RECOMBINANT HUMAN EOSINOPHIL CATIONIC PROTEIN - RIBONUCLEASE-ACTIVITYIS NOT ESSENTIAL FOR CYTOTOXICITY

Eosinophil cationic protein (ECP) is a toxin secreted by activated hum an eosinophils that has anti-parasitic, antibacterial, and neurotoxic activities; ECP also has ribonuclease activity and structural homology to other mammalian ribonucleases. To determine the relationship betwe en the ribonuclease activity and cytotoxicity of ECP, a method for pro ducing recombinant ECP (rECP) in a prokaryotic expression system was d evised. Periplasmic isolates from induced bacterial transfectants cont ained enzymatically active rECP; micromolar concentrations of rECP wer e shown to be toxic for Staphylococcus aureus (strain 502A). In contra st; recombinant eosinophil-derived neurotoxin, with 67% amino acid seq uence identity to ECP, had little to no toxicity for S. aureus; these findings are analogous to those obtained with purified, granule-derive d ECP and eosinophil-derived neurotoxin. Two single base pair mutation s were introduced into the coding sequence of rECP (Lys(38) to Arg and His(128) to Asp) to convert ribonuclease active-site residues into no n-functional counterparts. These mutations eliminated the ribonuclease activity of rECP but had no discernible effect on the antibacterial a ctivity of this protein, demonstrating that ribonuclease activity and cytotoxicity are, in this case, independent functions of ECP.