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.

Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

“…Furthermore, we have recently shown that N-glycosylation of the IL-6 receptor (IL-6R) is dispensable for its biological function, but rather influences limited proteolysis of the IL-6R by the metalloprotease ADAM17 [25]. In contrast, the receptors for C-X-C motif chemokine 12 (CXCL12), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF) or interleukin-5 require proper glycosylation for their biological functions [26-29]. …”

The Role of Glycosylation in Receptor Signaling

“…Complete removal of the glycosylation of IL5 leads to a loss of ligand binding. More detailed studies of the contributions of the N-glycosylation sites on IL5 revealed that Asn 196 is required for ligand binding (9). Loss of the other three sites by mutation had no effect on IL5 affinity and biological activity (B-cell proliferation assay).…”

“…Mechanistic studies have revealed that IL5 induces biological activity through a two step process in which IL5 binds to the IL5 subunit leading to interaction with the preformed c subunit (9,10). The c then induces the signaling cascade within the target cell through activation of a kinase cascade by way of associated JAK kinases.…”

“…Loss of the other three sites by mutation had no effect on IL5 affinity and biological activity (B-cell proliferation assay). Interestingly, mutation of Asn 196 led to a complete loss of binding and biological activity, suggesting that glycosylation of that residue is absolutely required for IL5 recognition (9). IL5R c subunit is also glycosylated.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Arginine-specific mono ADP-ribosylation in vitro of antimicrobial peptides by ADPribosylating toxins (Castagnini et al, 2012) Interleukin-5 receptor TOF Mutation of N-glycosylation sites to determine effect on function (Ishino et al, 2011) Interleukin-11 TOF (sinapinic), glycoprotein Introduction of O-glycans in the non-core region to investigate function (Yanaka et al, 2011) Lipoproteins…”