Tryptophan 415 Is Critical for the Cholesterol Transport Functions of Scavenger Receptor BI.
Tryptophan 415 Is Critical for the Cholesterol Transport Functions of Scavenger Receptor BI.
复制标题
DOI：
10.1021/acs.biochem.5b00804
复制
发表时间：
2016-01-12
期刊：
影响因子：
2.9
通讯作者：
Sahoo D
中科院分区：
文献类型：
--
作者：
Holme RL;Miller JJ;Nicholson K;Sahoo D
高密度脂蛋白（HDL）是抗动脉粥样硬化颗粒，主要是由于它们在胆固醇逆向转运途径中的作用，HDL通过与其受体清道夫受体BI （SR-BI）相互作用将胆固醇酯（CE）输送到肝脏排泄。我们设计了实验来验证SR-BI中8个高度保守的色氨酸（Trp； W）残基中的一个或多个对调节功能至关重要的假设。我们创建了一系列trp -to-苯丙氨酸（Phe， F）突变受体，以及不含trp的SR-BI （ΔW-SR-BI），并评估了它们介导胆固醇转运的能力。野生型（WT）或突变型SR-BI受体在COS-7细胞中短暂表达，并证实细胞表面表达。接下来，我们发现Trp-less和W415F-SR-BI结合HDL和促进HDL- ce选择性摄取的能力显著降低，尽管与WT-SR-BI相比，它们具有更高的选择性摄取效率。有趣的是，只有Trp-less-而不是W415F-SR-BI表现出介导游离胆固醇（FC）外排的能力受损。此外，基于对外源性胆固醇氧化酶缺乏敏感性，W415F-和Trp-less-SR-BI均不能重组FC的质膜池。将Trp 415恢复到Trp-less- sr - bi背景后，无法恢复Trp-less- sr - bi受损的功能，这表明Trp 415是关键的，但不足以使受体完全发挥功能。此外，除Trp 262外，单个细胞外Trp残基与Trp 415结合，恢复到Trp较少的SR-BI背景，部分恢复了SR-BI功能，这表明Trp 415必须与其他Trp残基结合，才能实现适当的胆固醇转运功能。
High density lipoproteins (HDL) are anti-atherogenic particles, primarily due to their role in the reverse cholesterol transport pathway whereby HDL delivers cholesteryl esters (CE) to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). We designed experiments to test the hypothesis that one or more of the eight highly conserved tryptophan (Trp; W) residues in SR-BI are critical for mediating function. We created a series of Trp-to-phenylalanine (Phe, F) mutant receptors, as well as Trp-less SR-BI (ΔW-SR-BI), and assessed their ability to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS-7 cells, and cell surface expression was confirmed. Next, we showed that Trp-less- and W415F-SR-BI had significantly decreased abilities to bind HDL and promote selective uptake of HDL-CE, albeit with higher selective uptake efficiency as compared to WT-SR-BI. Interestingly, only Trp-less-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC). Furthermore, both W415F- and Trp-less-SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity to exogenous cholesterol oxidase. Restoration of Trp 415 into the Trp-less-SR-BI background was unable to rescue Trp-less-SR-BI’s impaired functions, suggesting that Trp 415 is critical, but not sufficient for full receptor function. Furthermore, with the exception of Trp 262, restoration of individual extracellular Trp residues, in combination with Trp 415, into the Trp-less-SR-BI background partially rescued SR-BI function, indicating that Trp 415 must be present in combination with other Trp residues for proper cholesterol transport functions.