C3 was isolated from freshly prepared human plasma as described . For C5 isolation, a 1 ml N-hydroxysuccinimide (GE Healthcare, Little Chalfont, UK) column was loaded with the soft tick complement inhibitor OmCI that efficiently binds C5 . OmCI was kindly provided by Miles Nunn, Susan Lea and Matthijs Jore. The OmCI used in this study refers to pOmCI, a double mutant (N78Q/N102Q), which was expressed in Pichia methanolica and purified to homogeneity as described . The serum was diluted 1:1 with PBS, 10 mM EDTA was added, and loaded onto the OmCI-coupled column to allow C5 binding to the column. C5 was eluted with 0.1 M ethanolamine, 0.05 M NaCl (pH 11) that was immediately neutralized with 0.1 M HCl in 1 M phosphate, 10 mM NaCl buffer (pH 7.4). Fractions were analyzed by SDS-PAGE and dialyzed against PBS overnight at 4 °C. Activity was confirmed by CH50 analyses in depleted serum (Complement Technology, Inc., Tyler, TX, USA) and compared with commercially obtained C5 from Complement Technology, Inc. FB and FD were expressed in HEK293 cells stably expressing EBNA1 (HEK293E) as described  (U-Protein Express, Utrecht, The Netherlands). FB contained an N-terminal His-tag and was isolated from the expression medium via immobilized metal affinity chromatography (HiTrap chelating column, GE Healthcare). FD was isolated by size-exclusion chromatography (Superdex75, GE Healthcare).
Eculizumab was ordered via the pharmacy and kindly provided by Genmab (Utrecht, The Netherlands). SSL7 and the mutant of SSL7 that cannot bind C5 (SSL7ΔC5) were cloned and expressed in Escherichia coli and purified as described previously . The SSL7ΔC5 has the D147K mutation as described .
Biotinylation of C3b
Plasma-purified C3 was activated into C3b in the presence of a biotinylation agent that reacts with the cysteine residue of the C3b thioester as described previously [14–16] with some adaptations. A concentration of 1 mg/ml C3 was activated with 1.1 μg/ml trypsin, for 10 min (min) at 37 °C, in the presence of 100 μg/ml maleimide-PEG2-biotin (Thermo Scientific Pierce Protein Research, Rockford, IL, USA). The reaction was stopped by adding 5.5 μg/ml soybean trypsin inhibitor (SBTI) after which 20 mM iodoacetamide was added and incubated for 30 min on ice. The sample was 1:1 diluted into 20 mM phosphate, 10 mM NaCl buffer (pH 7.4) and applied to a 1 ml MonoQ anion-exchange column (GE Healthcare). C3b-biotin was eluted by a gradient to 55 % 20 mM phosphate, 500 mM NaCl buffer (pH 7.4) using AKTA FPLC (GE Healthcare). Fractions were analyzed by SDS-PAGE and successful biotinylation of the alpha chain was confirmed by Western blotting.
Preparation of C3b-coated beads
Streptavidin-coated magnetic beads (Dynabeads M-270 Streptavidin, Invitrogen (Carlsbad, CA, USA), 2.8 μm diameter) were washed twice and suspended in VBS-T+ (veronal buffered saline: 2 mM veronal, 145 mM NaCl, pH 7.4 (VBS, pH 7.4) containing 2.5 mM MgCl2 and 0.05 % Tween). Beads (4 × 106) were loaded with different concentrations of C3b-biotin by incubating a volume of 200 μl beads with 200 μl C3b-biotin (final concentrations as indicated) for 1 h at 4 °C under shaking conditions. For quantification of C3b on beads, pellets were incubated with FITC-conjugated C3 antibody (Protos Immunoresearch, Burlingame, CA, USA) for 30 min at 4 °C under shaking conditions. Beads were washed and analyzed by flow cytometry using a FACSVerse flow cytometer (Becton Dickinson, San Jose, CA, USA). Beads (total of 10,000 events) were gated based on forward/side scatter and the geometric mean fluorescence of the gated population was analyzed using FlowJo software.
