title: Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin.
abstract: Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.
authors.affiliation: Glycobiology Division, Institute of Chemistry, University of Natural Resources and Applied Life Sciences (Universität für Bodenkultur), Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: Monika
authors.initials: M
authors.lastname: Bencúrová
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hemmer
authors.forename: Margarete
authors.initials: M
authors.lastname: Focke-Tejkl
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001059:Apoproteins / Q000276:immunology*; D001514:Bee Venoms / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D019476:Insect Proteins / Q000276:immunology; D010843:Pichia; D010940:Plant Proteins / Q000276:immunology; D011134:Polysaccharides / Q000276:immunology*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000276:immunology*; D014994:Xylose / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001059:Apoproteins; D001514:Bee Venoms; D000939:Epitopes; D006023:Glycoproteins; D007074:Immunoglobulin G; D019476:Insect Proteins; D010940:Plant Proteins; D011134:Polysaccharides; D014168:Transferrin; C028284:apotransferrin; D005643:Fucose; D007073:Immunoglobulin E; D014994:Xylose