Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells

Abstract

IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cε3-domain are crucial for FcεR-binding. In order to further study structures of importance for the function of IgE, such as the carbohydrates, fragments with unmodified amino acid sequence were blunt-end cloned and expressed in baculovirus-infected Sf9 cells. Two fragments of human IgE, one encompassing the entire Fc-region (rCε2-4) and a smaller one comprising the second and third domain (rCε2-3), were produced and characterised with respect to epitope expression, glycosylation and FcεR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cε2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCε2-4 mainly formed a dimer, whereas rCε2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCε2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cε2-, Cε3- and Cε4-domains, whereas rCε2-3 only reacted with anti-Cε2 mAbs. Moreover, in FACS analysis rCε2-4 interacted with two cell-lines constitutively expressing FcεRI or FcεRII, whereas rCε2-3 lacked reactivity. A substantial reduction in the ability of rCε2-4, following endoglycosidase treatment, to react with recombinant α-chain of the high affinity receptor for IgE in sandwich ELISA, indicated a role of N-linked oligosaccharides in stabilising receptor binding structures. Taken together, our results show that rCε2-4, but not rCε2-3, will be useful in studies of structure–function relationships of IgE, including the role of N-glycosylation, since it demonstrated appropriate epitope expression, conformation and ability to bind Fcε-receptors.