Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.
Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.
复制标题
选择针对未糖基化的EGFRVIII胞外域的适体，细胞内递送降低膜结合的EGFRVIII并诱导细胞凋亡。
DOI：
10.1515/bc.2009.022
复制
发表时间：
2009-02
影响因子：
3.7
通讯作者：
Sullenger BA
中科院分区：
文献类型：
--
作者：
Liu Y;Kuan CT;Mi J;Zhang X;Clary BM;Bigner DD;Sullenger BA
表皮生长因子受体变异体III(EGFRvIII)是一种糖蛋白，仅在胶质母细胞瘤中表达，而在正常脑组织中不表达。为了开发脑肿瘤的靶向治疗方法，我们选择了针对组氨酸标记的EGFRvIII胞外区的RNA适配子，使用大肠杆菌系统进行蛋白质的表达和纯化。具有代表性的适体E21的解离常数(Kd)为33×10−9m，在EL ISA和表面等离子体共振分析中显示出与EGFRvIII的高亲和力和特异性。然而，所选择的适配子不能结合从真核细胞表达的相同的蛋白质，因为糖基化，一种仅存在于真核系统中的翻译后修饰，显著改变了目标蛋白质的结构。通过将EGFRvIII适配子导入细胞，我们发现膜结合的糖基化的EGFRvIII减少了，细胞凋亡的比例增加了。我们推测，转染适配子可以与新合成的EGFRvIII相互作用，破坏适当的糖基化，并减少到达细胞表面的成熟EGFRvIII的数量。我们的工作建立了用适配子原位干扰蛋白质翻译后修饰的可行性。这一发现有助于阐明经过各种修饰的目标蛋白的功能，以及剖析信号转导途径。
Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10−9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.