Li Fang, Feng Qiping, Lee Candace, Wang Shuzhan, Pelleymounter Linda L, Moon Irene, Eckloff Bruce W, Wieben Eric D, Schaid Daniel J, Yee Vivien, Weinshilboum Richard M
Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Mol Genet Metab. 2008 Jul;94(3):326-35. doi: 10.1016/j.ymgme.2008.03.013. Epub 2008 May 23.
Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT2 encodes a protein 73% identical in amino acid sequence to BHMT, but the function of BHMT2 remains unclear. We set out to identify and functionally characterize common genetic variation in BHMT and BHMT2. Specifically, we sequenced exons, exon-intron splice junctions and the 5'-flanking regions (5'-FRs) of BHMT and BHMT2 using 240 DNA samples from four ethnic groups. Twenty-five single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, and 39 SNPs, including 4 nonsynonymous, were observed in BHMT and BHMT2, respectively. BHMT wild type (WT) and variant allozymes were expressed in COS-1 cells. Variant allozymes showed no significant differences from WT in levels of enzyme activity or immunoreactive protein, but there were statistically significant differences in apparent K(m) values. Luciferase reporter gene constructs were created for the three most common BHMT 5'-FR haplotypes, and significant variation was observed in the ability of these constructs to drive transcription. Although BHMT2 mRNA has been observed in human liver and kidney, expression of the protein has not been reported. We were unable to express BHMT2 in mammalian cells, and the protein aggregated after bacterial expression. Furthermore, BHMT2 was rapidly degraded in a rabbit reticulocyte lysate, but it could be stabilized by cotransfection of COS-1 cells with BHMT and, after cotransfection, it coprecipitated with BHMT. These studies have defined common genetic variation in BHMT and BHMT2 and functionally characterized BHMT SNPs. They may also help to explain why BHMT2 has not previously been defined functionally.
甜菜碱-同型半胱氨酸甲基转移酶（BHMT）催化同型半胱氨酸的再甲基化反应。BHMT2编码一种氨基酸序列与BHMT有73%相同的蛋白质，但BHMT2的功能仍不清楚。我们着手鉴定BHMT和BHMT2中的常见基因变异并对其进行功能表征。具体而言，我们使用来自四个种族群体的240个DNA样本，对BHMT和BHMT2的外显子、外显子-内含子剪接接头以及5'侧翼区域（5'-FRs）进行了测序。在BHMT和BHMT2中分别观察到25个单核苷酸多态性（SNP），包括4个非同义SNP，以及39个SNP，包括4个非同义SNP。BHMT野生型（WT）和变异同工酶在COS-1细胞中表达。变异同工酶在酶活性水平或免疫反应性蛋白方面与WT没有显著差异，但在表观K(m)值上存在统计学显著差异。针对三种最常见的BHMT 5'-FR单倍型构建了荧光素酶报告基因构建体，并且观察到这些构建体驱动转录的能力存在显著差异。尽管在人类肝脏和肾脏中已观察到BHMT2 mRNA，但尚未有该蛋白质表达的报道。我们无法在哺乳动物细胞中表达BHMT2，并且该蛋白质在细菌表达后会聚集。此外，BHMT2在兔网织红细胞裂解物中迅速降解，但通过与BHMT共转染COS-1细胞可使其稳定，共转染后它与BHMT共沉淀。这些研究定义了BHMT和BHMT2中的常见基因变异，并对BHMT SNP进行了功能表征。它们也可能有助于解释为何之前未对BHMT2进行功能定义。
