Sialylation of IgE does not impact its interaction with FcεRI
Authors: Sayantan Banerjee, Colleen P. Phelan, Brian B. Reese, Michelle E. Conroy, Robert M. Anthony
Journal: Allergy (2024)

IgE binds FcεRI monomerically with high affinity.1-4 A recent manuscript interrogated interactions between IgE and FcεRI using sandwich ELISA and flow cytometry,5 methods which quantify the amount of IgE bound to FcεRI, but not interaction strength. The authors found more asialylated IgE bound FcεRI than sialylated IgE at saturating levels in both assays. Therefore, we asked whether removing sialic acid enabled binding of multiple IgE to FcεRI. We treated ⍺-OVA mIgE (Figure 1A) with neuraminidase and buffer (asialylated mIgE) or buffer-only (sialylated mIgE) as described.6 Digestions were confirmed by gel electrophoresis followed by lectin blotting with sialic acid-specific Sambucus Nigra Lectin (SNA) (Figure 1B). Sialylated and asialylated mIgE bound and saturated OVA similarly (Figure 1C). The mIgE preparations were tested in real-time human FcεRI (hFcεRI) binding assays by biolayer interferometry (BLI, Figure 1D). FcεRI binding was similar between sialylated (pink) and asialylated (yellow) mIgE, and modeling of both interactions was consistent with a 1:1 interaction (not shown). Next, we tested sialylated and asialylated mIgE in a model of passive cutaneous anaphylaxis (PCA), which is insensitive to serum half-life. Indeed, ears sensitized with sialylated mIgE exhibited robust inflammation, while inflammation in ears treated with asialylated IgE was markedly attenuated (Figure 1E).6 To extend these results, we generated sialylated and asialylated ⍺-OVA-human IgE (hIgE) as above (Figure 1F,G). The hIgE preparations were examined in ELISA and BLI experiments and found to interact similarly with OVA and hFcεRI (Figure 1H,I). The hIgE preparations were used to sensitize human LAD2 mast cells, and flow cytometry-based analysis revealed similar IgE loading on the cells (Figure 1J). We obtained similar results using hybridoma-derived ⍺-TNP mIgE following digestion and purification conditions reported elsewhere5 (Figure 2A,B, Figure S1). We next examined passive systemic anaphylaxis (PSA) 2 h after sensitization with sialylated or asialylated IgE. Mice sensitized with sialylated ⍺-TNP IgE had significant temperature loss, while those sensitized with asialylated IgE did not (Figure 2C). However, asialylated ⍺-TNP IgE serum titers 2 h after sensitization were significantly reduced compared to sialylated IgE (Figure 2D). Therefore, we blocked the asialoglycoprotein receptor (ASGPR) during PSA as previously described.5 This result phenocopied our previous result, with robust anaphylaxis in mice sensitized with sialylated ⍺-TNP IgE, but not asialylated IgE (Figure 2E). Serum ⍺-TNP mIgE titers were similar between sialylated and asialylated preparations 2 h after sensitization with ASGR blockade (Figure 2F). Indeed, PSA induced 24 h after sensitization was robust in mice sensitized with sialylated but not asialylated mIgE (Figure 2G), while no serum sialylated or asialylated IgE was detected at this timepoint (Figure 2H). Also, no differences in peritoneal mast cell surface IgE loading were observed 2 h following intraperitoneally sensitized with sialylated or asialylated ⍺-TNP IgE (Figure 2I). As ASGPR blockade impacted asialylated IgE serum titers 2 h after sensitization, we examined the interaction of sialylated and asialylated IgE with ASGR by BLI. Sialylated hIgE did not bind ASGPR, while asiaylated IgE bound ASGR with modest affinity (Figure 2J), albeit magnitudes lower than monomeric IgE-FcεRI binding. Together, our results indicate that the presence of sialic acid on IgE minimally impacts FcεRI interactions, allergen binding, or mast cell loading, but does influence the magnitude of anaphylaxis. The work was supported by NIH NIAID awards R01AI139669 to RMA and R01AI167933 to MEC. SB, CPP, and BBR conducted generated data, MEC and RMA obtained funding, supervised the research, and wrote the manuscript with SB. The authors declare no conflict of interests. If published, the manuscript will be submitted into PubMed Central, an open access source. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.