Salavaggione Oreste E, Wang Liewei, Wiepert Mathieu, Yee Vivien C, Weinshilboum Richard M
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine-Mayo Clinic, Rochester, Minnesota 55905, USA.
Pharmacogenet Genomics. 2005 Nov;15(11):801-15. doi: 10.1097/01.fpc.0000174788.69991.6b.
Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Genetic polymorphisms for TPMT are a major factor responsible for large individual variations in thiopurine toxicity and therapeutic effect. The present study investigated the functional effects of human TPMT variant alleles that alter the encoded amino acid sequence of the enzyme, TPMT*2, *3A, *3B, *3C and *5 to *13. After expression in COS-1 cells and correction for transfection efficiency, allozymes encoded by these alleles displayed levels of activity that varied from virtually undetectable (*3A,*3B and *5) to 98% (7) of that observed for the wild-type allele. Although some allozymes had significant elevations in apparent Km values for 6-mercaptopurine and S-adenosyl-L-methionine (i.e. the two cosubstrates for the reaction), the level of enzyme protein was the major factor responsible for variation in activity. Quantitative Western blot analysis demonstrated that the level of enzyme protein correlated closely with level of activity for all allozymes except TPMT5. Furthermore, protein levels correlated with rates of TPMT degradation. TPMT amino acid sequences were then determined for 16 non-human mammalian species and those sequences (plus seven reported previously, including two nonmammalian vertebrate species) were used to determine amino acid sequence conservation. Most human TPMT variant allozymes had alterations of residues that were highly conserved during vertebrate evolution. Finally, a human TPMT homology structural model was created on the basis of a Pseudomonas structure (the only TPMT structure solved to this time), and the model was used to infer the functional consequences of variant allozyme amino acid sequence alterations. These studies indicate that a common mechanism responsible for alterations in the activity of variant TPMT allozymes involves alteration in the level of enzyme protein due, at least in part, to accelerated degradation.
硫嘌呤 S-甲基转移酶（TPMT）催化硫嘌呤类药物的 S-甲基化反应。TPMT 的基因多态性是导致硫嘌呤毒性和治疗效果个体差异巨大的主要因素。本研究调查了人类 TPMT 变异等位基因（TPMT*2、*3A、3B、3C 和5 至13）对酶编码氨基酸序列的功能影响，这些变异等位基因会改变酶的编码氨基酸序列。在 COS-1 细胞中表达并校正转染效率后，由这些等位基因编码的同工酶所显示的活性水平各不相同，从几乎检测不到（*3A、3B 和5）到野生型等位基因活性的 98%（7）。尽管一些同工酶对 6-巯基嘌呤和 S-腺苷-L-甲硫氨酸（即该反应的两种共同底物）的表观 Km 值有显著升高，但酶蛋白水平是活性变化的主要因素。定量蛋白质印迹分析表明，除 TPMT5 外，所有同工酶的酶蛋白水平与活性水平密切相关。此外，蛋白质水平与 TPMT 的降解速率相关。随后测定了 16 种非人类哺乳动物物种的 TPMT 氨基酸序列，并利用这些序列（加上之前报道的 7 种，包括两种非哺乳动物脊椎动物物种）来确定氨基酸序列的保守性。大多数人类 TPMT 变异同工酶的残基在脊椎动物进化过程中高度保守。最后，基于假单胞菌的结构（这是目前唯一解析出的 TPMT 结构）创建了人类 TPMT 同源结构模型，并利用该模型推断变异同工酶氨基酸序列改变的功能后果。这些研究表明，导致变异 TPMT 同工酶活性改变的常见机制至少部分是由于加速降解导致酶蛋白水平发生改变。
