Fig. 1

Mechanisms of FH in the complement regulation, illustration  of FH variant structures and in vitro efficacy evaluation. GAG: Glycosaminoglycans, SA: Sialic acid, FH: Factor H, Bb: C3-binding fragment of complement factor B, FI: Factor I, FD: Factor D, Cr1: Complement C3b/C4b Receptor 1, CP: Classical Pathway, AP: Alternative Pathway, LP: Lectin Pathway. A The complement system is activated by three pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). These pathways converge in an amplification loop of the AP through the formation of C3b. FH binds to C3b and prevents the interaction of FB and its activated cleavage product Bb, thereby interrupting the amplification loop. This is known as the decay acceleration function because the C3 convertase (C3bBb) is not formed. In addition, FH acts as a cofactor for the serine protease FI that facilitates the breakdown of C3b into smaller cleavage products (iC3b, C3f). This process is crucial for the complete degradation of C3b by FI and its cofactors, such as CR1. B FH comprises of 20 CCP domains, with CCP1-4 and CCP19-20 mediating C3b binding. Binding to cell surfaces via glycosaminoglycans (GAG) primarily occurs through the CCP7 and CCP20 domains. The truncated FH variants used, consist of either the FH domains CCP1-4 and CCP19-20, which are connected by a glycine linker (FH1-4^19-20) or the domains CCP1-7 and CCP19-20 (FH1-7^19-20). Both truncated FH variants have a c-terminal signaling peptide for secretion and an n-terminal epitope (myc) tag. C FH inhibits the formation of C3 convertase (C3bBb) and accelerates C3bBb decay by binding the CCP1-4 domain to C3b and displacing Bb (upper part). Both FH1-4^19-20 and FH1-7^10–20 are capable of performing this FH function (lower part). Additionally, FH anchors to the cell surface through GAG binding, with FH1-4^19-20 having one such binding site and FH1-7^19-20 having two. D FH1-4^19-20 and FH1-7^19-20 were expressed and secreted by HEK293 cells. Both truncated FH variants were found with their predicted size in Western blot. EGFP was predominantly detected in cell lysates, corroborating that the FH1-4^19-20 and FH1-7^19-20 proteins were secreted and not released due to cell ruptures. Original documentation of the blots is added as Additional file 1: Fig. S1. E Upon addition of truncated FH variants to mouse serum, reduced C3b deposition was observed on AP-activating lipopolysaccharide-coated microtiter plates. This indicates that the truncated FH variants can mediate the decay acceleration function of FH. Each data point represents the mean ± SEM for n = 2 (biological replicates, shown is one of two technical replicates with similar results). F The evaluation of GAG binding revealed that FH1-7^19-20 exhibited the strongest binding affinity for immobilized heparin. In comparison to FH1-7^19-20, the reference miniFH of [57] demonstrated a lower binding capacity. FH1-4^19-20 did not show any interactions, likely due to a myc tag induced blockade in the CCP20 GAG binding domain. Each data point represents the mean ± SEM for n = 2 (biological replicates, shown is one of two technical replicates with similar results)