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A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor α
Journal article   Open access   Peer reviewed

A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor α

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, …
Biochemistry (Easton), v 50(35), pp 7546-7556
06 Sep 2011
DOI: https://doi.org/10.1021/bi2009135
PMID: 21770429
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1021/bi2009135View
Published, Version of Record (VoR)Open Access (License Unspecified),  Open

Abstract

Amino Acid Sequence Cell Line Protein Binding - genetics Protein Engineering - methods Humans Interleukin-5 Receptor alpha Subunit - genetics Molecular Sequence Data Glycosylation Interleukin-5 Receptor alpha Subunit - physiology Asparagine - physiology Genetic Variation Interleukin-5 Receptor alpha Subunit - chemistry Animals Ligands Mice Carbohydrate Conformation Drosophila melanogaster Asparagine - chemistry Asparagine - genetics
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.

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Domestic collaboration
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Web of Science research areas
Biochemistry & Molecular Biology
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