 Mn2+ and Mg2+ on C3bB and C3bBb (33, 34, 52), respectively. A previous study (33) documented that Mn2+ stabilizes C3bB in a form susceptible to FD cleavage, but C3bBb(Mn2+), once formed, is highly unstable and dissociates immediately. FB binding to C3b depends on elements in fragment Ba and on the metal ion-dependent adhesion site (MIDAS) motif in the von Willebrand type A domain of Bb (53). It has been proposed that Mn2+ induces low affinity conformation of the MIDAS in C3bBb that leads to the rapid dissociation of the C3 convertase complex (33). At variance, Mg2+ 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(Mg2+) to C3bBb(Mg2+) (38, 54). By using the new microplate/WB methods with ion-based selective stabilization, we could observe specific formation over time of C3bB(Mn2+) or C3bBb(Mg2+) 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 complement-mediated 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 (Kd C3b-FH, 1 μm, versus Kd 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(Mn2+) 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 (Mn2+ and Ni2+) 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 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 α′NT, MG2, and MG6–MG7 domains of C3b (56). Because the Ba fragment binds the α′NT 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 α′NT 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 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(Mg2+) 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 Mn2+ 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 user-friendly 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.

Author Contributions

S. B., R. D., and M. N. designed research, interpreted data, and wrote the paper; S. B. performed research and analyzed data; E. B. coordinated clinical data collection and patient recruitment; R. D. analyzed data; G. R. supervised research and revised the draft of the manuscript.

Acknowledgments

We are deeply indebted to Dr. Veronique Fremeaux-Bacchi (Assistance Publique des Hopitaux de Paris, Hopital Europeen Georges Pompidou, Laboratoire d'Immunologie, Paris, France) for purifying IgG fractions, for C3NeF activity measurements, and for useful discussion. Kerstin Mierke edited English style and grammar of the manuscript.

*This work was supported in part by European Union Seventh Framework Programme FP7-EURenOmics Project Number 305608. Part of this work was presented at the 24th International Complement Workshop, October 10–15, 2012, Crete, Greece; at the 14th European Meeting on Complement in Human Disease, August 17–21, 2013, Jena, Germany; and at the 15th European Meeting on Complement in Human Disease, June 26–30, 2015, Uppsala, Sweden. M. N. received honoraria from Alexion Pharmaceuticals for giving lectures and participating in advisory boards. None of these activities has had any influence on the results or interpretation in this article. G. R. has consultancy agreements with the following: AbbVie; Alexion Pharmaceuticals; Bayer Healthcare; Reata Pharmaceuticals; Novartis Pharma; AstraZeneca; Otsuka Pharmaceutical Europe; and Concert Pharmaceuticals. No personal remuneration was accepted, and compensation is paid to his institution for research and educational activities. None of these activities have had any influence on the results or interpretation in this article.

3The abbreviations used are:

aHUS
atypical hemolytic uremic syndrome
AP
alternative pathway
Ba
smaller cleavage subunit of FB
Bb
larger cleavage subunit of FB
C3b
activated fragment of C3
C3bB
C3 proconvertase
C3bBb
C3 convertase
C3G
C3 glomerulopathy
C3NeF
C3 nephritic factor
FB
factor B
FD
factor D
FH
factor H
HS
heparan sulfate
P
properdin
WB
Western blotting
SA
sialic acid
NHS
normal human serum
MIDAS
metal ion-dependent adhesion site.

