CN105651853A - 一种亚细胞结构的特征性n-连接糖链及其应用 - Google Patents
一种亚细胞结构的特征性n-连接糖链及其应用 Download PDFInfo
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Abstract
本发明公开了一种亚细胞结构的特征性N-连接糖链及其应用,属于糖生物学技术领域。本发明通过膀胱YTS1细胞为实验材料,使用差速离心的方法获得了四种亚细胞结构(微粒体、线粒体、细胞核和细胞质)的蛋白质组分,通过联合凝集素芯片和质谱(MS)的方法分别鉴定的亚细胞结构蛋白的糖基化修饰类型和结构的变化,显示亚细胞结构差异性糖链的分布。本发明得到了10种人膀胱YTS1细胞的亚细胞结构的特征性N-连接聚糖;利用特征性N-连接聚糖,可实现对相应亚细胞结构中其他目标物质的间接检测、定性或者定量检测。
Description
技术领域
本发明涉及一种亚细胞结构的特征性N-连接糖链及其应用,属于糖生物学技术领域。
背景技术
糖基化是最重要的一种蛋白质的翻译后修饰,大约有一半以上的天然蛋白质都被糖基化修饰。蛋白质的糖基化修饰可以促进蛋白质折叠,防止蛋白质被蛋白酶水解,并指导蛋白质运输。蛋白质的N-糖基化修饰可以调控蛋白质在细胞中的定位、功能、活性、寿命以及蛋白质的多样性。因此,细胞的糖链可以作为细胞生理学新的遗传密码。虽然目前糖生物学尚处在早期的研究阶段,但是越来越多研究集中在糖基化的结构和功能解析上。
真核细胞中的各种重要形式的糖基化是在内质网和高尔基体内合成的,许多研究亦表明,在细胞核和细胞质中存在着复合的糖缀合物,而且具有及其重要的作用,然而除了个别的描述的比较清楚地特定例子外,还缺乏细胞器中肯定性的糖链结构数据。目前对亚细胞的研究主要集中在单个蛋白糖基化修饰的解析,无法揭示亚细胞结构整体糖组的特征。
细胞分化和细胞的区室化使细胞环境的复杂性增加,有利于细胞适应各种环境,而理解细胞中每种分子功能对于科学家的挑战越来越大。通过初步分离和富集大分子物质(蛋白质,聚糖)的策略可以降低仪器检测中的最小阈值,有助于扩大检测样品(血清或组织)中目标分子的检测范围。通过该方法分析亚细胞结构中糖蛋白质糖组,可以更全面获得细胞中所有蛋白质的糖链结构。
因此,对细胞中的细胞器糖缀合物分布进行分析、确定更多的聚糖并找到一些亚细胞结构的聚糖标记物,是目前亟需解决的问题。
发明内容
为了解决上述问题,本发明通过膀胱YTS1细胞为实验材料,使用差速离心的方法获得了四种亚细胞结构(微小体、线粒体、细胞核和细胞质)的蛋白质组分,通过联合凝集素芯片和质谱(MS)的方法分别鉴定的亚细胞结构蛋白的糖基化修饰类型和结构的变化,显示亚细胞结构差异性糖链的分布。
本发明的第一个目的是提供10种亚细胞结构的特征性N-连接聚糖,结构式分别如下:
聚糖1(G1):1403.481(Fuc)1(Man)2+(Man)3(GlcNAc)2
聚糖2(G2):2401.862(Fuc)1(Neu5Ac)1(Gal)1(GlcNAc)4+(Man)3(GlcNAc)2
聚糖3(G3):2523.909(Fuc)2(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2
聚糖4(G4):1704.608(Gal)1(GlcNAc)3+(Man)3(GlcNAc)2
聚糖5(G5):2231.793(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2
聚糖6(G6):2256.825(Fuc)1(Gal)1(GalNAc)1(GlcNAc)3+(Man)3(GlcNAc)2,
聚糖7(G7):2272.280(Gal)2(GlcNAc)5+(Man)3(GlcNAc)2,
聚糖8(G8):2726.988(Fuc)2(Gal)3(GlcNAc)5+(Man)3(GlcNAc)2,
聚糖9(G9):1995.703(Neu5Ac)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2,
聚糖10(G10):2740.967(Neu5Ac)1(Gal)4(GlcNAc)4+(Man)3(GlcNAc)2,
结构式中,GlcNAc代表N-乙酰葡萄糖胺,Man代表甘露糖,Gal代表半乳糖,GalNAc代表N-乙酰半乳糖胺,Neu5Ac代表N-乙酰神经氨酸,Fuc代表岩藻糖。
具体地,各聚糖的结构如下:
其中,代表Man,代表GlcNAc,代表Fuc,代表Gal,代表Neu5Ac,代表GalNAc。