C5 convertase activity assay
C3b-coated beads were incubated with 20 μg/ml C5, 50 μg/ml FB and 5 μg/ml FD in absence or presence of inhibitory proteins (20 μg/ml eculizumab, SSL7, SSL7ΔC5) in a total volume of 100 μl VBS-T+ for 1 h at 37 °C while shaking constantly. After incubation, supernatants were collected and analyzed for the presence of C5a. Fluid-phase C5 convertase activity was assayed by incubating C3b-biotin together with 20 μg/ml C5, 50 μg/ml FB and 5 μg/ml FD in VBS-T+. CVF-based C5 convertase assays were performed in a total volume of 100 μl in VBS+-0.1 % BSA in which 0.01 μg/ml CVF was incubated with 20 μg/ml C5, 50 μg/ml FB and 5 μg/ml FD, in the absence or presence of inhibitory proteins (20 μg/ml eculizumab, SSL7, SSL7ΔC5) for 1 h at 37 °C. The release of C5a in supernatants was determined in a calcium mobilization assay . Stably transfected U937-C5aR cells were loaded with 2 μM Fluo-3-AM (Invitrogen), washed and re-suspended in RPMI/0.05 % HSA to a concentration of 1 × 106 cells/ml. The transient increase in free intracellular calcium concentration was measured by flow cytometry. Cells were gated based on scatter properties. The basal fluorescence level was monitored for 8 s, then stimulus (purified C5a or supernatants of convertase assays) was added and the sample tube was rapidly placed back to the sample holder and the fluorescence measurement continued up to 1 min. Absolute calcium mobilization (C5a flux) was calculated by subtracting background fluorescence from the fluorescence after stimulation. Standard curves were generated using different concentrations of purified C5a (10−11–10−7 M, Bachem, Bubendorf, Switzerland).
C5 binding to C3b beads
For analysis of C5 binding, C3b-coated beads were incubated with 50 μl 20 μg/ml C5 in absence or presence of inhibitory proteins (20 μg/ml eculizumab, SSL7, SSL7ΔC5) for 1 h at 37 °C. After washings in PBS-1 % BSA, bead pellets were incubated with 3 μg/ml rabbit anti-C5 (Dako, Carpinteria, CA, USA) in 50 μl PBS-1 % BSA for 45 min at 4 °C. After washing twice, pellets were incubated with FITC-conjugated goat anti-rabbit IgG (1:50, Sigma-Aldrich, St Louis, MO, USA) in 50 μl PBS-1 % BSA for 45 min at 4 °C.
Bead samples were heated at 95 °C for 5 min in 2× sample buffer (2 % SDS, 20 % glycerol, 20 mM Tris–HCl, pH 6.8 and 1 mg/ml bromophenol blue) with 50 mg/ml dithiothreitol (DTT). A volume of 10 μl of each sample was analyzed by SDS-PAGE (10 %) and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 4 % dried skim milk (ELK, Campina. Amersfoort, The Netherlands) in PBS-0.05 % Tween and incubated with peroxidase (PO)-conjugated streptavidin (to analyze C3b-biotinylation) or probed with goat antiserum against human C5 (1:300, Complement Technology, Inc.). The membranes were washed, incubated with PO-conjugated donkey anti-goat IgG (Bio-Connect, Huissen, The Netherlands) and developed by enhanced chemiluminescence (ECL, Fisher Emergo, Landsmeer, The Netherlands).
