Mutation of Leu25 and Val27 introduces CC chemokine activity into interleukin-8.
Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu25 in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val27 residue, which points out from the same side of the beta-sheet as Leu25. In RANTES, Val27 is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu25-->Tyr, Val27-->Tyr and the double mutant Leu25-->Tyr, Val27--> Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [125I]IL-8 competition assays. Additionally, the Leu25-->Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [125I]MIP-1 alpha by IL-8 Leu25-->Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1 alpha ligand with an affinity only 12-fold less than MIP-1 alpha itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.
Lusti-Narasimhan
M
M
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
Power
C A
CA
Allet
B
B
Alouani
S
S
Bacon
K B
KB
Mermod
J J
JJ
Proudfoot
A E
AE
Wells
T N
TN
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D018946:Chemokine CCL5; D002630:Chemotactic Factors / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000235:genetics; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins; D009154:Mutation; D014633:Valine / Q000235:genetics / Q000378:metabolism*
D018946:Chemokine CCL5; D002630:Chemotactic Factors; D016207:Cytokines; D018076:DNA, Complementary; D016209:Interleukin-8; D008222:Lymphokines; D018945:Monocyte Chemoattractant Proteins; D007930:Leucine; D014633:Valine; D002118:Calcium