Mechanism for activation of mouse mast cell tryptase: Dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6

Tryptase, a serine protease with trypsin-like substrate cleavage properties , is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex w ith heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for try ptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (m MCP-6), was produced in a mammalian expression system. The mMCP-6 fusion pr otein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6 x His-EK-mMCP-6). In the ab sence of heparin, barely detectable enzyme activity was obtained after ente rokinase cleavage of 6 x His-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6 x His-EK-mMCP-6 yielded active enzyme when ent erokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affin ity chromatography analysis showed that mMCP-6 bound strongly to heparin-Se pharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by F PLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, en zymatically active higher molecular weight complexes were formed, e.g., a d ominant similar to 200 kDa complex that may correspond to tryptase tetramer s. No formation of active tetramers was observed at neutral pH. When inject ed intraperitoneally, mMCP-6 together with heparin caused neutrophil influx , but no signs of inflammation were seen in the absence of heparin. The pre sent pager thus indicates a crucial role for heparin in the formation of ac tive mast cell tryptase.