CN112920264B - Tdp-43蛋白的o-糖基化突变体及其应用 - Google Patents
Tdp-43蛋白的o-糖基化突变体及其应用 Download PDFInfo
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Abstract
本发明公开了TDP‑43蛋白的O‑糖基化突变体及其构建方法和应用。所述突变体的氨基酸序列为在SEQ ID NO.3序列的第199位和/或233位突变成其他氨基酸。本发明还公开了上述突变体的编码序列、表达载体、宿主细胞及所述突变体在设计/制备神经退行性疾病的诊断试剂/诊断试剂盒/生物标志物，或者在制备神经退行性疾病相关的模式动物，或者在制备治疗/预防神经退行性疾病药物中的应用。本发明在首次证明了TDP‑43可被O‑糖基化，发现了TDP‑43主要是由OGT在T199和T233位点进行O‑糖基化，并基于此发现上述突变体可影响TDP‑43相关的RNA剪接功能，预防TDP‑43相关的蛋白质病变。
Description
技术领域
本发明属于生物技术领域，具体涉及TDP-43蛋白的O-糖基化突变体及其应用。
背景技术
Transactive response DNA-binding protein 43(TDP-43)是一种多功能的核蛋白，在转录、可变剪接、RNA稳定性和基因调控中发挥着重要的作用。TDP-43病理是大多数肌萎缩侧索硬化症(ALS)和约50％的额颞叶变性(FTLD)患者的疾病标志。编码TDP-43基因的突变与家族性和散发性ALS以及罕见的FTLD病例相关，并产生毒性。TDP-43主要定位在细胞核中，而病理的TDP-43和TDP-43截短片段主要存在于细胞质中，在病变的大脑和脊髓中形态为异常聚集。TDP-43在运动神经元中的胞质聚集物可能会表现出一种毒性的功能增益，获得异常的蛋白-蛋白和/或蛋白-RNA相互作用，同时伴有清除缺陷。然而，越来越多的证据支持TDP-43突变或聚集通过产生异常的RNA剪接或将核溶性TDP-43封存到细胞质内含物中，从而丧失剪接和转录活性，诱导功能的丧失。目前，TDP-43功能的调控机制仍然知之甚少。
TDP-43的翻译后修饰已被广泛研究，涉及TDP-43的聚集、稳定性和清除。TDP-43包含两个RNA识别基团(RRMs)，参与DNA或RNA结合；核输入和输出信号；以及一个C端富含甘氨酸的域，其中蕴藏着大多数与ALS相关的突变。特别是，TDP-43上的S409/S410位点的过度磷酸化被确定为疾病病理的标志，并在ALS和FTLD患者中普遍发现，与泛素化形式以及裂解的TDP-43C端片段相关，约35kDa和约25kDa。S409/S410位点的磷酸化可能会增强TDP-43的半衰期，并抑制蛋白酶体途径介导的降解，有助于聚集物的形成。最近，Cohen等发现TDP-43在氧化应激条件下可在RRM域内被乙酰化，导致TDP-43结合RNA的功能丧失，促进TDP-43聚集物在细胞中的积累。这些发现凸显了TDP-43翻译后修饰调控的新病理机制。
O-糖基化是一种蛋白质翻译后修饰，其中单糖N-乙酰葡糖胺(GlcNAc)通过O-连接的糖苷键连接到Ser/Thr残基上，据报道，O-糖基化调节多种细胞代谢，并参与神经退行性疾病。O-糖基化是一种高度动态可逆的、可诱导的修饰。存在于蛋白质上的糖链根据表达的组织和细胞不同而具有不同的结构。即使在同一细胞中，相同蛋白质的糖链结构也会根据发育过程的不同和外部环境的改变而产生极大的变化。因为蛋白质上O-糖基化修饰的高度动态性，导致了O-糖基化的质谱鉴定的困难，难以找到准确的O-糖基化位点，这些原因使得O-糖基化的研究进展缓慢。在脊椎动物中，O-糖基化是由一种唯一的催化酶负责修饰，被命名为OGT(O-linked N-acetylglucosamine transferase)，并由一种唯一的催化酶从蛋白质中移除，被称为OGA(O-GlcNAcase)。与神经退行性疾病相关的一个著名的例子是tau蛋白的O-糖基化，它广泛地发生在人类大脑中。增加O-GlcNAc水平可以阻断tau的磷酸化，减轻tau聚集的形成，这是tau相关神经退行性疾病的常见病理特征。然而，针对TDP-43蛋白的糖基化还未见报道。
发明内容
针对上述现有技术中存在的问题，发明人首次发现了TDP-43可以发生糖基化，本发明的目的之一是提供TDP-43蛋白的O-糖基化突变体，本发明发现并证明了TDP-43在特定位点的糖基化突变体能够影响TDP-43相关的蛋白病变和功能，并且与神经退行性疾病，尤其是ALS和FTLD的前期诊断和治疗有关。
为实现上述目的，本发明采用的技术方案是：
TDP-43蛋白的O-糖基化突变体，所述TDP-43蛋白的氨基酸序列如SEQ ID NO.3所示，所述突变体在SEQ ID NO.3序列的第199位和/或233位突变成非苏氨酸的其他氨基酸。
优选的，所述其他氨基酸为丙氨酸、酪氨酸、天冬氨酸或半胱氨酸。
虽然O-糖基化修饰已经被报道为一种普遍的修饰方式，但是由于实验技术的限制，被发现的大规模糖基化修饰组学研究并不多见。鉴于这种修饰形式非常不稳定，在质谱仪中很容易被离子冲击打断；另外Ser/Thr残基也是常见的磷酸化修饰位点，在鉴定中容易发生混淆。本发明通过富集HEK 293T细胞内过表达的TDP-43蛋白，运用一种称为ETD模式(Electron transfer dissociation)的特殊质谱鉴定技术，发现了T199、S212和T233三个修饰位点。通过氨基酸序列保守性比对分析，确定了T199和T233为TDP-43的主要糖基化修饰位点。
尽管上述背景技术中描述了O-糖基化质谱鉴定的困难，但本发明创造性地发现了TDP-43主要是由OGT在T199和T233位点进行O-糖基化修饰，通过构建T199和/或T233突变体，破坏TDP-43O-糖基化，损害了多个基因的前体mRNA剪接，同时还会调控信号传导、转录和细胞骨架功能。
优选地，所述非苏氨酸的其他氨基酸为丙氨酸，所述突变体的氨基酸序列如SEQID NO.4所示。
本发明的另一目的在于提供编码上述突变体的DNA序列。优选地，编码所述SEQ IDNO.4氨基酸序列的DNA序列如SEQ ID NO.2所示。
本发明的另一目的在于提供携带上述DNA序列的表达载体。
优选地，所述的表达载体的骨架质粒为pCS2质粒或pGEX-6p-1质粒。
本发明的另一目的在于提供含有上述的表达载体的细胞，其为HEK 293T细胞或SH-SY5Y细胞或Neuro 2a细胞。