C5 binding to pre-opsonized bacteria
To generate pre-opsonized bacteria, E. coli MG1655 were grown to midlog phase (OD660 ~ 0.5) in lysogeny broth (LB) and suspended in VBS containing 0.1 % BSA and 2.5 mM MgCl2 (VBS+). Bacteria (~5 × 108 cfu/ml) were pre-incubated for 1 h at 37 °C in 10 % C5 depleted serum (Complement Technology, Inc.), or 10 % normal serum that was heated at 56 °C for 30 min to eliminate complement activity as a control (HI serum). To allow decay of Bb, bacteria were incubated in PBS for 1.5 h at 37 °C. Then, bacteria were washed in VBS+ and incubated with 20 μg/ml C5 for 1 h at 37 °C under shaking conditions. After subsequent washing, binding of C5 was detected by flow cytometry. Bacterial cells (total of 10,000 events) were gated based on forward/side scatter and the geometric mean fluorescence of the gated population was analyzed using FlowJo software.
Preparation of differently coated C3b beads
To generate ‘self-amplified’ C3b beads, streptavidin-coated beads (Dynabeads M-270) were loaded with 0, 0.01 or 0.1 μg/ml C3b-biotin after which the bead pellets were incubated with 20 μg/ml FB and 5 μg/ml FD in a total volume of 125 μl for 10 min at 37 °C under shaking conditions. To prevent fluid-phase C3 conversion, 10 mM EDTA was added, and then 50 μg/ml C3. The beads were washed and the procedure was repeated for 5 rounds in total to allow self-amplification to completion. The C3b levels deposited on the beads were quantified by flow cytometry as described above (FITC-conjugated C3 antibody) and categorized in ‘low’ (for beads with 0 μg/ml C3b-biotin before amplification), ‘intermediate’ (0.01 μg/ml C3b-biotin before amplification) and ‘high’ (0.1 μg/ml C3b-biotin before amplification). Based on flow cytometry quantification, the ‘low’, ‘intermediate’ and ‘high’ self-amplified beads were then compared with beads loaded with 0 μg/ml, 0.3 μg/ml and 3 μg/ml C3b-biotin in a C5 convertase activity assay. For natural amplification in serum, C3b-coated beads were coated with 0.01 μg/ml C3b-biotin after which the beads were incubated in different concentrations of normal pooled human serum (as described ) in VBS containing 2.5 mM MgCl2 (VBS+).
To generate beads bearing C3b molecules in a random orientation, we used two types of tosyl-activated beads: Dynabeads® M-280 Tosylactivated and Dynabeads® M-280 Streptavidin, Invitrogen, 2.8 μm diameter. Both bead types were coated with C3b-biotin in PBS-0.05 % Tween overnight at 37 °C. After washing, beads were analyzed in the C5 convertase activity assay as described above.
To compare beads with different C3b densities but leaving total C3b level in the sample constant, we loaded different amounts of streptavidin-coated beads with 0.18 or 0.36 μg C3b-biotin in a total volume of 600 μl buffer. After washing, 1/3 of loaded beads were incubated with anti-C3 antibodies for flow cytometry analyses or Western blotting while 2/3 of beads were analyzed for C5 convertase activity as described above.
All molecular models were generated using UCSF Chimera . Convertase models were generated based on structures of the C3bBb-SCIN complex [PDB: 2WIN]  and the C5-CVF-SSL7 complex [PDB: 3PRX] . The coordinates of the C3b molecule from the C3bBb structure were superimposed on those of CVF from the C5-CVF-SSL7 structure, to model the hypothetical monomeric C3bBb convertase in complex with C5. The SSL7 binding site residues were selected as C5 residues within 6 Å of SSL7 from the crystallographic structure, and the eculizumab binding site residues were chosen based on the known C5 epitope . To examine C3b surface density at a molecular level, a 150 × 150 nm area representing a portion of the bead surface was modeled. The crystallographic structure of C3b [PDB: 2I07]  was oriented such that the long axis was perpendicular to the surface and the thioester (and in turn the biotin linker) was in contact with the surface. For each C3b density, the number of molecules per 150 × 150 nm surface area was calculated, and intermolecular distance was calculated assuming a uniform distribution of molecules. C3b molecules were then placed such that the center-to-center distance was equal to the calculated intermolecular distance.
Statistical analyses were performed using Prism (GraphPad Software).
The data sets supporting the results of this article are available in Additional file 1.