Gao Xia, Gu Hongmei, Li Ge, Rye Kerry-Anne, Zhang Da-Wei
Department of Pediatrics and Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Biochim Biophys Acta. 2012 Mar;1821(3):552-9. doi: 10.1016/j.bbalip.2011.07.012. Epub 2011 Jul 29.
The ATP-binding cassette transporter G1 (ABCG1) mediates free cholesterol efflux onto lipidated apolipoprotein A-I (apoA-I) and plays an important role in macrophage reverse cholesterol transport thereby reducing atherosclerosis. However, how ABCG1 mediates the efflux of cholesterol onto lipidated apoA-I is unclear. Since the crystal structure of ABCG family is not available, other approaches such as site-directed mutagenesis have been widely used to identify amino acid residues important for protein functions. We noticed that ABCG1 contains a single cysteine residue in its putative transmembrane domains. This cysteine residue locates at position 514 (Cys(514)) within the third putative transmembrane domain and is highly conserved. Replacement of Cys(514) with Ala (C514A) essentially abolished ABCG1-mediated cholesterol efflux onto lipidated apoA-I. Substitution of Cys(514) with more conserved amino acid residues, Ser or Thr, also significantly decreased cholesterol efflux. However, mutation C514A had no detectable effect on protein stability and trafficking. Mutation C514A also did not affect the dimerization of ABCG1. Our findings demonstrated that the sulfhydryl group of Cys residue located at position 514 plays a critical role in ABCG1-mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
ATP结合盒转运体G1（ABCG1）介导游离胆固醇向脂化载脂蛋白A-I（apoA-I）的流出，并在巨噬细胞逆向胆固醇转运中发挥重要作用，从而减轻动脉粥样硬化。然而，ABCG1如何介导胆固醇向脂化apoA-I的流出尚不清楚。由于ABCG家族的晶体结构尚未获得，其他方法如定点诱变已被广泛用于鉴定对蛋白质功能重要的氨基酸残基。我们注意到ABCG1在其假定的跨膜结构域中含有一个半胱氨酸残基。这个半胱氨酸残基位于第三个假定跨膜结构域内的第514位（Cys(514)），并且高度保守。用丙氨酸取代Cys(514)（C514A）基本上消除了ABCG1介导的胆固醇向脂化apoA-I的流出。用更保守的氨基酸残基丝氨酸或苏氨酸取代Cys(514)也显著降低了胆固醇流出。然而，C514A突变对蛋白质稳定性和运输没有可检测到的影响。C514A突变也不影响ABCG1的二聚化。我们的研究结果表明，位于第514位的半胱氨酸残基的巯基在ABCG1介导的胆固醇流出中起关键作用。本文是名为“高密度脂蛋白形成与代谢进展：向约翰·F·奥勒姆致敬（1945 - 2010）”特刊的一部分。