References

1. Walport M. J. (2001) Complement. First of two parts. N. Engl. J. Med. 344, 1058–1066 [PubMed]
2. Walport M. J. (2001) Complement. Second of two parts. N. Engl. J. Med. 344, 1140–1144 [PubMed]
3. Ricklin D., Hajishengallis G., Yang K., and Lambris J. D. (2010) Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797 [PMC free article] [PubMed]
4. Noris M., and Remuzzi G. (2009) Atypical hemolytic-uremic syndrome. N. Engl. J. Med. 361, 1676–1687 [PubMed]
5. Noris M., and Remuzzi G. (2013) Overview of complement activation and regulation. Semin. Nephrol. 33, 479–492 [PMC free article] [PubMed]
6. Rodríguez de Córdoba S., Harris C. L., Morgan B. P., and Llorca O. (2011) Lessons from functional and structural analyses of disease-associated genetic variants in the complement alternative pathway. Biochim. Biophys. Acta 1812, 12–22 [PubMed]
7. Bomback A. S., and Appel G. B. (2012) Pathogenesis of the C3 glomerulopathies and reclassification of MPGN. Nat. Rev. Nephrol. 8, 634–642 [PubMed]
8. Sethi S., Fervenza F. C., Zhang Y., Zand L., Vrana J. A., Nasr S. H., Theis J. D., Dogan A., and Smith R. J. (2012) C3 glomerulonephritis: clinicopathological findings, complement abnormalities, glomerular proteomic profile, treatment, and follow-up. Kidney Int. 82, 465–473 [PMC free article] [PubMed]
9. McHarg S., Clark S. J., Day A. J., and Bishop P. N. (2015) Age-related macular degeneration and the role of the complement system. Mol. Immunol. 67, 43–50 [PubMed]
10. Gruppo R. A., and Rother R. P. (2009) Eculizumab for congenital atypical hemolytic-uremic syndrome. N. Engl. J. Med. 360, 544–546 [PubMed]
11. Legendre C. M., Licht C., Muus P., Greenbaum L. A., Babu S., Bedrosian C., Bingham C., Cohen D. J., Delmas Y., Douglas K., Eitner F., Feldkamp T., Fouque D., Furman R. R., Gaber O., et al. (2013) Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N. Engl. J. Med. 368, 2169–2181 [PubMed]
12. Zuber J., Fakhouri F., Roumenina L. T., Loirat C., Frémeaux-Bacchi V., French Study Group for aHUS/C3G. (2012) Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat. Rev. Nephrol. 8, 643–657 [PubMed]
13. Daina E., Noris M., and Remuzzi G. (2012) Eculizumab in a patient with dense-deposit disease. N. Engl. J. Med. 366, 1161–1163 [PubMed]
14. Vivarelli M., and Emma F. (2014) Treatment of C3 glomerulopathy with complement blockers. Semin. Thromb. Hemost. 40, 472–477 [PubMed]
15. Nester C. M., and Smith R. J. (2013) Treatment options for C3 glomerulopathy. Curr. Opin. Nephrol. Hypertens. 22, 231–237 [PMC free article] [PubMed]
16. Leslie M. (2012) Immunology. The new view of complement. Science 337, 1034–1037 [PubMed]
17. Fearon D. T., and Austen K. F. (1975) Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J. Exp. Med. 142, 856–863 [PMC free article] [PubMed]
18. Hourcade D. E. (2006) The role of properdin in the assembly of the alternative pathway C3 convertases of complement. J. Biol. Chem. 281, 2128–2132 [PubMed]
19. Zipfel P. F., and Skerka C. (2009) Complement regulators and inhibitory proteins. Nat. Rev. Immunol. 9, 729–740 [PubMed]
20. Pangburn M. K., Schreiber R. D., and Müller-Eberhard H. J. (1977) Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein β1H for cleavage of C3b and C4b in solution. J. Exp. Med. 146, 257–270 [PMC free article] [PubMed]
21. Weiler J. M., Daha M. R., Austen K. F., and Fearon D. T. (1976) Control of the amplification convertase of complement by the plasma protein β1H. Proc. Natl. Acad. Sci. U.S.A. 73, 3268–3272 [PubMed]
22. Whaley K., and Ruddy S. (1976) Modulation of the alternative complement pathways by β1H globulin. J. Exp. Med. 144, 1147–1163 [PMC free article] [PubMed]
23. Conrad D. H., Carlo J. R., and Ruddy S. (1978) Interaction of β1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding. J. Exp. Med. 147, 1792–1805 [PMC free article] [PubMed]
24. Pangburn M. K., and Müller-Eberhard H. J. (1978) Complement C3 convertase: cell surface restriction of β1H control and generation of restriction on neuraminidase-treated cells. Proc. Natl. Acad. Sci. U.S.A. 75, 2416–2420 [PubMed]
25. Kazatchkine M. D., Fearon D. T., and Austen K. F. (1979) Human alternative complement pathway: membrane-associated sialic acid regulates the competition between B and β1H for cell-bound C3b. J. Immunol. 122, 75–81 [PubMed]
