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首页> 外文期刊>American Journal of Physiology >Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis.
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Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis.

机译：特定氨基酸残基在人硫胺素转运蛋白SLC19A2中的功能作用：突变分析。

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摘要

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.
机译：SLC19A2是在各种人体组织（包括胃肠道）中表达的膜硫胺素转运蛋白。目前对SLC19A2的结构/功能关系知之甚少。我们研究了在硫胺素反应性巨幼细胞性贫血综合征（TRMA）中发现的与SLC19A2相同的突变对转运蛋白的功能活性和膜表达的影响。我们还检查了在SLC19A2的跨膜（TM）域和假定的糖基化位点（N63，N314）的唯一保守的阴离子残基（E138）突变的影响。 Northern印迹分析显示，在用野生型或突变的SLC19A2转染的HeLa细胞中，SLC19A2 mRNA以相同水平表达。引入临床相关的突变（D93H，S143F，G172D）或SLC19A2的保守阴离子残基（E138A）处的突变会导致对硫胺素摄取的显着抑制（P <0.01）。 SLC19A2的两个潜在的N联糖基化位点（N63Q，N314Q）的突变不影响功能活性;但是，它们确实导致了蛋白质表观分子量的显着降低。蛋白质印迹分析显示，所有蛋白质（D93H除外）均在HeLa细胞的膜部分（而非细胞质）中表达。这些结果直接证实了在TRMA中观察到的SLC19A2的临床相关突变会导致转运蛋白功能异常和/或其翻译/稳定性缺陷。结果还表明，SLC19A2蛋白的保守TM阴离子残基对其功能至关重要。此外，天然SLC19A2被糖基化，但这对其功能并不重要。

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