本发明所述突变体采用pCS2质粒进行哺乳动物细胞转染，采用pGEX-6p-1质粒进行体外纯化制备突变体，上述突变体的体外纯化方法可采用现有方法。
本发明的另一目的在于提供所述的TDP-43突变体在设计/制备神经退行性疾病的诊断试剂/诊断试剂盒/生物标志物，或者在神经退行性疾病相关的模式动物，或者在制备治疗/预防神经退行性疾病药物中的应用。如：将所述的TDP-43突变体编码的DNA序列作为检测神经退行性疾病的标志物，通过转基因或基因编辑技术获得的表达所述TDP-43突变体的模式动物，将促进TDP-43糖基化修饰的物质作为治疗/预防神经退行性疾病的药物；所述的促进TDP-43糖基化修饰的物质包含小分子(N-乙酰葡萄糖胺)或生物大分子糖基化转移酶。
优选地，所述神经退行性疾病为肌萎缩侧索硬化症或额颞叶变性病。
发明人证明了，本发明的TDP-43蛋白的O-糖基化突变体可以改善幼虫和成蝇的运动缺陷，并延长成虫的寿命。
一种治疗/预防神经退行性疾病的药物，包含促进TDP-43糖基化修饰的物质。所述的促进TDP-43糖基化修饰的物质为小分子(N-乙酰葡萄糖胺)或生物大分子糖基化转移酶。
与现有技术相比，本发明的有益效果是：
本发明在首次证明TDP-43可被O-糖基化的基础上，创造性的发现了TDP-43由OGT在T199和T233位点进行O-糖基化，并基于此发现构建的TDP-43蛋白的O-糖基化突变体预防TDP-43相关的蛋白质病变，及影响TDP-43相关的RNA剪接功能中的应用；辅助高糖，可改善ALS相关TDP-43蛋白病引起的运动和寿命缺陷。
附图说明
图1为实施例1中检测从SH-SY5Y细胞免疫沉淀的TDP-43的O-糖基化信号。
图2为实施例1中检测用RL2抗体免疫沉淀的TDP-43的O-糖基化信号。
图3为实施例2中酵母细胞TDP-43蛋白聚集的观察。右图为左边免疫荧光图的统计数据。
图4为实施例2中GlcNA或/和EA处理细胞之后IP TDP-43，检测其糖基化水平和磷酸化水平。
图5为实施例2中SH-SY5Y细胞免疫荧光实验的代表图像。比例尺：10μm。
图6为图5中pTDP-43染色的信号强度(左)或每个细胞中蛋白质积聚点数及pTDP-43阳性细胞(右)的定量统计。
图7为实施例2中检测细胞在用EA处理或不处理的情况下，过表达OGT或其酶活突变体后TDP-43蛋白质不可溶积聚形式的水平。右图为左边WB图中不可溶TDP-43的定量图。
图8为实施例3质谱实验鉴定TDP-43蛋白糖基化T199位点。
图9为实施例3质谱实验鉴定TDP-43蛋白糖基化S212、T233位点。
图10为实施例4构建的糖基化位点突变体TDP-43序列与野生型序列比对。左图为T199A突变体的测序，右图为T233A突变体的测序。
图11为实施例4体外通过免疫印迹检测TDP-43蛋白的O-糖基化水平。
图12为实施例4在细胞内通过免疫沉淀检测TDP-43蛋白的O-糖基化水平。
图13为实施例5用普通蔗糖饮食(RS)或高蔗糖饮食(HS)饲喂成蝇，并检测指定基因型成蝇的存活率。Kaplan-Meier生存分析，每组n≥40。
图14为实施例5中不同基因型10日龄成年雄蝇的行走速度。平均值±SD。非配对双尾t检验，生物重复。对照组，n＝185，OGT，n＝148，TDP-43，n＝177，TDP-43/OGT，n＝187。
图15为实施例6通过PCR分析检查CFTR外显子9的剪接。数字代表外显子9跳跃(-)与保留(+)的相对比例(上图)。通过免疫印迹法(下图)检测指定样品中的内源性(endo.)和外源性(exo.)TDP-43蛋白水平。
图16为实施例6通过PCR分析检查STMN2的2a外显子的剪接。通过免疫印迹法检测样品中TDP-43的蛋白水平。
具体实施方式
下面将结合本发明中的附图，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，以下实施例是为了更好地说明阐述本发明的内容。本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是，本发明的保护和权利要求范围不限于所提供的案例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
1.细胞培养和试剂处理
HEK 293T细胞、SH-SY5Y细胞来自于中国典型培养物保藏中心(CCTCC)，以上细胞也可采用市售获得。HEK 293T细胞用DMEM培养基培养，SH-SY5Y细胞用DMEM/F12培养基培养，培养时均添加10％的FBS。试剂处理细胞所用浓度和时间：10μM TMG(MedChemExpressHY-12588)、50μM PUGNAc(Sigma，A7229)、10M OSMI-1(Sigma，SML1621)处理36小时；5mMGlcNAc(Sigma，A3286)或不同剂量的UDP-GlcNAc(Sigma，U4375)处理24小时；60μM EA(MedChemExpress，HY-B1640)处理24小时。
2.质粒构建和抗体信息
将TDP-43和OGT基因克隆到pCS2-3xFlag、pCS2-GFP或pGEX-6P-1载体上，并使用表1中列出的引物通过定点突变得到各个TDP-43突变体。为了产生UAS-TDP-43WT和突变体转基因，通过LR反应将人TDP-43WT和突变体克隆到Gateway兼容的pBID-UAS-G载体中，通过Gateway试剂盒克隆到pBID-UAS的Xho I/Xba I位点之间。抗体信息：TDP-43(Proteintech，10782-2-AP，1:1000用于免疫印迹；12892-1-AP用于免疫沉淀；Abcam，ab10423，1:500用于免疫荧光染色)、O-GlcNAc(CTD 110.6)来自Santa Cruz(sc-59623，1:1000)；Sigma(MABS1254，1:500)，O-GlcNAc(RL2)来自Abcam(ab2739，1:1000)，phospho-TDP-43 409/410(Cosmo Bio，425-4，1:2000)和GFP(Sungen，KM8009，1:3000)，GAPDH(Abclonal，AC002，1:5000)。
实施例1 TDP-43在体内可被O-糖基化修饰
本实施例首先检测TDP-43是否能够进行O-糖基化修饰。具体实验方法如下：
