Abstract 3860: Glycosylation is indispensable for ephrin A1 activity

Introduction: EphrinA1 belongs to the ephrin family of ligands that interact with the Eph-related tyrosine kinase receptors. ephrin/Eph regulatory pathways control a wide array of physiological cellular functions, but have also been implicated in the development and progression of malignancy. EphrinA1 specifically binds EphA2 receptor that is highly over-expressed in gliobastomas (GBM), but not in normal adult brain, inducing a receptor-mediated internalization and down-regulation with subsequent tumor suppressing action, making this an attractive target for the development of new anti-tumor therapeutics. Our laboratory documented that a soluble monomeric functionally active form of EphrinA1 is released from cancer cells. In the crystal structure of ephrinA1/EphA2 complex, ephrinA1 was shown to be glycosylated on Asparagine 26; however, there is no data available confirming the presence or explaining the importance of this glycosylation. We have thus initiated a study to investigate the role of glycosylation in ephrinA1 biological activity. Methods: EphrinA1-Fc tag was produced in Sf9 insect cells with the Baculovirus expression system. Proteins were purified by proteinG affinity and MonoQ ion exchange chromatography, using FPLC. The purity of the proteins was estimated by SDS-PAGE and Western blots (WB). EphrinA1 activity was evaluated on U-251 MG GBM cells by examining cell morphology and EphA2 receptor down-regulation. Enzymatic de-glycosylation with N-GlycosidaseF (PNGaseF) was performed as recommended by the manufacturer. Tunicamycin was used to inhibit N-linked glycosylation at the final concentration of 1 μg/ml. Results: Enzymatic de-glycosylation with PNGaseF caused a decrease in molecular weight of ephrinA1 detected by SDS-PAGE. The same was observed by WB on ephrinA1 immunoreactive band from PNGaseF treated U-251[ephrinA1+] conditioned media. Experiments using the drug tunicamycin on U-251[ephrinA1+] cells showed a clear shift in ephrinA1 gel mobility implying that the protein was expressed in an unglycosylated form. We compared glycosylated ephrinA1 to de-glycosylated ephrinA1 (Deg-ephrinA1) activities in both monomeric and dimeric forms on cells expressing EphA2 receptor. Deg-ephrinA1 lost almost all of cell rounding and EphA2 down-regulating abilities compared to the naturally glycosylated form of ephrinA1. Additionally, we mutagenized ephrinA1 glycosylation site to perform additional experiments trying to clarify the biological steps in which the glycosylation on ephrinA1 is critical for protein biological activity. Conclusions: EphrinA1glycosylation is essential for ephrinA1 activity through the activation of the EphA2 receptor. This observation was surprising, but it has important implications for the understanding of the ligand-receptor productive interactions and for the future production of novel ephrinA1-based, EphA2-targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3860. doi:1538-7445.AM2012-3860