title: A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor α.
abstract: 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 (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-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 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 IL5Rα, thus demonstrating a unique role for Asn(196) glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ishino
authors.forename: Nicoleta J
authors.initials: NJ
authors.lastname: Economou
authors.forename: Karyn
authors.initials: K
authors.lastname: McFadden
authors.forename: Meirav
authors.initials: M
authors.lastname: Zaks-Zilberman
authors.forename: Monika
authors.initials: M
authors.lastname: Jost
authors.forename: Sabine
authors.initials: S
authors.lastname: Baxter
authors.forename: Mark R
authors.initials: MR
authors.lastname: Contarino
authors.forename: Adrian E
authors.initials: AE
authors.lastname: Harrington
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Loll
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Pasut
authors.forename: Sam
authors.initials: S
authors.lastname: Lievens
authors.forename: Jan
authors.initials: J
authors.lastname: Tavernier
authors.forename: Irwin
authors.initials: I
authors.lastname: Chaiken
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D002236:Carbohydrate Conformation; D002460:Cell Line; D004331:Drosophila melanogaster; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D053666:Interleukin-5 Receptor alpha Subunit / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: C485918:IL5RA protein, human; D053666:Interleukin-5 Receptor alpha Subunit; D008024:Ligands; D001216:Asparagine