通过化学修饰鉴定钠耦合的中性氨基酸转运蛋白2（SNAT2）中的二硫键。,PLOS ONE

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通过化学修饰鉴定钠耦合的中性氨基酸转运蛋白2（SNAT2）中的二硫键。
PLOS ONE ( IF 2.9 ) Pub Date : 2016-06-29 , DOI: 10.1371/journal.pone.0158319
Chen Chen ₁ , Jiahong Wang ₁ , Ruiping Cai ₁ , Yanmeng Yuan ₁ , Zhanyun Guo ₂ , Christof Grewer ₃ , Zhou Zhang ₁

Affiliation

钠偶联的中性氨基酸转运蛋白2（SNAT2）属于溶质载体38（SLC38）转运蛋白家族，在哺乳动物组织中普遍表达并介导小的中性氨基酸的转运，例如丙氨酸（Ala，A）。尽管SNAT2在许多组织中起着至关重要的作用，但有关SNAT2的结构数据（包括固有的半胱氨酸残基与结构和功能的相关性）却很少。为了更好地定义内在半胱氨酸在SNAT2中形成二硫键的潜力，在有或没有有的情况下，均进行了诱变实验和N-乙基马来酰亚胺（NEM）和甲氧基-聚乙二醇马来酰亚胺（mPEG-Mal，MW 5000）进行的巯基特异性化学修饰还原剂二硫苏糖醇（DTT）处理。七个具有各种半胱氨酸（Cys，C）到丙氨酸（Ala，A）取代的单一突变转运蛋白，将C245,279A双突变体引入CNAT末端带有血凝素（HA）标签的SNAT2。结果表明，在存在DTT的情况下，表达C245A或C279A的细胞被一当量的mPEG-Mal标记，而野生型或所有其他Cys至Ala突变体则被两当量的mPEG-Mal修饰。此外，在存在或不存在DTT处理的情况下，C245,279A的分子量不变。结果表明SNAT2中Cys245和Cys279之间的二硫键对细胞表面运输以及转运蛋白功能没有影响。拟议的二硫键可能对描绘SNAT2和相关蛋白的细胞外结构域中的邻近度很重要。结果表明，在存在DTT的情况下，表达C245A或C279A的细胞被一当量的mPEG-Mal标记，而野生型或所有其他Cys至Ala突变体则被两当量的mPEG-Mal修饰。此外，在存在或不存在DTT处理的情况下，C245,279A的分子量不变。结果表明SNAT2中Cys245和Cys279之间的二硫键对细胞表面运输以及转运蛋白功能没有影响。拟议的二硫键对于描绘SNAT2和相关蛋白的胞外域中的邻近度可能很重要。结果表明，在存在DTT的情况下，表达C245A或C279A的细胞被一当量的mPEG-Mal标记，而野生型或所有其他Cys至Ala突变体则被两当量的mPEG-Mal修饰。此外，在存在或不存在DTT处理的情况下，C245,279A的分子量不变。结果表明SNAT2中Cys245和Cys279之间的二硫键对细胞表面运输以及转运蛋白功能没有影响。拟议的二硫键可能对描绘SNAT2和相关蛋白的细胞外结构域中的邻近度很重要。在有或没有DTT处理的情况下279A均未改变。结果表明SNAT2中Cys245和Cys279之间的二硫键对细胞表面运输以及转运蛋白功能没有影响。拟议的二硫键可能对描绘SNAT2和相关蛋白的细胞外结构域中的邻近度很重要。在有或没有DTT处理的情况下279A均未改变。结果表明SNAT2中的Cys245和Cys279之间的二硫键对细胞表面运输以及转运蛋白功能没有影响。拟议的二硫键可能对描绘SNAT2和相关蛋白的细胞外结构域中的邻近度很重要。

"点击查看英文标题和摘要"

Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification.

Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins.

更新日期：2019-11-01

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