核苷酸糖转运蛋白（NST）家族成员对微粒体UDP-葡萄糖醛酸摄取的分子与功能特性
Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.
作者信息
Kobayashi Tsutomu, Sleeman Judith E, Coughtrie Michael W H, Burchell Brian
机构信息
Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, DUNDEE DD1 9SY, Scotland, UK.
出版信息
Biochem J. 2006 Dec 1;400(2):281-9. doi: 10.1042/BJ20060429.
Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.
由于尿苷二磷酸葡萄糖醛酸转移酶（UGT）的催化位点位于内质网腔中，因此将共底物尿苷二磷酸葡萄糖醛酸（UDPGA）转运到内质网腔是葡萄糖醛酸化反应中的一个重要步骤。在本研究中，我们已将几种核苷酸糖转运蛋白（NST）和UGT的功能鉴定为葡萄糖醛酸化反应中UDPGA的潜在载体。在大鼠肝微粒体和从大鼠肝癌细胞系H4IIE制备的微粒体中均发现了依赖于UDP-N-乙酰葡糖胺（UDPGlcNAc）的UDPGA摄取。源自大鼠肝脏和表达UGT1A6的V79细胞的微粒体中UGT活性的潜伏性与甘露糖-6-磷酸酶的潜伏性密切相关，这证实重组细胞中的UGT保留了与大鼠肝微粒体相似的生理学特性。在本研究中，获得了四个编码NST的cDNA；两个先前已报道（UGTrel1和UGTrel7），两个是新鉴定的（huYEA4和huYEA4S）。NST在人类基因组序列中的定位表明，huYEA4S是huYEA4的可变剪接形式。所有克隆的NST均在V79（中国仓鼠成纤维细胞）细胞中稳定表达，并且在将分离的微粒体小泡用UDPGlcNAc预加载后能够转运UDPGA。UGTrel7的摄取最高，其V(max)约为大鼠肝微粒体的1%。用β-萘黄酮处理H4IIE细胞可诱导UGT蛋白表达，但不影响UDPGA摄取速率。此外，来自UGT1缺陷型Gunn大鼠肝脏的微粒体显示出与对照大鼠相似的UDPGA摄取。这些数据表明，NST可以作为葡萄糖醛酸化反应的UDPGA转运蛋白，并表明1A家族的UGT在微粒体中不作为UDPGA载体发挥作用。细胞系H4IIE是研究葡萄糖醛酸化反应中UDPGA转运蛋白的有用模型。
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