CHARACTERIZATION OF CHEMICALLY SYNTHESIZED HYBRID CXC CHEMOKINES. Tai Y. Fu, Yu Wai Chen, Yaoguang Luo, Ian Clark-Lewis and Gary D. Brayer, Department of Biochemistry and Molecular Biology, and the Protein Engineering Network of Centres of Excellence, 2146 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Interleukin-8 (IL-8) is a chemoattractant cytokine (chemokine) that stimulates the accumulation and activation of neutrophil leukocytes at sites of inflammation. This small protein (72 residues) has been linked to a wide variety of inflammatory events and is thought to be the causitive agent in their associated immuno-pathology. IL-8 is one of a family of related proteins termed the CXC chemokines that have four conserved cysteines, with the first two separated by one amino acid. Interferon-g inducible peptide-10 (IP-10; 74 residues) is also a member of the CXC chemokine family and is expressed by a variety of cell types on induction with interferon-g and lipopolysaccharide. Although IL-8 and IP-10 share 24% primary sequence identity, they appear to have very different physiological roles. IP-10 does not compete with IL-8 for binding to neutrophil receptors and has unique properties associated with T-cell mediated inflammatory reponses and host-mediated anti-tumor effects. To characterize the components of IL-8 that are responsible for its specific functions, four IL-8/IP-10 molecular hybrids were chemically synthesized in which portions of the sequence of IL-8 were replaced with the corresponding IP-10 sequence to determine the resultant functional and structural changes induced. Two of these hybrid CXC chemokines have activities comparable to wild-type IL-8, while the other two show only low activity. To fully understand the underlying structural principles behind functional activity shifts we have crystallized and solved the structures of all four hybrid proteins. Our results show that the overall polypeptide chain fold for these four hybrid proteins is similar to that of IL-8. However, also apparent are differential side chain interactions that appear to be correlated to observed functional differences. This has lead to a hypothesis as to which portions of the surface of IL-8 are essential for the expression of its activity.