Anaphylatoxins:From Supposed Toxin Anaphylactics to Effective Mediators of the Early Events of Inflammation

This review updates original data describing the experiments showing the complement origin of anaphylatoxins unexplainable submerged under the surface of the articles related to the subject. Next, recalls subsequent data describing the anaphylatoxins peptide nature and sequences, the cell receptors with which they interact and activate and the outcome of the cell responses. The review continues by highlighting the anaphylatoxin biological properties focusing on the unequivocal participation of these mediators in inflammation. The review concludes bringing data reinforcing the promising use of these peptides as molecular primers to create specific and efficient pharmacological antagonists.

Synergistic interaction between C5a and NOD2 signaling in the regulation of chemokine expression in RAW 264.7 macrophages

The innate immune response is a complex process involving multiple pathogen-recognition receptors, including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. Complement is also a critical component of innate immunity. While complement is known to interact with TLR-mediated signals, the interactions between NOD-like receptors and complement are not well understood. Here we report a synergistic interaction between C5a and Nod2 signaling in RAW 264.7 marophages. Long-term treatment with muramyl dipeptide (MDP), a NOD2 ligand, enhanced C5a-mediated expression of chemokine mRNAs in RAW 264.7 cells. This response was dependent on NOD2 expression and was associated with a decrease in expression of C5L2, a receptor for C5a which acts as a negative modulator of C5a receptor (C5aR) activity. MDP amplified C5a-mediated phosphorylation of p38 MAPK. Treatment of RAW264.7 cells with an inhibitor of p38 attenuated the synergistic effects of C5aon MDP-primed cells on MIP-2, but not MCP-1, mRNA. In contrast, inhibition of AKT prevented C5a stimulation of MCP-1, but not MIP-2, mRNA, in MDP-primed cells. Taken together, these data demonstrated a synergistic interaction between C5a and NOD2 in the regulation of chemokine expression in macrophages, associated with a down-regulation of C5L2, a negative regulator of C5a receptor activity.

Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases;but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists;for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.

Chemical synthesis and racemic crystallization of rat C5a-desArg

The deletion of the C-terminal arginine of the anaphylatoxin protein C5a reduces it receptor binding affinity.Understanding how C-terminal arginine affects the structure and bioactivity of C5a is important for the development of C5a C-terminal mimics as drug candidates.Herein,we report the total chemical synthesis of rat C5a and its D-enantiomer with its C-terminal arginine deleted,namely L-rC5a-desArg and D-rC5a-desArg.The structure of rC5a-desArg was then determined by racemic crystallography for the first time.The C-terminal residues of rC5a-Arg were found to expand from the fourth helix in a continuous helical confo rmation.This C-terminal conformation is significantly different from that of the previously reported full-length of C5a,indicating that the deletion of C-terminal arginine residue could result in the destruction of a positively charged surface formed by two adjacent Arg residues in C5a.