1.1培养SH-SY5Y细胞，收集细胞并用IP buffer在冰上裂解细胞30分钟，15％功率超声15秒使细胞核破裂，4℃高速离心10分钟，取上清用TDP-43抗体在4℃免疫沉淀(IP)过夜，IP后的样品用O-糖基化抗体进行Western blot(WB)检测。将IgG免疫沉淀作为阴性对照。
1.2实验方法同1.1类似。用RL2抗体进行IP，用TDP-43抗体进行WB检测。
从人SH-SY5Y神经母细胞中免疫沉淀出内源性TDP-43，并检测TDP-43的O-糖基化，结果如图1所示。另外，使用特异性的O-糖基化识别抗体RL2进行免疫沉淀检测，通过免疫印迹也可以检测到O-糖基化TDP-43，结果如图2所示。
实施例2小分子药物N-乙酰葡萄糖胺和糖基转移酶OGT抑制蛋白质积聚和TDP-43病理磷酸化修饰
为了研究TDP-43糖基化是否会影响TDP-43相关的蛋白质病变，本实施例使用一个成熟的酵母TDP-43系统来模拟人类TDP-43(hTDP-43)蛋白病的一些显著特征，包括细胞毒性和蛋白质聚集。具体实验方法如下：
2.1半乳糖诱导TDP-43表达：本实验是通过半乳糖诱导酵母内含Gal启动子的TDP-43蛋白的表达。接种该菌株时，液体培养基中的碳源为葡萄糖，TDP-43基因不会表达。在spot assay中，将培养基更换成以半乳糖及蔗糖为碳源的平板时可以诱导目的蛋白的表达，从而观察目的蛋白对于酵母生长的影响。进行实验时，将空载(Vec+Vec)、单独过表达TDP-43(TDP-43+Vec)、TDP-43与野生型OGT共转(TDP-43+OGT(WT))、TDP-43与酶活突变体OGT共转(TDP-43+OGT(H498N))四种酵母菌株提前一天接种目的酵母于以葡萄糖为碳源的Leu、Ura双缺培养基中，在30℃酵母培养摇床中培养过夜。第二天室温3000rpm离心酵母培养液5分钟。弃上清并用ddH2O洗酵母菌体3次，使葡萄糖彻底去除，置换成含半乳糖而不含葡糖糖的培养基中，在30℃酵母培养摇床中培养4小时，将四种酵母菌液滴在盖玻片上在荧光显微镜下观察TDP-43的聚集。其中TDP-43蛋白带绿色荧光蛋白标签(GFP)记为TDP-43(GFP)，OGT带红色荧光蛋白标签(mCherry)记为TDP-43(mCherry)。
2.2IP外源表达的Flag-TDP-43蛋白并检测其糖基化和磷酸化水平：在SH-SY5Y细胞中过表达Flag标签的TDP-43蛋白，转染24小时候用药物处理细胞，分组为：未表达Flag-TDP-43组、表达Flag-TDP-43未加药组、表达Flag-TDP-43后EA处理组、表达Flag-TDP-43后EA+GlcNAc联合处理组。处理24小时候收细胞，用Flag凝胶进行IP，IP后的样品用抗体检测其糖基化和磷酸化水平。
2.3免疫荧光实验：免疫荧光使用的玻片用4％多聚赖氨酸处理，实验时提前将SH-SY5Y细胞均匀地传到铺有玻片的12孔板里，用EA或/和N-乙酰葡萄糖胺药物处理细胞，分组为对照(Mock)、N-乙酰葡糖胺处理(GlcNAc)、利尿酸处理(EA)、N-乙酰葡糖胺和利尿酸联合处理(GlcNAc+EA)，处理24小时之后，用4％多聚甲醛固定细胞，0.3％TritonX-100通透细胞，含3％BSA的PBST(PBS+0.1％TritonX-100)封闭，分别孵育一抗(TDP-43和pTDP-43抗体)、二抗(绿色488荧光二抗和红色Cy3荧光二抗)、DAPI，最后加上抗荧光淬灭剂并封片在荧光显微镜下观察拍照和统计。
2.4TDP-43不可溶分析：在SH-SY5Y细胞中过表达OGT或酶活突变体(OGT H498N)，用EA处理细胞24小时，分组为：1.对照组，2.过表达Myc-OGT，3.过表达Myc-OGT H498N，4.EA处理，5.Myc-OGT+EA，6.Myc-OGT H498N+EA。处理结束后，收集细胞样品，沉淀加入裂解buffer(50mM Tris pH 8.0,150mM NaCl,1％NP-40,5mM EDTA,0.5％脱氧胆酸钠,0.1％SDS)混匀，冰上裂解处理15分钟。在裂解液中加4-5μL 10％NP-40，震荡混匀处理后放在冰上处理2分钟。在4℃离心机100,000g离心10-15分钟后取上清，为细胞可溶部分。沉淀使用PBS洗涤后离心去上清，重复三次后加入2×SDS sample buffer制样，得到的为细胞不可溶部分。
结果如图3所示，在酵母中诱导过表达TDP-43使蛋白质发生明显的积聚，而同时过表达糖基转移酶OGT使酵母中TDP-43聚集的减少；定量分析表明，含有>3个GFP聚集点的细胞群明显减少。因此，结果表明OGT在酵母中能够抑制TDP-43的积聚。
接下来，发明人测试在哺乳动物细胞中OGT是否可以通过拮抗TDP-43高磷酸化来缓解TDP-43蛋白质病变。发明人比较未表达Flag-TDP-43组、表达Flag-TDP-43未加药组、表达Flag-TDP-43后EA处理组、表达Flag-TDP-43后EA+GlcNAc联合处理组四组细胞的TDP-43病理情况，发明人发现，用乙醇酸(EA)处理的细胞导致磷酸化水平增加，而通过Western印迹观察到TDP-43的O-糖基化水平显著下降。然而，当用N-乙酰葡萄糖和EA同时处理细胞时，伴随着TDP-43的O-糖基化水平的恢复，TDP-43的磷酸化水平下降到与未处理对照细胞相当的水平(图4)，说明TDP-43糖基化可以拮抗其病理性磷酸化。TDP-43引起的病变也包括TDP-43形成不可溶的的蛋白质积聚。发明人在细胞水平用免疫荧光直观观察TDP-43蛋白质积聚情况，结果显示，在EA处理的细胞中，含磷酸化TDP-43的阳性细胞群和蛋白质病理积聚增加，但在仅用N-乙酰葡萄糖或EA和GlcNAc联合处理的细胞中则没有增加，如图5-6所示。说明TDP-43糖基化可以拮抗TDP-43磷酸化病理标志。图6中细胞数量：左图，"Mock"n＝58，"GlcNAc"n＝35，"EA"n＝45，"GlcNAc+EA"n＝52；右图，"Mock"n＝1167，"GlcNAc"n＝1051，"EA"n＝54 2，"GlcNAc+EA"n＝815。平均值±SD，非配对双尾t检验，P<0.0001。
发明人进一步检测糖基转移酶OGT对TDP-43不可溶颗粒的缓解功能。发明人用溶解度测定实验表明，野生型OGT的过表达显著减少内源性TDP-43的不溶性组分，且与其酶活性有关(图7，泳道2与泳道3)，即使在EA存在的情况下也是同样的现象(图7，泳道5与泳道6)。
