Protein Science, Vol 6, Issue 3 609-617, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

IL-8 single-chain homodimers and heterodimers: Interactions with the chemokine receptors CXCR1, CXCR2, and DARC

S. R. LEONG, H. B. LOWMAN, J. LIU, S. SHIRE, L. E. DEFORGE, B. L. GILLECE-CASTRO, R. MCDOWELL and C. A. HEBERT
Departments of Immunology, 460 Point San Bruno Boulevard, South San Francisco, California 94080

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or {beta}-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is functional in the single-chain variants.
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