Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.
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.
Liu
Yingmiao
Y
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Kuan
Chien-Tsun
CT
Mi
Jing
J
Zhang
Xiuwu
X
Clary
Bryan M
BM
Bigner
Darell D
DD
Sullenger
Bruce A
BA
D000818:Animals; D017209:Apoptosis; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D052156:SELEX Aptamer Technique
D052157:Aptamers, Nucleotide; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors