Glycobiology Advance Access originally published online on March 19, 2004
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Glycobiology vol 14 no 5 pp. 457-466, 2004
Glycobiology vol. 14 no. 5 © Oxford University Press 2004; all rights reserved.
Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin
2 Glycobiology Division, Institute of Chemistry, University of Natural Resources and Applied Life Sciences (Universität für Bodenkultur), Muthgasse 18, A-1190, Vienna, Austria; and 3 FAZ-Floridsdorf Allergy Center, Vienna, Austria
Received on October 23, 2003; revised on December 19, 2003; accepted on January 11, 2004
Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing ß1,2-xylose and core 
1,3-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-1, 3-fucosyltransferase (EC 2.4.1.214) and ß1,2-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 1,3-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.
1 To whom correspondence should be addressed; e-mail: friedrich.altmann{at}boku.ac.at
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