title: Close-up of the immunogenic α1,3-galactose epitope as defined by a monoclonal chimeric immunoglobulin E and human serum using saturation transfer difference (STD) NMR.
abstract: Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis.
authors: Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany.
authors: Melanie
authors: M
authors: Plum
authors: Yvonne
authors: Y
authors: Michel
authors: Katharina
authors: K
authors: Wallach
authors: Tim
authors: T
authors: Raiber
authors: Simon
authors: S
authors: Blank
authors: Frank I
authors: FI
authors: Bantleon
authors: Andrea
authors: A
authors: Diethers
authors: Kerstin
authors: K
authors: Greunke
authors: Ingke
authors: I
authors: Braren
authors: Thomas
authors: T
authors: Hackl
authors: Bernd
authors: B
authors: Meyer
authors: Edzard
authors: E
authors: Spillner
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005690:Galactose / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D051381:Rats; D020349:Surface Plasmon Resonance
chemical_list: D000939:Epitopes; D007073:Immunoglobulin E; D005690:Galactose