Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist.
Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.
Proudfoot
A E
AE
Glaxo Institute for Molecular Biology, Geneva, Switzerland. AEP6830@ggh.uk.co
Power
C A
CA
Hoogewerf
A J
AJ
Montjovent
M O
MO
Borlat
F
F
Offord
R E
RE
Wells
T N
TN
D018946:Chemokine CCL5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007457:Iodine Radioisotopes; D008715:Methionine / Q000378:metabolism*; D011869:Radioligand Assay; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D018946:Chemokine CCL5; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; C080633:macrophage inflammatory protein 1alpha receptor; D008715:Methionine