实施例2充分说明TDP-43糖基化修饰/促进糖基化修饰的小分子(N-乙酰葡萄糖胺)/糖基化转移酶都可以抑制TDP-43的病理性磷酸化修饰、缓解TDP-43的不可溶性蛋白质积聚，进而缓解由此引起的ALS病人症状，可能可以作为ALS疾病的治疗药物。
实施例3质谱实验鉴定TDP-43蛋白糖基化位点
本实施例利用质谱手段鉴定TDP-43潜在的糖基化位点。具体实验方法：
3.1在HEK 293T细胞中过表达Flag-TDP-43，免疫沉淀TDP-43蛋白，然后在SDS-PAGE凝胶中电泳。凝胶用考马斯亮蓝染色，切胶并进行胶内酶解。将多肽弹射到液相色谱中，然后进行质谱分析(LC-MS/MS)。用配备在线电喷雾离子源的质谱仪(Thermo FisherScientific，San Jose，CA)分析洗脱的肽段。用高能碰撞解离(HCD)和电子转移解离(ETD)组合检测样品。
为了确定TDP-43上潜在的OGT修饰位点，在GlcNAc存在下在HEK 293T细胞中表达Flag-TDP-43，用Flag树脂进行免疫沉淀，将富集的TDP-43洗脱液进行质谱(MS)分析。质谱数据显示，199位苏氨酸(T199)、212位丝氨酸212(S212)和233位苏氨酸(T233)是TDP-43的候选糖基化位点(如图8-9)。值得注意的是，T199和T233在高等真核生物中是保守的，这两个位点都位于第二个RRM结构域(RRM2)，而S212位点则是不保守的。
实施例4体内和体外糖基化实验证明TDP-43蛋白糖基化位点是T199和T233
为了确定T199和T233这两个保守残基是否是TDP-43中的主要O-糖基化位点，具体实验方法如下：
4.1质粒点突变
1)设计点突变引物。取突变位点前后各约20bp，引物序列如下表1。
表1
2)KOD酶PCR反应体系：
另配制一管加入相同体系但不加KOD酶，用作负对照。
3)PCR反应条件：
4)PCR完成后，加入1μL DpnI酶，37℃酶切4-6小时。
5)酶切完成后，取10μL转化DH5α细胞，在50μL感受态细胞中加入10μL酶切产物，混匀后冰上放置30分钟，42℃水浴锅热激45秒后，再置于冰上2分钟，超净工作台内涂到相应的抗性平板上。
6)次日，挑选单克隆，摇菌，质粒抽提后测序来检测目的碱基是否发生正确突变。
4.2GST标签蛋白纯化
挑取正确表达质粒(pGEX-GST-TDP-43及其突变体TDP-43 T199、TDP-43 T233A、TDP-43 2TA)的BL21菌株加入15mL含氨苄的LB培养基中37℃ 220rpm培养摇床培养过夜。将过夜培养的菌液转接到1L含氨苄的LB培养基中37℃ 200至220rpm震荡培养至OD600到达0.4至0.6。当OD600达到0.4至0.6时，将培养瓶取出放置于冰上快速冷却，加入0.2mM IPTG后培养箱中18℃、220rpm培养，诱导蛋白表达4至6个小时。提前洗干净收菌所需要的500mL离心瓶，倒入菌液，配平后在4℃离心机400rpm离心30分钟。用灭过菌的双蒸水重悬菌体转移至50mL离心管中，再次4℃ 4000rpm离心30分钟。弃上清用裂解buffer重悬菌体，加入溶菌酶、PMSF和蛋白酶抑制剂，冰上30分钟后超声裂菌。4℃ 4000rpm离心30分钟，弃沉淀再重复离心一次，上清在4℃孵育GST Affinity Resin Beads 4-6小时，用洗脱缓冲液将蛋白洗脱下来，置换到PBS buffer中，浓缩、定量，-80℃储存或直接用于后续实验。
4.3体外糖基化实验：在D100皿的HEK 293T细胞中过表达Flag-OGT并IP Flag得到有活性的OGT蛋白，将Flag-OGT和体外表达的TDP-43或其突变体TDP-43 T199A、TDP-43T233A、TDP-43 2TA蛋白片段配制20μL反应体系(TDP-43 5μg，Flag-OGT IP Beads 10μL，1MTris-HCl pH 7.6 50mM，MgCl2 12.5mM，DTT 1mM)，恒温振荡器中30℃、700rpm反应5小时。
4.4体内糖基化实验：方法同1.1，用GFP抗体IP，用糖基化抗体进行WB检测。
发明人构建了带有两个O-糖基化位点的GST标签的TDP-43(a.a.102-269)片段的质粒，构建了T199A、T233A和T199A/T233A双突变体(2TA)(图10)，使用大肠杆菌蛋白表达系统纯化出这些蛋白，然后在HEK 293T细胞里过表达Flag-OGT，通过免疫沉淀得到体外反应所需的酶(OGT)。在UDP-GlcNAc供体存在下，将来自HEK 293T细胞的免疫纯化的Flag标记的OGT与各种重组GST-TDP-43蛋白片段孵育。体外实验表明，T199A或T233A突变体的O-糖基化信号降低，而2TA突变体的O-糖基化信号则消失(如图11)。
发明人还构建了带GFP标签的TDP-43及T199A、T233A、2TA、S212A的点突变，T199和T233的点突变序列与野生型序列比对如图8所示，野生型TDP-43的DNA序列见SEQ ID NO.1，蛋白质序列见SEQ ID NO.3；突变得到的DNA序列见SEQ ID NO.2，蛋白质序列见SEQ IDNO.4。在293T细胞中转染这些质粒，然后用GFP的抗体进行免疫沉淀富集GFP-TDP-43蛋白，再通过CTD110.6检测TDP-43的O-糖基化信号，发现细胞中表达的带GFP标签的T199A或T233A突变体的O-糖基化水平明显降低，而2TA突变体的O-糖基化水平与WT相比几乎没有检测到(如图12)，但是同样位于RRM2中的TDP-43的S212A突变并没有影响其O-糖基化水平，这说明另外两个保守的O-糖基化位点具有特异性。这些结果表明，T199和T233是TDP-43的主要O-糖基化位点。
实施例5TDP-43的O-糖基化影响果蝇的运动和寿命
本实施例利用果蝇作为模型来研究TDP-43的O-糖基化在ALS特征中的影响。具体实验方法：
5.1果糖饮食果蝇寿命分析：转基因果蝇类型为：Control、过表达TDP-43野生型、过表达TDP-43 T199A突变体、过表达TDP-43 T233A突变体、过表达TDP-43 T199/233A突变体。进行实验时在果蝇管中挑选同性别的果蝇成虫，放在冰上麻醉。将麻醉的果蝇分成等数量的两组，分别转移到高糖培养基和普通培养基中。每天记录果蝇的存活率，每隔2至3天更换新的培养基避免果蝇意外死亡。待果蝇全部死亡，使用Kaplan-Meier生存分析绘制果蝇的生存曲线图。图11中RS为普通蔗糖饮食(4％蔗糖)，HS为高蔗糖饮食(16％蔗糖)。
