Structural requirements and mechanism for heparin-induced activation of a recombinant mouse mast cell tryptase, mouse mast cell protease-6 - Formation of active tryptase monomers in the presence of low molecular weight heparin

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase . Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombin ant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly depe ndent on anionic charge density and size. The dose-response curve for hepar in-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneou sly. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short olig osaccharides were unable to generate tryptase tetramers, but instead gave r ise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthe rmore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our resu lts suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active trypta se monomers that may be formed according to the mechanism described here.