Human Β-tryptase Research Articles

Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human β-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.

Structural Requirements and Mechanism for Heparin-dependent Activation and Tetramerization of Human βI- and βII-Tryptase

Tryptase, a tetrameric serine protease, is a main constituent of the secretory granules in human mast cells, where it is stored in complex with heparin or chondroitin sulfate proteoglycan. Human tryptase has been implicated in a variety of clinical conditions including asthma, but the mechanisms that lead to its tetramerization/activation have not been extensively investigated. Here we addressed the activation mechanisms for human betaI and betaII-tryptase, which differ in that betaI-tryptase is N-glycosylated at Asn102 whereas betaII-tryptase has a Lys residue at position 102, and consequently lacks the corresponding N-glycosylation. We found that both tryptases were dependent on heparin for activation/tetramerization, but whereas betaI-tryptase activation preferentially occurred at acidic pH, betaII-tryptase activation was less pH-dependent. Both betaI and betaII-tryptase bound strongly to heparin-Sepharose at acidic pH but with lower affinity at neutral pH. Further, while addition of heparin to betaI-tryptase predominantly resulted in formation of active tetrameric enzyme, betaII-tryptase showed a tendency to form inactive aggregates. betaI and betaII-tryptase were similar in that the minimal heparin size to induce activation was an octasaccharide and in that the interaction with heparin and structurally related polysaccharides was dependent on high anionic charge density rather than on specific structural motifs. Addition of decasaccharides to both betaI and betaII-tryptase resulted in the formation of active monomeric enzyme, whereas intact heparin promoted assembly of tetrameric enzyme. This, together with a bell-shaped dose response curve for heparin-induced activation, suggests that the mechanism for tetramerization involves bridging of individual tryptase monomers by heparin. Taken together, this study indicates a key role for heparin in the activation of human beta-tryptase.

Inhibition of human β-tryptase by Bowman–Birk inhibitor derived peptides: creation of a new tri-functional inhibitor

Bowman–Birk inhibitor proteins (BBIs), which are potent inhibitors of chymotrypsin-like proteases, do not inhibit human β-tryptase despite this protein having a chymotrypsin-like fold. We have reported previously that, in contrast, BBI-derived peptides (whose sequences incorporate the solvent exposed reactive site loop motif) are able to inhibit human β-tryptase. This is due to their small size, which allows them to access the restricted active site(s) of tryptase, which has an unusual tetrameric arrangement with four active sites flanking a central pore. In this paper, we have examined the possibility of creating additional interactions within this pore by adding extensions to the BBI–peptide motif. We have taken the core disulfide-bridged sequence SCTKSIPPQCY and examined a series of extensions, at both the C- and N-termini, that bear a second positively charged Lys residue at their end. The aim was to construct inhibitors that could make additional interactions in tryptase by spanning the gap between adjacent active sites in the enzyme, producing a double-headed inhibitor; a positively charged group was used as the dominant specificity of this enzyme is for a positively charged P1 residue. Both N- and C-terminal extensions are found to produce inhibitors of much increased potency, with a strong dependence of potency on chain length. Moreover, it was found that the C- and N-terminal extensions were able to synergise, with their combination on the same peptide producing an even better inhibitor with a potency 104-fold greater than the original sequence. We suggest that the C- and N-terminal extensions are picking up interactions with separate additional sites on the tryptase, making the doubly extended BBI peptide a tri-functional tryptase inhibitor.

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors.

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.

Formation of active monomers from tetrameric human beta-tryptase.

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.

Tryptase's potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions.

We reported previously that mast cell tryptase is a growth factor for dog tracheal smooth muscle cells. The goals of our current experiments were to determine if tryptase also is mitogenic in cultured human airway smooth muscle cells, to compare its strength as a growth factor with that of other mitogenic serine proteases, and to determine whether its proteolytic actions are required for mitogenesis. Highly purified preparations of human lung beta-tryptase (1-30 nM) caused dose-dependent increases in DNA synthesis in human airway smooth muscle cells. Maximum tryptase-induced increases in DNA synthesis far exceeded those occurring in response to coagulation cascade proteases, such as thrombin, factor Xa, or factor XII, or to other mast cell proteases, such as chymase or mastin. Irreversibly abolishing tryptase's catalytic activity did not alter its effects on increases in DNA synthesis. We conclude that beta-tryptase is a potent mitogenic serine protease in cultured human airway smooth muscle cells. However, its growth stimulatory effects in these cells occur predominantly via nonproteolytic actions.

