Abstract
Eosinophil cationic protein (ECP) is secreted from activated eosinophil at inflammatory site, and it functions in immune system to against pathogens such as bacteria and virus. ECP shows highly correlation with airway allergic diseases and has been used as biomarker for asthma in clinical. Recent study revealed that the cytotoxic activity of ECP toward Beas-2B human bronchial epithelial cell line depended on cell surface heparan sulfate proteoglycans (HSPGs) and the heparin binding motif of ECP with a basic amino acid cluster located at loop 3 (L3) is identified. However, the binding affinity between ECP and heparin is not fully elucidated. In this study, the binding constant between ECP and heparin was quantified by isothermal titration calorimeter (ITC), and the dissociation constant (Kd) was 1.3 x 10-6 M. A mutated MBP-ECP lacking of heparin binding L3 remained a weaker binding ability with a Kd value of 7.4 x 10-6 M, indicating the existence of additional heparin binding region in ECP. MBP-ECP single point mutants were used for identification of key heparin binding residues by ITC and affinity column chromatography. Two basic amino acid clusters were verified to be a novel heparin binding path on ECP. In addition, five heparin derivatives with difference modifications were used for determining the specificity of heparin binding activity of MBP-ECP. The results obtained from ITC and competitive heparin affinity chromatography indicated that the N-sufation on heparin predominantly contributed in ECP binding.