Standardized Enzyme Assay Promises More Reliable Prediction of Protein Immunogenicity

When the immune system encounters a foreign protein, one of the most consequential steps happens deep inside antigen-presenting cells, where endolysosomal compartments shred the protein into peptides that can be displayed to T cells. Which fragments survive this proteolytic gauntlet, and how quickly the parent protein is destroyed, largely determine whether an immune response is triggered at all. For roughly two decades, researchers have tried to capture this process in a test tube using the endolysosomal degradation assay, in which proteases isolated from dendritic cells are incubated with a protein of interest under acidic, reducing conditions. Now, a team at the University of Salzburg, working with colleagues at University Medical Centre Utrecht, has overhauled the method to eliminate the variability that has long plagued it, producing a standardized protocol that the authors say can serve as an orthogonal tool for predicting protein immunogenicity and T cell epitopes.

The problem the researchers set out to solve was stark. Published half-lives for the same protein, the major birch pollen allergen Bet v 1, ranged from two to sixteen hours across studies that used endolysosomal proteins from the same murine dendritic cell line, JAWSII. Such inconsistency undermines the assay’s core promise: a quantitative readout, the endolysosomal half-life, that links protein stability during antigen processing to immunogenicity. The underlying logic is that proteins with moderate stability are processed optimally for presentation on MHC class II molecules, whereas proteins that are either extremely stable or extremely fragile fail to generate the right peptides at the right time, resulting in lower immunogenicity.

Two culprits emerged as the main sources of variability. The first is pH. Endolysosomal maturation is tied to acidification, with the pH dropping from about 6.8 in early endosomes to 4.8 in late endolysosomal compartments, and protease activity is exquisitely sensitive to this gradient. Previous work showed that Bet v 1’s half-life changed roughly fourteen-fold when the assay pH shifted from 5.2 to 4.5. The Salzburg team found that even the way the citrate buffer stock is prepared matters: a one-molar citrate buffer adjusted to pH 4.8 drifted up to pH 5.1 when diluted to the working concentration of 0.1 molar, whereas a buffer prepared by mixing trisodium citrate and citric acid in a precise three-to-two ratio, without prior pH adjustment, landed exactly at pH 4.8 upon dilution. The polyanionic nature of citrate at high concentrations distorts electrostatic interactions and pH measurements, so the protocol now prescribes an exact preparation route.

The second culprit is the protein buffer in which the test protein is dissolved. Buffer simulation experiments showed that sodium phosphate at pH 6 preserved the required pH 4.8 across all tested concentrations and protein amounts, while buffers at pH 7 or above only maintained the correct pH at low molarities or high protein concentrations. Because a higher pH dampens proteolytic activity, an innocent-looking difference in the sample buffer can silently skew degradation kinetics. The optimized protocol therefore includes recommendations for buffer exchange or dilution, and advises researchers to verify the pH of a simulated degradation mixture whenever conditions fall outside the tested range.

The more fundamental fix, however, targets the enzyme preparation itself. The isolated endolysosomal fraction is a complex protein mixture in which active proteases are just one component, and its composition varies between batches. Using a fixed mass of endolysosomal protein, as earlier protocols did, therefore delivered wildly different catalytic doses. The team quantified cathepsin activity using the fluorogenic substrate Z-FR-AMC, which releases a fluorescent AMC group upon cleavage, and normalized measurements against an AMC standard curve. They then defined an endolysosomal constant of 10 micromolar cathepsin activity for degrading 5 micrograms of test protein, and derived a simple formula for calculating how much endolysosomal protein to add based on each batch’s measured activity.