5.2果蝇运动能力分析：转基因果蝇类型为：Control、过表达OGT、过表达TDP-43、OGT和TDP-43同时过表达、过表达TDP-43 T199/233A突变体、OGT和TDP-43 T199/T233A同时过表达。进行实验时，在果蝇管中挑选出同性别并且近三天内没有被麻醉过的果蝇成虫，转移到测量用的量筒中。使用相机拍摄果蝇从量筒底部爬完整个量筒的视频，每种基因型至少取80只果蝇分批拍摄。根据视频的时长计算出每只果蝇运动的速度，使用prism 7.0统计软件对果蝇的运动速度进行定量统计。
发明人研究发现，高糖饮食可以改善运动神经元TDP-43过度表达引起的运动和寿命缺陷。本实施例给表达hTDP-43的果蝇喂食普通(4％蔗糖，即4g蔗糖/100g培养基)或高糖(16％蔗糖，16g蔗糖/100g培养基)饮食，然后使用Kaplan-meier生存分析，绘制果蝇的生存曲线。由于蔗糖可以分解形成葡萄糖，直接进入六胺生物合成途径，并作为O-GlcNAc的底物，发明人观察到高蔗糖饮食减轻了由神经元表达WT hTDP-43引起的成虫运动功能缺陷。此外，与对照果蝇相比，在神经元中过量表达WT、T199A或T233A hTDP-43，但不是2TA突变体，显著缩短了果蝇的寿命，这表明它们具有较高的神经元毒性(图11)。有趣的是，发明人观察到，在WT、T199A或T233A hTDP-43过表达的果蝇中，高蔗糖饮食在一定程度上延长了果蝇的寿命。具体来说，用普通蔗糖饮食喂养WT表达的果蝇平均寿命为10天，用高蔗糖饮食喂养的果蝇寿命增加了60％(16天)。T199A和T233A表达的果蝇的寿命比WT长，用高蔗糖饮食喂养后，其平均寿命分别增加13％和3.7％。值得注意的是，表达TDP-43的2TA突变体的果蝇，即使喂食高糖饮食，寿命也没有差异，说明O-GlcNAylation发生在这两个位点的重要性(图13)。
接下来，发明人想验证这些指示TDP-43过量表达的幼虫或成蝇在高糖饮食时缓解ALS的特征是否来自于这两个部位的O-糖基化修饰。因此，hTDP-43和OGT在运动神经元中共表达，与hTDP-43单独表达相比，减轻了运动障碍，相反，OGT和2TA突变体的共表达与hTDP-43单独表达相比，不能进一步增强其运动障碍(图14)。这些结果表明，OGT对hTDP-43的O-糖基化可以部分缓解果蝇的那些表型，包括运动障碍和短寿，而所确定的两个O-糖基化位点对缓解ALS相关的神经毒性非常重要，TDP-43 2TA突变体由于无法糖基化，所以即使高糖饮食、共表达OGT仍然不能缓解神经毒性。
实施例6TDP-43 2TA突变体丧失了mRNA剪接功能
可变剪接是TDP-43最重要的功能之一。高通量测序表明，小鼠大脑中TDP-43的下调导致mRNA的不同剪接。为了测试O-糖基化是否调节TDP-43的mRNA剪接功能，发明人进行了基于细胞的核CFTR(囊性纤维化跨膜传导调节器)剪接实验(Buratti E,Baralle FE(2001)Characterization and functional implications of the RNA bindingproperties of nuclear factor TDP-43,a novel splicing regulator of CFTR exon9)。具体实施方法：
6.1准备HEK 293T细胞，铺在6孔板上，密度需要小一些。第二天待细胞密度长至30％-50％，进行siTDP-43转染(siTDP-43 RNA序列：5'-GGCUCAAGCAUGGA-UUCUA-3')，第二天转染对应的TDP-43质粒和CFTR mini基因质粒，两天后收细胞，一部分细胞用于WB检测，一部分细胞利用Trizol法抽取总RNA，反转录得到cDNA，接着，用cDNA为模板，用CFTR剪切引物进行PCR反应。在STMN2剪接实验中，在SH-SY5Y细胞中转染siTDP-43和对应的TDP-43质粒，用STMN2对应引物进行PCR反应。引物序列见下表2。
表2
| 引物名 | 序列(5’-3’) |
| CFTR-F | TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA |
| CFTR-R | CAACTTCAAGCTCCTAAGCCACTGC |
| STMN2-F | AGCTGTCCATGCTGTCACTG |
| STMN2-R | GCAGGCTGTCTGTCTCTCTC |
发明人采用了之前报道的TDP-43特异性siRNA(Tollervey JR,Curk T,Rogelj B,Briese M,Cereda M,Kayikci M,Konig J,Hortobagyi T,Nishimura AL,Zupunski V etal(2011)Characterizing the RNA targets and position-dependent splicingregulation by TDP-43.Nat Neurosci 14:452-458)，该siRNA可显著降低内源性TDP-43水平，而不干扰共转染抗RNAi的WT或其它TDP-43突变体的表达。结果显示，TDP-43敲除导致第9外显子未剪接转录物的积累，而与对照相比，回转WT TDP-43恢复了这种表型。然而，2TA突变体失去了mini-CFTR可变剪接能力(图15)，另外，我们发现将T199和T233位点突变成其它非苏氨酸的氨基酸如酪氨酸(Y)和半胱氨酸(C)也无法实行其RNA剪接的功能，这表明这两个位点的O-糖基化在RNA剪接的重要性。发明人在小鼠脑神经瘤细胞Neuro 2a细胞中也发现TDP-43 2TA突变体丧失其剪接功能。
