N-glycosylation influences epitope expression and receptor binding structures in human IgE

Although human IgE is relatively rich in carbohydrates, there are few studies concerning their structural and functional importance. The low serum concentration of IgE has limited carbohydrate characterisation to a few IgE myeloma proteins. Four to six of the seven potential N-glycosylation sites in the constant region of the epsilon chain seem occupied together with some residual microheterogeneity. We have used a panel of 28 anti-Cepsilon2, 7 anti-Cepsilon3 and 18 anti-Cepsilon4 domain-specific anti-IgE mAbs, and rFcepsilonRIalpha to examine the effect of N-glycosylation on epitope expression of human IgE. Myeloma proteins IgE(DES)-kappa, IgE(ND)-lambda and IgE(UD)-kappa as well as polyclonal IgE were deglycosylated with PNGF and/or sialidase and tested in different ELISA. In all ELISA approaches, the reactivity of most domain-specific anti-IgE mAbs was independent of the glycosylation state of IgE(DES), except for one-third of the anti-Cepsilon2 mAbs. These mAbs reacted better with deglycosylated IgE(DES) in the order of treatment PNGF/sialidase > PNGF > or = sialidase > buffer control. In sharp contrast, the reactivity of IgE(DES) with rFcepsilonRIalpha was not influenced by sialidase but markedly reduced following PNGF or PNGF/sialidase treatment. These findings were neither myeloma restricted nor caused by aggregation, since monomeric IgE demonstrated the same reactivity pattern. Thus. N-glycosylation seems to influence both structure and function of human IgE. The oligosaccharides modulate epitope expression, mainly in the Cepsilon2-domain, as revealed by a subset of mAbs. They also promote subtle changes in the Cepsilon3-domain, leading to a reduced FcepsilonRIalpha binding. These findings suggest physiological implications of carbohydrates in human IgE.