其中,微小体的标记性N-连接聚糖为聚糖1、聚糖2、聚糖3;线粒体的特征性N-连接聚糖为聚糖4、聚糖5、聚糖6、聚糖7、聚糖8;细胞核的特征性N-连接聚糖为聚糖9、聚糖10。
本发明的第二个目的是提供一种试剂盒,所述试剂盒中含有定量或者定性检测或者分离特征性N-连接聚糖的试剂和标签;标签上记载该试剂盒用于定量或者定性检测目标物质;所述目标物质存在于特征性N-连接聚糖所对应的亚细胞结构中。
所述特征性N-连接聚糖为聚糖1、聚糖2或聚糖3时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为聚糖4、聚糖5、聚糖6、聚糖7或聚糖8时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为聚糖9或聚糖10时对应的亚细胞结构为细胞核。
在本发明的一种实施方式中,所述亚细胞结构是来自离体的人膀胱YTS1细胞。
本发明的第三个目的是提供一种利用特征性N-连接聚糖检测相应亚细胞结构中目标物质的方法,所述目标物质存在于亚细胞结构中,通过特征性N-连接聚糖的检测实现目标物质的定性检测;或者是所述目标物质存在于亚细胞结构中且其含量与特征性N-连接聚糖相关,通过定量检测特征性N-连接聚糖实现目标物质的定量检测。
所述N-连接聚糖为聚糖1、聚糖2或聚糖3时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为聚糖4、聚糖5、聚糖6、聚糖7或聚糖8时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为聚糖9或聚糖10时对应的亚细胞结构为细胞核。
在本发明的一种实施方式中,所述亚细胞来自离体的人膀胱YTS1细胞。
在本发明的一种实施方式中,所述N-连接聚糖的检测是采用质谱检测或者凝集素芯片检测或者其他任何可实现N-连接聚糖的检测的方法进行检测。
在本发明的一种实施方式中,所述N-连接聚糖的检测是先使用差速离心法分离提取亚细胞结构蛋白,然后利用超滤膜分离糖蛋白,进而对糖蛋白上的N-连接聚糖进行检测。
在本发明的一种实施方式中,所述分离提取亚细胞结构蛋白,具体是:
本发明的第四个目的是提供一种检测或区分或鉴别亚细胞结构的方法,是对亚细胞结构中的N-连接聚糖进行检测;N-连接聚糖为聚糖1、聚糖2或聚糖3时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为聚糖4、聚糖5、聚糖6、聚糖7或聚糖8时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为聚糖9或聚糖10时对应的亚细胞结构为细胞核。
所述亚细胞结构是来自离体的人膀胱YTS1细胞。
在本发明的一种实施方式中,所述对亚细胞结构中的N-连接聚糖进行检测,是指先使用差速离心法分离亚细胞结构蛋白,然后利用超滤膜分离糖蛋白,进而对糖蛋白上的N-连接聚糖进行检测。
本发明的有益效果:
得到了10种人膀胱YTS1细胞的亚细胞结构的特征性N-连接聚糖;利用特征性N-连接聚糖,可实现对相应亚细胞结构中其他目标物质的间接检测、定性或者定量检测。
附图说明
图1:YTS1亚细胞结构蛋白分离的实验流程图以及蛋白marker的鉴定;A:YTS1亚细胞结构的分离方法的实验流程图;B:SDS-PAGE鉴定分离YTS1亚细胞结构的蛋白;C:Westernblot验证YTS1亚细胞结构蛋白marker(M:marker蛋白1:线粒体蛋白2:细胞核蛋白3:微粒体蛋白4:细胞质蛋白);
图2:YTS1细胞的四种亚细胞结构N-连接糖链的质谱图;
图3:四种亚细胞结构N-连接聚糖的相对峰度和糖型;
图4:三种亚细胞结构N-连接聚糖的相对峰度和糖型;
图5:两种亚细胞结构N-连接聚糖的相对峰度和糖型;
图6:一种亚细胞结构N-连接聚糖的相对峰度和糖型。
具体实施方式
实验材料:经离体培养的人膀胱YTS1细胞。
实施例1:细胞破碎总蛋白的提取
将15mL离心管、移液器等试验用材置于生物安全柜内紫外灭菌15min;取细胞培养瓶倒去旧培养基,加入1.5mL胰酶迅速清洗瓶底,吸出后重新加入1.5mL胰酶,置于37℃细胞培养箱计时反应3min,反应完成后镜检确保细胞充分消化解除贴壁;加入1.5mL细胞培养基解除酶效,充分吹吸培养瓶底面,混匀后吸出移至15mL离心管1000r/min离心5min;离心完成后去掉上清液,加入5mL1×PBS,小心吹吸重新悬浮细胞作清洗,随后1000r/min离心5min,重复3次;获得的细胞沉淀加入1mLT-PER细胞裂解液,10μLPMSF,1μL抑肽酶,冰浴计时30min;12000g离心15min,取上清液即所提取的细胞总蛋白质溶液。