Structural Requirements and Mechanism for Heparin-induced Activation of a Recombinant Mouse Mast Cell Tryptase, Mouse Mast Cell Protease-6

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 recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results 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 tryptase monomers that may be formed according to the mechanism described here.

Bivalent inhibition of human β-tryptase

Background: Human β-tryptase is a mast cell specific trypsin-like serine protease that is thought to play a key role in the pathogenesis of diverse allergic and inflammatory disorders like asthma and psoriasis. The recently resolved crystal structure revealed that the enzymatically active tetramer consists of four quasi-identical monomers. The spatial display of the four identical active sites represents an ideal basis for the rational design of bivalent inhibitors. Results: Based on modeling experiments homobivalent inhibitors were constructed using (i) 6 A,6 D-dideoxy-6 A,6 D-diamino-β-cyclodextrin as a rigid template to bridge the space between the two pairs of identical active sites and (ii) 3-(aminomethyl)benzene as a headgroup to occupy the arginine/lysine specific S1 subsites. A comparative analysis of the inhibitory potencies of synthetic constructs that differ in size and type of the spacer between headgroup and template revealed that the construct contained two 3-(aminomethyl)benzenesulfonyl-glycine groups linked to the 6 A,6 D-diamino groups of β-cyclodextrin as an almost ideal bivalent inhibitor with a cooperativity factor of 1.9 vs. the ideal value of 2. The bivalent binding mode is supported by the inhibitor/tetramer ratio of 2:1 required for inactivation of tryptase and by X-ray analysis of the inhibitor/tryptase complex. Conclusion: The results obtained with the rigid cyclodextrin template underlined the importance of a minimal loss of conformational entropy in bivalent binding, but also showed the limitations imposed by such rigid core molecules in terms of optimal occupancy of binding sites and thus of enthalpic strains in bidentate binding modes. The main advantage of bivalent inhibitors is their high selectivity for the target enzyme that can be achieved utilizing the principle of multivalency.

Regulation of Human Mast Cell β-Tryptase: Conversion of Inactive Monomer to Active Tetramer at Acid pH

Abstract At neutral pH, human mast cell β-tryptase is stabilized in its enzymatically active, tetrameric form by heparin, and resists inhibition by biologic protease inhibitors. After dissociation of β-tryptase from heparin, active tetramers rapidly convert to inactive monomers in an isotonic, neutral pH environment. Although reversible transition states probably exist during this conversion, once inactive monomers form, addition of heparin fails to reconstitute active tetramer at neutral pH. The current study shows that complete reactivation of inactive monomers can occur at acidic pH in a heparin-independent manner. The respective rate-determining steps for formation of tetramer and active enzyme from inactive monomers exhibit second and first order kinetics based on an analysis of initial reaction rates. The optimal pH for tetramer formation and reactivation is about 6, suggesting His residues play a critical role. The optimal ionic strength equivalent is 160 mM NaCl; and the optimal temperature range is 22°C to 37°C. We propose a sequential three-step reactivation process at acidic pH, dimerization of monomers (rate-determining second order step), rapid formation of inactive tetramers, and slow formation of active tetramers (overall rate-determining first order step). Whether reactivation of human β-tryptase occurs at extracellular or intracellular sites, where the pH is acidic in vivo, should be considered.

Human beta-tryptase is a ring-like tetramer with active sites facing a central pore.

Human tryptase, a mast-cell-specific serine proteinase that may be involved in causing asthma and other allergic and inflammatory disorders, is unique in two respects: it is enzymatically active only as a heparin-stabilized tetramer, and it is resistant to all known endogenous proteinase inhibitors. The 3-A crystal structure of human beta-tryptase in a complex with 4-amidinophenyl pyruvic acid shows four quasi-equivalent monomers arranged in a square flat ring of pseudo 222 symmetry. Each monomer contacts its neighbours at two different interfaces through six loop segments. These loops are located around the active site of beta-tryptase and differ considerably in length and conformation from loops of other trypsin-like proteinases. The four active centres of the tetramer are directed towards an oval central pore, restricting access for macromolecular substrates and enzyme inhibitors. Heparin chains might stabilize the complex by binding to an elongated patch of positively charged residues spanning two adjacent monomers. The nature of this unique tetrameric architecture explains many of tryptase's biochemical properties and provides a basis for the rational design of monofunctional and bifunctional tryptase inhibitors.

346 Expression and isolation of non-glycosylated recombinant human (rh)β-tryptase

346 Expression and isolation of non-glycosylated recombinant human (rh)β-tryptase