Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Abstract

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␣ (MIP-1␣) 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 [ RANTES is a member of a large family of cytokines, known as chemokines, which have the ability to recruit and activate a wide variety of proinflammatory cell types (1). They are small polypeptides of 8 -10 kDa and have been further classified into CXC or CC chemokines based on the spacings of the cysteine residues proximal to the amino terminus. CXC chemokines primarily activate neutrophils, whereas CC chemokines have effects on several leucocyte cell types. RANTES is a CC chemokine, and in vitro it can produce chemotaxis and activation of monocytes, eosinophils, and T cells, particularly CD4 ϩ CD45RO ϩ (memory) T cells (2), but not neutrophils. These results imply a role for RANTES in diseases such as allergen induced late phase skin reactions or in allergic asthma. This hypothesis is strengthened by the fact that large amounts of RANTES are found in nasal polyp tissues, which are rich in infiltrating eosinophils (3). In addition, injection of RANTES into dog skin has been shown to induce a large eosinophilic infiltrate in vivo (4), and migration of human T lymphocytes was observed on injection of human RANTES into a human/severe combined immune deficiency mouse model (5).MIP-1␣ shares an overlapping cell-type specificity with RANTES in vitro (6, 7) and has been shown to elicit an inflammatory response mediated through mast cell degranulation in vivo (8). A common receptor for these two CC chemokines has been cloned (9, 10) and is a member of the seven transmembrane G-protein linked receptor family. Recombinant expression of the receptor has shown that it can transduce a functional response on stimulation by both chemokines.We report the purification of human RANTES expressed heterologously in Escherichia coli. In this system, the protein retains its initiating methionine residue, which renders it inactive as an agonist, while enabling it to antagonize effects induced both by RANTES and MIP-1␣. It is able to compete for binding of both the radiolabeled ligands on THP-1 cells and to the recombinant RANTES/MIP-1...

Characterisation of the RANTES/MIP‐1α receptor (CC CKR‐1) stably transfected in HEK 293 cells and the recombinant ligands

“…Both proteins were estimated as > 98% pure by SDS-PAGE and reverse phase HPLC analyses. In order to produce the RANTES protein with the correct amino terminus, it was necessary to express the protein with a cleavable leader sequence since retention of the initiating methionine when the cDNA encoding the mature form of the protein is expressed yields an inactive protein [23]). Amino terminal sequencing showed that the initiating methionine was removed from MIP-I~.…”

Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

“…The activation event following ligand tethering is postulated to occur in close proximity to the cell membrane and involve the disruption of interactions between side chains of neighbouring TM helices by the chemokine N terminus [13,14]. Supportive of this postulate, truncation or addition at the chemokine N terminus by one or two amino acids has been shown to produce potent receptor antagonists [21,27] and recently described small molecule agonists of the chemokine receptors CCR3 and CCR8 have been shown to bind within an intrahelical pocket [28,29].…”

“…This pocket is of limited accessibility; indeed, extension of the N terminus of CCL5 by a single methionine generates a potent receptor antagonist [21]. To directly address the activation of CXCR6 by CXCL16, we generated a recombinant protein in Escherichia coli, in which the N terminus of mature CXCL16 was extended by 24 amino acids (Fig.…”

Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

“…In the case of IL-8, the N-terminal residues 4, 5, and 6 are essential for receptor binding and triggering function (35,36). RANTES loses agonistic potency and becomes a potent antagonist of chemokine binding when the first amino acid residue is modified artificially by addition of methionine or treatment with aminooxypentane (30,32,33). The naturally cleaved forms of MCP-2 and RANTES are also devoid of bioactivity (31,34).…”