title: Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity.
abstract: Eosinophil cationic protein (ECP) is a toxin secreted by activated human 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 between the ribonuclease activity and cytotoxicity of ECP, a method for producing recombinant ECP (rECP) in a prokaryotic expression system was devised. Periplasmic isolates from induced bacterial transfectants contained enzymatically active rECP; micromolar concentrations of rECP were shown to be toxic for Staphylococcus aureus (strain 502A). In contrast, recombinant eosinophil-derived neurotoxin, with 67% amino acid sequence identity to ECP, had little to no toxicity for S. aureus; these findings are analogous to those obtained with purified, granule-derived ECP and eosinophil-derived neurotoxin. Two single base pair mutations were introduced into the coding sequence of rECP (Lys38 to Arg and His128 to Asp) to convert ribonuclease active-site residues into non-functional counterparts. These mutations eliminated the ribonuclease activity of rECP but had no discernible effect on the antibacterial activity of this protein, demonstrating that ribonuclease activity and cytotoxicity are, in this case, independent functions of ECP.
authors.affiliation: Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: H F
authors.initials: HF
authors.lastname: Rosenberg
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001798:Blood Proteins / Q000494:pharmacology* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D003603:Cytotoxins / Q000494:pharmacology / Q000633:toxicity; D047091:Eosinophil Granule Proteins; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology* / Q000633:toxicity; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D012260:Ribonucleases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D003603:Cytotoxins; D047091:Eosinophil Granule Proteins; D018836:Inflammation Mediators; D011994:Recombinant Proteins; D012260:Ribonucleases