Enzyme histochemistry of tryptase in stomach mucosal mast cells of the mouse.

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.     

Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

Expression and characterization of recombinant mast cell tryptase

Tryptase, a serine protease, is the major protein component in mast cells. In an animal model of asthma, tryptase has been established as an important mediator of inflammation and late airway responses induced by antigen challenge. Human tryptase is notable for its tetrameric structure, requirement of heparin for stability, and resistance to endogenous inhibitors. Human protryptase was expressed as a recombinant protein in Pichia pastoris. The recombinant protein consisted of two forms of protryptase, one containing the entire propeptide and the other containing only the Val-Gly dipeptide at its amino terminus. Isolation of active recombinant tryptase required a two column purification protocol and included a heparin- and dipeptidyl peptidase I-dependent activation step. Purified recombinant tryptase migrated as a tetramer on a gel filtration column and displayed kinetic parameters identical to those of a native tryptase obtained from HMC-1 cells, a human mast cell line. Recombinant and HMC-1 tryptase exhibited comparable sensitivities to an array of protein and low-molecular-weight inhibitors, including one that is highly specific for tryptase (APC-1167). Similarly, the recombinant enzyme cleaved both alpha- and beta-chains of fibrinogen to generate fibrinogen fragments indistinguishable from those generated by HMC-1-derived tryptase. Thus, recombinant tryptase expressed in P. pastoris displays physical and enzymatic properties essentially identical to the native enzyme. This system provides a cost-effective and easy to manipulate expression system that will enable the functional characterization of this unique enzyme.     

Heparin antagonists are potent inhibitors of mast cell tryptase

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.     

Biology of mast cell tryptase. An inflammatory mediator

In 1960, a trypsin-like activity was found in mast cells [Glenner GG & Cohen LA (1960) Nature 185, 846-847] and this activity is now commonly referred to as 'tryptase'. Over the years, much knowledge about mast cell tryptase has been gathered, and a recent (18 January 2006) PubMed search for the keywords 'tryptase + mast cell*' retrieved 1661 articles. However, still very little is known about its true biological function. For example, the true physiological substrate(s) for mast cell tryptase has not been identified, and the potential role of tryptase in mast cell-related disease is not understood. Mast cell tryptase has several unique features, with perhaps the most remarkable being its organization into a tetrameric state with all of the active sites oriented towards a narrow central pore and its consequent complete resistance towards endogenous macromolecular protease inhibitors. Much effort has been invested to elucidate these properties of tryptase. In this review we summarize the current knowledge of mast cell tryptase, including novel insights into its possible biological functions and mechanisms of regulation.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Restricted ability of human mast cell tryptase to activate proteinase-activated receptor-2 in rat aorta

We investigated the potential of human mast cell tryptase to induce relaxation of rat aorta. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated robust relaxation of phenylephrine-precontracted rat aortic rings. However, human lung tryptase (1-100 nM) either in the presence or absence of heparin failed to induce any significant relaxation. Notwithstanding, incubation of the aorta with tryptase (100 nM), following the addition of a peptide corresponding to the cleavage/activation sequence of rat PAR2 (rPAR2), resulted in relaxation of precontracted tissue due to the proteolytic release of the PAR2-AP SLIGRL/ from the parent peptide. Thus, tryptase was enzymatically active in the bioassay system. Preincubation of aorta with neuraminidase to remove cell-surface sialic acid unmasked the ability of tryptase to induce relaxation of the aorta, but had no effect on relaxation induced by trypsin, SLIGRL-NH2, or acetylcholine (Ach). Like trypsin and SLIGRL-NH2, the tryptase-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Interestingly, tryptase in the presence of heparin failed to induce relaxation of precontracted neuraminidase-treated rat aorta. We conclude that tryptase-induced relaxation of rat aorta, most likely via PAR2, is tightly regulated by heparin and cell-surface sialic acid.     

Histidines are critical for heparin-dependent activation of mast cell tryptase

Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.     

Evaluation of human peripheral blood leukocytes for mast cell tryptase

Murine monoclonal and goat polyclonal antibodies against tryptase, the dominant neutral protease and protein component in secretory granules of human mast cells, were used to assess the presence of tryptase in peripheral leukocytes. Carnoy's fluid-fixed cytocentrifuge preparations of enriched populations of lymphocytes, monocytes, eosinophils, and neutrophils showed no reactivity with anti-tryptase antibodies by a sensitive indirect immunoperoxidase procedure. Dispersed human lung mast cells showed strong granular cytoplasmic staining with both antibodies, whereas only approximately 50% of the peripheral blood basophils detectable with Wright's stain were detected with anti-tryptase antibodies, and these showed a staining pattern that was faint, granular, and cytoplasmic at high concentrations of antibody. At lower antibody concentrations mast cell staining was still intense, whereas basophils were not stained. Extracts of neutrophils and lymphocytes of up to 90% purity had undetectable amounts of tryptase by an ELISA sandwich immunoassay, as well as undetectable enzymatic activity with tosyl-L-gly-pro-lys-p-nitroanilide (a sensitive substrate for tryptase) in the presence of soybean trypsin inhibitor. Extracts of basophil-enriched (6 to 50% purity) preparations contained 0.046 +/- 0.013 pg of tryptase per basophil by the immunoassay along with 2 X 10(-9) +/- 0.8 X 10(-9) U of tryptase-like enzyme activity per basophil, compared with corresponding values of 12 pg, 480 X 10(-9) U of tryptase per human lung mast cell. Thus very small amounts of tryptase are present in human basophils (approximately 0.4% of that found in mast cells), but not in other peripheral leukocytes.     

