Abstract
Human eosinophil cationic protein (hECP) is a basic and cytotoxic granular protein released from activated eosinophils. ECP interacts with cellular surface heparan sulfate proteoglycans (HSPGs) and internalizes into cells through lipid raft-associated macropinocytosis. Three heparin binding regions (HBRs) on ECP, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), have been recently identified. In this study, binding energy of amino acids interacting with haprin hexasaccharide was estimated by docking simulation, and Arg34, Gln40, His64 and Arg105 in ECP were predicted to contribute the most. To determine the roles of these residues in heparin binding to ECP, mutant ECPs with single alanine replacement were generated, and heparin binding affinities of wild type and mutant ECPs were measured and compared by isothermal titration calorimetry. Further, cell ELISA showed that Arg34, Arg36 and Lys38 within HBR1 acted as key residues for heparin binding in ECP. In addtion a novel cell penetrating peptide (CPPecp) spanning residues 32 to 41 in ECP was recently demonstrated to possess multiple biological functions. CPPecp was able to increase rhodamine-labeled liposome (RL) penetration into human cells. Moreover, cytotoxicity of liposomal formulated drug (LFD) significantly enhanced in the presence of CPPecp in the cells. Taken together, we have demonstrated key residues in HBRs of ECP for heparin binding. Membrane HSPGs and phospholipid binding drive CPPecp to enhance cellular uptake of liposomes, which in turn may facilitate novel design and application for LFD delivery.