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.

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

“…Earlier reports showed that xylose is not always bound equally well by specific antibodies (31), and one study suggested that presence of ␣1,3-mannose on M3X(F) structures might interfere sterically with binding (20). In our study, three-dimensional-glycan modeling provided a first correlative and plausible explanation for altered xylose recognition.…”

“…Previously, van Ree et al (20) concluded that xylose-specific IgE antibodies that bound to bromelain M2XF (MUXF) N-glycan did not recognize closely related M3XF (MMXF) structures of PHA-L or HRP because of sterical hindrance caused by the presence of ␣1,3-mannose in case of M3XF (MMXF), whereas Bencúrová et al (31) concluded that bromelain is not useful for detection of xylose-specific antibodies because of an absence of ␣1,3-mannose from M2X (MUX).…”

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

“…The role of CCDs as a cause of allergic symptoms still is controversial (4). IgE against classical CCDs has been shown to be clinically relevant (5)(6)(7)(8), but artificial or recombinant glycoproteins did not show clear cut effects in mediator release assays or skin prick tests (9,10).…”

<i>N</i>-Glycan structures of glycoproteins in suspension-cultured <i>Arabidopsis thaliana</i> T87 cells

“…However, mammalian N-glycans generally have α(1,6)-Fuc residues linked to Man 3 GlcNAc 2 instead, but not have β(1,2)-Xyl. Plant-specific sugar residues have been suggested to be potentially immunogenic or allergenic (Altmann 2007;Bencúrová et al 2004;Jin et al 2008). Previously, we examined N-glycan structures derived from endogenous and soluble proteins of suspensioncultured cells of tobacco (Nicotiana tabacum) BY2 (Misaki et al 2001;Palacpac et al 1999) and A. thaliana MM2d (Fujiyama et al 2007), and showed that M3FX is the predominant structure.…”