The catalytic properties of a proteinase isolated from sheep abomasal mucosal mast cells

The catalytic properties of a sheep mast cell proteinase (SMCP), isolated from abomasal mucosal mast cells, were investigated. The enzyme was shown to have chymotrypsin-like esterase activity, with no detectable amide activity, using a range of low molecular weight substrates. Maximal activity, against Benzyloxycarbonyl-L-tyrosine-4-nitrophenol ester, was determined to be in the range pH 7.6-8.0. Inhibitor studies showed that, unlike chymotrypsin, a serine proteinase, SMCP was found to be susceptible to the action of thiol blocking agents and chelating agents, but to be unaffected by diisopropylphosphofluoridate, a serine proteinase inhibitor.     

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

Hormone-responsive alkaline proteinase in rat skeletal muscle is not a mast cell-derived enzyme

Proteinase activity was determined in myofibrils from intact rat skeletal muscle and from skeletal muscle myocytes grown in culture. In vivo administration of the mast cell degranulator compound 48/80 abolished the alkaline proteinase activity in myofibrils obtained from normal or streptozotocin-diabetic rats. Exposure of myocytes to compound 48/80 in cell cultures had no effect on their myofibrillar proteinase activity, nor did it affect the rate of overall protein degradation in these cells. Co-incubation of cultured mast cells (line P815Y) with myocytes followed by sonication of the cell mixture resulted in a marked reduction of the proteinase activity in the pellet fraction, suggesting that the mast cells contain inhibitor(s) of myofibrillar proteinase activity. It is suggested that the myofibril-bound alkaline proteinase activity is not a mast cell-derived enzyme but a genuine component of muscle cells. The in vivo 48/80-induced reduction of muscle myofibrillar proteinase activity appears to be due to release of a soluble inhibitory activity rather than removal of mast cell proteinase from the tissue by degranulation.     

Differential inhibition of rat mast cell proteinase I and II by members of the alpha-1-proteinase inhibitor family of serine proteinase inhibitors

Rat mast cell proteinase II (RMCP II) from mucosal mast cells was titrated into rat serum, and the resulting serine proteinase inhibitor (serpin)-enzyme complex was purified by affinity chromatography on anti-RMCP II-Sepharose 4B and by Mono-Q anion-exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCP II-Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) that inhibit RMCP II in rat serum. The kinetic constants characterizing the interaction between RMCP II and RSI and RSII are ka, 2.2 x 10(5) and 1.65 x 10(5) M-1 s-1, respectively; Ki, 3.6 x 10(-10) and 1.0 x 10(-9) M; and kd, 7.9 x 10(-5) and 1.65 x 10(-4) s-1. Amino-terminal sequence analysis indicated that RSI and RSII are distinct, differing at the amino-terminal residues, and are products of the rat Spi-1 locus. Rat mast cell proteinase I (RMCP I) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited.     

Enzyme histochemistry of rat mast cell tryptase

Fixation and staining conditions for rat mast cell tryptase and its histochemical distribution in different rat tissues were investigated. Prostate, skin, lung, gut, stomach and salivary glands were fixed in either aldehyde or Carnoy fixatives and then frozen or embedded in paraffin wax. Preservation of tryptase enzymic activity against peptide substrates required aldehyde fixation and frozen sectioning. Of the peptide substrates examined, z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthylamide proved the most effective for the demonstration of tryptase. Double staining by enzyme cytochemistry followed by immunological detection of tryptase showed that, in all tryptase-containing mast cells, the enzyme is at least in part active. Conventional dye-binding histochemistry was used to confirm the identity of mast cells. Aldehyde-fixed mucosal mast cells required a much shorter staining time with Toluidine Blue if tissue sections were washed directly in t-butyl alcohol. Double staining by enzyme cytochemistry and dye binding showed that tryptase is absent from mucosal and subepidermal mast cells, which are also smaller in size and appear to contain fewer granules than connective tissue mast cells. This study demonstrates that rat mast cell tryptase, unlike tryptases in other species, is a soluble enzyme. It is stored in an active form and is absent from some mast cell subpopulations in mucosa, skin and lung. © 1998 Chapman & Hall     

Cloning of the cDNA and gene of mouse mast cell protease-6. Transcription by progenitor mast cells and mast cells of the connective tissue subclass

The cDNA and gene for mouse mast cell protease-6 (MMCP-6) have been sequenced and show MMCP-6 to be translated as a prepro-enzyme with a 21-amino acid hydrophobic leader peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The mature form of the enzyme has 73% amino acid sequence identity with human and dog mast cell tryptases. The MMCP-6 gene includes 6 exons, with a total span of 1.8 kilobases. A 208-base pair intron was defined which separates the 5'-untranslated sequence of MMCP-6 from the translation initiation codon, thereby presenting a gene organization which distinguishes tryptic serine proteases from chymotryptic serine proteases of the mast cell secretory granule. By RNA blot analysis with a gene-specific probe, MMCP-6 has a unique subclass distribution in being transcribed in mouse connective tissue mast cells but undetectable in mucosal mast cells. MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populations in mast cell-deficient mice.     

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.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

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.     

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of atrial natriuretic factor (ANF).

