Supported lipid bilayer platforms for investigating complement activation, termination and regulation
Yorulmaz Avsar, Saziye
Date of Issue2017-05-12
School of Materials Science and Engineering
The complement system serves as the frontline of host defense for the human body against foreign entities, and is triggered via one or more of several signaling cascades that consist of interactions between soluble and cell surface-bound complement proteins. In this thesis, the objective is to explore how lipid membranes influence the initiation, amplification, termination and regulation of complement activation, utilizing supported lipid bilayer platforms as a model experimental system. The overall hypothesis of this work is that the supported lipid bilayer platform provides a useful model system to recapitulate key protein-lipid and protein-protein interactions involved in complement activation at lipid membrane interfaces, thereby establishing an experimental framework to decipher key trigger factors (e.g., membrane surface charge) as well as evaluate candidate inhibitors acting against complement convertases and membrane attack complex assemblies. Within this scope, four different experimental studies were conducted, leading to new insights into the mechanisms by which lipid membranes influence complement activation. In the first study, the influence of membrane surface change on the adsorption properties of different pattern recognition proteins involved in initiation of complement activation was investigated. In the second study, the effects of covalent and noncovalent tethering strategies on the self-assembly of the alternative pathway C3 convertase was monitored. It was identified that the C3b immobilization scheme is a critical factor governing convertase assembly, further enabling successful evaluation of a clinically relevant complement inhibitor, compstatin. In the third study, the assembly of the complement-terminating membrane attack complex was monitored onto supported lipid bilayer platforms, with particular attention to the influence of membrane surface charge. It was determined that the membrane attack complex assembly preferentially forms on negatively charged lipid membranes, indicating the importance of membrane electrostatics. In the fourth study, the inhibitory action of vitronectin and clusterin against the membrane attack complex assembly was evaluated, offering insight into the different mechanisms by which these two regulatory proteins inhibit complement activation. In summary, the findings presented in this thesis significantly contribute to the fundamental understanding of complement activation occurring at lipid membrane interfaces, and offer a new platform to characterize the mechanisms of action for a wide range of complement inhibitors including small molecules, antibodies, and peptides.