Aspects of N-glycosylation in human IgE

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medicine

Sammanfattning: The medical interest of allergy in industrialised societies has generated a much greater knowledge about the pathophysiology of IgE-mediated responses than about its normal physiological role. Because of the central role of IgE in allergic inflammation, neutralisation of effector functions and inhibition of IgE-synthesis are attractive means of therapy. Further understanding of the IgEstructure and conformations involved in receptor binding is therefore of great interest. Thus, the main objective of this thesis was to examine the importance of N-linked carbohydrates for the structure and receptor-binding properties of human lgE. Native and deglycosylated purified IgE proteins from patients with myceloma as well as recombinant IgE-Fc fragments were studied by cellular and immunological methods. Enzymatic deglycosylation of IgE revealed that N-glycosylation affects expression of epitopes in the C-epsilon-2-domain either by masking them or by stabilising their conformation. In contrast, we found no major influence on structures in the C-epsilon-3- and C-epsilon-4-domains with monoclonal antibodies. To further elucidate the structural implications of carbohydrates in IgE, it was necessary to establish a modelsystem. Production of a recombinant fragment of an entire lgE-Fc, without any amino acid extensions or substitutions in baculovirus-infected insect cells demonstrated the usefulness of this expression system. Interestingly, enzymatic removal of carbohydrates markedly reduced the ability of lgE and IgEFc fragments to react with both recombinant and cell-surface expressed ct-chain of the high affinity receptor for IgE (Fc-epsilon-RI). Selective removal of N-glycosylation sites by mutagenesis in the classical IgE-Fc fragment suggested that the oligosaccharides at Asn394 are of substantial importance in stabilising Fc-epsilon-Rl-binding structures. In contrast, fragments mutated in the two other glycosylation sites of IgE-Fc, Asn265 and/or Asn371, demonstrated correct overall conformation both in terms of epitope expression and Fc-epsilon-RI-binding. As no major structural changes were found in the receptor binding part of IgE with monoclonal antibodies, the sugars at Asn394 in IgE-Fc must have some subtle effects on receptor-binding structures. In addition, sequential enzymatic removal of sugar residues in one mutant only containing the carbohydrates at Asn394 indicated that the three innermost sugar residues were sufficient to maintain conformational integrity of the C-epsilon-3-domain. Moreover, a fragment, encompassing the Fc-epsilon-region of the secreted tail-piece isoform, demonstrated similar molecular properties as classical lgE-Fc including N-glycosylation and Fareceptor-binding. However, monoclonal antibodies detected conformational differences in the C- epsilon-4-domain of the tail-piece variant that could be of biological significance. We have used in vitro techniques to show that oligosaccharide heterogeneities affect structural and Fc-epsilon-RI-binding properties of IgE. As natural variability of glycosylation sites exist in native IgE, it still remains to determine if our findings have any clinical correlates. Congenital disorders of glycosylation type 1 involve severe N-glycosylation defects and even though we found normal scrum levels with a tendency of some high IgE levels, the immunoglobulins may have carbohydrate alterations of biological importance. Obviously, further studies are warranted to elucidate the physiological and clinical relevance of IgE glycoforms.

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