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Fig. 1 | BMC Biology

Fig. 1

From: Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes

Fig. 1

A novel bead-based assay model for purified alternative pathway (AP) C5 convertases. a Proposed model for assembly of C5 convertases in the AP. Surface-bound C3 convertase (C3bBb) cleaves multiple C3 molecules into C3b that covalently binds to target surfaces via the reactive thioester (red dot). Association of deposited C3b molecules with the existing C3 convertase gives rise to multimeric complexes (C3b-C3bn) that, together with Bb, can convert C5. The precise arrangement of surface-specific C5 convertases is currently unknown. In the novel C5 convertase assay model described in this study, C3b molecules are site-specifically biotinylated via the thioester and loaded on bacteria-sized streptavidin beads (2.8 μm) to mimic their natural density and orientation on target surfaces. b Loading of streptavidin beads with biotinylated C3b was analyzed by flow cytometry or immunoblotting (below). c C5 convertase activity of C3b-coated beads that were incubated with factor B (FB), factor D (FD) (together needed to form Bb) and C5. Conversion of C5 was determined by measuring release of C5a in the supernatant using a calcium mobilization assay with U937-C5aR cells. Values represent absolute C5a flux (mean fluorescence of stimulated cells subtracted by the mean fluorescence before stimulus). d C5 convertase activity of C3b molecules on beads versus C3b molecules in solution. The amount of C3b molecules in solution was adjusted to the levels of C3b loaded onto the beads (relative C3b-biotin levels) and both were incubated with FB, FD and C5. bd Data of three independent experiments, presented as means ± standard deviation (SD). Immunoblot is a representative of three independent experiments

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