Biological Activity of the Growth Factor-Induced Cytokine N51: Structure-Function Analysis Using N51/Interleukin-8 Chimeric Molecules

Abstract

The immediate-early gene N51/KC encodes a protein which following expression in the baculovirus system and purification to apparent homogeneity is able to induce chemotaxis and intracellular Ca²⁺ flux, to compete for ¹²⁵I-labeled interleukin-8 (IL-8) binding, and upon iodination, to bind specifically to human neutrophils. The activity of N51/KC can be distinguished from that of IL-8 by a number of criteria. First, at equivalent concentrations, the specific binding of [¹²⁵I]N51/KC to human neutrophils is about 10 times less than that of [¹²⁵I]IL-8. Second, the competition studies of [¹²⁵I]IL-8 with IL-8 define a single class of high-affinity receptors, while the presence of both a high- and a low-affinity class of receptors is defined by N51/KC. Third, although the changes in intracellular Ca²⁺ of fura-2/AM-preloaded human neutrophils elicited by N51/KC and IL-8 are similar, pretreatment of the cells with N51/KC did not result in a loss of response to a subsequent treatment with IL-8; in contrast, treatment with IL-8 did result in the subsequent desensitization to N51/KC. To further characterize N51/KC, mutants and hybrids of N51/KC and IL-8 were produced and analyzed for the ability to compete for [¹²⁵I]IL-8 binding and elicit intracellular Ca²⁺ changes in human neutrophils. Two important observations came from these studies. First, the N51/IL-8I hybrid in which the N51/KC sequence between cysteines 2 and 3 (or first disulfide bond) is replaced by the corresponding sequence in IL-8 shows IL-8-like properties, indicating that this region is important for specific receptor recognition. Second, the N51ΔIII and IL-8ΔIII C-terminus deletion mutants were biologically inactive, but the hybrid molecules N51/IL-8III and IL-8/N51III, in which the C termini were exchanged, had biological activities similar to that of the wild-type molecules, demonstrating that the presence of the C terminus is essential for the biological activity of these chemokines but does not confer receptor specificity.