Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter
Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter
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DOI：
10.1038/s41598-019-54367-9
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发表时间：
2019-12-02
影响因子：
4.6
通讯作者：
Yuasa, Hiroaki
中科院分区：
文献类型：
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作者：
Yamashiro, Takahiro;Yasujima, Tomoya;Yuasa, Hiroaki
人类质子偶联叶酸转运体(hPCFT/SLC46A1)最近被发现被杨梅素以一种持久的机制抑制，这引起了人们的担忧，即这种抑制可能导致肠道对叶酸的吸收不良，而hPCFT在肠道中作用于叶酸的上皮吸收。然而，最近发现杨梅素不能抑制大鼠PCFT(RPCFT)。在这一发现的推动下，我们试图通过比较hPCFT和rPCFT的分析来确定其中涉及的氨基酸残基。在最初的分析中，检测由hPCFT和rPCFT制备的嵌合构建体对杨梅素的敏感性，以确定与敏感性有关的hPCFT片段。聚焦于由此确定的第83至186个氨基酸残基的片段，将hPCFT突变体的指定氨基酸残基替换为rPCFT中的对应氨基酸残基，用于随后的分析。其中，只有G158N取代的hPCFT被转化为对杨梅素不敏感，相应地，N158G取代的rPCFT被转化为对杨梅素敏感。这些结果表明Gly(1)(58)在hPCFT的杨梅素敏感性中起关键作用。这一发现将有助于阐明杨梅素抑制hPCFT的机制，并管理由此产生的潜在风险。
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly(1)(58) in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.