A novel interaction between complement product C4a and the receptor for advanced glycation end products (RAGE)

Abstract Complement proteins (C) including their split products play important roles in host defense and homeostasis. Anaphylatoxins C3a and C5a, by binding to their receptors on immune and non-immune cells, mediate cell recruitment and the release of proinflammatory molecules. The receptor and function of C4a remain unclear. C3a, however, in addition to its conventional receptor C3aR, is known to bind to the receptor for advanced glycation end products (RAGE), a receptor implicated in initiating and sustaining inflammatory responses. This interaction can also be mediated by the TLR9 agonist, CpG. C4a is similar to C3a in its structure and net charge. Therefore, we hypothesize that C4a may interact with RAGE. We performed co-immunoprecipitation experiment on 293T cell lysate expressing C4a and RAGE and found that RAGE did not co-immunoprecipitate with C4a, indicating that C4a does not directly interact with RAGE. Using fluorescently labelled C4a protein, we found that there was no significant C4a binding to 293T cells irrespective of RAGE overexpression by flow cytometry. However, by preincubating C4a with CpG, we detected elevated C4a binding to RAGE-expressing 293T cells compared to control cells. These findings suggest that CpG mediates interaction between C4a and RAGE. Our ongoing investigations focus on determining the binding properties and the functional impact on inflammatory response of this novel interaction.