1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg- containing substrate that resembles the cleavage site for activation of pro-ANF. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg- containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 (J. biol. Chem. 263, 9515-9519) found an identical enzyme would cleave ANF from pro-ANF. 4. These results suggest therefore that mast cell tryptase may be involved in the activation of ANF from pro-ANF.     

Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization.

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.     

The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: effects of secretagogues and anti-allergic drugs on histamine secretion

Despite the apparent involvement of gastrointestinal mast cells in hypersensitivity reactions in the mucosa, remarkably little information is available concerning the characteristics of these cells from man and higher animals. To study the characteristics of gastrointestinal mast cells from nonhuman primates, a previously described technique which uses a combination of mechanical and enzymatic methods to obtain mast cells from the tissues of rodents required modification to permit the successful dispersion of normal gastrointestinal tissues of higher animals. This modified procedure, as described in this report, appears to be relatively selective for mast cells located in the mucosal site, and typically yields ca 9 X 10(5) mast cells per gram of tissue. The mucosal mast cells obtained comprised ca 2% of the total nucleated cells, contained approximately 1 pg of histamine per cell, and stained metachromatically with toluidine blue only at low pH. The cells exhibited a dose-dependent release of histamine on challenge with goat anti-human IgE or the ionophores A23187 and Br-x537A but were refractory to the action of compound 48/80. IgE-mediated histamine release from monkey intestinal mast cells differed from that observed from rat intestinal mast cells in that release was inhibited not only by quercetin but also by theophylline. Disodium cromoglycate gave variable results. The data indicate that viable nonhuman primate mucosal mast cells can be obtained for study, and that these cells, although sharing some characteristics of mucosal mast cells from lower species, have distinct and unique properties. The availability of this nonhuman primate model for the study of mast cell function in higher animals should contribute to the understanding of mast cell-mediated diseases in man.     

Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.     

Mast cell heterogeneity

Increasing evidence for the existence of inter- and intra-species mast cell heterogeneity has expanded the potential biological role of this cell. Early studies suggesting that mast cells at mucosal sites differ morphologically and histochemically from connective tissue mast cells have been confirmed using isolated intestinal mucosal mast cells in the rat and more recently in man. These studies also established that mucosal mast cells are functionally distinct from connective tissue mast cells. Thus, mucosal and connective tissue mast cells differ in their responsiveness to a variety of mast cell secretagogues and antiallergic agents. Speculation about the therapeutic use of antiallergic drugs in disorders involving intestinal mast cells cannot, therefore, be based on extrapolation from studies of their effects on mast cells from other sites. Regulatory mechanisms for mast cell secretion may also be heterogeneous since mucosal mast cells differ from connective tissue mast cells in their response to a variety of physiologically occurring regulatory peptides. The development of techniques to purify isolated mast cell subpopulations will facilitate future analysis of the biochemical basis of the functional heterogeneity of mast cells.     

The role of PAR1 in protective action of activated protein C during nonimmune activation of mast cells

Activated protein C (APC) regulates the functional activity of mast cells by reducing release of β-hexosaminidase, the marker of mast cell degranulation. APC modulated not only spontaneous secretion from mast cells, but also secretion induced by the degranulators, proteinase-activated receptor agonist peptide (PAR1-AP) and compound 48/80. PAR1 desensitization by thrombin abolished the decrease of β-hexosaminidase secretion induced by low APC concentrations (≤1.5 nM). APC inactivated by phenylmethylsulfonyl fluoride (PMSF), did non mimic the enzyme action on mast cells. Duodenase (the duodenal proteinase) activated peritoneal mast cell via PAR1. APC abolished the proinflammatory effect of duodenase and PAR1-AP by reducing release of mast cell mediators. The effect of APC could be attributed to nitric oxide generation by mast cells because in the presence of L-NAME the secretory function restored. These data suggest involvement of mast cell PAR1 into regulatory mechanism responsible for the anti-inflammatory effect of APC.     

Long term increase of mucosal mast cells in the rat induced by administration of Compound 48/80

Summary Administration of Compound 48/80 to rats for 5 days resulted in an increase of the specific type of mucosal mast cell, while connective tissue mast cells elsewhere were almost completely degranulated. The number of mucosal mast cells increased slowly for another 5 days and then returned to the control level, in an exponential manner. The half life of the newly formed mast cells was calculated to be about 40 days. This value may be taken as an estimate of the half life of mucosal mast cells. These cells, therefore, constitute a fraction of mast cells with rapid turnover. Available evidence indicates that the classical connective tissue mast cell has a very long life span, without significant turnover in terms of cell death and cell renewal. We suggest that the increase of mucosal mast cells is an indirect effect of Compound 48/80, related to its effect on other mast cells and mediated by material(s) released from these cells during the secretory process.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Isolation and properties of an elastase-like proteinase from horse blood leucocytes

The rapid, two steps method of purification of an elastase-like proteinase from cytoplasmic granules of horse leucocytes is described. This enzyme called the proteinase 1 is released easily from isolated granules in the low ionic strength solutions in opposite to the other two molecular forms of which one differs slightly in isoelectric point from the other. The enzyme is a typical neutral proteinase of a broad substrate specificity and wide pH optimum. In a physiological conditions the enzyme is built of two polypeptide chains of molecular weight about 30000 and 20000, respectively.