Silencing ?1,2-xylosyltransferase in Transgenic Tomato Fruits Reveals xylose as Constitutive Component of Ige-Binding Epitopes

Abstract

Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called “carbohydrate cross-reactive determinants” reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT), and core α1,3-fucosyltransferase (FucT) are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of β1,2-xylose-containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi) in transgenic plants. Fruits harvested from these transgenic plants were analyzed for accumulation of XylT mRNA, abundance of β1,2-xylose epitopes and their allergenic potential. Based on quantitative real-time PCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of β1,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of β1,2-xylose-containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of β1,2-xylose-containing epitopes in vivo.

Fc glycans of therapeutic antibodies as critical quality attributes

“…However, the clinical relevance of these reactive IgE remains unclear. Strasser et al used RNAi to successfully silence XylT, the gene coding for β1,2-xylotransferase, in tomato plants and demonstrated a patient-specific reduction in IgE reactivity in their studies ( Paulus et al 2011 ). Arabidopsis thaliana knockouts of the XylT (encoding β1,2-xylotransferase) and FucT (encoding α1,3 fucosyl transferase) genes have also been generated that are viable with no obvious phenotype.…”

Multiple xylosyltransferases heterogeneously xylosylate proteinN‐linked glycans inChlamydomonas reinhardtii

“…Three putative genes, of which the translated amino acid sequences showed 12–16% of identity with XTA , were identified as Cre10.g458950, Cre13.g588750 and Cre08.g361250. XTA, XTB and the three additional putative C. reinhardtii xylosyltransferases were compared with core β(1,2)‐xylosyltransferases previously functionally characterized in A. thaliana (Strasser et al , ), Solanum lycopersicum (Paulus et al , ), Oryza sativa (Shin et al , ), Lemna minor (Cox et al , ), Medicago sativa (Sourrouille et al , ) and Physcomitrella patens (Koprivova et al , ; Figure ). The phylogenic tree showed that C. reinhardtii and plant xylosyltransferases are distributed in two distinct clades (Figure a).…”

Definition und Design hypoallergener Nahrungsmittel

“…Mehrere Tomatenallergene konnten zwischenzeitlich mittels RNAi-Silencing supprimiert werden [51,54,55]. Die reduzierte oder nicht vorhandene IgE-Reaktivität der allergenreduzierten Tomaten konnte auch in den nachfolgenden Tochtergenerationen bestätigt werden, was darauf hinweist, dass das RNAi-Silencing über mehrere Generationen stabil bleibt.…”

“…Bezüglich der Reduktion der Allergenität von Sola l 2 (Invertase) wurde ein anderer Ansatz gewählt [55]: Es konnte gezeigt werden, dass das IgEbindende Epitop im Tomatenallergen Sola l 2 eine β-1,2-gebundene Xylose enthält. In P anzen wird der Transfer der Xylose von Uridindiphosphat(UDP)-Xylose an die Kern-Mannose ("core mannose") von N-Glykanen durch das Enzym β-1,2-Xylosyltransferase vermittelt.…”

“…Während ein RNAi-Silencing von β-1,2-Xylosyltransferase und daraus resultierendem hypoallergenem Sola l 2 sowie von Sola l 3 in der Tomatenp anze und -frucht ohne morphologische Veränderungen möglich war [54,55], war bei Sola l 1-inhibierten P anzen ein deutlich vermindertes P anzenwachstum und ein reduzierter Fruchtansatz festzustellen [51] (Abb. 2), was auf die physiologische Bedeutung von Pro lin und seiner Funktion für eine Regulation des Zytoskeletts der P anze hinweist.…”