Insights into the molecular IgE-IPSE/alpha-1 interaction responsible for basophil activation

Abstract text

IPSE/alpha-1 (IL-4 inducing principle of S. mansoni eggs) is a glycoprotein secreted by the eggs of the blood trematode S. mansoni, the causative agent of schistosomiasis, an important neglected tropical disease. Natural IPSE occurs as a dimer, in which an unpaired cysteine (C132) is responsible for homodimerization. IPSE has been shown to bind to IgE, resulting in the release of IL-4 and IL-13 from basophils and mast cells. The classical mechanism of IgE-dependent activation consists of cross-linking IgE by allergen binding to the antigen-recognition variable region of the corresponding immunoglobulin. While IPSE must occur as homodimer for successful IgE binding, this protein appears to activate basophils by binding to IgE without any typical cross-linking.

The aim of this study is to investigate the molecular details underlying this unique interaction between IPSE/alpha-1 and IgE.

Using site-directed mutagenesis, we created several mutants, based on the knowledge that neither IPSE monomers nor the T92Y/R127L mutant, are able to activate basophils. Proteins were expressed in HEK293-6E suspension cells, followed by affinity chromatography for purification. The ability of all IPSE forms to activate basophils by binding IgE was evaluated using humanized RS-ATL8 rat basophilic leukemia (RBL) reporter cells. Cells were sensitized with either IgE-containing sera or different IgE truncates and luciferase expression was measured after stimulation with IPSE. Ancillary ELISAs using similar truncated forms of IgE and WT IPSE were performed in order to further determine the binding region.

Our results show that all the mutations have an impact on IPSE"s capability to interact with IgE, thus lowering the activation of the reporter cells. Only the double mutant T92Y/R127L hampers cell activation completely, leading us to the conclusion that both amino acids must be key residues involved in IgE interaction. Another reason could be that these mutations cause a conformational change of the protein, resulting in incorrect binding to IgE. Furthermore, we show that WT IPSE does not bind to all truncated forms of IgE, suggesting that IPSE needs all heavy chain domains, with or without light chains, to successfully bind to IgE.

The proper folding of the IPSE mutants will be validated by thermal shift assay. Ultimately, Negative Staining of the ternary IgE/IPSE/FcεRIα complex are expected to reveal a detailed model of interaction.