The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations.
The chemokines are a family of immune mediators involved in a wide range of inflammatory processes, most importantly as chemoattractants of monocytes, neutrophils, lymphocytes, and fibroblasts to sites of inflammation. Nuclear magnetic resonance and x-ray crystallographic studies have shown that IL-8 and macrophage-inflammatory protein-1 beta (MIP-1 beta) form noncovalent dimers and that platelet factor-4 (PF-4) forms noncovalent dimers and tetramers, leading to the assumption that, as a family, the chemokines would form multimeric structures. In this study, we analyze the association states of the chemokines IL-8, monocyte chemoattractant protein-1 (MCP-1), and I-309, by using a combination of size exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical cross-linking. We find that the association states of MCP-1 and IL-8 are characterized by an equilibrium between monomers and dimers: although dimers predominate at concentrations above 100 microM, these chemokines are almost exclusively monomeric at the nanomolar concentrations at which they display maximal chemotactic activity. I-309, by contrast, remains a monomer at all concentrations tested. I-309 contains two additional cysteine residues (C26 and C68) that are not found in any other members of the chemokine family. We used cyanogen bromide and trypsin digestion strategies to demonstrate that these two residues are linked in a unique intramolecular disulfide bond. Furthermore, by using site-directed mutagenesis, we show that the integrity of this bond is crucial for protein secretion.
Paolini
J F
JF
Department of Immunology, Duke University Medical Center, Durham, NC 27710.
Willard
D
D
Consler
T
T
Luther
M
M
Krangel
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D002460:Cell Line; D018932:Chemokine CCL2; D019742:Chemokines, CC; D002630:Chemotactic Factors / Q000737:chemistry*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004220:Disulfides; D016209:Interleukin-8 / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014162:Transfection / Q000379:methods; D014357:Trypsin; D014461:Ultracentrifugation
D001704:Biopolymers; D018932:Chemokine CCL2; D019742:Chemokines, CC; D002630:Chemotactic Factors; D003432:Cross-Linking Reagents; D004220:Disulfides; D016209:Interleukin-8; D014357:Trypsin; D003488:Cyanogen Bromide