CN108715601B - 一种同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用与编码该多肽的基因 - Google Patents
一种同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用与编码该多肽的基因 Download PDFInfo
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Abstract
本发明公开了一种同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用与编码该多肽的基因。本发明的同时具有抗氧化和抑制Aβ42聚集特性的多肽的氨基酸序列为Trp‑Pro‑Pro‑Lys‑Asn。本发明的具有抗氧化和抑制Aβ42聚集特性的多肽,具有很好的抗氧化效果,同时能明显减少细胞表达Aβ42聚集点,有效起到Aβ42聚集的抑制作用,应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品,进而能对包括AD疾病的神经退行疾病进行有效预防和治疗,改善神经退行疾病医疗状况,具有重大社会和经济效益。
Description
技术领域
本发明涉及多肽技术领域,具体涉及同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用。
背景技术
β-淀粉样蛋白(amyloid-β,Aβ)是由淀粉样前体蛋白(amyloid precursorprotein,APP)经β-和γ-分泌酶的蛋白水解作用而产生的含有39~43个氨基酸的多肽。其中,Aβ42是人体内Aβ最常见的亚型,在阿尔茨海默病患者中大量存在。它容易聚集,形成Aβ沉淀的核心,引发神经毒性,被认为是阿尔兹海默症早发性生物标志物。
阿尔茨海默病(Alzheimer’s disease,AD)是一种伴有记忆障碍、失语、失认、视空间技能损害、执行功能障碍等功能失常的进行性神经退行疾病,是老年痴呆最常见的一种形式,其病因迄今尚未完全清楚。据调查,该疾病的患者集中在65岁以上人群中。根据我国第六次人口普查,国内65岁以上的老年人口已有1.19亿,占总人口的8.87%。因此,我国存在大量老年人患上AD疾病的风险。鉴于AD患者晚期会丧失独立生活能力,需要他人看护和悉心照料。因此,这种情况会给社会和家庭带来沉重的经济负担,影响我国社会和经济健康可持续发展。鉴于此,有效的AD疾病预防和治疗的需要极其迫切,同时也是一个重大的挑战。据所知,AD疾病的病理特征是:大脑皮质弥漫萎缩,沟回增宽,脑室扩大,神经元大量减少,形成可见的老年斑(Senile Plaques,SP)、神经纤维缠结(Neurofibrillary tangles,NFT)等脑内病变。在大脑皮层和海马中,大量出现老年斑和神经纤维缠结,是诊断AD的两个主要依据。SP主要是由β-淀粉样蛋白(Amyloidβ,Aβ)的沉积形成,而NFT的主要成分为异常磷酸化的tau蛋白。迄今为止,AD的病因和发病机制仍未能彻底阐明,目前临床上对治愈AD患者的药物尚没有任何特异性,极大部分处于研究阶段的治疗药物属于指标不治本,只能尽量改善临床症状和延缓病情发展。因此,对预防和治疗AD药物的研究仍需要在广度和深度上大量投入。
在AD发展早期,Aβ42就已被发现在大脑中沉积,而Aβ42的异常聚集和沉淀会对神经细胞产生毒性,这种毒性能使大脑皮层内发生复杂的级联变化,甚至会引起神经细胞的凋亡,导致的最终结果是产生AD病症。在正常生理条件下,Aβ42的产生和降解是处于动态平衡的,然而在基因突变或者降解Aβ42的酶功能出现紊乱的条件下,Aβ42产生和清除之间的动态平衡就会被打破,由此会引发Aβ42异常聚集沉淀,进而发生纤维化,甚至引发包括Tau蛋白过度磷酸化等一连串的复杂反应,如突触变化、神经递质丢失、炎症反应等。此外,Aβ42能明显加剧神经元和脑组织的脂质、核酸以及蛋白质的过氧化水平,诱导大量的过氧化物和自由基的生成,从而导致神经细胞的死亡。相关的研究结果显示,Aβ42聚集和形成纤维过程与自由基的产生有着非常密切的关系。在APP代谢过程中,金属离子催化的氧化作用可诱导Aβ42聚集,并在此过程中产生氧自由基,而自由基又可以促进Aβ42转向β折叠的构象,从而聚集形成纤维。