26. Tortajada A., Montes T., Martínez-Barricarte R., Morgan B. P., Harris C. L., and de Córdoba S. R. (2009) The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity. Hum. Mol. Genet. 18, 3452–3461 [PMC free article] [PubMed]
27. Hourcade D. E., Mitchell L. M., and Medof M. E. (1999) Decay acceleration of the complement alternative pathway C3 convertase. Immunopharmacology 42, 167–173 [PubMed]
28. Makou E., Herbert A. P., and Barlow P. N. (2013) Functional anatomy of complement factor H. Biochemistry 52, 3949–3962 [PubMed]
29. Schmidt C. Q., Herbert A. P., Hocking H. G., Uhrín D., and Barlow P. N. (2008) Translational mini-review series on complement factor H: structural and functional correlations for factor H. Clin. Exp. Immunol 151, 14–24 [PubMed]
30. Rodríguez de Córdoba S., Esparza-Gordillo J., Goicoechea de Jorge E., Lopez-Trascasa M., and Sánchez-Corral P. (2004) The human complement factor H: functional roles, genetic variations and disease associations. Mol. Immunol. 41, 355–367 [PubMed]
31. Hourcade D. E., Wagner L. M., and Oglesby T. J. (1995) Analysis of the short consensus repeats of human complement factor B by site-directed mutagenesis. J. Biol. Chem. 270, 19716–19722 [PubMed]
32. Hourcade D. E., Mitchell L., Kuttner-Kondo L. A., Atkinson J. P., and Medof M. E. (2002) Decay-accelerating factor (DAF), complement receptor 1 (CR1), and factor H dissociate the complement AP C3 convertase (C3bBb) via sites on the type A domain of Bb. J. Biol. Chem. 277, 1107–1112 [PubMed]
33. Hourcade D. E., and Mitchell L. M. (2011) Access to the complement factor B scissile bond is facilitated by association of factor B with C3b protein. J. Biol. Chem. 286, 35725–35732 [PMC free article] [PubMed]
34. Fishelson Z., and Müller-Eberhard H. J. (1982) C3 convertase of human complement: enhanced formation and stability of the enzyme generated with nickel instead of magnesium. J. Immunol. 129, 2603–2607 [PubMed]
35. Kouser L., Abdul-Aziz M., Nayak A., Stover C. M., Sim R. B., and Kishore U. (2013) Properdin and factor H: opposing players on the alternative complement pathway “see-saw”. Front. Immunol. 4, 93. [PMC free article] [PubMed]
36. Frémeaux-Bacchi V., Weiss L., Brun P., and Kazatchkine M. D. (1994) Selective disappearance of C3NeF IgG autoantibody in the plasma of a patient with membranoproliferative glomerulonephritis following renal transplantation. Nephrol. Dial. Transplant. 9, 811–814 [PubMed]
37. Torreira E., Tortajada A., Montes T., Rodríguez de Córdoba S., and Llorca O. (2009) 3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase. Proc. Natl. Acad. Sci. U.S.A. 106, 882–887 [PubMed]
38. Harris C. L., Abbott R. J., Smith R. A., Morgan B. P., and Lea S. M. (2005) Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55). J. Biol. Chem. 280, 2569–2578 [PubMed]
39. Morgan H. P., Schmidt C. Q., Guariento M., Blaum B. S., Gillespie D., Herbert A. P., Kavanagh D., Mertens H. D., Svergun D. I., Johansson C. M., Uhrín D., Barlow P. N., and Hannan J. P. (2011) Structural basis for engagement by complement factor H of C3b on a self- surface. Nat. Struct. Mol. Biol. 18, 463–470 [PMC free article] [PubMed]
40. Alcorlo M., Tortajada A., Rodríguez de Córdoba S., and Llorca O. (2013) Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin. Proc. Natl. Acad. Sci. U.S.A. 110, 13504–13509 [PubMed]
41. Jelezarova E., Schlumberger M., Sadallah S., Späth P. J., Schifferli J. A., and Lutz H. U. (2001) A C3 convertase assay for nephritic factor functional activity. J. Immunol. Methods 251, 45–52 [PubMed]
42. Paixão-Cavalcante D., López-Trascasa M., Skattum L., Giclas P. C., Goodship T. H., de Córdoba S. R., Truedsson L., Morgan B. P., and Harris C. L. (2012) Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation. Kidney Int. 82, 1084–1092 [PMC free article] [PubMed]
43. Lavigne K. M., Rapin L. A., Metzak P. D., Whitman J. C., Jung K., Dohen M., Lœvenbruck H., and Woodward T. S. (2015) Left-dominant temporal-frontal hypercoupling in schizophrenia patients with hallucinations during speech perception. Schizophr. Bull. 41, 259–267 [PMC free article] [PubMed]
