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 with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose 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 FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.

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Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of ␣-chymases from different species indicated that certain rodent ␣-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the ␣-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His 6 purification tag and an enterokinasesusceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serineprotease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-AlaAla-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1-(Xaa), and P2-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat ␣-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human ␣-chymase.Chymases constitute a family of mast cell (MC) 1 serine proteases with chymotrypsin-like substrate specificity, i.e. they cleave a substrate at the C-terminal side of aromatic amino acids. When comparing the structures of a variety of leukocyte serine proteases, chymases form a separate branch, indicating a common ancestor for this subfamily. Based on the phylogenetic tree, chymases can be subdivided into ␣-and ␤-chymases (1). A single gene encoding an ␣-chymase is present in most investigated mammalian species, but genes encoding ␤-chymases seem to be present in rodents only. The rodent ␣-chymases rat mast cell protease 5 (rMCP-5) (2) (initially designated rMCP-3 (3)) and mouse mast cell protease 5 (mMCP-5) (4) are 95% identical at the protein level and have similar expression patterns, indicating that they are functional homologues (3, 5). mMCP-5 and rMCP-5 are predominantly expressed in connective tissue MCs but also early in MC development (6 -8).The substrate specificities of mMCP-5/rMCP-5 have never been studied, and thus, the biological function of the rodent ␣-chymases is unknown. In fact, neither of these chymases has been purified to allow any biochemical characterization. Instead, most data today on ␣-chymases originate from studies on the human chymase (HC). HC expressi...

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.