Functional immunoproteomic modification of human antigen processing enzymes by bactericidal small molecule and allergen methylisothiazolinone

Objective: Contact allergies to isothiazolinones in humans are a common health problem worldwide, affecting hundreds of thousands of consumers and workers in their daily quality of life. Adaptive immune responses to the widely used preservative methylisothiazolinone (MI) require cellular uptake and the involvement of antigen processing and presentation pathways. Electrophilic MI molecules form disulfide bonds with cysteine thiols of self-proteins (haptenation) which are processed to generate T-cell epitopes. In the case of antigen processing cysteine proteases, such as lysosomal cathepsins, cysteine thiol residues provide structural protein integrity to ensure catalytic activity. Possible MI-induced disruption of these structures may impair homeostatic antigen processing and lead to unexpected immunomodulation.

In this immunoproteomic study, we examined (1) potential exogenous, post-translational MI modifications of cysteine-proteases of the cathepsin family (B, S), which play a crucial role in antigen processing and presentation; and (2) cathepsin enzyme activity and enzyme kinetics, potentially affected by transient or persistent MI-protein modifications.

Methods: Classical Orbi-Trap LC-MS/MS analyses were applied to identify position-specific MI-modified cysteine residues on cathepsin B (& S), which is essential for MHC class II peptide loading. Data of chemical-specific modifications were analyzed in more detail by Peaks software. Complementary functional enzyme assays were performed and kinetic characteristics compared in the presence or absence of MI-protein modifications. Moreover, complementary immune cell effects were studied (FACS analyses).

Results: (1) LC-MS/MS studies revealed several specific binding sites for MI, apart from the catalytic center. (2) MI-protein modification demonstrated significant cathepsin B (& S) enzyme inhibition, which was not neutralized by lysosomal and physiological concentrations of reducing agent glutathione (GSH). (3) Enzyme kinetics with moderate MI concentrations (MI-protein modification) indicated e.g. a non-competitive inhibition of cathepsin B by MI, which supported the LC-MS/MS observation (1).

Conclusion: MI-protein modifications of antigen processing enzymes may cause altered immune cell responses due to novel generated T cell epitopes, independent of initially modified self-proteins, by directly interfering with the homoeostatic antigen processing machinery in human immune cells. In particular, the MI-specific inhibition of cathepsin B (& S) might contribute to novel T cell epitopes by (1) generation of non-homoeostatic immunopeptidomes and (2) by loading incompletely processed peptides into MHC class II molecules for antigen presentation (neo-antigens, immunogenic peptides, low affinity peptides) to variable human T cell receptors.