实施例2:亚细胞结构蛋白的提取
本发明采用的亚细胞结构的分离方法在以前的分离方法的基础上修改后确立的实验方案。细胞(~1×107)用胰酶消化下来,预冷的PBS(0.01M磷酸盐缓冲液,0.15MNaCl,pH7.4)清洗两次。加入500ul匀浆液(50mMHEPESpH7.4,3mMMgCl2,1mMEGTA)冰上进行超声(超声时间3s,间隙时间15s,工作次数10次,功率400w)。离心720g15min,上清液被转移到新管中,记为(S1)。500μL250mMSTMDPS缓冲液(250mM蔗糖,50mMTris-HCl)加入沉淀中,离心720g15min,上清液被转移到新管中,记为(S2)。沉淀中加入2MSTMDPS缓冲液(2M蔗糖,50mMTris-HCl(pH7.4),5mMMgCl2,1mMDTT,1mMPMSF,25mg/ml精胺,25mg/mL亚精胺),冰上超声破碎,4℃100000g离心1h。沉淀加500μLNE缓冲液(20mMHEPES(pH7.9),1.5mMMgCl2,0.5MNaCl,0.2mMEDTAand20%甘油),500μLNET缓冲液(20mMHEPES(pH7.9),1.5mMMgCl2,0.5MNaCl,0.2mMEDTA和20%甘油,1%Triton-X100,1mMDTT,1mMPMSF),冰上孵育30分钟,8000g35min离心,上清液为细胞核蛋白。S1和S2混合后离心6000g30min,上清液转移到新的管子中,记为S3。沉淀加入500μLHDP缓冲液(10mMHEPES(pH7.9),1mMDTT和1mMPMSF),500μLME缓冲液(20mMTris-HCl(pH7.8),0.4MNaCl,15%甘油,1mMDTT,1mMPMSF,1.5%Triton-X100),冰上孵育30min,离心9000g30min,上清液为线粒体蛋白。S3离心100000g1h,上清液转移到新的管子中,记为胞质蛋白。沉淀加入500μL缓冲液(20mMTris-HCl(pH7.8),0.4MNaCl,15%甘油,1mMDTT,1mMPMSF和1.5%Triton-X100),4℃轻摇孵育30min,9000g离心30min,上清液记为微粒体蛋白。用BCA蛋白浓度测定试剂盒(Beyotimebiotechnology,China)测定各蛋白浓度,然后通过SDS-PAGE和Western印迹法鉴定亚细胞结构的marker蛋白。包括线粒体marker蛋白(压力依赖性通道蛋白1,VDAC1,Abcam,Cambridge,MA,USA)检测,细胞核marker(组蛋白H4,H4,SantaCruz,Dallas,TX,USA),微粒体蛋白marker(溶酶体相关的膜蛋白2,LAMP2,SantaCruz),胞质蛋白marker(β肌动蛋白,Sigma-Aldrich,St.Louis,MO,USA)。
使用差速离心法分离亚细胞结构蛋白,整个分离实验流程如图1A所示。SDS-PAGE结果显示微粒体和细胞核的蛋白的丰度比线粒体和细胞质的蛋白相对较低(图1B)。蛋白的富集水平和亚细胞结构的分离方法的特异性通过使用细胞器标记蛋白特异性结合的抗体的Western法评估。结果显示这些marker蛋白质只出现在相应的细胞器中,如LAMP2只出现在微粒体蛋白,VDAC1只出现在线粒体蛋白,H4只出现在细胞核蛋白,而β-肌动蛋白只出现在细胞质蛋白(图1C),从而证明本发明改进的分离细胞中四种细胞器的方法具有很好的特异性。
实施例3:YTS1亚细胞结构的N-连接聚糖的质谱分析
(1)亚细胞结构蛋白样品中酶法提取糖链唾液酸的乙酰肼修饰及释放
取10kD超滤膜装配好超滤过程中收集废液的收集管后,加入含蛋白量1mg对应体积的样品蛋白溶液,用8M尿素补齐各管液面,充分混匀。14000g离心15min,溶液浓缩至超滤膜管底,弃去流出液;加入300μL的8mol/L尿素,14000g离心15min,再加200μL的8M尿素,离心,弃去流出液;加入150μL的10mMDTT溶液充分混匀,于干式恒温器中设定56℃孵育45min,反应结束后于台式离心机内14000g离心15min,弃去流出液;加入150μL20mmol/LIAM溶液充分吹吸混匀,注意IAM的避光操作,混匀后将超滤管置于黑暗环境中静置反应20min,反应完成后14000g离心15min,弃去流出液;加入150μL超纯水充分混匀,14000g离心15min,此步骤重复三次以清洗掉溶液中的IAM避免影响之后反应;清洗完成后加入100μL1mol/L乙酰肼,20μL1mol/L盐酸,20μL2mmol/LEDC,充分吹吸混匀,将超滤管置于120转摇床保证蛋白悬浮反应,于室温下反应4小时;反应完成后于14000g离心15min,弃去流出液,加入150μL40mmol/LNH4HCO3溶液,充分吹吸混匀后14000g离心15min,用NH4HCO3溶液重复清洗3次;将超滤管取出,转移至洁净的收集管中,用1μLPNGase-F释放糖链,37℃孵育10~12h;完成后14000g离心15min,保留流出液,再用150μL超纯水加入超滤膜吹吸重悬沉淀的蛋白,15000g离心15min,重复两次,充分收集N-连接糖糖链,保留收集管内的流出液,冷冻干燥。
