PMC

The immunoblot was prepared with extracts of wild-type (wt) and GNTI-silenced (GTI) tubers and developed with the CCD-specific rabbit antiserum (α-CCD). Sizes of known glycoprotein allergens Sola t 1 and Sola t 2 are indicated. The protein-stained blot confirms equal loading and reveals band shifts around 40–43 kDa in GTI extracts, likely due to different Sola t 1 (patatin) isoforms.

Comparison of basophils stimulated with potato tuber (left panels) or tomato fruit extracts (right panels) of either wild-type (wt) or GTI plants. In all tests, horseradish peroxidase (HRP, dotted line) served as control for CCD-dependent stimulation. The percentage of activated basophils was calculated by subtracting values of spontaneous CD203c expression (negative control, PBS) from the values obtained with the particular allergen challenge. A: Potato/tomato-allergic patient without CCD-sIgE; B: Potato/tomato-allergic patients with CCD-sIgE; C: Hymenoptera venom-allergic patients with CCD-sIgE but no allergic symptoms to potato or tomato (for patient details, see ).

A: Immunoblots prepared with whole fruit extracts (pulp with peel) of wild-type (wt), Le2, and GTI plants were developed with CCD-positive potato/tomato-allergic patient sera (PT-02(+) and PT-06(+)). Arrows point to a faint band around 52 kDa missing in Le2 (for Lyc e 2 size, compare and ). Protein staining is shown for equal loading. B: Basophil activation test of PT-02(+) with indicated tomato fruit extracts. Horseradish peroxidase (HRP), a vacuolar glycoprotein with plant-specific CCD epitopes, was used as control for CCD-dependent stimulation (dotted line).

A: Immunoblots of potato tuber and tomato fruit extracts (pulp with peel) were incubated with selected patient sera (NA: non-allergic control; PT: potato/tomato food-allergic; BW: bee/wasp hymenoptera venom-allergic) and developed for detection of human IgE. CCD-sIgE negative patients are labeled (−) and CCD-sIgE positive patients (+) (compare ). B: After sensitive ECL detection of IgE binding, blots were additionally subjected to colorimetric development for visualizing bound IgG₄. Sera that differentiate between wild-type (wt) and CCD-reduced (GTI) plant extracts at the IgG₄ level are labeled pink.

A: Immunoblots prepared with tomato fruit extracts of wild-type (wt), Lyc e 2-silenced (Le2), or GNTI-silenced (GTI) lines were developed either with α-Le2 or α-CCD polyclonal rabbit antiserum. Protein staining is shown as loading control for the blot developed with α-CCD. Note that the CCD pattern of Le2 is similar to wt, except for a faint band corresponding to Lyc e 2. Consistently with the immunoblots, enzymatic activity of vacuolar β-fructofuranosidase (invertase) was undetectable in Le2-fruit extracts (data not shown). Sizes of glycoprotein allergens are indicated: Lyc e 2 (∼52 kDa), PG (polygalacturonase 2A, 46 kDa), and PME (pectin(methyl)esterase, ∼35 kDa). B: Le2 and GTI tomato plants compared to wt. Note that both transformants are viable and form mature fruits.

Comparison of sIgE values in three different patient groups. A: Potato, commercial (f35) versus in-house-made wild-type (wt). B: Tomato, commercial (f25) versus in-house-made wt. C: Potato, in-house-made wt versus GTI. D: Tomato, in-house-made wt versus GTI. (Black circles: CCD-negative potato/tomato-allergic patients, red squares: CCD-positive potato/tomato-allergic patients, yellow triangles: CCD-positive hymenoptera venom-allergic patients). For sIgE values compare . Note that sIgE values of CCD-negative food-allergic patients (black circles) match more or less the bisecting line and those of CCD-positive patients (red squares and yellow triangles) shift downwards with GTI extracts (especially obvious for tomato). For better illustration, zero values were set to 0.01. Horizontal and vertical lines indicate the 0.35 kU/l threshold for sIgE positivity, bisecting lines congruency. Correlation coefficient r was calculated by the Spearman's rank correlation test, r = +1/−1 would be ideal (*: p<0.05; **: p<0.01).

A: Flow chart of conducted experimental analyses. The impact of single glycoallergen Lyc e 2 in tomato allergy was studied by establishing Lyc e 2-reduced tomato fruits (plants further referred to as Le2). To evaluate contribution of carbohydrate- versus peptide-specific determinants in reactivity to foodstuff, CCD-reduced tomato and potato plants were created by silencing N-acetylglucosaminyltransferase-I (GNTI, plants further also referred to as GTI), catalyzing the crucial step leading to CCD formation in the Golgi apparatus. B: Predominant protein-bound N-glycan structures prevailing in wild-type (wt) and in GTI transformants (no CCD epitopes). N-glycan structures are depicted according to the proglycan system (www.proglycan.com). Note that terminal GlcNac (N-acetylglucosamine) residues are not present on fully trimmed wild-type N-glycans (dotted lines and brackets). C: RNAi-expression cassette used for Lyc e 2- or GNTI-silencing; restriction sites are indicated (destroyed ones in brackets). (BHI: BamHI; B33: tuber/fruit-specific promoter; CCD: cross-reactive carbohydrate determinants; polyA: polyadenylation signal; 35S: constitutive promoter of Cauliflower Mosaic Virus).