CXCL8((3-73))K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8.
We recently reported that CXCL8((3-73))K11R is a high affinity agonist of neutrophil activation and chemotactic responses. In this report we employed CXCL8((3-73))K11R as a template to generate CXCL8/IL-8 analogues with antagonist activities, using site-directed mutagenesis to introduce conservative amino acid substitutions into the first turn within the molecule's beta-pleated sheet region (G31P, P32G) and, in association with these, into the putative receptor-recognition site (T12S, H13F, F17S). We then examined their impact on the analogues' biological activities and found that a G31P substitution rendered CXCL8((3-73))K11R a high affinity antagonist of CXCL8/IL-8. The ranking (in the order of decreasing CXCL8/IL-8 antagonist activities) of the CXCL8((3-73))K11R analogues we generated was, G31P>T12S/G31P>H13F/G31P>T12S/H13F/G31P>>P32G approximately T12S/P32G approximately H13F/P32G>T12S/H13F/P32G; CXCL8((3-73))K11R/F17S did not inhibit CXCL8/IL-8-dependent responses. CXCL8((3-73))K11R/G31P had no discernible agonist (beta-glucuronidase release, chemotactic) activity, but at 12.5 ng/ml it bound to purified neutrophils more avidly than did 1.25 microg/ml CXCL8/IL-8. Furthermore, CXCL8((3-73))K11R/G31P competitively antagonized the binding of CXCR1- and CXCR2-specific antibodies to these receptors. Taken together, these data thus provide further impetus to the study of the potential efficacy of CXCL8((3-73))K11R/G31P as a broad-spectrum antagonist of the ELR-CXC chemokines in experimental and clinical settings.
Li
Fang
F
Department of Veterinary Microbiology, 52 Campus Dr., University of Saskatchewan, Saskatoon, SK, Canada.
Zhang
Xiaobei
X
Gordon
John R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019743:Chemokines, CXC / Q000737:chemistry / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D023062:Receptors, Interleukin-8A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D023063:Receptors, Interleukin-8B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016415:Sequence Alignment
C457773:CXCL8(3-73)K11R,G31P, bovine; D019743:Chemokines, CXC; D016209:Interleukin-8; D008024:Ligands; D010446:Peptide Fragments; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B