晚期的糖基化终末产物(Advanced glycation end products,AGEs)与活性氧种类(Reactive oxygen species,ROS)的产生有关,而AGEs修饰的Aβ42不断累积,导致可溶性纤维的Aβ42聚集,同时,糖基化后的Aβ42能够抵抗蛋白酶的水解作用和巨噬细胞的吞噬作用。因此,控制ROS的过度生成有利于AGEs的减少,从而降低Aβ42聚集水平。在自由基和Aβ42相互关联过程中,一方面,自由基促进Aβ42的生成、聚集和沉积;另一方面,Aβ42也加入了自由基的氧化损伤过程中。当Aβ42诱导细胞膜上的脂质和蛋白质氧化修饰时,它会致使ROS生成增多,并改变细胞膜的通透性,使Ca2+内流增大,从而激活Ca2+依赖性蛋白激酶、酯酶,使胞质自由基合成增多,进而损伤细胞器。此外,Aβ42可下调细胞周期蛋白激酶Cdk5和糖原合成激酶GSK3beta活性,影响着Tau蛋白磷酸化作用,致使自由基生成增多。另外,近年来研究发现,胞内Aβ42累积能导致线粒体形体变化、抑制电子传递链上酶的活性、降低氧化磷酸化水平,造成线粒体结构和功能性破坏,使细胞中ATP生成变少而ROS增多。ROS大量的产生和积聚显然会造成器官的损伤,从而导致神经元无法进行正常的有氧代谢获取能量而功能严重受损。由此,Aβ42的过度产生聚集和氧化应激损伤之间的恶性循环,会严重损坏神经元的正常生理功能,诱发AD的产生和发展。
对于Aβ42和氧化应激对AD的发病机制和其发展过程中起到极其重要的作用,基于此可知,减少Aβ42的过度生成或者抑制Aβ42聚集沉淀,恢复Aβ的生成和清除之间的动态平衡,并完善自由基清除体系达到抗氧化效果,对AD的预防和治疗具有重要作用。在AD预防和治疗药物研究中,以Aβ为治疗靶点的药物开发是该领域研究热点。它存在病理结果清楚、支持Aβ假说的证据较多以及针对Aβ治疗的方式明确等优点。目前以Aβ为治疗靶点的药物包括有:(1)降解Aβ42的生成;(2)促进Aβ在脑内清除,阻碍Aβ的集聚过程并降低其毒性;(3)结合免疫治疗。根据这些药物化学特征的不同可以分三大类:即是有机化合物、抗体和多肽。目前,具有抗氧化作用的维生素E已经作为治疗AD的临床药物,展示出抗氧化剂对预防和治疗AD具有良好的应用前景。近年来,针对Aβ42的肽类抑制物研究备受关注,大量的活性肽被报道具有抗氧化效果而且兼有营养价值。如果抗氧化肽同时能够抑制Aβ42聚集,那么它在AD预防和治疗中存在潜力而发挥重要作用。此外,活性肽为氨基酸脱水缩合而成,易于化学合成,且具有易吸收、毒副作用小等优点,在生物制药中利于发挥作用,尤其在研制AD预防和治疗药物中有着良好的发展应用前景。
发明内容
本发明的目的在于针对现有技术的不足,提供了一种同时具有抗氧化和抑制Aβ42聚集特性的多肽。该多肽同时具有抗氧化性和抑制Aβ42聚集的特性,能有效应用于制备预防和治疗神经系统疾病药物,尤其应用于制备预防或治疗阿尔兹海默症的药物或食品,进而应用于预防和治疗AD疾病。
本发明的目的还在于提供一种编码所述同时具有抗氧化和抑制Aβ42聚集特性的多肽的基因。
本发明的另一目的还在于提供所述的一种同时具有抗氧化和抑制Aβ42聚集特性的多肽的应用。
本发明的目的通过如下技术方案实现。
一种同时具有抗氧化和抑制Aβ42聚集特性的多肽,氨基酸序列为Trp-Pro-Pro-Lys-Asn(简称WPPKN),如SEQ ID No:1所示,名称为WN-5;
其中,Trp为色氨酸(Tryptophan)的氨基酸相应残基(简称W),Pro为脯氨酸(Proline)的氨基酸相应残基(简称P),Lys为赖氨酸(Lysine)的氨基酸相应残基(简称K),Asn为天冬酰胺(Asparagine)的氨基酸相应残基(简称N)。
一种编码所述的同时具有抗氧化和抑制Aβ42聚集特性的多肽的基因,核酸序列如SEQ ID No:2所示;
其中,TGG为色氨酸的氨基酸相应基因密码子,CCA或CCC为脯氨酸的氨基酸相应基因密码子,AAG为赖氨酸的氨基酸相应基因密码子,AAC为天冬酰胺的氨基酸相应基因密码子。
所述的一种同时具有抗氧化和抑制Aβ42聚集特性的多肽的应用,包括应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品。
