Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4
Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4
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人单羧酸转运蛋白 1、2 和 4 中保守的必需细胞外天冬氨酸的鉴定
DOI：
10.1016/j.bbrc.2020.06.068
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Kobayashi M
中科院分区：
文献类型：
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作者：
Yamaguchi A;Narumi K;Furugen A;Iseki K;Kobayashi M
人单羧酸转运蛋白（hmct） 1-4在质膜上转运单羧酸盐，如乳酸和丙酮酸，以及H+。hMCT1、2和4在能量平衡、pH稳态中起重要作用。然而，这些转运体的分子机制，特别是它们的pH依赖性，仍然是未知的。本研究的目的是鉴定与hMCT1、2和4的pH依赖性有关的残基。首先，我们重点研究了hMCT1细胞外酸的作用。l-乳酸摄取试验和定点诱变显示，hMCT1的天冬氨酸（hMCT1 D414）是MCT1、2和4 （hMCT2 D398和hMCT4 D379）中保守的重要残基。由于hmct2介导的l-乳酸转运的功能特征尚未报道，我们利用非洲爪蟾卵母细胞构建了hmct2表达系统。hMCT2与辅助蛋白embigin的共表达增强了转运活性，hMCT2介导的乳酸摄取动力学分析显示，表观km值（0.32±0.02 mM）低于hMCT1和4介导的。最后，我们研究了hMCT2和4中保守的天冬氨酸，发现这些残基对l-乳酸转运至关重要。这些发现表明，保存在hMCT1、2和4上的细胞外天冬氨酸在转运活性和pH依赖性中发挥重要作用，可以作为底物和H+识别和从细胞外区域转运到细胞内的第一步。这些发现有助于加深我们对hMCT1、2和4的转运过程的理解。
Human monocarboxylate transporters (hMCTs) 1–4 transport monocarboxylates, such asl-lactate and pyruvate, as well as H+across the plasma membrane. hMCT1, 2, and 4 play important roles in energy balance, pH homeostasis. However, the molecular mechanism of these transporters, especially their pH dependency, remains unknown. The aim of this study was to identify the residues involved in the pH dependence of hMCT1, 2, and 4. Firstly, we focused on the effects of extracellular acids of hMCT1.l-Lactate uptake assay and site-directed mutagenesis revealed that the aspartic acid of hMCT1 (hMCT1 D414) was an important residue conserved in MCT1, 2, and 4 (hMCT2 D398 and hMCT4 D379). Because the functional characteristic of hMCT2-mediatedl-lactate transport has not been reported, we built a hMCT2-expressing system using Xenopus laevis oocytes. The transport activity of hMCT2 was enhanced by co-expression with embigin, an ancillary protein, and kinetic analysis of hMCT2-mediatedl-lactate uptake revealed that the apparent Kmvalue (0.32 ± 0.02 mM) was lower than that mediated by hMCT1 and 4. Finally, we investigated the conserved aspartic acids of hMCT2 and 4, and revealed that these residues were essential forl-lactate transport. These findings suggested that the extracellular aspartic acids conserved in hMCT1, 2, and 4 played important roles in transport activity and pH dependency, and can function as a first step of substrate and H+recognition and transport from the extracellular to the intracellular region. These findings contributed to enhance our understanding of the transport process of hMCT1, 2, and 4.