(2)初级N-糖糖链样品的除盐(Cleanup)处理
取1.5mL离心管加入100μLSepharose4B,加入1:1的甲醇:水(V/V)溶液1mL,9000g离心5min,重复清洗5次;加入5:1:1的正丁醇:甲醇:水(V/V)溶液1mL,充分混匀,9000g离心5min,重复清洗3次,向糖链样品中加入500μL的5:1:1正丁醇:甲醇:水(V/V)溶液,溶解管底浓缩结晶的糖链样品,充分溶解后将溶液上样至预处理好的Sepharose4B凝胶中,充分混匀,于80r/min摇床室温振荡反应1h,9000g离心5min,小心吸取弃去上清,用700μL的5:1:1正丁醇:甲醇:水(V/V)溶液重复清洗3次;清洗完成后,加入500μL的1:1甲醇:水(V/V)溶液,充分混匀室温振荡20min,洗脱N-连接糖链,收集上清液,重复1次,冷冻干燥。
(3)N-连接糖链样品的MALDI-TOF-质谱解析
用BrukerDaltonics公司的UltrafleXtremeMALDI-TOF/TOF串联质谱解析各亚细胞器中的N-连接聚糖。将糖链质谱数据在flexAnalysis软件中获取,取信噪比大于5,且被至少三次试验鉴定到的质谱峰做后续分析。结合Glycoworkbench软件同时手动分析糖链结构。
为了获得更多唾液酸化聚糖的信息,本发明利用超滤膜分离糖蛋白并且对蛋白质的唾液酸化糖链进行乙酰肼修饰,被标记的聚糖随后在超滤膜上进行释放和并用质谱检测。用GlycoWorkbench软件对信噪比>4的N-连接聚糖的质谱数据进行注释。
本发明得到的YTS1亚细胞结构的N-连接糖链分布的质谱数据分析如下:
(1)发现在所有四种亚细胞结构总共有40种N-连接聚糖(图2至图6);
(2)在所有40种N糖中,其中有10种糖链结构出现在所有的4种亚细胞结构中(图3),有15种糖链结构出现在3种亚细胞结构中(图4),有5种糖链结构在两种亚细胞结构中被检测(图5),仅仅有10种糖链结构只在一种亚细胞结构中被检测到(图6);
(3)m/z1419.476,1581.528,1743.581,1905.634为高甘露聚糖型,在微粒体和细胞核中含量比较高;m/z1688.613,1809.639,1891.692,2053.745,2069.740,2215.798是核心岩藻糖基化N-连接聚糖,在微粒体和线粒体含量更高;m/z1403.481,2401.862,2523.909只在微粒体中检测到;m/z1704.608,2231.793,2256.825,2272.820,2726.988只在线粒体检测到;m/z1995.703,2740.967只在细胞核检测到。只在一种亚细胞结构中检测到的聚糖是这些亚细胞结构的N-连接聚糖标记物。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (9)
1.一种试剂盒,其特征在于,所述试剂盒中含有定量检测或者定性检测或者分离特征性N-连接聚糖的试剂和标签;标签上记载该试剂盒用于定量或者定性检测目标物质;所述目标物质存在于特征性N-连接聚糖所对应的亚细胞结构中。
2.根据权利要求1所述的试剂盒,其特征在于,所述特征性N-连接聚糖为m/z1403.481(Fuc)1(Man)2+(Man)3(GlcNAc)2、2401.862(Fuc)1(Neu5Ac)1(Gal)1(GlcNAc)4+(Man)3(GlcNAc)2、或者2523.909(Fuc)2(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为m/z1704.608(Gal)1(GlcNAc)3+(Man)3(GlcNAc)2、2231.793(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2、2256.825(Fuc)1(Gal)1(GalNAc)1(GlcNAc)3+(Man)3(GlcNAc)2、2272.280(Gal)2(GlcNAc)5+(Man)3(GlcNAc)2或者2726.988(Fuc)2(Gal)3(GlcNAc)5+(Man)3(GlcNAc)2时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为m/z1995.703(Neu5Ac)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2或者2740.967(Neu5Ac)1(Gal)4(GlcNAc)4+(Man)3(GlcNAc)2时,所对应的亚细胞结构为细胞核。
