Allergenic mites excrete cathepsins B and C as active cysteine proteases distinct from group 1 allergens

Allergenic mites are a major source of indoor allergens and are linked to the increasing prevalence of allergic diseases, posing a growing global health concern. Protease allergens released by mites play a key role in the development of hypersensitivity by disrupting the epithelial barrier and inducing immune responses through their proteolytic activity. In this study, we show that cysteine proteases significantly contribute to the overall proteolytic capacity of allergenic mites, particularly in house dust mites, and are involved in the degradation of human extracellular matrix proteins. Using a functional proteomics approach, combining specific substrates, inhibitors, and activity-based imaging probes, we profiled the component proteases in the species Dermatophagoides farinae and Acarus siro. This analysis identified two novel types of abundant, active cysteine proteases related to cathepsins: cathepsin B from D. farinae (DfCB) and cathepsin C from A. siro (AsCC), which are excreted into the environment via mite feces. Both cathepsins are clearly distinct from canonical cysteine proteases belonging to mite group 1 allergens, as demonstrated by comprehensive biochemical and functional characterizations, together with sequence and phylogenetic analyses. We also validated selective substrates for the detection of DfCB and AsCC in complex proteomes. Our findings suggest that DfCB and AsCC function as digestive enzymes in mites, and that their potent proteolytic activity, retained after excretion, may contribute to interactions with human tissues involved in allergic sensitization.