The results were striking. When endolysosomal activity was normalized to the 10 micromolar constant, seven independent Bet v 1 measurements yielded a consistent half-life of one hour, plus or minus 0.1 hours. In contrast, following the old protocol with a fixed 7.5 micrograms of endolysosomal protein from a batch with 2.7-fold higher cathepsin activity compressed the half-life to 0.6 hours. The same pattern held for the second model allergen, Aes h 1, a recently identified 5.8-kilodalton plant defensin from horse chestnut stabilized by four disulfide bonds: normalization produced a reproducible half-life of 16 hours, whereas the unadjusted protocol skewed it to 12 hours. In practical terms, a 2.1-fold difference in cathepsin activity had been producing a 1.7-fold difference in measured half-life, an error the new constant eliminates.

The team also addressed practical workflow concerns. Endolysosomal proteins had always been isolated fresh, one day before each experiment, a time-consuming requirement. Storage tests at minus 70 degrees Celsius showed that cathepsin activity remained stable for up to 63 days, with degradation kinetics, half-lives, and peptide profiles indistinguishable from freshly prepared material. Half-life calculations were likewise standardized by fitting densitometric gel data to a one-phase association model constrained to start at zero and plateau at complete degradation, replacing the earlier practice of eyeballing a linear segment crossing the fifty percent mark, which could not properly model the underlying kinetics.

On the peptide side, the researchers introduced two innovations. First, for proteins stabilized by disulfide bonds, they added a reduction and alkylation step after degradation, using TCEP and iodoacetamide to prevent artificial disulfide scrambling that renders cysteine-containing peptides invisible to mass spectrometry. For Aes h 1, this was decisive: without treatment, only a single peptide lacking cysteine was identified, whereas reduction and alkylation revealed 28 peptides, many of them cysteine-containing. Second, they built PeptideStudio, an open-source, browser-based visualization tool that maps peptides onto the protein sequence with color-coded intensity values, displays each time point on separate tracks, and allows users to annotate validated T cell epitopes, filter by length and intensity, and export publication-quality figures. The application runs locally, so no sequence or intensity data leaves the user’s machine.

The biological readouts validate the approach. Bet v 1’s degradome contained ten peptide clusters spanning the entire protein, and experimentally validated T cell epitopes fell within the dominant clusters, including the immunodominant epitope Bet v 1 142-156, whose peptides appeared early and persisted for several hours, consistent with the idea that early and sustained peptide availability enhances MHC class II loading. Aes h 1, analyzed for the first time, yielded two prominent clusters, one encompassing the region homologous to the immunodominant T cell epitope of the mugwort allergen Art v 1, with peptides peaking at 24 hours and long fragments covering up to 92 percent of the tiny protein persisting to 72 hours, mirroring its high proteolytic stability.

The authors acknowledge limitations, including the use of a murine cell line rather than human antigen-presenting cells, although prior work showed similar kinetics between the two, and the semi-quantitative nature of mass spectrometric peptide intensities. Still, the payoff is a robust, reproducible assay that can compare proteins across laboratories and batches, distinguishing high- and low-allergenic variants within protein families and flagging potential T cell epitopes before any clinical exposure. For novel foods, protein-based immunotherapeutics, and synthetic proteins where a priori safety knowledge is scarce, a standardized window into how the endolysosomal system will process them could prove an invaluable early warning system.

Subject of Research: Optimization and standardization of the endolysosomal degradation assay for predicting protein immunogenicity and T cell epitopes

Article Title: The optimized endolysosomal degradation assay with standardized enzyme activity represents an orthogonal method for predicting protein immunogenicity and T cell epitopes

Article References: Öztemiz, E., Regl, C., Brandstetter, H., Huber, C. G., Verhoeckx, K., & Gadermaier, G. (2026). The optimized endolysosomal degradation assay with standardized enzyme activity represents an orthogonal method for predicting protein immunogenicity and T cell epitopes. Frontiers in Allergy, 7, Article 1936335. https://doi.org/10.3389/falgy.2026.1936335

Image Credits: AI Generated

DOI: 10.3389/falgy.2026.1936335

Keywords: endolysosomal degradation, protein immunogenicity, T cell epitopes, cathepsin activity, allergens, Bet v 1, Aes h 1, mass spectrometry, antigen processing, PeptideStudio, allergy, novel foods