有报道发现，STMN2是神经轴突生长和更新的重要调节因子，而TDP-43可以调节STMN2的剪切(Klim JR,Williams LA,Limone F,Guerra San Juan I,Davis-DusenberyBN,Mordes DA,Burberry A,Steinbaugh MJ,Gamage KK,Kirchner R et al(2019)ALS-implicated protein TDP-43sustains levels of STMN2,a mediator of motor neurongrowth and repair.Nat Neurosci 22:167-179)。为了测试TDP-43的O-糖基化是否会影响STMN2的剪接，发明人在SH-SY5Y细胞中敲低内源性TDP-43并回转TDP-43野生型和糖基化位点突变体，通过PCR检测外显子2a的表达。TDP-43的敲低诱导了外显子2a的产生。在TDP-43敲低细胞中回转表达WT TDP-43，以及T199A和Q331K突变体，STMN2发生正常剪接，抑制了外显子2a的表达。相反，T233A或2TA突变体表达的细胞中仍然检测出较高的外显子2a水平(图16)。这些结果表明，TDP-43的O-糖基化是STMN2正确的mRNA剪接所必需的，通过对STMN2的剪接从而在神经元生长和轴突再生中发挥作用，糖基化位点突变体(2TA)由于不能发生糖基化，其剪接功能丧失，无法正常调控STMN2的剪接，或可引起神经退行性疾病ALS的发生。
总结上述结果，发明人发现TDP-43糖基化修饰/促进糖基化修饰的小分子(N-乙酰葡萄糖胺)/糖基化转移酶都可以抑制TDP-43的病理性磷酸化修饰、缓解TDP-43的不可溶性蛋白质积聚，进而缓解由此引起的ALS病人症状，可能可以作为ALS疾病的治疗药物；果蝇高糖饮食可缓解过表达TDP-43引起的神经毒性；TDP-43 2TA丧失了RNA剪接功能，引起STMN2不正常剪接，可能引起ALS疾病。
本发明所述TDP-43蛋白的O-糖基化突变体在设计/制备神经退行性疾病的诊断试剂/诊断试剂盒/生物标志物，或者在制备神经退行性疾病相关的模式动物，或者在制备治疗/预防神经退行性疾病药物中应用，所述的神经退行性疾病尤其指肌萎缩侧索硬化症或额颞叶变性病。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 武汉大学
<120> TDP-43蛋白的O-糖基化突变体及其应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1251
<212> DNA
<213> Homo sapiens
<400> 1
atgtctgaat atattcgggt aaccgaagat gagaacgatg agcccattga aataccatcg 60
gaagacgatg ggacggtgct gctctccacg gttacagccc agtttccagg ggcgtgtggg 120
cttcgctaca ggaatccagt gtctcagtgt atgagaggtg tccggctggt agaaggaatt 180
ctgcatgccc cagatgctgg ctggggaaat ctggtgtatg ttgtcaacta tccaaaagat 240
aacaaaagaa aaatggatga gacagatgct tcatcagcag tgaaagtgaa aagagcagtc 300
cagaaaacat ccgatttaat agtgttgggt ctcccatgga aaacaaccga acaggacctg 360
aaagagtatt ttagtacctt tggagaagtt cttatggtgc aggtcaagaa agatcttaag 420
actggtcatt caaaggggtt tggctttgtt cgttttacgg aatatgaaac acaagtgaaa 480
gtaatgtcac agcgacatat gatagatgga cgatggtgtg actgcaaact tcctaattct 540
aagcaaagcc aagatgagcc tttgagaagc agaaaagtgt ttgtggggcg ctgtacagag 600
gacatgactg aggatgagct gcgggagttc ttctctcagt acggggatgt gatggatgtc 660
ttcatcccca agccattcag ggcctttgcc tttgttacat ttgcagatga tcagattgcg 720
cagtctcttt gtggagagga cttgatcatt aaaggaatca gcgttcatat atccaatgcc 780
gaacctaagc acaatagcaa tagacagtta gaaagaagtg gaagatttgg tggtaatcca 840
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aacaatcaag gtagtaatat gggtggtggg atgaactttg gtgcgttcag cattaatcca 960
gccatgatgg ctgccgccca ggcagcacta cagagcagtt ggggtatgat gggcatgtta 1020
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agggagccaa accaggcctt cggttctgga aataactctt atagtggctc taattctggt 1140
gcagcaattg gttggggatc agcatccaat gcagggtcgg gcagtggttt taatggaggc 1200
tttggctcaa gcatggattc taagtcttct ggctggggaa tgaatcacta g 1251
<210> 2
<211> 1251
<212> DNA
<213> Homo sapiens
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atgtctgaat atattcgggt aaccgaagat gagaacgatg agcccattga aataccatcg 60
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cttcgctaca ggaatccagt gtctcagtgt atgagaggtg tccggctggt agaaggaatt 180
ctgcatgccc cagatgctgg ctggggaaat ctggtgtatg ttgtcaacta tccaaaagat 240
aacaaaagaa aaatggatga gacagatgct tcatcagcag tgaaagtgaa aagagcagtc 300
cagaaaacat ccgatttaat agtgttgggt ctcccatgga aaacaaccga acaggacctg 360
