Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal α-helix were synthesized using 9- fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (Δ24-CCL15) converts the slightly active 92-residue Δ0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from Δ24-CCL15 to Δ29-CCL15, and re-increases from Δ29-CCL15 to Δ30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for Δ24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal α-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Quantum proteolytic activation of chemokine CCL15 by neutrophil granulocytes modulates mononuclear cell adhesiveness

Monocyte infiltration into inflammatory sites is generally preceded by neutrophils. We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreactivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (t R) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce Δ23 and Δ26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a Δ21 isoform. Compared with full-length CCL15, Δ23 and Δ26 isoforms displayed a significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes and to induce adhesiveness of mononuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteoliytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G. Copyright © 2005 by The American Association of Immunologists, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe