期刊
BIOLOGICAL CHEMISTRY
卷 390, 期 2, 页码 137-144出版社
WALTER DE GRUYTER GMBH
DOI: 10.1515/BC.2009.022
关键词
EGFRvIII; glycosylation; RNA aptamer; SELEX
资金
- NIH [U54-CA-119343, R37 CA 011898-38]
- NINDS [5P50 NS20023-25]
- NIH SPORE [R37 CA 011898-38, 5P50 CA108786-05]
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 (K-d) of 33 X 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.
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