CN115960160A - 活细胞膜整合素αvβ3聚糖的原位糖链延长方法 - Google Patents
活细胞膜整合素αvβ3聚糖的原位糖链延长方法 Download PDFInfo
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Abstract
本发明涉及一种活细胞膜整合素αvβ3聚糖的原位糖链延长方法。通过偶联αvβ3的特异性识别多肽(Pep)和半乳糖氧化酶(GAO)，构建聚糖重构探针Pep‑GAO。在K4[Fe(CN)6]存在下，酶活性被抑制的Pep‑GAO可以结合活细胞表面的整合素αvβ3，然后加入K3[Fe(CN)6]激活GAO，使其氧化αvβ3糖链末端的半乳糖或N‑乙酰半乳糖胺，生成可以发生生物正交反应的醛基。该基团可与酰肼功能化的糖分子发生连接反应，生成腙键，从而实现αvβ3上糖链的延长。该方法在活细胞水平上实现了对特定蛋白聚糖结构的编辑，为细胞膜蛋白聚糖的功能研究和活体聚糖调控提供了有力的工具。
Description
一、技术领域
本发明介绍了一种活细胞膜整合素αvβ3聚糖的原位糖链延长方法。该方法通过构建特异性识别αvβ3的聚糖重构探针，氧化活细胞表面αvβ3糖链的末端聚糖生成醛基，进一步与酰肼修饰糖分子反应，实现细胞膜αvβ3聚糖的糖链延长。
二、背景技术
糖基化是普遍存在于真核生物中的蛋白质翻译后修饰模式。细胞表面的聚糖参与了一系列重要的生物过程。异常的糖基化与多种疾病的发生和发展密切相关，因此对活细胞表面特定蛋白的聚糖结构进行编辑，是一种重要的疾病干预手段，已被用于增强赫赛汀对乳腺癌的治疗效果。
整合素αvβ3是细胞膜表面由α亚基(CD51)和β3亚基(CD61)形成的跨膜异源二聚体糖蛋白。αvβ3介导细胞与细胞外基质间的双向信号传递，是一个高度糖基化的蛋白。αvβ3的聚糖结构与细胞存活、迁移、侵袭等细胞行为密切相关，被认为是有潜力的KRAS突变相关肿瘤的治疗靶标。然而目前尚无在活细胞水平特异性编辑αvβ3糖链的方法。
本发明结合可以特异性识别αvβ3的多肽和可以在细胞外部特异性氧化糖链末端半乳糖/N-乙酰半乳糖胺(Gal/GalNAc)的半乳糖氧化酶(GAO)，构建活细胞聚糖重构探针，通过对探针的酶活性进行程序性抑制-激活，特异性氧化活细胞表面整合素αvβ3的末端Gal/GalNAc生成醛基，进一步与酰肼修饰的糖分子发生连接反应，实现αvβ3糖链的延长。该方法无需进行基因操作，从活细胞外部简单快捷地编辑糖链结构，为在活细胞上原位研究特定蛋白上聚糖的生物学功能提供了工具，对于发展聚糖干预技术用于疾病治疗具有重要意义。
三、发明内容
本发明的内容是：构建特定蛋白的聚糖重构探针，在活细胞表面原位靶向整合素αvβ3后特异性氧化其糖链末端的Gal/GalNAc，生成可以发生生物正交反应的醛基。进而利用醛基与酰肼修饰糖分子的偶联反应实现αvβ3上聚糖的糖链延长。
该方法首先以琥珀酰亚胺酯和马来酰亚胺双修饰的聚乙二醇1000(NHS-PEGl000-MAL)作为连接分子，偶联巯基修饰的c(RGDfK)多肽(Pep)和GAO的氨基，制备聚糖重构探针Pep-GAO，如图1所示。
本发明提出的活细胞膜整合素αvβ3聚糖的原位糖链延长方法如图2所示，将Pep-GAO与K4[Fe(CN)6]混合后加入活细胞，探针可在Pep的引导下特异性结合αvβ3，而在此过程中探针的GAO活性被K4[Fe(CN)6]抑制。识别结束后洗掉未结合探针，加入K3[Fe(CN)6]激活GAO，由于GAO被限域在αvβ3上，只能特异性氧化αvβ3糖链末端的Gal/GalNAc生成生物正交基团醛基。通过己二酸二酰肼与糖分子反应制得酰肼修饰的糖分子，进而与活细胞表面αvβ3糖链上生成的醛基发生连接反应生成腙键，实现整合素αvβ3聚糖的原位糖链延长。
本发明通过以下技术方案来实现：
1)如图1所示，以特异性识别整合素αvβ3的多肽c(RGDfK)作为识别引导基团，以GAO作为聚糖重构工具，以NHS-PEG1000-MAL为连接分子，在25℃，800rpm的条件下恒温振荡2h制备Pep-GAO，通过超滤的方法进行纯化。
2)如图2所示，将Pep-GAO与K4[Fe(CN)6]混合，抑制探针的GAO活性。将活细胞与混合液在4℃下孵育30min，使探针靶向细胞表面整合素αvβ3。用磷酸盐缓冲液(PBS，pH7.4)洗细胞后加入K3[Fe(CN)6]激活GAO活性，局域在αvβ3的GAO氧化αvβ3糖链末端的Gal/GalNAc，生成可以在细胞膜表面发生生物正交反应的醛基。
3)在80℃磁力搅拌下，将己二酸二酰肼与糖分子(24mM∶2.4mM)在乙腈/水溶液(体积比1∶1)中冷凝回流反应8h后，用硅胶柱层析得到酰肼修饰的糖分子，洗脱液为体积比为6∶3∶1∶1的异丙醇，水，氨水和乙腈的混合液。
