Native chemical ligation (NCL) has been used to synthesize full-length human complement protein C3a, specifically modified at the N-terminus by appending diethylenetriaminepentaacetic acid (DTPA) that is a good chelator of europium. The human C3a sequence was divided into three 20-30 residue fragments: DTPA−C3a[1−22]−NHNH<sub>2</sub> (1), Thz-C3a[24-48]-COSR (2), and H−C3a[49−77]−OH (3), which were individually prepared on resin by standard Fmoc−solid phase peptide synthesis (SPPS) using a peptide synthesizer. Full assembly of reduced C3a was accomplished by consecutively joining the three polypeptides in the C- to N- direction. After folding and chelation of Europium, the europium labelled product (Eu−DTPA−hC3a) was obtained. Time-resolved fluorescence analysis has demonstrated that Eu−DTPA−hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. The potency of Eu−DTPA−hC3a was further validated by competitive binding experiments and used to measure affinities of potent C3aR-specific agonists (TR16, BR103) and antagonists (SB290157, BR111) via displacement of Eu−DTPA−hC3a from hC3aR. The macromolecular conjugate Eu−DTPA−hC3a is a novel nonradioactive probe suitable for studying ligand−C3aR interactions and discovering high affinity ligands for C3aR. Antagonists of this receptor show potent anti-inflammatory activity via C3aR on human and rodent cells and in animal models of disease.

Chemical synthesis and applications of a novel fluorescent probe for human complement C3a receptor

256th National Meeting (2018)

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