aaagagtatt ttagtacctt tggagaagtt cttatggtgc aggtcaagaa agatcttaag 420
actggtcatt caaaggggtt tggctttgtt cgttttacgg aatatgaaac acaagtgaaa 480
gtaatgtcac agcgacatat gatagatgga cgatggtgtg actgcaaact tcctaattct 540
aagcaaagcc aagatgagcc tttgagaagc agaaaagtgt ttgtggggcg ctgtgcagag 600
gacatgactg aggatgagct gcgggagttc ttctctcagt acggggatgt gatggatgtc 660
ttcatcccca agccattcag ggcctttgcc tttgttgcat ttgcagatga tcagattgcg 720
cagtctcttt gtggagagga cttgatcatt aaaggaatca gcgttcatat atccaatgcc 780
gaacctaagc acaatagcaa tagacagtta gaaagaagtg gaagatttgg tggtaatcca 840
ggtggctttg ggaatcaggg tggatttggt aatagcagag ggggtggagc tggtttggga 900
aacaatcaag gtagtaatat gggtggtggg atgaactttg gtgcgttcag cattaatcca 960
gccatgatgg ctgccgccca ggcagcacta cagagcagtt ggggtatgat gggcatgtta 1020
gccagccagc agaaccagtc aggcccatcg ggtaataacc aaaaccaagg caacatgcag 1080
agggagccaa accaggcctt cggttctgga aataactctt atagtggctc taattctggt 1140
gcagcaattg gttggggatc agcatccaat gcagggtcgg gcagtggttt taatggaggc 1200
tttggctcaa gcatggattc taagtcttct ggctggggaa tgaatcacta g 1251
<210> 3
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Asp Ala Gly Trp Gly Asn Leu Val Tyr Val Val Asn Tyr Pro Lys Asp
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Asn Lys Arg Lys Met Asp Glu Thr Asp Ala Ser Ser Ala Val Lys Val
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Lys Arg Ala Val Gln Lys Thr Ser Asp Leu Ile Val Leu Gly Leu Pro
100 105 110
Trp Lys Thr Thr Glu Gln Asp Leu Lys Glu Tyr Phe Ser Thr Phe Gly
115 120 125
Glu Val Leu Met Val Gln Val Lys Lys Asp Leu Lys Thr Gly His Ser
130 135 140
Lys Gly Phe Gly Phe Val Arg Phe Thr Glu Tyr Glu Thr Gln Val Lys
145 150 155 160
Val Met Ser Gln Arg His Met Ile Asp Gly Arg Trp Cys Asp Cys Lys
165 170 175
Leu Pro Asn Ser Lys Gln Ser Gln Asp Glu Pro Leu Arg Ser Arg Lys
180 185 190
Val Phe Val Gly Arg Cys Thr Glu Asp Met Thr Glu Asp Glu Leu Arg
195 200 205
Glu Phe Phe Ser Gln Tyr Gly Asp Val Met Asp Val Phe Ile Pro Lys
210 215 220
Pro Phe Arg Ala Phe Ala Phe Val Thr Phe Ala Asp Asp Gln Ile Ala
225 230 235 240
Gln Ser Leu Cys Gly Glu Asp Leu Ile Ile Lys Gly Ile Ser Val His
245 250 255
Ile Ser Asn Ala Glu Pro Lys His Asn Ser Asn Arg Gln Leu Glu Arg
260 265 270
Ser Gly Arg Phe Gly Gly Asn Pro Gly Gly Phe Gly Asn Gln Gly Gly
275 280 285
Phe Gly Asn Ser Arg Gly Gly Gly Ala Gly Leu Gly Asn Asn Gln Gly
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Ser Asn Met Gly Gly Gly Met Asn Phe Gly Ala Phe Ser Ile Asn Pro
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Ala Met Met Ala Ala Ala Gln Ala Ala Leu Gln Ser Ser Trp Gly Met
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Asn Gln Asn Gln Gly Asn Met Gln Arg Glu Pro Asn Gln Ala Phe Gly
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Ser Gly Asn Asn Ser Tyr Ser Gly Ser Asn Ser Gly Ala Ala Ile Gly
