Epsilon Open Archive

Abstract

Mast cells play an important role in our immune defense against bacteria and parasites but are also key effector cells in various inflammatory diseases. They act by releasing inflammatory mediators from intracellular granules. Tryptase, one of the most abundant mast cell proteases, is stored in its active form and may therefore act immediately after mast cell degranulation. In this thesis, the activation mechanism of mast cell tryptase has been addressed. Further, the interaction between heparin and tryptase has been thoroughly investigated. We found that the mouse tryptase, mMCP-6, is critically dependent on heparin and acidic pH for its activation. The critical role of heparin for tryptase activation indicated that displacement of heparin might inactivate tryptase. Indeed, we proved that heparin antagonists, protamine and Polybrene, were potent inhibitors of mMCP-6 and purified human lung tryptase. A closer study of the structural requirements of heparin revealed that its capacity to activate tryptase is dependent on size and high anionic charge density. Further, these studies led to a novel finding in the demonstration of an active tryptase monomer. The dependence of mMCP-6 activation on acidic pH suggested that histidines were involved in heparin binding. Site-directed mutagenesis of four selected histidines (H35, H106, H108 and H238) demonstrated that H106, positioned closest to the interface, contributed most to heparin binding, indicating that this region may be particularly important. Generally, the single mutants displayed subtle defects compared to when several mutations were combined, which produced large defects in activation, tetramerization and heparin binding. The heparin-induced activation of human β-tryptase was dependent on the size and high anionic charge density of the activator and closely resembled the structural requirements of mMCP-6 for its interaction with heparin. Altogether, we showed that the mechanism for activation of human β-tryptase was very similar to that of mMCP-6. This indicates that the mouse system is a highly relevant model for the analysis of the biological role of tryptase in human mast cell-related diseases.