Journal: eLife
Article Title: The allosteric modulation of complement C5 by knob domain peptides
doi: 10.7554/eLife.63586
Figure Lengend Snippet: ( A ) shows an abridged schematic for classical pathway (CP) activation. Following activation of C1q via antibody Fc, C4 and C2 are cleaved and form C4bC2a (the CP C3 convertase) which cleaves C3 into C3a (not shown) and C3b. At high C3b concentrations, C4bC2aC3b (the CP C3 convertase) forms and cleaves C5 into C5a and C5b. C5b associates with C6 and forms the membrane attack complex (MAC) with C7, C8, and multiple copies of C9. ( B ) shows an abridged schematic for surface phase alternative pathway (AP) activation in assays (where generation of C3b from the CP/LP is virtually excluded); tick-over of C3 generates C3a (not shown) and C3b. In the presence of factor B and factor D, C3bBb (the AP C3 convertase) generates additional C3b, prompting formation of C3bBbC3b (the AP C5 convertase), which cleaves C5 into C5a and C5b, driving MAC formation. CP-driven ELISAs ( C ) and AP-driven ELISAs ( D ) are shown. For both pathways, the inhibition of C3d (the surface-associated domain of C3b, which is upstream of C5 inhibition), C5a release, and C5b neo-epitope formation and C9 deposition were tracked within the MAC. Haemolysis assays with sheep erythrocytes, for the CP ( E ), and rabbit erythrocytes, for the AP ( F ), show that K57 is a potent and efficacious inhibitor of both pathways. K92 is selective, partial antagonist of the AP, while K8 is a weak antagonist of the CP but did not show efficacy in the AP haemolysis assay, below 10 µM. For the AP assays, 5% serum (v/v) gives a putative C5 concentration of 20 nM. For the CP assays, 1% serum (v/v) gives a putative C5 concentration of 4 nM, based on a reported C5 serum concentration of 397 nM/75 µg/mL .
Article Snippet: Complement activation was assessed through detection of deposited complement activation factors using specific antibodies against C3b (rat anti-human C3d HM2198, Hycult, Uden, The Netherlands) and C9 (goat anti-human C9, A226, Complement Technologies Tyler, TX) at a 1:1000 dilution.
Techniques: Activation Assay, Inhibition, Haemolysis Assay, Concentration Assay
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