Structural and functional characterisation of properdin and properdin-binding complement inhibitors from ticks

The complement system is a major effector of the immune response which can lead to opsonisation and inflammation, as well as the removal of immune complexes and damaged self-cells. The complement system requires many regulatory proteins to prevent inappropriate activation towards healthy self-cells and to only target invading pathogens. This thesis focuses on a complement regulator/activator called properdin, which has primarily been associated with the alternative pathway (AP). Properdin functions by stabilising the AP C3 convertase (C3bBb). In this thesis, I investigated the molecular basis of AP inhibition by a novel set of properdin-binding complement inhibitors from the salivary glands of the tick Rhipicephalus pulchellus, CIRp-A1 and homologs. These proteins were recombinantly expressed in insect and bacterial systems for functional and structural characterisation. ELISA-based complement activation assays showed that CIRp-A1 specifically inhibits the AP and has an IC50 of 13 nM. AP haemolysis assays using different animal sera showed that CIRp-A1 and homologs have different inhibitory activity profiles in different animals. To understand the molecular basis of host specificity, crystal structures of CIRp-A1 and homologs were solved. The structures of these inhibitors, in combination with mutagenesis studies of CIRp-A1, suggested that a loop contributes to the inhibitory activity of CIRp-A1. Pulldown assays using CIRp-A1 and mammalian cell-expressed recombinant properdin suggested that CIRp-A1 binds to the final domain of properdin (TSR6), which had been implicated in C3b-properdin interaction. Pulldown assays using C3b showed that CIRp-A1 inhibits the binding of properdin to C3b and displaces properdin already bound to C3b. By binding to TSR6, CIRp-A1 might sterically hinder the interaction between properdin and C3b, or change the conformation of properdin such that it can no longer interact with C3b. The exact molecular mechanism behind properdin inhibition by CIRp-A1 remains to be elucidated in future studies, particularly with structural studies of the properdin-CIRp-A1 complex.