优选的,所述预防或治疗阿尔兹海默症的药物包括药学上可接受的载体,或者以丸剂、胶囊剂、片剂或注射剂制剂的形式存在的促血脑屏障穿越因子。
应用制备的药物或食品对神经退行疾病有潜在作用,尤其对于AD疾病具有明显效果。
与现有技术相比,本发明具有如下优点和有益效果:
本发明的具有抗氧化和抑制Aβ42聚集特性的多肽,具有很好的抗氧化效果,同时能明显减少细胞表达Aβ42聚集点,有效起到Aβ42聚集的抑制作用,应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品,进而能对包括AD疾病的神经退行疾病进行有效预防和治疗,改善神经退行疾病医疗状况,具有重大社会和经济效益。
附图说明
图1为实施例1合成的同时具有抗氧化和抑制Aβ42聚集特性的多肽WN-5的高效液相色谱(HPLC)图;
图2为实施例1合成的同时具有抗氧化和抑制Aβ42聚集特性的多肽WN-5的电喷雾离子化质谱(ESI-MS)图;
图3为实施例4中添加多肽WN-5后H2O2损伤SH-SY5Y细胞的线粒体超氧化合物的定量分析图;
图4a、图4b、图4c和图4d分别为实施例5中空白组、mCherry组、多肽0.05mmol/mL组和多肽0.5mmol/mL组对HEK-293细胞表达Aβ42聚集的抑制作用荧光图;
图5为实施例5中多肽WN-5对HEK-293细胞表达Aβ42聚集的抑制作用的Aβ42聚集点统计柱状图。
具体实施方式
以下结合具体实施例及附图对本发明技术方案作进一步详细的描述,但本发明的具体实施方式及保护范围不限于此。
具体实施例中,如无特殊说明,所涉及的试剂均为市售产品,均可通过商业渠道购买获得。实施例中所采用的神经细胞氧化损伤模型的细胞为SH-SY5Y,诱导产生超氧化物的氧化剂为H2O2,氧化剂H2O2能产生大量氧自由基引起细胞产生氧化应激反应,是一种常用的细胞氧化应激诱导剂;实施例中对抑制Aβ蛋白聚集模型所采用的细胞为HEK-293,该细胞广泛用于诱导产生神经退行性疾病的细胞模型,当被注入Aβ42-mCherry基因后,HEK-293能同时表达Aβ42和红色荧光蛋白mCherry,而只被注入mCherry基因后,则只表达红色荧光蛋白mCherry。
具体实施例中,本发明的一种同时具有抗氧化和抑制Aβ42聚集特性的多肽,名称为WN-5,氨基酸序列为Trp-Pro-Pro-Lys-Asn(简称WPPKN),氨基酸序列的序列表如SEQ IDNo:1所示;
其中,Trp为色氨酸(Tryptophan)的氨基酸相应残基(简称W),Pro为脯氨酸(Proline)的氨基酸相应残基(简称P),Lys为赖氨酸(Lysine)的氨基酸相应残基(简称K),Asn为天冬酰胺(Asparagine)的氨基酸相应残基(简称N);
分子结构式如下:
具体实施例中,本发明同时具有抗氧化和抑制Aβ42聚集特性的多肽可通过多肽固相合成法合成,或通过基因工程技术合成。
其中,通过基因工程技术合成时,将编码基因接入到载体中,再将载体转录到原核表达体系大肠杆菌中或真核表达体系酵母中进行表达,然后对目标多肽进行分离纯化,得到具有抗氧化和抑制Aβ42聚集特性的多肽。
编码上述的同时具有抗氧化和抑制Aβ42聚集特性的多肽的基因的核酸序列如SEQID No:2所示;
其中,TGG为色氨酸的氨基酸相应基因密码子,CCA或CCC为脯氨酸的氨基酸相应基因密码子,AAG为赖氨酸的氨基酸相应基因密码子,AAC为天冬酰胺的氨基酸相应基因密码子。
实施例1
多肽固相合成法合成同时具有抗氧化和抑制Aβ42聚集特性的多肽WN-5,具体如下:
采用标准Fomc方案,选用Wang树脂,按照氨基酸序列Trp-Pro-Pro-Lys-Asn的序列特征,使肽链从C端逐个向N端延伸,各氨基酸的用量为0.1mol,加入0.3mol Fmoc保护氨基酸,每步缩合都加入HOBt活化保护氨基酸的羧基,每步缩合采用20%哌啶/N,N-二甲基甲酰胺(DMF)溶液(15ml/g)处理20min,去除Fmoc保护基;肽侧链合成后,将含有树脂的肽链加入到体积比99:1的二氯甲烷:三氟乙酸混合液中,将肽链从树脂上切割下来;再次将肽加入到体积比94.5:2.5:2:1的三氟乙酸:酒石酸乙二胺:蒸馏水:胰蛋白酶抑制剂(TIS)的混合液中反应2h,脱去侧链保护基。
