title: Role of unique basic residues in cytotoxic, antibacterial and antiparasitic activities of human eosinophil cationic protein.
abstract: Eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin are members of the RNase A superfamily, which play a crucial role in host defense against various pathogens as they are endowed with several biological activities. Some of the biological activities possessed by ECP have been attributed to its strong basic character. In the current study, we have investigated the role of five unique basic residues, Arg22, Arg34, Arg61, Arg77 and His64 of ECP in its catalytic, cytotoxic, antibacterial and antiparasitic activities. These residues were changed to alanine to generate single and double mutants. None of the selected residues was found to be involved in the RNase activity of ECP. The substitution of all five residues individually was detrimental for the cytotoxic, antibacterial and antiparasitic activities of ECP; however, mutation of Arg22 and Arg34 resulted in the most significant effects. The double mutants also had reduced biological activities. All ECP mutants that had significantly reduced toxicity also had reduced membrane destabilization activity. Our study demonstrates that Arg22, Arg34, Arg61, Arg77 and His64 of ECP are crucial for its membrane destabilization activity, which appears to be the underlying mechanism of its cytotoxic, antibacterial and antiparasitic activities.
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Anubha
authors.initials: A
authors.lastname: Singh
authors.forename: Janendra K
authors.initials: JK
authors.lastname: Batra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000977:Antiparasitic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D055162:Biocatalysis; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D047150:Eosinophil Cationic Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
chemical_list: D000900:Anti-Bacterial Agents; D000977:Antiparasitic Agents; D003603:Cytotoxins; D047150:Eosinophil Cationic Protein