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The role of N-glycosylation in kiwi allergy.

Garrido-Arandia M, Murua-García A, Palacin A, Tordesillas L, Gómez-Casado C, Blanca-Lopez N, Ramos T, Canto G, Blanco C, Cuesta-Herranz J, Sánchez-Monge R, Pacios LF, Díaz Perales A - Food Sci Nutr (2014)

Bottom Line: The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen.With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2.Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Biotechnology and Genomics U.P.M. - I.N.I.A., Campus de Montegancedo Pozuelo de Alarcón, Madrid, Spain.

ABSTRACT
The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen. In this sense, N-glycosylation is an exclusive characteristic of plant allergens not present in mammals and it could be implied in allergenic sensitization. With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2. The natural allergen, Act d 2, was deglycosylated by trifluoromethanesulfonic acid treatment; the N-glycan fraction was obtained by extended treatment with proteinase K. N-glycan- and protein- fractions were recognized by specific IgE of kiwi-allergic patients. By contrast, the sugar moiety showed a reduced capacity to activate basophils and T cells, but not dendritic cells derived from patients' monocytes. Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety. Thus, the sugar moiety plays a significant role in sensitization, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions.

No MeSH data available.


Related in: MedlinePlus

Comparison of Act d 2 and dAct d 2 (TMSF-treated Act d 2). (A) Coomassie-stained immunodetection with serum pool from kiwi-allergic patients (serum pool), or with rabbit polyclonal antibodies against thaumatin-like proteins (anti-TLP), plant complex glycans (anti-N-gly), xylose or fucose (anti-xyl and anti-fuc). Act d 2 (Ad2) and dAct d 2 (dAd2). (B) Secondary structure composition of Act d 2 and dAct d 2, calculated from their far-ultraviolet circular dichroism data. (C) β-Glucanase activity as a function of carboxymethylated-Pachyman concentration in a Lineweaver–Burke plot. (D) Antifungal activity. Spores from Plectospharella cucumerina were grown under standard conditions. Time corresponds to days after inoculation. Growth was measured as absorbance at 660 nm.
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fig01: Comparison of Act d 2 and dAct d 2 (TMSF-treated Act d 2). (A) Coomassie-stained immunodetection with serum pool from kiwi-allergic patients (serum pool), or with rabbit polyclonal antibodies against thaumatin-like proteins (anti-TLP), plant complex glycans (anti-N-gly), xylose or fucose (anti-xyl and anti-fuc). Act d 2 (Ad2) and dAct d 2 (dAd2). (B) Secondary structure composition of Act d 2 and dAct d 2, calculated from their far-ultraviolet circular dichroism data. (C) β-Glucanase activity as a function of carboxymethylated-Pachyman concentration in a Lineweaver–Burke plot. (D) Antifungal activity. Spores from Plectospharella cucumerina were grown under standard conditions. Time corresponds to days after inoculation. Growth was measured as absorbance at 660 nm.

Mentions: To determine the role of the N-glycan fraction in the allergenic activity of Act d 2, the natural protein purified from kiwi was deglycosylated (dAct d 2) by means of TMSF (Fig. 1). When subjected to SDS-PAGE, we found that both forms—Act d 2 and dAct d 2—migrated as a band with a similar molecular mass (Fig. 1A). The IgE-binding capacities of both forms were compared by Western blot using a pool of sera from kiwi-allergic patients (Fig. 1A, serum pool). In addition, epitopes recognized by anti-TLP antibodies were also retained after treatment (Fig. 1A, anti-TLP). However, no recognition was detected in dAct d 2 by antibodies produced against N-plant complex glycans (anti-N-gly), antibodies against xylose (anti-xyl) or by antibodies against fucose (anti-fuc), suggesting that deglycosylation treatment successfully removed the glycan fractions (Fig. 1A, anti-Ngly, anti-xyl, and anti-fuc).


The role of N-glycosylation in kiwi allergy.

Garrido-Arandia M, Murua-García A, Palacin A, Tordesillas L, Gómez-Casado C, Blanca-Lopez N, Ramos T, Canto G, Blanco C, Cuesta-Herranz J, Sánchez-Monge R, Pacios LF, Díaz Perales A - Food Sci Nutr (2014)

Comparison of Act d 2 and dAct d 2 (TMSF-treated Act d 2). (A) Coomassie-stained immunodetection with serum pool from kiwi-allergic patients (serum pool), or with rabbit polyclonal antibodies against thaumatin-like proteins (anti-TLP), plant complex glycans (anti-N-gly), xylose or fucose (anti-xyl and anti-fuc). Act d 2 (Ad2) and dAct d 2 (dAd2). (B) Secondary structure composition of Act d 2 and dAct d 2, calculated from their far-ultraviolet circular dichroism data. (C) β-Glucanase activity as a function of carboxymethylated-Pachyman concentration in a Lineweaver–Burke plot. (D) Antifungal activity. Spores from Plectospharella cucumerina were grown under standard conditions. Time corresponds to days after inoculation. Growth was measured as absorbance at 660 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048612&req=5

fig01: Comparison of Act d 2 and dAct d 2 (TMSF-treated Act d 2). (A) Coomassie-stained immunodetection with serum pool from kiwi-allergic patients (serum pool), or with rabbit polyclonal antibodies against thaumatin-like proteins (anti-TLP), plant complex glycans (anti-N-gly), xylose or fucose (anti-xyl and anti-fuc). Act d 2 (Ad2) and dAct d 2 (dAd2). (B) Secondary structure composition of Act d 2 and dAct d 2, calculated from their far-ultraviolet circular dichroism data. (C) β-Glucanase activity as a function of carboxymethylated-Pachyman concentration in a Lineweaver–Burke plot. (D) Antifungal activity. Spores from Plectospharella cucumerina were grown under standard conditions. Time corresponds to days after inoculation. Growth was measured as absorbance at 660 nm.
Mentions: To determine the role of the N-glycan fraction in the allergenic activity of Act d 2, the natural protein purified from kiwi was deglycosylated (dAct d 2) by means of TMSF (Fig. 1). When subjected to SDS-PAGE, we found that both forms—Act d 2 and dAct d 2—migrated as a band with a similar molecular mass (Fig. 1A). The IgE-binding capacities of both forms were compared by Western blot using a pool of sera from kiwi-allergic patients (Fig. 1A, serum pool). In addition, epitopes recognized by anti-TLP antibodies were also retained after treatment (Fig. 1A, anti-TLP). However, no recognition was detected in dAct d 2 by antibodies produced against N-plant complex glycans (anti-N-gly), antibodies against xylose (anti-xyl) or by antibodies against fucose (anti-fuc), suggesting that deglycosylation treatment successfully removed the glycan fractions (Fig. 1A, anti-Ngly, anti-xyl, and anti-fuc).

Bottom Line: The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen.With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2.Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Biotechnology and Genomics U.P.M. - I.N.I.A., Campus de Montegancedo Pozuelo de Alarcón, Madrid, Spain.

ABSTRACT
The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen. In this sense, N-glycosylation is an exclusive characteristic of plant allergens not present in mammals and it could be implied in allergenic sensitization. With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2. The natural allergen, Act d 2, was deglycosylated by trifluoromethanesulfonic acid treatment; the N-glycan fraction was obtained by extended treatment with proteinase K. N-glycan- and protein- fractions were recognized by specific IgE of kiwi-allergic patients. By contrast, the sugar moiety showed a reduced capacity to activate basophils and T cells, but not dendritic cells derived from patients' monocytes. Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety. Thus, the sugar moiety plays a significant role in sensitization, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions.

No MeSH data available.


Related in: MedlinePlus