3.根据权利要求1所述的试剂盒,其特征在于,所述亚细胞结构是来自离体的人膀胱YTS1细胞。
4.一种利用特征性N-连接聚糖检测相应亚细胞结构中目标物质的方法,其特征在于,所述目标物质存在于亚细胞结构中,通过特征性N-连接聚糖的检测实现目标物质的定性检测;或者是所述目标物质存在于亚细胞结构中且其含量与特征性N-连接聚糖相关,通过定量检测特征性N-连接聚糖实现目标物质的定量检测。
5.根据权利要求4所述的方法,其特征在于,所述特征性N-连接聚糖为m/z1403.481(Fuc)1(Man)2+(Man)3(GlcNAc)2、2401.862(Fuc)1(Neu5Ac)1(Gal)1(GlcNAc)4+(Man)3(GlcNAc)2,或者2523.909(Fuc)2(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为m/z1704.608(Gal)1(GlcNAc)3+(Man)3(GlcNAc)2、2231.793(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2、2256.825(Fuc)1(Gal)1(GalNAc)1(GlcNAc)3+(Man)3(GlcNAc)2、2272.280(Gal)2(GlcNAc)5+(Man)3(GlcNAc)2或者2726.988(Fuc)2(Gal)3(GlcNAc)5+(Man)3(GlcNAc)2时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为m/z1995.703(Neu5Ac)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2或者2740.967(Neu5Ac)1(Gal)4(GlcNAc)4+(Man)3(GlcNAc)2时,所对应的亚细胞结构为细胞核。
6.根据权利要求4所述的方法,其特征在于,所述亚细胞来自离体的人膀胱YTS1细胞。
7.根据权利要求4所述的方法,其特征在于,所述N-连接聚糖的检测是采用质谱检测方法进行检测。
8.一种检测或区分或鉴别亚细胞结构的方法,其特征在于,所述方法是对亚细胞中的N-连接糖链进行检测;当N-连接糖链为m/z1403.481(Fuc)1(Man)2+(Man)3(GlcNAc)2、2401.862(Fuc)1(Neu5Ac)1(Gal)1(GlcNAc)4+(Man)3(GlcNAc)2,或者2523.909(Fuc)2(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2时,所对应的亚细胞结构为微小体;所述特征性N-连接聚糖为m/z1704.608(Gal)1(GlcNAc)3+(Man)3(GlcNAc)2、2231.793(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2、2256.825(Fuc)1(Gal)1(GalNAc)1(GlcNAc)3+(Man)3(GlcNAc)2、2272.280(Gal)2(GlcNAc)5+(Man)3(GlcNAc)2或者2726.988(Fuc)2(Gal)3(GlcNAc)5+(Man)3(GlcNAc)2时,所对应的亚细胞结构为线粒体;所述特征性N-连接聚糖为m/z1995.703(Neu5Ac)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2或者2740.967(Neu5Ac)1(Gal)4(GlcNAc)4+(Man)3(GlcNAc)2时,所对应的亚细胞结构为细胞核。
9.根据权利要求8所述的方法,其特征在于,所述亚细胞结构是来自离体的人膀胱YTS1细胞。
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