Papain reproduces the prothrombin-dependent PAR1/PAR4 signalling mechanism of the house dust mite allergen Der p 1

Despite triggering IgE-dependent hypersensitivity and being related to the clinically important group 1 allergens of house dust mites (HDMs), the archetypal CA clan cysteine protease, papain, is not an officially denominated allergen. Though lacking formal classification, its ancestral relationship and commercial availability have nevertheless made papain an inviting investigational tool with which to study the role of protease activity in allergy development. However, papain differs sufficiently in substrate specificity from the HDM group 1 allergens for the legitimacy of this premise to be examined. Here, we studied papain as an initiator of a sentinel innate response in a relevant model system whose reactions to the HDM allergome are known. Cultured keratinocytes were loaded with dihydrorhodamine to measure intracellular reactive oxidant species (ROS) production due to papain. Well-characterised pharmacological tools were then used to explore the network activated by this process. Papain evoked an increase in ROS production which required the activation of thrombin and the stimulation of protease-activated receptors 1 and 4 - a finding that was surprising because papain-induced fibrin gelation is not generally attributed to thrombin. Other signalling network components invoked by papain involved epidermal growth factor receptor stimulation, purinoceptor activation, and ligation of both Toll-like receptor (TLR) 1/2 heterodimers and TLR4. The close qualitative parallels between the responses evoked by papain and HDM group 1 allergens indicate that papain engages a shared biosensory and effector network. These findings predict that papain itself may promote allergic sensitisation in humans via the same innate mechanisms.