Abstract
Eosinophil cationic protein (ECP) is degranulated by eosinophils and released into the extracellular space to defense against pathogens. With another eosinophil granule protein, eosinophil derived neurotoxin (EDN), high level of ECP and EDN expression has been detected in asthma patients, and thus considered as a biomarker for diagnosis of disease severity. Both of ECP and EDN belong to human ribonuclease A (RNase A) superfamily and are able to enter several cells. Endocytosis of ECP into bronchial epithelial cells was greatly dependent on the cell surface glycosaminoglycan (GAGs), specifically heparan sulfate proteoglycans (HSPGs). The present study focuses on identification and characterization of the essential heparin/heparan sulfate (HS) binding motifs on ECP. HS binding motif has been reported to be mainly correlated with positive charge and aromatic residues, so each of the arginine (R), trytophan (W) and lysine (K) residues on ECP was specifically mutated to alanine, and the cell surface binding activity of mutant ECP was evaluated employing cell-enzyme-linked immunosorbent assay (ELISA). It was found that ECP-R22A, R34A, R36A, K38A, R45A, R73A and R77A showed significantly decreased HS binding activity, and the major heparin/HS binding motif in ECP was located at 34RWRCK38. The sequence composition and pattern of this motif were different from those of conventional heparan binding motifs (XBBXXB). In addition, de-2-O-sulfated heparin could not inhibit the cell binding ability of ECP as well as other heparin derivatives, indicating that ECP had high affinity to 2-O-sulfation of heparin. On the other hands, heparin binding peptide (ECP32-41) could bind BEAS-2B cells in a time- and concentration-dependent manner. This discovery provided the first direct evidence in biological functions and molecular interactions between ECP and heparin sulfate.