Endolysosomal Degradation of Allergenic Ole e 1-Like Proteins: Analysis of Proteolytic Cleavage Sites Revealing T Cell Epitope-Containing Peptides

Abstract

Knowledge of the susceptibility of proteins to endolysosomal proteases provides valuable information on immunogenicity. Though Ole e 1-like proteins are considered relevant allergens, little is known about their immunogenic properties and T cell epitopes. Thus, six representative molecules, i.e., Ole e 1, Fra e 1, Sal k 5, Che a 1, Phl p 11 and Pla l 1, were investigated. Endolysosomal degradation and peptide generation were simulated using microsomal fractions of JAWS II dendritic cells. Kinetics and peptide patterns were evaluated by gel electrophoresis and mass spectrometry. In silico MHC (major histocompatibility complex) class II binding prediction was performed with ProPred. Cleavage sites were assigned to the primary and secondary structure, and in silico docking experiments between the protease cathepsin S and Ole e 1 were performed. Different kinetics during endolysosomal degradation were observed while similar peptide profiles especially at the C-termini were detected. Typically, the identified peptide clusters comprised the previously-reported T cell epitopes of Ole e 1, consistent with an in silico analysis of the T cell epitopes. The results emphasize the importance of the fold on allergen processing, as also reflected by conserved cleavage sites located within the large flexible loop. In silico docking and mass spectrometry results suggest that one of the first Ole e 1 cleavages might occur at positions 107–108. Our results provided kinetic and structural information on endolysosomal processing of Ole e 1-like proteins.

Distinct epitope structures of defensin‐like proteins linked to proline‐rich regions give rise to differences in their allergenic activity

“…As differences in the primary structure might provide distinct cleavage sites for different proteases, we additionally investigated the proteolytic stability to cathepsin S, a major component of the endolysosomal protease cocktail . Similar degradation kinetics was observed as compared with the endolysosomal extract and, in general, cathepsin S cleavage sites were conserved (Fig.…”

Laser‐facilitated epicutaneous immunotherapy with hypoallergenic beta‐glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

“…Carbohydrate concentration in conjugates was estimated using anthrone method 18 and OVA concentration was determined by amino acid analysis 19 . The hydrodynamic radius of conjugates was analyzed by dynamic light scattering using a Zetasizer Nano ZS with a DTS1070 capillary cell (Malvern Instruments).…”

Laser-facilitated epicutaneous immunotherapy with hypoallergenic beta-glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

“…Carbohydrate concentration in conjugates was measured using anthrone method 18 and OVA concentration was determined by amino acid analysis 19 . The hydrodynamic radius of conjugates was analyzed by dynamic light scattering using a Zetasizer Nano ZS with a DTS1070 capillary cell (Malvern Instruments).…”