TY - JOUR
T1 - Silencing β1,2-xylosyltransferase in transgenic tomato fruits reveals xylose as constitutive component of IgE-binding epitopes
AU - Paulus, Kathrin Elisabeth
AU - Mahler, Vera
AU - Pabst, Martin
AU - Kogel, Karl Heinz
AU - Altmann, Friedrich
AU - Sonnewald, Uwe
PY - 2011
Y1 - 2011
N2 - Complex plant N-glycans containing (31,2-xylose and core a1,3-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, (31,2-xylosyltransferase (XylT), and core a1,3-fucosyltransferase (FucT) are responsible for the transfer of (31,2-linked xylose and core a1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of (31,2-xylose-containing epitopes, expression of the tomato (31,2-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 (31,2-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-specifc IgG antibodies revealed a strong reduction of (31,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specifc reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of (31,2-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 (31,2-xylose-containing epitopes in vivo.
AB - Complex plant N-glycans containing (31,2-xylose and core a1,3-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, (31,2-xylosyltransferase (XylT), and core a1,3-fucosyltransferase (FucT) are responsible for the transfer of (31,2-linked xylose and core a1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of (31,2-xylose-containing epitopes, expression of the tomato (31,2-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 (31,2-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-specifc IgG antibodies revealed a strong reduction of (31,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specifc reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of (31,2-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 (31,2-xylose-containing epitopes in vivo.
KW - 2-xylosyltransferase
KW - CCDs
KW - N -glycosylation
KW - RNA interference
KW - Tomato allergy
KW - β
UR - http://www.scopus.com/inward/record.url?scp=84891928331&partnerID=8YFLogxK
U2 - 10.3389/fpls.2011.00042
DO - 10.3389/fpls.2011.00042
M3 - Article
AN - SCOPUS:84891928331
SN - 1664-462x
VL - 2
JO - Frontiers in Plant Science
JF - Frontiers in Plant Science
IS - AUG
M1 - 42
ER -