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.     

Lectin histochemistry of the mast cell: Heterogeneity of rodent and human mast cell populations

Summary Mast cell granules contain a variety of N-linked saccharides. Heterogeneity of the expression of these saccharides in mast cells was studied in rodent and human tissues which were so selected as to contain all the mast cell subsets previously identified using other criteria. Dermal and intestinal mucosal mast cells were stained with lectins using an avidin-biotin system. It was found that dermal and subepidermal mast cells in the rat and mouse, and mucosal and dermal human mast cells showed very similar lectin binding properties to each other, with a fine variation in the inlensity of staining with certain lectins. Rat mucosal mast cells, however, showed a distinctive pattern of lectin binding which was not seen in mast cells from any other tissue studied. The chemical basis of the differences seen were deduced and the possible significance of these structural variations is discussed.     

Interactions of human mast cell tryptase with biological protease inhibitors.

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.     

Activation of latent rheumatoid synovial collagenase by human mast cell tryptase

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells

Two murine monoclonal antibodies were prepared against tryptase, the major neutral protease and protein component of human mast cells. The antibodies were termed G5 (IgG2B-kappa) and H4 (IgG1-kappa). They were specific for tryptase by an enzyme-linked immunosorbent assay and an immunotransblot technique. The latter procedure showed that H4 and G5 each bind to the 35,000 and 37,000 m.w. subunits of tryptase, indicating immunologic cross-reactivity between the subunits. The monoclonal antibodies reacted only with tryptase subunits in an extract of dispersed lung cells. By immunofluorescence microscopy, tryptase was further identified to be present only in cytoplasmic granules of Alcian Blue-stained mast cells in dispersed pulmonary cell preparations. No evidence for a mast cell subtype lacking tryptase was detected. In addition, a procedure for the purification of tryptase to homogeneity from dispersed pulmonary cells containing less than 10% mast cells was developed; this procedure involved high salt extraction, ammonium sulfate precipitation, and sequential chromatography with decyl-agarose, DEAE-agarose, and heparin-agarose. The procedure resulted in a higher yield even with less pure starting material than reported previously. Tryptase is a selective marker for mast cells in dispersed pulmonary cells, and can be detected with specific anti-tryptase antibodies.     

Inactivation of human high molecular weight kininogen by human mast cell tryptase

Tryptase, the major neutral protease of human pulmonary mast cell secretory granules, rapidly inactivates human high m.w. kininogen (HMWK) in vitro. HMWK (5600 nM) lost 50% of its capacity to release kinin in response to kallikrein after a 5-min incubation with tryptase (31 nM), even though kinin activity was neither generated nor, when bradykinin was incubated with tryptase, destroyed by tryptase. The procoagulant activity of HMWK (51 nM) and the purified procoagulant chain (40 nM) that is derived from HMWK were each 72% inactivated after 7 min of incubation with tryptase (0.04 nM and 0.02 nM, respectively). Human urinary and pancreatic kallikrein did not inactivate this procoagulant activity under conditions in which kinin generation occurs. Complete cleavage of native single-chain HMWK by tryptase occurred in less than 10 min as analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. The major products formed during the initial 2 min were proteins of 100,000 and 95,000 apparent m.w., and by 10 to 30 min were fragments of 74,000 and 67,000 apparent m.w. Reduction of these cleavage products yielded two major fragments of 67,000 and 66,000 apparent m.w. that were both present by 0.17 min. The presence of lower m.w. products, thought to be primarily from the carboxy-terminal procoagulant region of HMWK, were also detected with and without reduction. The capacity of tryptase to inactivate HMWK is consistent with the ability of other mast cell-derived mediators, such as heparin proteoglycan and prostaglandin D2, to suppress blood coagulation and thrombosis, and may play an important role in the biology of mast cell-dependent events in vivo.     

The heterogeneity of mast cell tryptase from human lung and skin.

There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9-12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis-Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues.     

Human lung mast cell tryptase fails to activate procollagenase or degrade proteoglycan

Pig synovial and human skin fibroblast procollagenases were treated with highly purified tryptase, the major proteinase of human mast cells, to determine whether this trypsin-like proteinase could activate the latent form of collagenase and so be involved in connective tissue breakdown. No significant activation of either human or pig procollagenase was found, but the highest concentration of tryptase partially destroyed procollagenase. Tryptase did not degrade type I collagen or proteoglycan. These data indicate that human mast cell tryptase does not contribute to connective tissue breakdown via procollagenase activation or via proteoglycan degradation.