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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
The optimized endolysosomal degradation assay with standardized enzyme activity represents an orthogonal method for
Elif Öztemiz1, Christof Regl1, Hans Brandstetter1,2
1Department of Biosciences and Medical Biology, University of Salzburg, Salzburg, Austria.
Introduction:
The endolysosomal degradation assay mimics endolysosomal processing in antigen-presenting cells in vitro and can be used to evaluate protein immunogenicity and T cell epitopes. As previous endolysosomal degradation results demonstrated significant variability, we developed an optimized protocol to ensure the reproducibility and reliability of the assay.
Methods:
Different buffers were tested regarding their influence on the assay pH. Cleavage of ZFR-AMC to evaluate cathepsin activity was performed prior to endolysosomal degradation. Allergens from birch pollen (Bet v 1) and horse chestnut (Aes h 1) were degraded by endolysosomal proteins from the murine dendritic cell line JAWSII using an activity-standardized protocol. Long-term storage of endolysosomal proteins was evaluated, the endolysosomal half-life was calculated with a one-phase association model using densitometric gel analyses. Aes h 1 peptides were alkylated and all peptides analyzed by HPLCMS/MS. A browser-based application for visualizing the endolysosomal degradome was developed.
Results:
The content of the degradation and protein buffers had a significant impact on the assay pH conditions. Measurements using Z-FR-AMC showed varying proteolytic activities of different endolysosomal protein preparations. We therefore defined an endolysosomal constant of 10 μM, which was incorporated into the optimized protocol. After adjusting the endolysosomal protease amount, comparable degradation kinetics and half-lives were observed, providing consistency across different batches. Stable protease activity was observed for 63-day storage of endolysosomal proteins. Bet v 1 exhibited a half-life of one hour and multiple peptide clusters with high peptide signal intensities, some of which harbored validated T cell epitopes, were detected. In comparison, the half-life of Aes h 1 was 16 h, indicating higher proteolytic stability. Notably, alkylation of Aes h 1 endolysosomal peptides enabled superior detection of cysteine-containing fragments. Two prominent peptide clusters were identified, one harboring the region of the homologous Art v 1 T cell epitope. PeptideStudio incorporates visualization of peptide intensities and allows feature modifications to enhance the visualization of endolysomal degradomes.
Discussion:
The optimized endolysosomal degradation process integrates standardized protease activity and enables verified analyses of cysteine-containing peptides using PeptideStudio. This robust, orthogonal tool can support the prediction of protein immunogenicity, thereby contributing to the safety assessment of novel foods or immunogenic proteins.
