C5aR-dependent cell activation by physiological concentrations of C5a-desArg: Insights from a novel label-free cellular assay

Abstract

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 G α 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 Asn 64 and partial cysteinylation at Cys 27 . 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.

Structural insights into ligand-recognition, activation, and signaling-bias at the complement C5a receptor, C5aR1

“…Interestingly, previous studies have suggested that the removal of terminal arginine (Arg 74 ) in C5a in-vivo by the action of a carboxypeptidase yields C5a des-Arg , and it displays significantly reduced binding affinity and potency at C5aR1 when tested using recombinant ligand 10,16,25,26 . The extensive interaction of Arg 74 in C5a with multiple residues in C5aR1 such as Tyr259 6.51 , Gly263 6.55 , Ile266 6.58 and Val287 7.38 , which are critical for stabilizing carboxyl-terminal conformation of C5a in the orthosteric pocket, and will be absent in case of C5a des-Arg , may help rationalize its lower potency at the receptor (Figure S10B).…”