Honchel R, Aksoy I A, Szumlanski C, Wood T C, Otterness D M, Wieben E D, Weinshilboum R M
Department of Pharmacology, Mayo Medical School, Mayo Clinic, Rochester, Minnesota 55905.
Mol Pharmacol. 1993 Jun;43(6):878-87.
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Levels of TPMT activity in human tissue are controlled by a common genetic polymorphism that is an important factor responsible for individual variation in thiopurine drug toxicity and therapeutic efficacy. Our goal was to purify, to obtain a partial amino acid sequence for, and to clone and express cDNA for human TPMT as a first step in determining the molecular basis for this genetic polymorphism. Human kidney TPMT was purified, the protein was subjected to limited proteolysis, and amino acid sequence information was obtained from the resultant peptide fragments. Primers based on the amino acid sequence information were used to amplify a unique sequence from human liver cDNA by use of the polymerase chain reaction. Because TPMT has been reported to be present in the colon, T84 human colon carcinoma cells were studied and were found to express TPMT activity with biochemical properties similar to those of the human kidney and liver enzymes. Oligonucleotide probes based on the human kidney TPMT amino acid sequence were then used to screen a T84 human colon carcinoma cell cDNA library. A 2.7-kilobase cDNA clone was isolated that contained an open reading frame of 735 nucleotides, which encoded a protein of 245 amino acids. The deduced amino acid sequence of the encoded protein included one 24- and two separate 12-amino acid sequences identical to those obtained by sequencing proteolytic fragments of purified human kidney TPMT. Transcripts were made in vitro from the open reading frame of the cDNA clone. These transcripts were translated in a rabbit reticulocyte lysate system, and the resulting translation product comigrated with human kidney TPMT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T84 cell cDNA clone, truncated within the 3' untranslated region at an Sstl restriction site, was then used to create an expression construct with the eukaryotic expression vector P91023(B), and this construct was used to transfect COS-1 cells. The transfected cells expressed a high level of TPMT enzymatic activity, and this activity displayed a pattern of inhibition by TPMT inhibitors identical to that of human kidney and T84 human colon carcinoma cell TPMT. Cloning of cDNA for this important drug-metabolizing enzyme may make it possible to define the molecular basis of the TPMT genetic polymorphism in humans.
硫嘌呤甲基转移酶（TPMT）催化硫嘌呤类药物（如6-巯基嘌呤）的S-甲基化反应。人体组织中TPMT的活性水平受一种常见的基因多态性控制，这种多态性是导致硫嘌呤类药物毒性和治疗效果个体差异的重要因素。我们的目标是纯化人TPMT、获得其部分氨基酸序列，并克隆和表达人TPMT的cDNA，作为确定这种基因多态性分子基础的第一步。纯化了人肾TPMT，对该蛋白质进行了有限的蛋白酶解，并从所得的肽片段中获得了氨基酸序列信息。基于氨基酸序列信息设计的引物用于通过聚合酶链反应从人肝cDNA中扩增出一个独特的序列。由于据报道TPMT存在于结肠中，因此对T84人结肠癌细胞进行了研究，发现其表达的TPMT活性具有与人类肾脏和肝脏酶相似的生化特性。然后，基于人肾TPMT氨基酸序列的寡核苷酸探针用于筛选T84人结肠癌细胞cDNA文库。分离出一个2.7千碱基的cDNA克隆，其包含一个735个核苷酸的开放阅读框，编码一个245个氨基酸的蛋白质。所编码蛋白质的推导氨基酸序列包括一个与通过对纯化的人肾TPMT的蛋白水解片段测序获得的24个氨基酸序列以及两个单独的12个氨基酸序列相同。从cDNA克隆的开放阅读框进行体外转录。这些转录本在兔网织红细胞裂解物系统中进行翻译，所得翻译产物在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中与人肾TPMT共迁移。然后，使用在3'非翻译区的SstI限制性位点处截短的T84细胞cDNA克隆与真核表达载体P91023(B)构建表达构建体，并使用该构建体转染COS-1细胞。转染后的细胞表达高水平的TPMT酶活性，并且这种活性表现出与人类肾脏和T84人结肠癌细胞TPMT相同的TPMT抑制剂抑制模式。克隆这种重要的药物代谢酶的cDNA可能有助于确定人类TPMT基因多态性的分子基础。