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Trp Gly Ser Ala Ser Asn Ala Gly Ser Gly Ser Gly Phe Asn Gly Gly
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Phe Gly Ser Ser Met Asp Ser Lys Ser Ser Gly Trp Gly Met Asn His
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<210> 4
<211> 416
<212> PRT
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1 5 10 15
Glu Ile Pro Ser Glu Asp Asp Gly Thr Val Leu Leu Ser Thr Val Thr
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Trp Lys Thr Thr Glu Gln Asp Leu Lys Glu Tyr Phe Ser Thr Phe Gly
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Glu Val Leu Met Val Gln Val Lys Lys Asp Leu Lys Thr Gly His Ser
130 135 140
Lys Gly Phe Gly Phe Val Arg Phe Thr Glu Tyr Glu Thr Gln Val Lys
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Val Met Ser Gln Arg His Met Ile Asp Gly Arg Trp Cys Asp Cys Lys
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Glu Phe Phe Ser Gln Tyr Gly Asp Val Met Asp Val Phe Ile Pro Lys
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Pro Phe Arg Ala Phe Ala Phe Val Ala Phe Ala Asp Asp Gln Ile Ala
225 230 235 240
Gln Ser Leu Cys Gly Glu Asp Leu Ile Ile Lys Gly Ile Ser Val His
245 250 255
Ile Ser Asn Ala Glu Pro Lys His Asn Ser Asn Arg Gln Leu Glu Arg
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Phe Gly Asn Ser Arg Gly Gly Gly Ala Gly Leu Gly Asn Asn Gln Gly
290 295 300
Ser Asn Met Gly Gly Gly Met Asn Phe Gly Ala Phe Ser Ile Asn Pro
305 310 315 320
Ala Met Met Ala Ala Ala Gln Ala Ala Leu Gln Ser Ser Trp Gly Met
325 330 335
Met Gly Met Leu Ala Ser Gln Gln Asn Gln Ser Gly Pro Ser Gly Asn
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Ser Gly Asn Asn Ser Tyr Ser Gly Ser Asn Ser Gly Ala Ala Ile Gly
370 375 380
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cacatccccg tactgagcga agaactcccg 30
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cctttgttgc atttgcagat gatcaga 27
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agctgtccat gctgtcactg 20
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gcaggctgtc tgtctctctc 20
Claims (9)
1.TDP-43蛋白的O-糖基化突变体，所述TDP-43蛋白的氨基酸序列如SEQ ID NO.3 所示，其特征在于，所述突变体在SEQ ID NO.3 序列的第199位和/或233位突变成非苏氨酸的其他氨基酸；所述其他氨基酸为丙氨酸、酪氨酸、天冬氨酸或半胱氨酸。
2.根据权利要求1所示的TDP-43蛋白的O-糖基化突变体，其特征在于，所述非苏氨酸的其他氨基酸为丙氨酸，所述突变体的氨基酸序列如SEQ ID NO.4 所示。
3.编码权利要求1或2所述的突变体的DNA序列。
4.一种携带权利要求3所述的DNA序列的表达载体。
5.根据权利要求4所述的表达载体，其特征在于：其骨架质粒为pCS2质粒或pGEX-6p-1质粒。
6.一种含有权利要求4所述的表达载体的细胞。
7.根据权利要求6所述的细胞，其特征在于：为含有权利要求4所述的表达载体的HEK293T细胞或SH-SY5Y细胞或Neuro 2a细胞。
8.权利要求1或2所述的突变体在设计/制备神经退行性疾病的诊断试剂/诊断试剂盒/生物标志物，或者在制备神经退行性疾病相关的模式动物，或者在制备治疗/预防神经退行性疾病药物中的应用，其特征在于：所述神经退行性疾病为肌萎缩侧索硬化症或额颞叶变性病。
9.根据权利要求8所述的应用，其特征在于：当所述突变体应用于制备治疗/预防神经退行性疾病药物时，所述药物包括N-乙酰葡萄糖胺。
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