IgE production and stability regulated by Glycosylation in vivo

Abstract

Abstract Rationale: Essential functions of IgE have been attributed to specific glycosylations such as oligomannose (Fcer1a binding) and sialylation (pathogenicity). However, no study has examined how glycosylation affects IgE stability in vivo. Methods: Forward genetic screening for IgE specific phenotypes was performed using N-ethyl-N-nitrosourea (ENU) mutagenized mice. One low IgE phenotype, named benadryl, linked to a mutation in Mpi, which is essential for N-linked glycosylation. Benadrylwas validated by CRISPR knock-in . Immunological studies were performed to determine the causative mechanism (passive/ active anaphylaxis, mast cell analysis, conditional knockout in T, B and dendritic cells, antibody production in vivoand in vitro, using IgE-Venus, IgE-KO, and IL-4 GFP mice). Results: Benadrylmice had low total and papain-specific IgE responses with resistance to active anaphylaxis. In vitroand in vivomast cell activation studies resulted in no difference in response to passive sensitization anaphylaxis. Both, in vivo and in vitro cellular staining in the conditional knockout showed no decrease in IgE B cells. IgE from Mpi deficient mice was produced and secreted from B cell culture. When de-glycosylated IgE was injected to IgE KO mice, it was cleared 10-fold faster than native IgE. Conclusions: Glycosylation status determines not only IgE functionality but in vivo stability. Mpi deficient mice produce IgE hypoglycosylated in nature and therefore it is cleared faster escaping anaphylaxis. Modulating IgE glycosylation represents a novel way to reduce IgE below pathogenic levels. NA