TmT1 serine protease from cowshed dust extracts generates biologically active C5a in murine bronchoalveolar lavages

Introduction: Intranasal application of cowshed dust extract (CDE) during sensitisation in a murine model of OVA-allergy leads to significant alleviation of the clinical parameters of allergic asthma. However, neither the underlying immunological mechanisms nor the protective substances have been described yet. Methods: CDE was fractionated by size exclusion chromatography and anion exchange chromatography. Fractions with proteolytic activity were characterized by zymography and proteolytic bands were identified by mass spectrometric analysis. Murine BALFs were incubated with either CDE or with purified protease for 30min. at 37°C and C5a concentration was measured by ELISA. Identity of C5a was verified by Western-Blot analysis and biological activity of the released C5a was determined with chemotaxis-under agarose assay. Results: The proteolytic activity in CDE was caused by TmT1 from the midgut of tenebrio molitor. Incubation of murine bronchoalveolar lavage fluids (BALFs) with CDE led to a dose dependent release of C5a. These results were confirmed by incubation of BALFs with purified TmT1 from tenebrio molitor indicating that C5 is cleaved directly by C5 convertase activity of TmT1. Inhibiting proteolytic activity with aprotinin partially inhibited generation of C5a in BALFs. Identity of C5a was confirmed by Western-Blot and biological activity was verified by chemotaxis assay. Discussion: Tenebrio molitor is known as stored product pest. Digestive proteinases from this organism are therefore not unusual in dust samples from cowsheds. TmT1 did not further degrade C5a in murine BALFs, thereby acting like a C5-convertase from complement system. Since C5a has recently been described to play a regulatory role during the sensitisation phase, we suggest that the release of C5a caused by TmT1 in CDE might be one possible mechanism underlying the protective effect of CDE.