Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing

Abstract

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

Keywords: Amb a 1; Bet v 1; Der p 1; Der p 2; allergen proteolysis; degradome assay; proteases from B cells; proteases from dendritic cells; proteases from macrophages.

Figure 1

(a) 15% SDS-PAGE of purified allergens; 1 μg recombinant proteins were loaded per lane; (b) The protease activity of endolysosomal fractions was determined using the fluorescent substrate Z-Phe-Arg-AMC.

Figure 2

Fifteen percent SDS-PAGE analyses of proteolytically processed allergens. The bands representing intact protein were scanned using the Bio-Rad Chemidoc™ MP Imaging System and evaluated with the Bio-Rad Image Lab Software tool. (a) Bet v 1, (b) Amb a 1, (c) proDer p 1, and (d) Der p 2 were digested with endolysosomal proteases from different APCs.

Figure 3

Endolysosomal degradations of purified allergens were analyzed by mass spectrometry (MS) after 6 h. Peptides identified by MS are represented as bars. Protein sequence areas, which contain published T cell epitopes, are highlighted in orange. The following protein sequences were used: Bet v 1.0101 (a), Amb a 1.0301 (b), proDer p 1.0102 (c), and Der p 2.0103 (d).