以上过程均在SYMPHONY型12通道多肽合成仪中完成,所合成的多肽经SHIMADZU高效液相色谱仪纯化(HPLC图如图1所示),纯度达到99.0694%,并经ESI-MS鉴定结构(ESI-MS图如图2所示),合成的多肽为目标肽,即同时具有抗氧化和抑制Aβ42聚集特性的多肽WN-5。
实施例2
合成的多肽WN-5的氧化自由基吸收能力测定
氧化自由基吸收能力(Oxygen radical absorbance capacity,ORAC)是一种基于氢原子转移机理评价抗氧化能力的方法,被认为接近生物体抗氧化功能的方法。ORAC反应在37℃的75mM磷酸盐缓冲溶液环境中进行,合成的多肽WN-5、还原性谷胱甘肽(GSH)、荧光素和AAPH自由基均溶解在75mM的磷酸盐缓冲溶液,其中缓冲溶液的pH值为7.4,其终浓度分别为1.30mM、3.38mM、78nM和9.95mM。以Trolox作为标准抗氧化物,其终浓度梯度分为0.5μM、1.0μM、2μM和4.2μM,并以GSH作为阳性对照。将合成的多肽WN-5、GSH分别与荧光素混合,并在37℃条件下保温20分钟,之后加入偶氮化合物AAPH,在激发波长485nm,发射波长538nm处检测荧光强度,检测时长为3h,获取荧光衰退曲线。抗氧化剂的氧自由基吸收能力ORAC值通过荧光衰退曲线的保护面积与标准抗氧化物质(Trolox)的保护面积相比得到。ORAC值以Trolox当量表达,结果如表1所示。
表1合成的多肽WN-5和GSH的ORAC结果
由表1可知,合成的多肽WN-5比GSH更具有ORAC活性,显示出合成的多肽WN-5更容易通过氢转移方式吸收氧化自由基。
实施例3
合成的多肽WN-5的DPPH·清除活性测定
DPPH自由基清除活性是一种基于电子转移机理评价抗氧化能力的方法。DPPH自由基清除活性反应在37℃条件下进行,合成的多肽WN-5、抗坏血酸和DPPH分别溶于95vol%乙醇中,用移液器准确量取各浓度(4mM,8mM,12mM,16mM和20mM)合成肽、抗坏血酸溶液100μL放入96孔酶标板中,之后加入DPPH溶液100μL,DPPH的终浓度为0.2mM,并在酶标仪中振动30s后保温孵育15min,之后在517nm处检测溶液吸光值,合成肽或抗坏血酸的DPPH自由基清除活性计算如下:
DPPH·清除能力(%)=[(Acontrol-Asample)/Acontrol]×100%
其中,Acontrol和Asample分别是未添加与添加合成的多肽WN-5或抗坏血酸的溶液在517nm处的吸光值。
合成的多肽WN-5以及抗坏血酸的DPPH清除活性并以半抑制浓度IC50值表示,结果如表2所示。
表2合成的多肽WN-5和抗坏血酸的DPPH自由基清除活性结果
由表2可知,合成的多肽WN-5的DPPH自由基清除活性与抗氧化剂抗坏血酸相当,表明此合成多肽具有较好的抗氧化性。
实施例4
合成的多肽WN-5在神经细胞中的抗氧化应用
在细胞内,线粒体是活性氧产生的主要来源,线粒体内的超氧化物含量水平是良好的氧化应激显示剂。采用具有细胞渗透性的荧光染料MitoSOX红来测量SH-SY5Y细胞的线粒体内的超氧化物含量水平,染料MitoSOX红以线粒体为靶标很容易被氧化生成发红色荧光的物质。
其测试方法如下:SH-SY5Y细胞培养在96微孔板中,合成的多肽WN-5溶于磷酸缓冲盐溶液(PBS缓冲溶液)中形成多肽液;当细胞融合达到约80%时,以浓度为0.5mM的多肽溶液处理细胞,并在37℃条件下孵育24小时,同时以0.5mM可自然产生的抗氧化剂GSH作为阳性对照。
孵育完毕之后,在37℃条件下用2mM的H2O2作用细胞1小时来产生细胞氧化应激反应,然后细胞用PBS溶液清洗三次,紧接着在37℃条件下用MitoSOX Red染料标记30min。随后,细胞再次用PBS溶液清洗,为IncuCyte Zoom仪器检测荧光做准备;采用IncuCyte Zoom并通过相对荧光强度检测线粒体超氧化物的含量水平,每次实验采用15000个细胞(n=3),采用单因素方差分析(ANOVA)来检验含多肽WN-5与不含肽对照组统计学差异,其中,ns表示P>0.05;***表示P<0.001。
