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The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

Al-Ghouleh A, Johal R, Sharquie IK, Emara M, Harrington H, Shakib F, Ghaemmaghami AM - PLoS ONE (2012)

Bottom Line: Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent.The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations.Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Medical Sciences, Division of Immunology, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.

ABSTRACT
Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

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A. The MFI ± SEM readings for the uptake of different concentrations of nDer p 1 and rDer p 1 by immature DCs compared to the periodate treated preparations. Both nDer p 1 and rDer p 1 were treated with periodate for 30 mins and 1 hr, then their uptake was measured. The results show a significant decrease in uptake of periodate treated preparations compared with the untreated ones.*** P value<0.001, all Der p 1 preparations were labelled with FITC. B. Confocal images showing the uptake of periodate treated Der p 1 by immature DCs.
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pone-0033929-g010: A. The MFI ± SEM readings for the uptake of different concentrations of nDer p 1 and rDer p 1 by immature DCs compared to the periodate treated preparations. Both nDer p 1 and rDer p 1 were treated with periodate for 30 mins and 1 hr, then their uptake was measured. The results show a significant decrease in uptake of periodate treated preparations compared with the untreated ones.*** P value<0.001, all Der p 1 preparations were labelled with FITC. B. Confocal images showing the uptake of periodate treated Der p 1 by immature DCs.

Mentions: Periodate oxidation was used to deglycosylate both natural and recombinant Der p 1 preparations by using sodium metaperiodate. Periodate has been used in the literature to deglycosylate protein preparations [42]–[45] and it is known to remove mannose and fucose from proteins. Natural and recombinant Der p 1 were incubated with periodate in the dark at room temperature for 30 and 60 mins. A western blot against GNA (anti 1–2,3,6 mannose) was performed on the samples before and after periodate treatment (Fig. 7) to confirm that demannosylation had worked. All these glycoforms retained their reactivity with anti-Der p 1 5H8 monoclonal antibody (Fig. 8), thereby ascertaining their structural integrity. A commassie blue stained gel of natural Der p 1 before and after deglycosylation with periodate showed a slight decrease in deglycosylated Der p 1 MW as to be expected (Fig. 9). The preparations were then labelled with FITC and the uptake by DCs was measured against untreated preparations (Fig. 10 A).


The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

Al-Ghouleh A, Johal R, Sharquie IK, Emara M, Harrington H, Shakib F, Ghaemmaghami AM - PLoS ONE (2012)

A. The MFI ± SEM readings for the uptake of different concentrations of nDer p 1 and rDer p 1 by immature DCs compared to the periodate treated preparations. Both nDer p 1 and rDer p 1 were treated with periodate for 30 mins and 1 hr, then their uptake was measured. The results show a significant decrease in uptake of periodate treated preparations compared with the untreated ones.*** P value<0.001, all Der p 1 preparations were labelled with FITC. B. Confocal images showing the uptake of periodate treated Der p 1 by immature DCs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316510&req=5

pone-0033929-g010: A. The MFI ± SEM readings for the uptake of different concentrations of nDer p 1 and rDer p 1 by immature DCs compared to the periodate treated preparations. Both nDer p 1 and rDer p 1 were treated with periodate for 30 mins and 1 hr, then their uptake was measured. The results show a significant decrease in uptake of periodate treated preparations compared with the untreated ones.*** P value<0.001, all Der p 1 preparations were labelled with FITC. B. Confocal images showing the uptake of periodate treated Der p 1 by immature DCs.
Mentions: Periodate oxidation was used to deglycosylate both natural and recombinant Der p 1 preparations by using sodium metaperiodate. Periodate has been used in the literature to deglycosylate protein preparations [42]–[45] and it is known to remove mannose and fucose from proteins. Natural and recombinant Der p 1 were incubated with periodate in the dark at room temperature for 30 and 60 mins. A western blot against GNA (anti 1–2,3,6 mannose) was performed on the samples before and after periodate treatment (Fig. 7) to confirm that demannosylation had worked. All these glycoforms retained their reactivity with anti-Der p 1 5H8 monoclonal antibody (Fig. 8), thereby ascertaining their structural integrity. A commassie blue stained gel of natural Der p 1 before and after deglycosylation with periodate showed a slight decrease in deglycosylated Der p 1 MW as to be expected (Fig. 9). The preparations were then labelled with FITC and the uptake by DCs was measured against untreated preparations (Fig. 10 A).

Bottom Line: Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent.The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations.Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Medical Sciences, Division of Immunology, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.

ABSTRACT
Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

Show MeSH
Related in: MedlinePlus