Wide tolerance to amino acids substitutions in the OCTN1 ergothioneine transporter.
Wide tolerance to amino acids substitutions in the OCTN1 ergothioneine transporter.
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DOI：
10.1016/j.bbagen.2016.03.021
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发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
Longo N
中科院分区：
文献类型：
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作者：
Frigeni M;Iacobazzi F;Yin X;Longo N
有机阳离子转运体在质膜上转移带正电荷的溶质。新型有机阳离子转运体1 （OCTN1）和2 （OCTN2）分别转运麦角硫因和肉碱。编码OCTN2的SLC22A5基因突变导致原发性肉毒碱缺乏症，这是一种隐性疾病，导致肉毒碱水平低和脂肪酸氧化缺陷。编码OCTN1的SLC22A4基因变异与类风湿关节炎和克罗恩病有关。在这里，我们利用融合OCTN1和OCTN2 cdna的不同部分构建的嵌合转运体来评估OCTN1转运体的功能特性。通过western blot分析和共聚焦显微镜观察它们的相对丰度和亚细胞分布。用OCTN2的相应残基取代OCTN1的c端部分，产生的嵌合OCTN转运体在运输麦角硫因方面比野生型OCTN1更活跃。在嵌合OCTN转运体中引入额外的单氨基酸取代进一步增加麦角硫因的转运活性。动力学分析表明，运输活性的增加是由于Vmax的增加，Km对麦角硫因的变化不大。我们的研究结果表明，OCTN1转运体耐受广泛的氨基酸取代。这与通过进化选择具有高功能活性的OCTN2肉毒碱转运蛋白形成鲜明对比，几乎所有的取代都降低了肉毒碱转运活性。OCTN1对氨基酸替换的广泛耐受性表明，相应的SLC22A4基因可能来自SLC22A5基因的近期重复，可能尚未确定其生理作用。
Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease. Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy. Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased Vmax, with modest changes in Km toward ergothioneine. Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity. The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role.