A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein
A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein
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DOI：
10.1111/j.1600-0854.2007.00650.x
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发表时间：
2007-12-01
期刊：
影响因子：
4.5
通讯作者：
Chang, Margaret Dah-Tsyr
中科院分区：
文献类型：
--
作者：
Fan, Tan-Chi;Chang, Hao-Teng;Chang, Margaret Dah-Tsyr
嗜酸性粒细胞阳离子蛋白（ECP）是人RNA酶A超家族成员，与哮喘病理密切相关，内吞后对支气管上皮细胞具有毒性。ECP内化到细胞中的机制知之甚少。在这项研究中，我们表明，细胞表面结合的硫酸乙酰肝素蛋白多糖作为ECP内化的主要受体。通过肝素酶去除细胞表面硫酸乙酰肝素或通过氯酸盐减少聚糖硫酸化显著降低ECP与人支气管上皮Beas-2B细胞的结合。此外，与野生型细胞相比，糖胺聚糖缺陷细胞的ECP摄取和相关细胞毒性降低。此外，药物治疗结合siRNA敲低鉴定了网格蛋白和小窝蛋白独立的内吞途径作为ECP内化的主要途径。该途径受Rac 1和ADP-核糖基化因子6 GTP酶调节。它需要胆固醇、肌动蛋白细胞骨架重排和磷脂酰肌醇-3-激酶活性，并且与筏依赖性巨胞饮作用的特征相容。因此，我们的研究结果定义了ECP内化的早期事件，并可能对ECP相关疾病的新治疗设计产生影响。
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.