Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye
Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye
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DOI：
10.1074/jbc.m405334200
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发表时间：
2004-11-19
影响因子：
4.8
通讯作者：
Bernstein, PS
中科院分区：
文献类型：
--
作者：
Bhosale, P;Larson, AJ;Bernstein, PS
叶黄素类胡萝卜素在视网膜中的吸收、代谢和稳定被认为是由特定的叶黄素结合蛋白(XBP)介导的。采用离子交换层析和凝胶排斥层析的方法，从人黄斑组织中分离纯化出一种膜结合的XBP。双向凝胶电泳法得到23 kDa的显著斑点，等电点为5.7。利用质谱学测序方法和公共NCBI数据库，将其鉴定为人谷胱甘肽S转移酶的PI异构体。饲料中(3R，3‘R)-玉米黄质亲和力最强，表观K-d为0.33um，其次为(3R，3’-S-中位)-玉米黄质，表观K-d为0.52um。(3R，3‘R，6’R)-叶黄素与GSTP1无高亲和力结合。其他重组人谷胱甘肽S转移酶蛋白GSTA1和GSTM1只与叶黄素有低亲和力结合。(3R，3‘S-Meso)-玉米黄质是一种存在于人黄斑中的非膳食叶黄素类胡萝卜素，它与人黄斑XBP结合显示出强烈的诱导CD谱，与GSTP1诱导的CD谱几乎相同。同样，饮食中的(3R，3‘R)-玉米黄质在其CD光谱中显示出与GSTP1和XBP相关的变化。其他哺乳动物叶黄素载体蛋白如微管蛋白、高密度脂蛋白、低密度脂蛋白、白蛋白和β-乳球蛋白等不能与玉米黄质高亲和力结合，也不能诱导或改变叶黄素CD光谱。人黄斑切片上的GSTP1抗体免疫细胞化学显示，外层和内层的GSTP1标记最高。这些结果表明，GSTP1是人黄斑中一种特异的XBP，它与(3R，3‘S-Meso)-玉米黄质和饮食(3R，3’R)-玉米黄质相互作用，而与(3R，3‘R，6’R)-叶黄素的相互作用明显较弱。
Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.7. Using mass spectral sequencing methods and the public NCBI database, it was identified as a Pi isoform of human glutathione S-transferase (GSTP1). Dietary (3R,3'R)-zeaxanthin displayed the highest affinity with an apparent K-d of 0.33 muM, followed by (3R,3'S-meso)-zeaxanthin with an apparent K-d of 0.52 muM. (3R,3'R,6'R)-Lutein did not display any high-affinity binding to GSTP1. Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (3R,3'S-meso)-Zeaxanthin, an optically inactive non-dietary xanthophyll carotenoid present in the human macula, exhibited a strong induced CD spectrum in association with human macular XBP that was nearly identical to the CD spectrum induced by GSTP1. Likewise, dietary (3R,3'R)-zeaxanthin displayed alterations in its CD spectrum in association with GSTP1 and XBP. Other mammalian xanthophyll carrier proteins such as tubulin, high-density lipoprotein, low-density lipoprotein, albumin, and beta-lactoglobulin did not bind zeaxanthins with high affinity, and they failed to induce or alter xanthophyll CD spectra to any significant extent. Immunocytochemistry with an antibody to GSTP1 on human macula sections showed highest labeling in the outer and inner plexiform layers. These results indicate that GSTP1 is a specific XBP in human macula that interacts with (3R,3'S-meso)-zeaxanthin and dietary (3R,3'R)-zeaxanthin in contrast to apparently weaker interactions with (3R,3'R,6'R)-lutein.