Formation of active monomers from tetrameric human beta-tryptase

Abstract

Tryptase is a serine protease that is stored at low pH in the mast cell secretory granules in complex with heparin proteoglycan. When mast cells are activated, e.g. during allergic responses, the tryptase/heparin complexes are released together with a variety of other preformed inflammatory mediators. Previous crystallization of human beta-tryptase revealed a unique tetrameric structure with all of the active sites facing a central pore that has a limited accessibility both for potential substrates as well as for protease inhibitors. In this study we examined whether human beta-tryptase, in addition, could form active monomers. Incubation of recombinant tetrameric human beta-tryptase at neutral pH and 37 degrees C, followed by gel-filtration analysis using a running buffer containing pig mucosal heparin, led to the formation of enzymically active compounds that were of a size compatible with tryptase monomers in complex with heparin. The monomers were, in contrast to tryptase in the tetrameric form, inhibited by bovine pancreatic trypsin inhibitor. Further, the monomers, but not the tetramers, degraded fibronectin. Formation of active monomers was more pronounced at pH 7.5 than at pH 6.0 and was not detected at room temperature or at high heparin/tryptase ratios. The present findings thus introduce the possibility that human beta-tryptase, after mast cell degranulation and exposure to neutral pH in the tissue, may dissociate into active monomers with properties that are distinct from the tetrameric counterpart. Possibly, some of the biological activities of human tryptase may be attributable to active tryptase in its monomeric rather than tetrameric form.

Active monomers of human β-tryptase have expanded substrate specificities

“…However, whether short heparin glycosaminoglycans are available to β-tryptase in vivo is uncertain. Formation of active monomers using recombinant human β-tryptase was also noted [74]. β-rTryptase (2 μg in 20 μl of 10 mM Mes, pH 6.1 containing 2 M NaCl) was diluted 10-fold in PBS, pH 7.4, and incubated at 37°C for 30 min.…”

Human Mast Cell β-Tryptase Is a Gelatinase

“…We have recently shown that human ␤-tryptase, when exposed to neutral pH and 37°C, can dissociate into heparin-dependent active monomers (23). We next wanted to test whether gelatin and type I collagen were substrates for both tryptase tetramers and monomers.…”

“…We next wanted to test whether gelatin and type I collagen were substrates for both tryptase tetramers and monomers. Tetrameric and (active) monomeric tryptase were isolated by size-exclusion gel chromatography using a running buffer containing heparin, as previously described (23). Gelatin or type I collagen was incubated (at room temperature and 37°C, respectively) with tetrameric or monomeric tryptase for different time periods, followed by SDS-PAGE analysis.…”

“…The relatively few known macromolecular substrates for human ␤-tryptase include fibrinogen (20), single-chain urinary-type PA (pro-urokinase) (21), and fibronectin (22). However, recent data indicate that human ␤-tryptase can undergo dissociation into proteolytically active monomers, and it was shown that only the monomeric form of ␤-tryptase degrades fibronectin (23). Previous reports have also suggested that ␤-tryptase can activate proMMP-3 (24).…”

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

“…The ␤-tryptase tetramer is proteolytically active in both acidic and neutral pH buffers, though greater fibrinogenolytic activity occurs at acidic than neutral pH. In contrast, a heparin-stabilized monomeric form of mature ␤-tryptase has been detected at low concentrations of ␤-tryptase, which is inactive at neutral pH but active at acidic (pH 6 -6.5) conditions (22,40). High concentrations of ␤-tryptase monomers can be formed with the anti-␣␤-tryptase mAb, B12, as well as the B12 Fab fragment, which dissociates heparin-stabilized ␤-tryptase tetramers to monomers that, like free monomers, are inactive at neutral pH but active at acidic pH (23).…”