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. 2015 Apr 6;212(4):457-67.
doi: 10.1084/jem.20142182. Epub 2015 Mar 30.

A single glycan on IgE is indispensable for initiation of anaphylaxis

Kai-Ting C Shade  1 , Barbara Platzer  2 , Nathaniel Washburn  3 , Vinidhra Mani  1 , Yannic C Bartsch  1 , Michelle Conroy  1 , Jose D Pagan  1 , Carlos Bosques  3 , Thorsten R Mempel  1 , Edda Fiebiger  2 , Robert M Anthony  4
Affiliations

A single glycan on IgE is indispensable for initiation of anaphylaxis

Kai-Ting C Shade et al. J Exp Med. .

Abstract

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.

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Figures

Figure 1.
Figure 1.
N-linked glycosylation is essential for IgE-mediated allergic inflammation. (a and b) Quantified vascular leakage and representative ear images after PCA with PBS, WT, or PNG–poly-mIgE specific for OVA or peanut extracts (n = 4 ears; 2 independent experiments; a) or monoclonal mIgE specific for OVA, DNP, or TNP (n = 8; 2, 3, and 3 independent experiments for OVA, DNP, and TNP; b). Bar, 1 cm. (c) Histograms and mean fluorescent intensity (MFI) of WT or PNG-αOVA-mIgE binding to mBMMCs determined by A647-OVA and mFcεRI (n = 3; 2 independent experiments). (d) β-Hexosaminidase activity after OVA stimulation of LAD2 cell sensitized with WT or PNG-treated αOVA-hIgE (n = 3; 5 independent experiments). (e) Quantified OVA-induced PCA in mFcεRI−/− or hFcεRI+mFcεRI−/− mice sensitized by WT or PNG-αOVA-hIgE (n = 4; 2 independent experiments). (f) FACS of WT or PNG-αOVA-hIgE to hFcεRI+-HeLa cells assessed by A647-OVA and α-hFcεRI-PE (n = 2; 2 independent experiments). Means and SEM are plotted; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.
Figure 2.
Figure 2.
Glycosylation of IgE Cε3 is required for anaphylaxis and FcεRI binding. (a) Quantified OVA-specific PCA by PBS, WT, or αOVA-mIgE domain mutants (n = 8; 3 independent experiments). Bar, 1 cm. (b) β-Hexosaminidase activity after OVA-induced degranulation in LAD2 cells sensitized with WT or αOVA-hIgE glycomutants (n = 3; 3 independent experiments). (c) WT-αOVA-hIgE or Cε1- and Cε3-αOVA-hIgE glycosylation mutant binding to hFcεRI+-HeLa cells assessed by A647-OVA and α-hFcεRI-PE FACS (n = 2; 2 independent experiments). Mean and SEM are plotted; ****, P < 0.0001; ***, P < 0.001; *, P < 0.05; ns, not significant.
Figure 3.
Figure 3.
A single N-linked glycosylation site is indispensable for anaphylaxis and hFcεRI binding. (a) Quantified OVA-induced PCA with PBS, WT, or αOVA-mIgE Cε3 domain glycomutants (n = 8; 3 independent experiments). (b) Scatter plots of CD45+c-Kit+CD11b− mast cells recovered from WT or mFcεRI−/− ears injected with A488-WT and A568-WT αOVA-mIgE or A488-WT and A568-N384Q αOVA-mIgE the previous day. Percentage of A488 and A568 double-positive mast cells are shown (n = 2; 2 independent experiments). (c) Histograms and MFI of WT or N384Q αOVA-mIgE binding to mBMMCs determined by A647-OVA and mFcεRI (n = 3; 2 independent experiments). (d) OVA-induced degranulation assayed by β-hexosaminidase in LAD2 cells sensitized with WT or αOVA-hIgE glycosylation mutants (n = 3; 4 independent experiments). (e) OVA-induced PCA vascular leakage in hFcεRI+/mFcεRI−/− mice by PBS, WT, or N394Q αOVA-hIgE (n = 3; 2 independent experiments). (f) Binding of WT or αOVA-hIgE mutants to hFcεRI+-HeLa cells as assessed by A647-OVA (n = 2; 2 independent experiments). (g) Binding of increasing concentrations of WT or N394Q αOVA-hIgE to immobilized hFcεRIα in vitro (2 independent experiments.) Mean and SEM are plotted; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant.
Figure 4.
Figure 4.
Removal of the IgE oligomannose glycan abrogates anaphylaxis. (a and b) αOVA-mIgE (a) and αOVA-hIgE (b) schematics with N-linked glycosylation consensus sites are shown. Percentages of glycan structures identified by glycopeptide mass spectroscopy at each site are plotted. Glycans are composed of fucose (red), N-acetylglucosamine (GlcNAc; blue), mannose (green), galactose (yellow circles), N-acetylgalactosamine (GalNAc; yellow squares), and sialic acid (pink); representative of two experiments. (c) PCA quantified after PBS, WT, or EndoF1-mIgE specific for OVA, DNP, or TNP (n = 8; 2 independent experiments). (d) Histograms and MFI of WT-, EndoF1-, and EndoF1 buffer only–αOVA-mIgE bound to mBMMCs determined by A647-OVA and mFcεRI FACS (n = 3; 2 independent experiments). (e) Scatter plots of dermal mast cells recovered from ears injected with A488-WT- and A568-WT- or A488-WT- and A568-EndoF1-αOVA-mIgE (n = 2; 2 independent experiments). (f) β-Hexosaminidase activity after stimulation by OVA, streptavidin, or anti–human kappa light chain in LAD2 cells sensitized with αOVA-hIgE, biotinylated hIgE, or allergic human serum IgE or hIgE treated with EndoF1 or EndoF1 buffer only (n = 3; 2 independent experiments). (g) Binding of WT, EndoF1, or EndoF1 buffer only biotinylated hIgE to hFcεRI-HeLa cells as assessed by FACS (n = 3; 2 independent experiments). (h) Quantitation of WT or EndoF1-treated αOVA-hIgE bound to immobilized hFcεRIα (2 independent experiments). (i) Size exclusion chromatography profile of αOVA-mIgE or hIgE treated with EndoF1 or buffer only. (j) CD spectra of WT, EndoF1-treated, or EndoF1 buffer control hIgE (representative of two experiments). Mean and SEM are plotted; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.
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