Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution

Abstract

Skp derived from Escherichia coli attracts leukocytes as a pure chemotactic ligand of the C5a receptor. We identified the submolecular region of Skp that binds and activates the C5a receptor to be -Gln103-Asp104-Arg105- using synthetic peptide fragments and site-directed mutants of Skp. As the C5a amino acid residue equivalent to Gln103 of Skp is Leu72, we prepared a Gln103Leu-Skp mutant as a recombinant protein. With this mutation, Skp gained secretagogue functions including induction of the respiratory burst and granule release reactions and leukotriene generation, in addition to the chemoattraction displayed by C5a. However, when we substituted Leu72 with Gln in C5a, the L72Q-C5a mutant largely lost its secretagogue function. These functional conversions were reproduced using synthetic peptides mimicking the receptor-binding/-activating regions of the recombinant proteins. Receptor-binding assays using the mimicking peptides demonstrated only a small difference between the Leu72-C5a and Gln72-C5a peptides. Consistently, L72Q-C5a apparently antagonized C5a secretagogue function. These results indicate that the difference between a chemotactic response and a combined chemotactic/secretory response can be attributed not to the nature of the receptor but to guidance by the ligand, at least in the case of C5a receptor-mediated leukocyte responses.

The Chemokine Receptor CXCR2 Ligand KC (CXCL1) Mediates Neutrophil Recruitment and Is Critical for Development of Experimental Lyme Arthritis and Carditis

“…However, despite success in animal models, clin- (8,35,52). Although this may be attributable to the perceived promiscuity and redundancy of chemokine receptors and their ligands, it was recently reported that a single amino acid change in a chemoattractant ligand could alter the functional outcome of binding to a single receptor (25). Further studies are necessary to define the functional in vivo specificity of chemokine ligand/receptor interactions.…”

Inhibitory effects of C4a on chemoattractant and secretagogue functions of the other anaphylatoxins via Gi protein-adenylyl cyclase inhibition pathway in mast cells

“…Recently, we found that C5a, mutated at Leu72 to Gln, maintained its chemoattractant capacity but lost its secretagogue capacity in the context of neutrophil stimulation. Additionally, we found that cytoplasmic Ca 2+ influx was absent during the chemotactic response induced in neutrophils exposed to Leu72Gln-C5a [15]. These data suggest that while C5a and Leu72Gln-C5a both ligate the C5a receptor and induce chemotactic migration in neutrophils, the intracellular signaling pathways differ between these ligands.…”

“…Although Leu72Gln mutation of C5a allows this protein to maintains its chemoattractant properties, this mutation abolishes its secretagogue and Ca 2+ mobilization capacities in the context of neutrophil stimulation [15]. Here, we examined the properties of this mutant in the context of HMC-1 cell stimulation.…”

“…Recombinant C5a and Leu72Gln-C5a were prepared as described previously [15,17]. Recombinant C3a and C4a were prepared in a manner similar to C5a.…”

New aspects of the C5a receptor

“…We suggested another role of find-me signals in the initiation phase. Their receptors are commonly coupled with Gαiβγ protein (GiPCRs) and/or Gαqβγ protein (GqPCRs), which are classical but not pure chemoattractant receptors [6]. Classical chemoattractant receptors commonly mediate chemotaxis and secretion of at least macrophages and neutrophils, signaling through downstream extracellular signal-regulated kinase 1/2 (ERK1/2), and leading to an enhancement in cytoplasmic calcium mobilization.…”