C5aR-dependent cell activation by physiological concentrations of C5adesArg: Insights from a novel label-free cellular assay

摘要

The complement anaphylatoxins C3a, C5a, and C5a^desArg play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3aR and C5aR. The role of C5a^desArg in inducing cell activation has been often neglected, since the affinity of C5a^desArg for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5a^desArg to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5a^desArg induce significant levels of cell activation that are even higher than that achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, since it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of Gα proteins upon C5aR engagement by C5a or C5a^desArg. Further, mass spectrometric characterization of plasma-derived C5a and C5a^desArg provided important insight into the post-translational modification pattern of these anaphylatoxins, which includes glycosylation at Asn⁶⁴ and partial cysteinylation at Cys²⁷. While the context-specific physiological contribution of C5a^desArg has to be further explored, our data suggest that C5a^desArg acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.