Remodeling of extracellular matrix is an important component in a variety of inflammatory disorders as well as in normal physiological processes such as wound healing and angiogenesis. Previous investigations have identified the various matrix metalloproteases, e.g., gelatinases A and B, as key players in the degradation of extracellular matrix under such conditions. Here we show that an additional enzyme, human mast cell beta-tryptase, has potent gelatin-degrading properties, indicating a potential contribution of this protease to matrix degradation. Human beta-tryptase was shown to degrade gelatin both in solution and during gelatin zymographic analysis. Further, beta-tryptase was shown to degrade partially denatured collagen type I. beta-Tryptase bound strongly to gelatin, forming high molecular weight complexes that were stable during SDS-PAGE. Mast cells store large amounts of preformed, active tryptase in their secretory granules. Considering the location of mast cells in connective tissues and the recently recognized role of mast cells in disorders in which connective tissue degradation is a key event, e.g., rheumatoid arthritis, it is thus likely that tryptase may contribute to extracellular matrix-degrading processes in vivo.
► "Here we show that an additional enzyme, human mast cell β-tryptase, has potent gelatin-degrading properties, indicating a potential contribution of this protease to matrix degradation. Human β-tryptase was shown to degrade gelatin both in solution and during gelatin zymographic analysis. Further, β-tryptase was shown to degrade partially denatured collagen type I."
► "Tissue-remodeling processes take place in many physiological and pathological conditions, e.g., embryonic development, wound healing, rheumatoid arthritis, and tumor invasion. In these processes degradation of the extracellular matrix (ECM)3 is required to allow the proliferation and/or migration of different cell types."
► "At present, matrix metalloproteinases (MMPs) are being recognized as the main players in the ECM degradation associated with various tissue-remodeling processes. [...]
Together the MMPs have the ability to degrade all the different types of macromolecules present in the extracellular matrix, e.g., collagens, laminin, fibronectin, and proteoglycans."
► "Mast cells synthesize and store large amounts of various serine proteases of two subclasses: tryptases and chymases. In connective tissue-type mast cells, both the chymases and tryptases are packaged in the secretory granules in tight complexes with heparin proteoglycan (8). When mast cells are activated [...] the mast cells degranulate and release large amounts of serine proteases and other inflammatory mediators, e.g., histamine and TNF-α (9)."
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The large amounts of proteases that are being released by mast cells during inflammatory conditions together with the fact that the mast cell serine proteases are stored and released in their active form make these proteases
likely candidates for involvement in connective tissue degradation. Indeed, it has been indicated previously that
mast cell chymase may promote ECM degradation by processing proMMP-9 (pro-gelatinase B) to its active form (10, 11) and
by directly degrading various connective tissue components, e.g., fibronectin (12)."
►"No detectable degradation was observed for type I collagen when incubations were performed at room temperature (not shown). In contrast, when incubations were conducted at 37°C [98.6°F], a marked degradation of type I collagen was observed at pH 6.0... and, to a lesser extent, at pH 7.5 (not shown).
The degradation of type I collagen at 37°C, but not at room temperature, suggests that the elevated temperature causes partial unfolding of the collagen into a form that is susceptible to degradation by tryptase. This is in agreement with earlier reports that demonstrated denaturation of intact type I collagen at temperatures >36°C [96.8°F] (28, 29). Moreover, control experiments showed that the type I collagen preparation was susceptible to degradation (at 37°C) by pancreatic trypsin, an enzyme that is not recognized as a collagenase (not shown). These results indicate that
β-tryptase appears to have the ability to degrade partially denatured, but not native, type I collagen."
► "However, our results indicate that denatured collagen, obtained after unfolding of the triple helix, readily enters the central pore of tetrameric tryptase. It is important to note that the active tryptase monomers, similarly to the tetramers, were not able to degrade intact collagen type I. This indicates that the failure of tetrameric β-tryptase to degrade intact collagen may not only be a result of the restricted accessibility to its active sites, but may also be due to an absolute requirement for collagen unfolding to unmask susceptible peptide bonds."
► "The present findings are thus in agreement with the well-established idea that
only the collagenases, i.e., interstitial collagenase, neutrophil collagenase, collagenase-3, and cathepsin K,
are able to destroy the highly ordered structure of fibrillar collagens, and that subsequent degradation is conducted by other proteases (2, 5) (41)."
► "We may thus propose that
mast cell β-tryptase could act in a similar fashion as the previously described gelatinases (e.g., MMP-2 and -9), i.e.,
once collagen strands are disordered, tryptase would be able to continue the degradation process. Importantly, mast cells reside in the connective tissue and contain large stores of preformed active tryptase that, once released, could act early in the ECM degradation process."
► "Hence,
degradation of ECM by tryptase may be a comparably long-lived event following mast cell degranulation, insensitive to, e.g., the protease inhibitors that escape into the tissue due to the plasma leakage that occurs during an inflammatory process."
► "Heparin, the physiological ligand to tryptase in the secretory granule (9), is a well-known stabilizing agent for tryptase and has also been implicated in the assembly of the tryptase tetramer (45). The interaction of tryptase with heparin is promoted by the low pH that is present in the secretory granules (∼5.5), and it is thought that exocytosed tryptase is regulated by its dissociation from heparin due to a higher extracellular pH, leading to tryptase monomerization. The released monomers will rapidly lose activity, although it is possible that they may possess enzymatic activity for a short time period (23). If, however, the monomers are rescued by binding to heparin, they will regain enzymatic activity (46, 47), predominantly by the generation of active tryptase monomers (23)."
► "According to the results presented here,
tryptase may thus, after dissociation from heparin, interact strongly with collagens (or proteoglycans) in the ECM. A strong binding of tryptase to ECM is also supported by a previous study in which human lung tryptase was found to bind to bovine cartilage (47). This interaction could serve to target tryptase to its potential substrate in the ECM."
