| Summary: | 博士 === 國立清華大學 === 分子與細胞生物研究所 === 96 === Eosinophil cationic protein (ECP) is currently used as a biomarker of airway inflammation. It is a heparin-binding ribonuclease (RNase) released by activated eosinophils and is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In first part of the study, we show that cell surface-bound heparan sulfate proteoglycans (HSPGs) serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells as compared to their wild-type counterparts. Furthermore, pharmacological treatment combined with si-RNA knock-down identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and Arf6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and PI3K activities, and is compatible with the characteristics of raft-dependent macropinocytosis. In terms of specific polysaccharide binding activity, ssynthetic heparin with 5 or higher monosaccharide units showed strong inhibition of ECP binding to cell surface. The heparin binding site in ECP is determined by site-directed mutagenesis and synthetic peptide segment derived from ECP. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that the charged and aromatic residues were involved in ECP binding to heparin and cell surface. Such binding motif is located in the loop L3 region between helix 脉2 and strand 刍1, outside the RNA binding domain. The peptide derived from the loop L3 region displayed strong pentasaccharide binding affinities and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin act as the primary step for protein endocyosis. These results provide new insights into the structure and function of ECP regarding specific molecular interaction with heparan sulfate. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.
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