Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein.
Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.
Carreras
Esther
E
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain.
Boix
Ester
E
Rosenberg
Helene F
HF
Cuchillo
Claudi M
CM
Nogués
M Victòria
MV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000235:genetics / Q000494:pharmacology*; D024361:Amino Acids, Basic / Q000235:genetics / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects*; D047091:Eosinophil Granule Proteins; D047208:Eosinophil-Derived Neurotoxin; D004926:Escherichia coli / Q000187:drug effects; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008567:Membranes, Artificial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000494:pharmacology; D012260:Ribonucleases / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000187:drug effects
D024322:Amino Acids, Aromatic; D024361:Amino Acids, Basic; D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D008081:Liposomes; D008567:Membranes, Artificial; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic