Oligomerisation of the complement regulator, properdin

The complement system provides protection against invading pathogens through lysis, opsonisation and by triggering the inflammatory immune response. The alternative pathway, one of the three activation pathways of the complement system, activates spontaneously on the surfaces of pathogen and depends on regulators to protect host cells. Properdin is the only positive regulator of complement. It stabilises a key enzyme called the alternative pathway C3 convertase, extending its half-life by up to 10-fold. Properdin consists of seven thrombospondin type-I repeat (TSR) domains and circulates as dimers, trimers, and tetramers of polypeptides connected to each other via head-to-tail contacts. Properdin oligomerisation is necessary for function.

The aim of this project is to understand how properdin oligomerises. To address this question, the full-length properdin as well as truncated fragments were produced in both mammalian and bacterial expression systems. The results show that the full-length properdin produced in Chinese hamster ovary cells oligomerises in the same way as the native protein. A truncated form comprising a fusion of TSR-0 and TSR-6, the putative oligomerisation TSR domains, dimerises, confirming that these domains probably mediate oligomerisation in the full-length protein. However, when this fragment is expressed in E. coli, which lacks the enzymes responsible for post-translational modifications, the truncated form is monomeric, suggesting that one or more of the modifications that occur after synthesis are necessary for self-association. Analysis of these post-translational modifications suggests that C-mannosylation, a rare modification often found in proteins containing thrombospondin type-I repeat domains, is essential for the efficient oligomerisation of properdin. A model is proposed to explain how C-mannosylation promotes self-association of properdin polypeptides.