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

作者：Ishino Tetsuya; Economou Nicoleta J; McFadden Karyn; Zaks Zilberman Meirav; Jost Monika; Baxter Sabine; Contarino Mark R; Harrington Adrian E; Loll Patrick J; Pasut Gianfranco; Lievens Sam; Tavernier Jan; Chaiken Irwin^*

来源：Biochemistry, 2011, 50(35): 7546-7556.

DOI：10.1021/bi2009135

Human interleukin-5 receptor alpha (IL5R alpha) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5R alpha resulted in complete loss of IL5 binding To localize the functionally important carbohydrate moieties, we employed site directed mutagenesis at the N-glycosylation sites (Asn(15), Asn(111), Asn(196), and Asn(224)). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5R alpha and other cytokine receptors. These approaches were most successful at Asn(15), Asn(111), and Asn(224). In contrast, any replacement at Asn(196) 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 Asn(196) 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 Asn(196) while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5R alpha, thus demonstrating a unique role for Asn(196) glycosylation in IL5R alpha function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.

• 出版日期2011-9-6