Abstract
Abstract Eosinophil cationic protein (ECP) is secreted by activated granular eosinophilic leukocytes. It belongs to human ribonuclease (RNase) A superfamily and is also named as RNase 3. Endocytosis of ECP into Beas-2B, human bronchial epithelial cells, depends on the interaction between protein and cell surface glycosaminoglycan (GAGs), especially heparin sulfate proteoglycans (HSPGs), and this process results in cell death which in turn leads to airway inflammatory disease such as asthma. In general, heparin/ heparan sulfate on the surface of mammalian cells has been reported to interact with positively charged residues in heparin binding proteins. In previous studies, three putative heparin binding regions (HBRs) (34RWRCK38, 75RSRFR79, and 101RPGRR105) of ECP were identified by bioinformatics methods and point mutation analysis. To further examine functional characteristics of these HBRs on ECP, seven mutant ECPs with different HBRs were constructed in this study. The binding ability of each mutant ECP to LMWH and Beas-2B cell surface was evaluated by fluorescence-assisted carbohydrate electrophoresis (FACE) and cell based enzyme-link immunosorbent assay (ELISA), respectively. The results show that HBR2 (75RSRFR79) and HBR3 (101RPGRR105) are involved in cellular binding ability of ECP, whereas HBR1 (34RWRCK38) plays a crucial role on cell penetration ability of ECP based on ECP uptake assay. Furthermore, cell viability assay indicates that HBR3 (101RPGRR105) acts as the key component contributing the cytotoxicity of ECP towards Beas-2B cells. The present study provides a novel insight in functional characteristics of three HBRs of ECP.