44. Blaum B. S., Hannan J. P., Herbert A. P., Kavanagh D., Uhrín D., and Stehle T. (2015) Structural basis for sialic acid-mediated self-recognition by complement factor H. Nat. Chem. Biol. 11, 77–82 [PubMed]
45. Nilsson U. R., Storm K. E., Elwing H., and Nilsson B. (1993) Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface. Mol. Immunol. 30, 211–219 [PubMed]
46. Roumenina L. T., Loirat C., Dragon-Durey M. A., Halbwachs-Mecarelli L., Sautes-Fridman C., and Fremeaux-Bacchi V. (2011) Alternative complement pathway assessment in patients with atypical HUS. J. Immunol. Methods 365, 8–26 [PubMed]
47. Mollnes T. E., Jokiranta T. S., Truedsson L., Nilsson B., Rodriguez de Cordoba S., and Kirschfink M. (2007) Complement analysis in the 21st century. Mol. Immunol. 44, 3838–3849 [PubMed]
48. Jokiranta T. S., Westin J., Nilsson U. R., Nilsson B., Hellwage J., Löfås S., Gordon D. L., Ekdahl K. N., and Meri S. (2001) Complement C3b interactions studied with surface plasmon resonance technique. Int. Immunopharmacol. 1, 495–506 [PubMed]
49. Goicoechea de Jorge E., Harris C. L., Esparza-Gordillo J., Carreras L., Arranz E. A., Garrido C. A., López-Trascasa M., Sánchez-Corral P., Morgan B. P., and Rodríguez de Córdoba S. (2007) Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc. Natl. Acad. Sci. U.S.A. 104, 240–245 [PubMed]
50. Hui K. M., Magnadóttir B., Schifferli J. A., and Inal J. M. (2006) CRIT peptide interacts with factor B and interferes with alternative pathway activation. Biochem. Biophys. Res. Commun. 344, 308–314 [PubMed]
51. Janssen B. J., Huizinga E. G., Raaijmakers H. C., Roos A., Daha M. R., Nilsson-Ekdahl K., Nilsson B., and Gros P. (2005) Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437, 505–511 [PubMed]
52. Torreira E., Tortajada A., Montes T., Rodríguez de Córdoba S., and Llorca O. (2009) Coexistence of closed and open conformations of complement factor B in the alternative pathway C3bB(Mg2+) proconvertase. J. Immunol. 183, 7347–7351 [PubMed]
53. Milder F. J., Gomes L., Schouten A., Janssen B. J., Huizinga E. G., Romijn R. A., Hemrika W., Roos A., Daha M. R., and Gros P. (2007) Factor B structure provides insights into activation of the central protease of the complement system. Nat. Struct. Mol. Biol. 14, 224–228 [PubMed]
54. Forneris F., Ricklin D., Wu J., Tzekou A., Wallace R. S., Lambris J. D., and Gros P. (2010) Structures of C3b in complex with factors B and D give insight into complement convertase formation. Science 330, 1816–1820 [PMC free article] [PubMed]
55. Servais A., Noël L. H., Roumenina L. T., Le Quintrec M., Ngo S., Dragon-Durey M. A., Macher M. A., Zuber J., Karras A., Provot F., Moulin B., Grünfeld J. P., Niaudet P., Lesavre P., and Frémeaux-Bacchi V. (2012) Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int. 82, 454–464 [PubMed]
56. Wu J., Wu Y. Q., Ricklin D., Janssen B. J., Lambris J. D., and Gros P. (2009) Structure of complement fragment C3b-factor H and implications for host protection by complement regulators. Nat. Immunol. 10, 728–733 [PMC free article] [PubMed]
57. Pangburn M. K. (2000) Host recognition and target differentiation by factor H, a regulator of the alternative pathway of complement. Immunopharmacology 49, 149–157 [PubMed]
58. Jokiranta T. S., Cheng Z. Z., Seeberger H., Jòzsi M., Heinen S., Noris M., Remuzzi G., Ormsby R., Gordon D. L., Meri S., Hellwage J., and Zipfel P. F. (2005) Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site. Am. J. Pathol. 167, 1173–1181 [PubMed]
59. Lehtinen M. J., Rops A. L., Isenman D. E., van der Vlag J., and Jokiranta T. S. (2009) Mutations of factor H impair regulation of surface-bound C3b by three mechanisms in atypical hemolytic uremic syndrome. J. Biol. Chem. 284, 15650–15658 [PMC free article] [PubMed]
60. Janssen B. J., Gomes L., Koning R. I., Svergun D. I., Koster A. J., Fritzinger D. C., Vogel C. W., and Gros P. (2009) Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex. EMBO J. 28, 2469–2478 [PubMed]
61. Janssen B. J., Christodoulidou A., McCarthy A., Lambris J. D., and Gros P. (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444, 213–216 [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology