1 CLOSE-UP OF THE IMMUNOGENIC ALPHA-1,3-GAL EPITOPE AS DEFINED BY A MONOCLONAL CHIMERIC IGE AND HUMAN SERUM USING SATURATION TRANSFER DIFFERENCE (STD) NMR Melanie Plum 1,* , Yvonne Michel 1,* , Katharina Wallach 2,* , Tim Raiber 1 , Simon Blank 1 , Frank I. Bantleon 1 , Andrea Diethers 1 , Kerstin Greunke 1 , Ingke Braren 3 , Thomas Hackl 2 , Bernd Meyer 2 , and Edzard Spillner 1 From the Institute of Biochemistry and Molecular Biology 1 , and Institute of Organic Chemistry 2 University of Hamburg, Hamburg, Germany and the Hamburg Center for Experimental Therapy Research (HEXT) 3 University Medical Center Hamburg-Eppendorf, Hamburg, Germany Running head: Close-up of the alpha-Gal epitope * These authors contributed equally to this work Address correspondence to: Edzard Spillner, Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany, Phone.: 0049-40-42838-6982; Fax: 0049-40-42838-7255; E-mail: spillner@chemie.uni- hamburg.de CAPSULE Background: Antibody binding to xenobiotic alpha-Gal structures mediates anaphylaxis. Result: Humanized IgE antibodies exhibit recognition footprints similar to serum immunoglobulins but have no capability of effector cell activation. Conclusion: Recognition of the alpha-Gal epitope is based on the terminal disaccharide, but interaction does not imply cellular activation. Significance: These data provide first insights into the recognition of carbohydrate IgE epitopes. SUMMARY Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen alpha-1,3-Gal (alpha-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 alpha-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 alpha-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 STD NMR analyses. The alpha- 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 alpha-1,3- Gal epitope fine structure of both the recombinant IgE and affinity-purified serum were defined by STD NMR revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying alpha-1,3- Gal-mediated anaphylaxis. The complemen- tary 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. INTRODUCTION Circulating concentrations of IgE, the antibody class responsible for allergic hyper- sensitivity, are linked to the development of http://www.jbc.org/cgi/doi/10.1074/jbc.M111.291823 The latest version is at JBC Papers in Press. Published on October 11, 2011 as Manuscript M111.291823 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on January 19, 2017 http://www.jbc.org/ Downloaded from