Endocytosis of extracellular superoxide dismutase into endothelial cells - Role of the heparin-binding domain
Endocytosis of extracellular superoxide dismutase into endothelial cells - Role of the heparin-binding domain
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DOI：
10.1161/01.atv.0000234921.88489.5c
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发表时间：
2006-09-01
影响因子：
8.7
通讯作者：
Heistad, Donald D.
中科院分区：
文献类型：
--
作者：
Chu, Yi;Piper, Robert;Heistad, Donald D.
目的-细胞外超氧化物歧化酶（EC-SOD）是一种分泌型抗氧化酶，通过其肝素结合结构域（HBD）与细胞外质膜和细胞外基质结合。携带一种常见的EC-SOD遗传变异体（EC-SODR 213 G，在HBD内）的人血浆EC-SOD浓度更高，患血管疾病的风险也更高。在本研究中，我们使用共聚焦荧光显微镜检查EC-SOD的内吞机制，以确定EC-SOD是否易位到内皮细胞的核中，并检验EC-SOD而不是EC-SODR 213 G被内吞到内皮细胞中的假设。或HBD缺失的EC-SOD（EC-SOD Δ HBD）。仅用EC-SOD观察到与MS- 1的结合，而不是EC-SODR 213 G或EC-SOD Δ HBD。在MS- 1细胞与EC-SOD和BSA-德克萨斯红（BSA-TR）（其标记内体和溶酶体）共孵育后监测EC-SOD的内吞作用。只有EC-SOD被内吞，与BSA-TR共定位. EC-SOD还与早期内吞体抗原1（EEA- 1）共定位，EEA-1是内吞作用的特异性标志物。氯丙嗪可抑制EC-SOD的内吞作用，而甲基-β-环糊精和制霉菌素则不抑制，说明EC-SOD的内吞作用是由网格蛋白介导的，而不是由小窝介导的。EC-SOD在MS- 1细胞核中的定位被检测到，但不是EC-SODR 213 G，我们的研究结果表明，EC-SOD，是通过网格蛋白介导的途径内吞到内皮细胞，但不易位到细胞核。我们推测，R213 G携带者的内吞功能受损可能导致EC-SODR 213 G的高血浆水平。
Objective - Extracellular superoxide dismutase ( EC-SOD) is a secreted antioxidant enzyme that binds to the outer plasma membrane and extracellular matrix through its heparin-binding domain ( HBD). Carriers of a common genetic variant of EC- SOD ( EC- SODR213G, within the HBD) have higher plasma concentration of EC- SOD and increased risk for vascular disease. In the present study, we used confocal fluorescence microscopy to examine mechanisms of endocytosis of EC- SOD to determine whether EC- SOD translocates to the nucleus of endothelial cells, and to test the hypothesis that EC- SOD, but not EC- SODR213G, is endocytosed into endothelial cells.Methods and Results - Mouse endothelial cells ( MS- 1) were incubated with EC- SOD, EC- SODR213G, or HBD- deleted EC- SOD ( EC- SOD Delta HBD). Binding to MS- 1 was observed only with EC- SOD, but not EC- SODR213G or EC-SOD Delta HBD. Endocytosis of EC-SODs was monitored after coincubation of MS- 1 cells with EC-SODs and BSA-Texas Red ( BSA- TR), which marks endosomes and lysosomes. Only EC- SOD was endocytosed, colocalizing with BSA- TR. EC- SOD also colocalized with early endosome antigen 1 ( EEA- 1), a specific marker for endocytosis. Endocytosis of EC- SOD was inhibited by chlorpromazine, but not by methyl-beta-cyclodextrin or nystatin, which suggests that endocytosis of EC- SOD is mediated by clathrin but not by caveolae. Minimal or no localization of EC- SOD in the nucleus of MS- 1 cells was detected.Conclusions - Our findings indicate that EC- SOD, but not EC- SODR213G, is endocytosed into endothelial cells through clathrin-mediated pathway, but does not translocate to the nucleus. We speculate that impairment of endocytosis may contribute to high plasma levels of EC- SODR213G in R213G carriers.