Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR
Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR
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DOI：
10.1152/ajpgi.1997.272.1.g16
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发表时间：
1997-01-01
影响因子：
4.5
通讯作者：
Sugiyama, Y
中科院分区：
文献类型：
--
作者：
Ito, K;Suzuki, H;Sugiyama, Y
多种有机阴离子通过胆小管多特异性有机阴离子转运体(CMOAT)排出到胆汁中，该转运蛋白在卫材高胆红素血症大鼠(EHER)和tr(-)大鼠等突变大鼠中存在遗传缺陷。在本研究中，我们根据与人多药耐药相关蛋白(HMRP)的同源性，从SD大鼠肝脏的cDNA文库中克隆了cMOAT。CMOAT基因全长4623个碱基对，与hMRP的同源性分别为53.0%和46.3%。推导的氨基酸序列与在Wistar大鼠(C.C.Paulusma，P.J.Bosma，G.J.Zaman，C.T.Bakker，M.Otter，G.L.Sceffer，P.Borst)中克隆的氨基酸序列相同。和R.P.Oude Elferink。科学洗手间。DC 271：1126,1996)，除了四个氨基酸替换。通过对文库的筛选，获得了3种具有相同开放阅读框、不同3‘非翻译区长度(0.2、1.5和3.5kbP)的cMOAT基因。Northern印迹分析表明，在EHBR中有多条带(类似于5、6和8kb)的表达存在缺陷，这可能与这些cDNA物种的存在有关。CMOAT几乎只在肝脏中表达，在十二指肠、肾脏和空肠中的表达程度较低。逆转录聚合酶链式反应(RT-FCR)和随后对EHBR肝、肾、十二指肠和空肠的序列分析表明，2564位核苷酸的1个碱基从G到A的替换导致了所有组织中过早终止密码子的引入。该突变不同于在tr(-)(C.C.Paulusma、P.J.Bosma、G.J.Zaman、C.T.Bakker、M.Otter、G.L.Sceffer、P.Borst和R.P.Oude Elferink)中观察到的突变。科学洗手间。DC 271：1126,1996)。由于EHBR和tr(-)是等位基因突变体，而且两株菌在胆汁中有机阴离子的排泄都表现出常染色体隐性遗传，因此，这一特定蛋白的表达受损与突变动物高胆红素血症的发病机制有关。
Several organic anions are excreted into the bile via a canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats, such as the Eisai hyperbilirubinemic rat (EHER) and TR(-) rat. Ln the present study, we cloned cMOAT from the Sprague-Dawley rat liver cDNA library based on the homology with human multidrug resistance-associated protein (hMRP). cMOAT was encoded by 4,623-base pair (bp) cDNA with a homology of 53.0 and 46.3% with hMRP at the cDNA and deduced amino acid level, respectively. The deduced amino acid sequence was the same as that cloned in Wistar rats (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst. and R. P. Oude Elferink. Science Wash. DC 271: 1126, 1996) except for four amino acid substitutions. By screening the library, three kinds of cDNA species for cMOAT with the same open reading frame and different 3'-untranslated region lengths (0.2, 1.5, and 3.5 kbp) were isolated. The Northern blot analysis of poly(A)I RNA from the liver revealed that the expression of plural bands (similar to 5, 6, and 8 kb) was defective in EHBR, and this may be due to the presence of these cDNA species. Expression of cMOAT was observed almost exclusively in the liver and to a lesser extent in the duodenum, kidney, and jejunum. Reverse transcription-polymerase chain reaction (RT-FCR) and subsequent sequence analysis of EHBR liver, kidney, duodenum, and jejunum revealed that 1-bp replacement from G to A at nucleotide 2564 resulted in the introduction of the premature stop codon in all tissues examined. This mutation was different from that observed in TR(-) (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst, and R. P. Oude Elferink. Science Wash. DC 271:1126, 1996). Because EHBR and TR(-) are allelic mutants and both strains exhibit an autosomal recessive inheritance in the biliary excretion of organic anions, it was concluded that the impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant animals.