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A molecular switch of chemokine receptor selectivity. Chemical modification of the interleukin-8 Leu25 --> Cys mutant.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1996 Feb 09; Vol. 271 (6), pp. 3148-53. - Publication Year :
- 1996
-
Abstract
- Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu25 to cysteine. The protein folds correctly with two disulfide bonds and a free thiol group at Cys25. This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor binding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leucine. However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2-N-methyl, N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminoethyl)acetamido (NBD) group lowers potency in neutrophil chemotaxis and affinity in IL-8 receptor binding assays by 2 orders of magnitude. This Leu25 --> Cys-NBD mutant introduces monocyte chemoattractant activity and the ability to displace 125I-labeled macrophage inflammatory protein-1 alpha from the recombinant CC CKR-1 receptor. Additionally, we show a specific interaction between the fluorescent mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specificity between CXC and CC chemokines. Circular dichroism spectra of the IL-8 mutants having CC chemokine-like activity show a consistent drop in alpha-helical content compared with the spectra for wild-type IL-8. This suggests that distortion of the C-terminal helix may play a role in chemokine receptor-ligand selectivity.
- Subjects :
- 4-Chloro-7-nitrobenzofurazan
Amino Acid Sequence
Base Sequence
Binding Sites
Binding, Competitive
Chemokine CCL5 pharmacology
Chemotaxis, Leukocyte drug effects
Cloning, Molecular
Cysteine
DNA Primers
HL-60 Cells
Humans
Interleukin-8 chemistry
Interleukin-8 pharmacology
Molecular Sequence Data
Monocytes physiology
Mutagenesis, Site-Directed
Neutrophils drug effects
Neutrophils physiology
Point Mutation
Polymerase Chain Reaction
Protein Structure, Secondary
Receptors, Interleukin-8A
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Recombinant Proteins pharmacology
Substrate Specificity
Transfection
Antigens, CD metabolism
Interleukin-8 metabolism
Leucine
Receptors, Interleukin metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 271
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8621714
- Full Text :
- https://doi.org/10.1074/jbc.271.6.3148