Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8

Human interleukin 8 (hIL-8), a neutrophilactivating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophildriven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seventransmembrane helical G proteincoupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their Ntermini resulted in fulllength hIL-8 and the specifically carboxyfluoresceinlabelled analogue [K-69(CF)]hIL-8(1-77). [K-69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the Cterminus of IL-8 is accessible to solvent in the receptorbound state. Thus, we introduce here a powerful approach that allows the sitespecific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.