Ritter J K, Crawford J M, Owens I S
Section on Genetic Disorders of Drug Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Jan 15;266(2):1043-7.
We report the isolation and characterization of two human liver cDNA clones, HUG-Br1 and HUG-Br2; each encodes a UDP-glucuronosyltransferase enzyme which glucuronidates bilirubin IX alpha to form both the IX alpha C8 and IX alpha C12 monoconjugates and a diconjugate. HUG-Br1 cDNA (2351 base pairs) and HUG-Br2 cDNA (2368 base pairs) encode proteins with 533 and 534 amino acid residues, respectively, with a typical membrane-insertion signal peptide, membrane-spanning domain, and 3 or 5 potential asparagine-linked glycosylation sites. At the nucleic acid and deduced amino acid sequence levels the two clones are 82% similar overall, 66% similar in the amino termini, and identical after codon 287, thus encoding proteins with the same carboxyl terminus. The mRNA encoding HUG-Br1 is of high abundance, and the one encoding HUG-Br2 is of low abundance; both are 2.6 kilobases in length. Both messages (2.6 kilobases) were present in the explanted liver of a Type I Crigler-Najjar patient, although the level for that of HUG-Br1 was reduced 4.5-fold. Northern blot analysis of poly(A)+ RNA isolated from the liver of an untreated and a phenobarbital-treated Erythrocebus patas monkey with 5'-specific probes for each clone indicated that the HUG-Br2-encoded message is induced two fold, but that for HUG-Br1 is not. These data indicate that bilirubin is glucuronidated by at least two different proteins, most likely present in very different amounts. These cDNAs which encode functional bilirubin UDP-glucuronosyltransferases will allow the isolation of an appropriate gene to develop a gene therapy model for patients which have the totally deficient trait.
我们报道了两个人类肝脏cDNA克隆HUG-Br1和HUG-Br2的分离与特性鉴定；每个克隆编码一种UDP-葡萄糖醛酸基转移酶，该酶可将胆红素IXα葡萄糖醛酸化，形成IXα C8和IXα C12单缀合物以及一种双缀合物。HUG-Br1 cDNA（2351个碱基对）和HUG-Br2 cDNA（2368个碱基对）分别编码含有533和534个氨基酸残基的蛋白质，具有典型的膜插入信号肽、跨膜结构域以及3个或5个潜在的天冬酰胺连接糖基化位点。在核酸和推导的氨基酸序列水平上，这两个克隆总体上相似性为82%，氨基末端相似性为66%，在密码子287之后相同，因此编码具有相同羧基末端的蛋白质。编码HUG-Br1的mRNA丰度高，而编码HUG-Br2的mRNA丰度低；两者长度均为2.6千碱基。在一名I型克里格勒-纳贾尔患者的移植肝脏中均存在这两种信息（2.6千碱基），尽管HUG-Br1的水平降低了4.5倍。用针对每个克隆的5'特异性探针，对从未经处理和经苯巴比妥处理的赤猴肝脏中分离的聚腺苷酸加尾RNA进行Northern印迹分析表明，HUG-Br2编码的信息诱导了两倍，但HUG-Br1的信息未被诱导。这些数据表明胆红素由至少两种不同的蛋白质进行葡萄糖醛酸化，很可能它们的含量差异很大。这些编码功能性胆红素UDP-葡萄糖醛酸基转移酶的cDNA将有助于分离合适的基因，从而为具有完全缺陷性状的患者开发基因治疗模型。
