Amino-terminal processing of chemokine ENA-78 regulates biological activity

Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a pote nt stimulator of neutrophils, inducing a variety of biological responses su ch as chemotaxis, enzyme release, up-regulation of surface receptors, and i ntracellular calcium mobilization. Proteolysis of ENA-78 with cathepsin G a nd chymotrypsin yielded a time-dependent increase in elastase-releasing act ivity, predicting the formation of truncation products with higher potency than native ENA-78. To investigate the biological implications of progressi ve truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E. coli. When tested in the neutrop hil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3- fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold le ss potent. In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fo ld and ENA(9-78) a 2-fold higher potency than native ENA-78, ENA(10-78), co nversely, was 10-fold less potent, but reached a comparable efficacy to ENA -78 at 10(-7) M concentration. In summary, the rank order in potency with r espect to elastase release was ENA(9-78) 1 ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78), V ariant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency a t concentrations of 1-100 nM. The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N- terminal proteolysis during inflammatory processes in vivo.