*3.2. Treatment of HEK293 and HeLa Cells with Cysteine Proteases*

Many allergens have biological activities, including enzymatic ones. Exposure to proteases via the respiratory tract can induce the release of UA into the airway lumen, and promote type 2 immune response [53]. In this study, two allergens with cysteine protease activity, papain and actinidin, were employed as stressor agents for the induction of UA release as an endogenous danger signal molecule. Papain is a potent proteolytic enzyme [54] (EC 3.4.22.2) and allergen from papaya. Actinidin is a major allergen from kiwifruit (Actinidia deliciosa), which was purified under native conditions. The purified enzyme preserved cysteine protease activity [54] and was employed for cell treatment. Detection of UA was performed in the respective cell culture medium after the treatment of HEK293 cells as well as HeLa cells with papain and actinidin after 0, 6, 12, and 24 h, respectively. Papain was a potent inducer of UA release from both cell lines in a timedependent manner, while actinidin did not induce detectable amounts of the UA molecule. Although the molecular mechanisms for UA release upon allergen treatment are not clarified in detail, papain exhibits five times higher proteolytic (caseinolytic) activity than actinidin [54].