4)如图2所示，在10mM苯胺的催化下，细胞表面上生成的醛基与300μM酰肼修饰糖分子在4℃条件下反应1h，实现活细胞膜整合素αvβ3聚糖的原位糖链延长。
本发明与现有技术相比，具有以下特点：
本发明设计首次提出了活细胞膜整合素αvβ3聚糖的原位糖链延长方法，通过合成可以特异性识别αvβ3，基于GAO的聚糖重构探针，结合酶活性的程序性控制方法，在细胞膜αvβ3的聚糖糖链末端特异性地生成醛基，进一步与酰肼修饰糖分子偶联，实现αvβ3聚糖的原位糖链延长。
与基于糖修饰酶的聚糖编辑技术相比，本方法由于在糖修饰酶上修饰了可以特异性识别目标蛋白的多肽，赋予了方法蛋白特异性；与基于聚糖新陈代谢技术的糖编辑技术相比，本方法无需经过细胞内的代谢过程，耗时大大缩短，同时方法具有蛋白特异性。同时本方法合成的聚糖重构探针具有良好的细胞相容性，方法可以在活细胞上进行。
本方法对给体糖分子的结构没有限制，在对糖分子进行一步酰肼功能化后，即可通过与目标蛋白上氧化生成的醛基的生物正交化学反应快速连接在目标蛋白糖链上。
该方法实现了对活细胞表面目标蛋白糖链的编辑，为理解聚糖的生物学功能，通过干预聚糖进行疾病治疗提供了工具。
四、附图说明
图1.聚糖重构探针Pep-GAO制备示意图
图2.活细胞膜整合素αvβ3聚糖的原位糖链延长方法示意图
图3.酰肼修饰甘露三糖制备示意图
五、具体实施方式
实施例1：结合图1，制备聚糖重构探针Pep-GAO
在pH 7.4的PBS(10mM)中按照浓度比10μM∶1mM∶1mM混合GAO、NHS-PEG1000-MAL和Pep，在25℃，800rpm/h的条件下恒温振荡2h，之后用PBS超滤8次(超滤管截留分子量：30kDa)，制得Pep-GAO。
实施例2：结合图2，乳腺癌细胞系MDA-MB-231膜蛋白整合素αvβ3聚糖的糖链延长
将1×104个MDA-MB-231细胞种于细胞培养皿，加入含Pep-GO(等价酶浓度：0.1mg/mL)和100mM K4[Fe(CN)6]的PBS溶液100μL，在4℃下孵育30min。用PBS洗3次后，加入含10mMK3[Fe(CN)6]的PBS溶液孵育30min，从而将细胞表面αvβ3糖链末端的Gal/GalNAc氧化生成醛基。用PBS洗细胞3次后，加入含300μM酰肼修饰糖分子、10mM苯胺和5％胎牛血清的PBS溶液在4℃反应1h，用PBS洗涤3次后，完成αvβ3上的糖链延长。
实施例3：结合图3，制备酰肼修饰的甘露三糖
将己二酸二酰肼和甘露三糖溶解在等体积混合的乙腈/水溶液中，使其浓度分别为24mM和2.4mM。在80℃磁力搅拌下将混合物冷凝回流反应8h，然后用硅胶柱层析制得酰肼修饰的甘露三糖(Man3-H)。洗脱液为将异丙醇，水，氨水和乙腈按体积比6∶3∶1∶1配制的混合液。
实施例4：根据实施例2和3，乳腺癌细胞系MDA-MB-231膜蛋白整合素αvβ3聚糖上连接甘露三糖
将含300μM Man3-H、10mM苯胺和5％胎牛血清的PBS溶液加入已在αvβ3糖链末端氧化生成醛基的细胞，在4℃反应1h，用PBS洗涤3次后，实现αvβ3上的糖链的甘露三糖连接。αvβ3上连接甘露三糖的验证：用整合素αvβ3的抗体通过免疫沉淀技术将细胞裂解液中的αvβ3提取出来，再利用伴刀豆凝集素A进行凝集素印迹。
Claims (4)
1.一种活细胞膜整合素αvβ3聚糖的原位糖链延长方法，其特征在于偶联整合素αvβ3特异性识别多肽(Pep)和半乳糖氧化酶(GAO)，制备聚糖重构探针Pep-GAO，该探针在被K4[Fe(CN)6]抑制酶活性的条件下，识别活细胞表面αvβ3，然后加入K3[Fe(CN)6]激活GAO活性，特异性氧化αvβ3糖链上的半乳糖/N-乙酰半乳糖胺(Gal/GalNAc)生成可以发生生物正交反应的醛基，该基团与酰肼功能化的糖分子发生连接反应，实现αvβ3糖链的延长。
2.根据权利要求1所述的方法，其特征在于以琥珀酰亚胺酯和马来酰亚胺双修饰的聚乙二醇1000(NHS-PEG1000-MAL)为连接分子，偶联GAO的氨基和巯基修饰的c(RGDfK)多肽，制备聚糖重构探针Pep-GAO。
3.根据权利要求1所述的方法，其特征在于在K4[Fe(CN)6]存在下，Pep-GAO特异性结合细胞表面αvβ3，过程中GAO活性被抑制不能催化氧化反应，而在洗掉未结合探针，加入K3[Fe(CN)6]后，激活的GAO由于被限域在αvβ3上，只能特异性氧化αvβ3糖链末端的Gal/GalNAc生成生物正交基团醛基。
4.根据权利要求1所述的方法，其特征在于通过己二酸二酰肼与糖分子反应制得酰肼修饰的糖分子，进而与活细胞表面αvβ3糖链上生成的醛基发生连接反应生成腙键，实现整合素αvβ3聚糖的原位糖链延长。
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