Liu Yingmiao, Kuan Chien-Tsun, Mi Jing, Zhang Xiuwu, Clary Bryan M, Bigner Darell D, Sullenger Bruce A
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Biol Chem. 2009 Feb;390(2):137-44. doi: 10.1515/BC.2009.022.
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 33x10(-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.
表皮生长因子受体变体III（EGFRvIII）是一种仅在胶质母细胞瘤中表达而在正常脑组织中不表达的糖蛋白。为了开发针对脑肿瘤的靶向治疗方法，我们使用大肠杆菌系统进行蛋白质表达和纯化，筛选针对组氨酸标签化EGFRvIII胞外结构域的RNA适体。代表性适体E21的解离常数（Kd）为33×10（-9）m，并且在ELISA和表面等离子体共振分析中对EGFRvIII表现出高亲和力和特异性。然而，所选适体不能结合真核细胞表达的相同蛋白质，因为糖基化（仅存在于真核系统中的一种翻译后修饰）会显著改变靶蛋白的结构。通过将EGFRvIII适体转染到细胞中，我们发现膜结合的、糖基化的EGFRvIII减少，并且细胞凋亡百分比增加。我们推测转染的适体可以与新合成的EGFRvIII相互作用，破坏正常糖基化，并减少到达细胞表面的成熟EGFRvIII的量。我们的工作确立了用适体原位破坏蛋白质翻译后修饰的可行性。这一发现对于阐明具有各种修饰的感兴趣蛋白质的功能以及剖析信号转导途径很有用。
