大鼠中性氨基酸转运蛋白HA-SNAT3融合蛋白的构建SNAT1和SNAT2中二硫键的鉴定

【作者】 陈琛；

【导师】 张舟；

【作者基本信息】 上海师范大学 ， 细胞生物学， 2015， 硕士

【摘要】 钠离子偶联的中性氨基酸转运蛋白（Sodium-coupled Neutral Amino Acid Transporter,SNAT）属于SLC38家族,共有11个成员,分别为SNAT1-11。它们在机体内分布广泛,参与体内许多重要的生理活动。SNAT3是System N的成员之一,主要在脑部和视网膜的星形胶质细胞中表达,在脑部和肝脏的谷氨酸-谷氨酰胺循环中发挥重要作用。SNAT3的最适底物为谷氨酰胺和组氨酸,在转运氨基酸的同时协同转运1个Na⁺,反向转运1个H⁺,对Li⁺具有耐受性。SNAT3含有504个氨基酸残基,分子量约为55 k D。为了方便检测SNAT3在细胞膜上的表达和定位,从而进一步研究SNAT3的结构和功能,我们采用PCR扩增和酶切、连接的方法将HA标签序列连接到大鼠SNAT3的N端,并将融合蛋白构建在真核生物表达载体p BK-CMVΔ（1098-1300）上,获得p BK-CMVΔ-HA-SNAT3质粒。用脂质体转染法将该表达载体瞬时转染进人胚肾细胞（HEK293T）,通过Western blot的方法检测HA-SNAT3融合蛋白的表达。结果表明,HA-SNAT3能够正确在细胞膜上表达。由于目前还没有有效的SNAT3的商业化抗体,p BK-CMVΔ-HA-SNAT3融合蛋白表达载体的成功构建对于进一步研究SNAT3的结构和功能提供了有利的工具。SNAT1和SNAT2属于System A,广泛存在于哺乳动物的各个组织中,负责转运小的中性氨基酸,在转运氨基酸的同时协同转运一个Na⁺。SNAT1和SNAT2在大脑谷氨酸-谷氨酰胺循环中发挥重要作用,其功能紊乱会导致许多神经退行性疾病。二硫键是蛋白质多肽链的两个半胱氨酸残基之间形成的共价键,对于蛋白质空间结构的稳定性和活性功能的维持具有极其重要的作用。SNAT1中含有10个半胱氨酸,SNAT2中含有7个半胱氨酸。我们利用半胱氨酸特异性修饰剂NEM和m PEG₅000-Mal对野生型和10个半胱氨酸单突变体SNAT1分别进行化学修饰,用Western blot进行检测,结果显示,SNAT1野生型及C245A和C262A两个单突变体在先用NEM进行封闭、再经过DTT处理、然后进行m PEG₅000-Mal修饰后,分子量增加到约120 k D,而其它8个半胱氨酸单突变体和C245A,C262A双突变体在经过和不经过DTT处理时分子量不变,表明SNAT1中含有1对二硫键,由Cys245和Cys262形成。用同样的方法对SNAT2的野生型、7个半胱氨酸单突变体及C245A,C279A双突变体进行修饰,Western blot结果显示SNAT2野生型及C245A和C279A单突变体在先用NEM进行封闭、再经过DTT处理、然后进行m PEG₅000-Mal修饰后,分子量增加到约110 k D,而其它5个半胱氨酸单突变体及C245A,C279A双突变体在经过和不经过DTT处理时分子量不变,这表明SNAT2中也含有1对二硫键,由Cys245和Cys279形成。免疫染色的结果表明SNAT1和SNAT2中的半胱氨酸残基及二硫键不参与蛋白在细胞膜上的定位。SNAT1和SNAT2中二硫键的成功鉴定有助于我们进一步研究它们的功能并进行药物筛选,同时为确定其他膜蛋白中的二硫键提供了新的思路和方法。更多还原

【Abstract】 Sodium-coupled Neutral Amino Acid Transporter（SNAT） belongs to the Solute Carrier 38 family（SLC38）, which contains 11 members, namely SNAT1-11. They are ubiquitously expressed in mammalian tissues and have critical physiological functions.SNAT3 is a classical member of System N, which transports Gln and His predominantly. SNAT3 has the ability to counter transport both Na⁺ and H⁺, and reveals tolerance to replacement of Na⁺ by Li⁺. Abundant expression of SNAT3 protein is detected in astrocytes throughout the retina and brain. It has been suggested to be responsible for glutamine transport in glutamate-glutamine cycle in the brain and liver. SNAT3 contains 504 amino acid residues with a predicted molecular mass of 55 k D. In order to easily detect the expression and localization of SNAT3 on the membrane, we constructed a eukaryotic expression plasmid with an HA tag at the N-terminus of rat SNAT3 in the mammalian expression vector p BK-CMVΔ（1098-1300） by PCR and double restriction endonuclease digestion techniques. After transiently transfected the plasmid into human embryonic kidney cells（HEK293T）, expression of HA-SNAT3 fusion protein was detected by Western bolt. The result suggested that the HA-SNAT3 fusion protein could normally express on the membrane. The plasmid p BK-CMVΔ（1098-1300）-HA-SNAT3 will provide a useful tool for the investigation of structure and function of SNAT3 in the future.SNAT1 and SNAT2 are members of System A, which are responsible for transporting small, aliphatic amino acids. They transport substrates coupled to the uptake of Na with a stoichiometry of 1:1. Both SNAT1 and SNAT2 are involved in the significant glutamate/glutamine cycle in neurons. The dysfunction of SNAT1 and SNAT2 may cause some neurodegenerative diseases.There are 10 cysteine residues in SNAT1 and 7 in SNAT2. In this study, we performed thiol-specific chemical modification by sequential treatment of NEM, DTT, and m PEG₅000-Mal to wildtype, 10 single C to A mutants and the C245 A,C262A double mutant of SNAT1. The results of Western blot suggested that the apparent molecular weight of single mutants C245 A and C262 A were shifted to about 120 k D, as well as SNAT1 wildtype. On the contrary, the protein molecular weight of 8 other single mutants and C245 A,C262A double mutant remained to be 55 k D. The results suggested that Cys245 and Cys262 formed a disulfide bond in SNAT1. The same way of chemical modification was used to identify the disulfide bridge in SNAT2. The results indicated that the apparent molecular weight of SNAT2 wildtype and C245 A and C262 A single mutants was shifted to about 110 k D, while that of 5 other single mutants and C245 A,C279A double mutant was still 55-70 k D. The disulfide bond in SNAT2 was thus identified between Cys245 and Cys279. Besides, the results of Immunofluorescence staining showed that both the cysteine residues and the disulfide bridge in SNAT1 and SNAT2 had no effect on their trafficking to the cell surface.The successful identification of disulfide bonds in SNAT1 and SNAT2 will be helpful to the study of their structure and function. It also provides an excellent method to identify the disulfide bond in other transmembrane proteins.更多还原