Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1
Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1
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DOI：
10.1152/ajpcell.00019.2008
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发表时间：
2008-09-01
影响因子：
5.5
通讯作者：
Inui, Ken-ichi
中科院分区：
文献类型：
--
作者：
Yonezawa, Atsushi;Masuda, Satohiro;Inui, Ken-ichi
核黄素的吸收是由转运蛋白介导的(S)。然而，哺乳动物的核黄素转运蛋白尚未确定。本研究基于我们的大鼠肾脏基因表达数据库(Horiba N，Masuda S，Takeuchi A，Saito H，Okuda M，Inui K.Kidney Int 66：29-45,2004)，鉴定了新的人和大鼠核黄素转运蛋白hRFT1和rRFT1。HRFT1和rRFT1有一个开放阅读框，分别编码448个和450个氨基酸的蛋白质，它们之间有81.1%的同源性和96.4%的相似性。此外，还克隆了hRFT1的非活性剪接变异体hRFT1sv。HRFT1sv基因编码167个氨基酸的蛋白质，在hRFT1的外显子2和3之间保留一个内含子。实时荧光定量聚合酶链式反应显示hRFT1和hRFT1sv的mRNAs在胎盘和小肠中均有表达，并在所有受检组织中均有表达。此外，hRFT1和hRFT1sv在人胚胎肾(HEK)-293和Caco-2细胞中均有表达。转染绿色荧光蛋白标记的hRFT1和rRFT1的HEK-293细胞在细胞膜上有荧光信号。过表达hRFT1和rRFT1，而不是hRFT1sv，增加了细胞内[H-3]核黄素的积累。同时针对hRFT1和hRFT1sv的小干扰RNA显著降低HEK-293和Caco-2细胞对[H-3]核黄素的摄取。核黄素的转运不依赖于Na+、电位和pH。动力学分析表明HEK-293和Caco-2细胞摄取的米氏常数分别为28.1和63.7 nm。我们认为hRFT1和rRFT1是新的哺乳动物核黄素转运蛋白，属于一个新的哺乳动物核黄素转运蛋白家族。
Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, the novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified on the basis of our rat kidney mRNA expression database (Horiba N, Masuda S, Takeuchi A, Saito H, Okuda M, Inui K. Kidney Int 66: 29-45, 2004). hRFT1 and rRFT1 cDNAs have an open reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to one another. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFT1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in human embryonic kidney (HEK)-293 and Caco-2 cells. HEK-293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [H-3] riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [H-3] riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is Na+, potential, and pH independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK-293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family.