A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein.
Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface-bound heparan sulfate proteoglycans 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 compared with their wild-type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP-ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol-3-kinase activities, and is compatible with the characteristics of raft-dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.
Fan
Tan-Chi
TC
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.
Chang
Hao-Teng
HT
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.
Chen
I-Wen
IW
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.
Wang
Hsiu-Yiu
HY
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.
Chang
Margaret Dah-Tsyr
MD
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.
D000818:Animals; D001692:Biological Transport; D001980:Bronchi / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D047150:Eosinophil Cationic Protein / Q000737:chemistry*; D004804:Eosinophils / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006497:Heparan Sulfate / Q000494:pharmacology*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010873:Pinocytosis
D047150:Eosinophil Cationic Protein; D006497:Heparan Sulfate; D019869:Phosphatidylinositol 3-Kinases