LIMITED AND DEFINED TRUNCATION AT THE C-TERMINUS ENHANCES RECEPTOR-BINDING AND DEGRANULATION ACTIVITY OF THE NEUTROPHIL-ACTIVATING PEPTIDE-2 (NAP-2) - COMPARISON OF NATIVE AND RECOMBINANT NAP-2 VARIANTS

We have previously described a C-terminally truncated variant of the chemokine neutrophil-activating peptide 2 (NAP-2) that exhibited higher neutrophil-stimulating capacity than the full-size poly peptide. To investigate the impact of the NAP-2 C terminus on biological activity and receptor binding, we have now purified the novel molecule to homogeneity. Furthermore, we have cloned, expressed in Escherichia coli, and purified full-size recombinant NAP-2 (rNAP-2-(1-70)) and a series of C-terminally deleted variants (rNAP-2-(1-69) to rNAP-2-(1-64)), Biochemical and immunochemical analyses revealed that the natural NAP-2 variant was structurally identical to the rNAP-2-(1-66) isoform, As compared with their respective native and recombinant full-size counterparts, both molecules exhibited similar to 3-4-fold enhanced potency in the induction of neutrophil degranulation as well as 3-fold enhanced binding affinity for specific receptors on these cells. All other variants were considerably less active. The natural occurrence of a NAP-2 variant truncated by exactly four residues at the C terminus suggests that limited and defined proteolysis at this site plays a role in the regulation of the biological function of the chemokine.