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A Protein Engineering Approach Differentiates the Functional Importance of Carbohydrate Moieties of Interleukin-5 Receptor α

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posted on 06.09.2011, 00:00 by Tetsuya Ishino, Nicoleta J. Economou, Karyn McFadden, Meirav Zaks-Zilberman, Monika Jost, Sabine Baxter, Mark R. Contarino, Adrian E. Harrington, Patrick J. Loll, Gianfranco Pasut, Sam Lievens, Jan Tavernier, Irwin Chaiken
Human interleukin-5 receptor α (IL5Rα) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5Rα resulted in complete loss of IL5 binding. To localize the functionally important carbohydrate moieties, we employed site-directed mutagenesis at the N-glycosylation sites (Asn15, Asn111, Asn196, and Asn224). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability-preserving changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5Rα and other cytokine receptors. These approaches were most successful at Asn15, Asn111, and Asn224. In contrast, any replacement at Asn196 severely reduced stability, with the N196T mutant having a reduced binding affinity for IL5 and diminished biological activity because of the lack of cell surface expression. Lectin inhibition analysis suggested that the carbohydrate at Asn196 is unlikely involved in direct ligand binding. Taking this into account, we constructed a stable variant, with triple mutational deglycosylation (N15D, I109V/V110T/N111D, and L223R/N224Q). The re-engineered protein retained Asn196 while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5Rα, thus demonstrating a unique role for Asn196 glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.

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