Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234.
Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234.
复制标题
兔 LAT1 氨基酸 219 和 234 的定点诱变。
DOI：
10.1046/j.1471-4159.2003.01622.x
复制
发表时间：
2003
影响因子：
4.7
通讯作者：
Pardridge,WilliamM
中科院分区：
文献类型：
--
作者：
Boado,RubenJ;Li,JianYi;Pardridge,WilliamM
脑内氨基酸的可用性受血脑屏障（BBB）大中性氨基酸转运蛋白1型（LAT1）异构体的调节，其特点是对底物大中性氨基酸具有高亲和力（低km）。目前的研究通过血脑屏障LAT1的定点诱变验证了单核苷酸多态性可以改变脑氨基酸运输活性的假设。与人类或大鼠血脑屏障的低钾中性氨基酸转运体相比，兔具有高kmlat1大中性氨基酸转运体。从兔脑毛细管cDNA文库中克隆了兔LAT1基因。对兔、人和大鼠LAT1的氨基酸序列进行比对，发现兔与大鼠或人的LAT1有两个不同的自由基氨基酸残基。G219D突变对克隆兔LAT1在青蛙卵母细胞中转运色氨酸的kmandvmax有一定影响，而W234L突变使kmandvmax减少64%，vmax96%。相反，当产生双突变W234L/G219D变体时，色氨酸或苯丙氨酸的LAT1转运几乎是正常的。这些研究表明，LAT1的亲和力和容量的显著变化是由单核苷酸多态性引起的，并且可以通过双突变恢复表型。
The availability of amino acids in the brain is regulated by the blood–brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (lowKm) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site‐directed mutagenesis of the BBB LAT1. The rabbit has a highKmLAT1 large neutral amino acid transporter, as compared to the lowKmneutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on theKmandVmaxof tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced theKmby 64% and theVmaxby 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.