IncuCyte Zoom仪测试分析添加多肽WN-5后H2O2损伤SH-SY5Y细胞的线粒体超氧化合物的定量分析图如图3所示,显而易见,与不含H2O2处理的SH-SY5Y细胞相比,采用H2O2处理但不含有多肽液孵育处理的SH-SY5Y细胞的MitoSox红荧光强度明显增大。然而,当同样采用H2O2处理,但用谷胱甘肽(GSH)和多肽液孵育处理的SH-SY5Y细胞的MitoSox红荧光强度大大减小,并且多肽液孵育处理的SH-SY5Y细胞的MitoSox红荧光强度减少程度与GSH相当,说明合成的多肽WN-5具有强的抗氧化作用。
实施例5
合成的多肽WN-5对HEK-293细胞表达Aβ的抑制作用
采用能表达Aβ42和mCherry的HEK-293细胞,正常培养情况下,该细胞携带的基因能顺利表达Aβ42和mCherry,当激活mCherrys发红光时,若Aβ42产生聚集,会出现局部特别亮的红点,即为聚集点。因此,这类聚集点数量越大反映出Aβ42产生的聚集越多。鉴于此,利用聚集点的多少来指示Aβ42聚集情况。具体测试如下:HEK-293细胞密度为5000/孔;种板24小时后,加入0.05mM和0.5mM的合成多肽WN-5进行孵育,48小时后,加入终浓度为10μg/ml的四环素诱导mCherry,72小时后,在荧光显微镜下拍照,统计聚集点个数,并以不表达Aβ42而只表达红色荧光蛋白mCherry的HEK-293细胞作为对比。
空白组(能表达Aβ42和mCherry的HEK-293细胞组)、mCherry组(只表达mCherry的HEK-293细胞组)、多肽0.05mmol/mL组(多肽WN-5的浓度为0.05mmol/mL)和多肽0.5mmol/mL组(多肽WN-5的浓度为0.5mmol/mL)对HEK-293细胞表达Aβ42聚集的抑制作用荧光图如图4a~图4d所示;
多肽WN-5对HEK-293细胞表达Aβ42聚集的抑制作用的Aβ42聚集点统计柱状图如图5所示,其中,空白代表能表达Aβ42和mCherry的HEK-293细胞;mCherry代表只表达mCherry的HEK-293细胞,每个实验进行3次,采用单因素ANOVA来检验与空白组的统计学差异,ns表示P>0.05;***代表P<0.001;
由图4a~图4d以及图5可知,在四环素诱导下,能同时表达Aβ42和mCherry的HEK-293细胞,其聚集点比只表达红色荧光蛋白mCherry的HEK-293细胞呈现的要多。当采用合成肽孵育能同时表达Aβ42和mCherry的HEK-293细胞时,其聚集点明显地减少,表明此时的HEK-293细胞表达Aβ42产生聚集数量变少,即合成多肽对Aβ42聚集具有显著性抑制作用。
以上实施例仅为本发明较优的实施方式,仅用于解释本发明,而非限制本发明,本领域技术人员在未脱离本发明精神实质与原理下所作的任何改变、替换、组合、简化、修饰等,均应为等效的置换方式,均应包含在本发明的保护范围内。
序列表
<110> 华南理工大学
<120> 一种同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用与编码该多肽的基因
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 五肽(Pentapeptide)
<400> 1
Trp Pro Pro Lys Asn
1 5
<210> 2
<211> 15
<212> DNA/RNA
<213> 五肽(Pentapeptide)
<400> 2
tggccaccca agaac 15
Claims (2)
1.一种同时具有抗氧化和抑制Aβ42聚集特性的多肽的应用,其特征在于,应用于制备抗Aβ42蛋白聚集的药物,或者应用于制备预防或治疗阿尔兹海默症的药物,所述多肽的氨基酸序列为Trp-Pro-Pro-Lys-Asn,如SEQ ID No:1所示,名称为WN-5;
其中,Trp为色氨酸的氨基酸相应残基,Pro为脯氨酸的氨基酸相应残基,Lys为赖氨酸的氨基酸相应残基,Asn为天冬酰胺的氨基酸相应残基。
2.根据权利要求1所述的应用,其特征在于,所述预防或治疗阿尔兹海默症的药物包括药学上可接受的载体,所述药物以丸剂、胶囊剂、片剂或注射剂制剂的形式存在。
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