Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells.
IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cvarepsilon3-domain are crucial for FcvarepsilonR-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 (rCvarepsilon2-4) and a smaller one comprising the second and third domain (rCvarepsilon2-3), were produced and characterised with respect to epitope expression, glycosylation and FcvarepsilonR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cvarepsilon2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCvarepsilon2-4 mainly formed a dimer, whereas rCvarepsilon2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCvarepsilon2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cvarepsilon2-, Cvarepsilon3- and Cvarepsilon4-domains, whereas rCvarepsilon2-3 only reacted with anti-Cvarepsilon2 mAbs. Moreover, in FACS analysis rCvarepsilon2-4 interacted with two cell-lines constitutively expressing FcvarepsilonRI or FcvarepsilonRII, whereas rCvarepsilon2-3 lacked reactivity. A substantial reduction in the ability of rCvarepsilon2-4, following endoglycosidase treatment, to react with recombinant alpha-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 rCvarepsilon2-4, but not rCvarepsilon2-3, 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 Fcvarepsilon-receptors.
Björklund
J E
JE
Department of Medicine, Division of Clinical Immunology and Allergy, Karolinska Institutet and Hospital, Stockholm, Sweden.
Schmidt
M
M
Magnusson
C G
CG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D006023:Glycoproteins; D006031:Glycosylation; D006801:Humans; D007073:Immunoglobulin E / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011499:Protein Processing, Post-Translational; D017455:Receptors, IgE; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology; D018411:Spodoptera / Q000166:cytology
D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E