Al-Ghouleh, Abeer
(2011)
The role of glycosylation in allergen recognition.
PhD thesis, University of Nottingham.
Abstract
This project is an attempt to have a better understanding of the role of carbohydrates in recognition and uptake of allergens by the innate immune system. Glycosylation analysis of different allergens like Der p 1, Fel d 1, Ara h 1, Ber e 1, Der p 2, Bla g 2, Can f 1, Bromelain and Papain was made using labelled lectins that can detect specific carbohydrate moieties on proteins to reveal their pattern of glycosylation. These experiments showed that all major allergens are glycosylated and this represented a major first step towards demonstrating a link between glycosylation and allergen recognition by the innate immune system. N- and O-glycosylation patterns were predicted in different allergens and a difference in mannosylation and fucosylation was detected between allergens and non-allergenic proteins. We found that the main dominant sugars on allergens are 1,2 1,3 and 1,6 mannose, as detected by GNA lectin. We have also showed that Der p 1 and Der p 2 possess 1,3 fucose in their natural forms, thus concluding that Der p1 and Der p 2 have part of the CCDs which are epitope structures for IgE. O-glycosylation in allergens was also studied giving a better understanding of the whole glycan structure in allergens.
The role of mannosylation in allergen recognition by the immune system was investigated further. Different methods, including recombinant expression, enzymatic and chemical deglycosylation, were optimised to produce glycoforms of Der p 1. A recombinant preparation of Der p 1 produced in Pichia pastoris was used as a hypermannosylated form of the allergen. These glycoforms served as useful tools in addressing the nature of glycoallergen recognition by looking at the uptake of hyper- and hypo-glycosylated preparations by DCs, with confocal microscopy, ELISA and FACS as readouts. Results indicate that deglycosylated forms of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant allergen. Comparative analysis of the hypermannosylated preparation of Der p 1 and its natural counterpart, possessing less mannan, showed that the recombinant form was taken up more readily by DCs at 37°C and at 4°C. We also showed that these glycoforms bind to the MR subfragment CTLD 4-7-FC, the C-type lectin carbohydrate recognition domain. This binding significantly decreased when the Der p 1 allergen was deglycosylated.
These results were confirmed further using confocal microscopy imaging which also showed that recombinant Der p 1 uptake is immediate, starting at 5 mins of incubation and that a higher quantity accumulates inside the DC compared to natural allergen. Recombinant and natural Der p 1 both co-localised with MR, DC-SIGN and LAMP-2 lysosomal marker, suggesting a key role for these receptors in allergen uptake and a common fate for these preparations inside the DC.
Further experiments were done to show the effect of Der p 1 and Der p 1 glycoforms on TSLP secretion by epithelial cells, which is known to induce Th2 driven immune responses. The results show that TSLP secretion decreases significantly when epithelial cells are challenged with deglycosylated preparations of the same allergen. This may indicate a change in the outcome of adaptive immune responses when a deglycosylated allergen challenges epithelial cells.
In conclusion, this work has demonstrated a link between allergenicity and glycosylation patterns in allergens. It therefore appears that mannosylation is the dominant sugar moiety associated with allergen uptake and recognition by humans DCs, and this is in line with MR being the main receptor involved in allergen binding by these cells.
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