Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A gene encoding human chymase was cloned and sequenced. The protein-coding exons reveal a preproenzyme with a 19-amino acid signal peptide, an acidic 2-amino acid propeptide, and a 226-amino acid catalytic domain. The mature enzyme is predicted to be cationic (net charge of +13) and to be modified by N-glycosylation at two sites. The amino acid sequence is identical to the 35 residues of NH2-terminal amino acid sequence reported for human skin chymase and is identical to 29 of 31 residues of NH2-terminal and internal amino acid sequence reported for human heart chymase. The full predicted sequence of the catalytic domain reveals a high level of sequence identity to dog mast cell chymase (83%) and a lower level of identity to the sequences of rodent chymases (58-62%). In the phase and placement of introns, the organization of this human chymase gene is similar to that of several other granule-associated leukocyte serine proteases, including rat chymase II, lymphocyte granzymes, and neutrophil cathespin G and elastase. However, the gene organization differs from that of mast cell tryptase, providing additional evidence that the major mast cell serine proteases are separated by substantial evolutionary distance. Amplification of chymase gene-specific fragments from hamster/human hybrid cell line DNA suggests localization of the chymase gene to human chromosome 14. High stringency hybridization of chymase DNA to a human genomic DNA blot suggests the possibility of more than one human chymase gene. Evidence that the chymase gene is expressed in human tissues was obtained by the amplification of chymase-specific DNA from skin and placental cDNA libraries.
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PMID:Structure, chromosomal assignment, and deduced amino acid sequence of a human gene for mast cell chymase. 207 82

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.
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PMID:Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family. 218 93

We have used fiberoptic bronchoscopy to obtain endobronchial biopsies in which mast cells and eosinophils were enumerated using monoclonal antibodies directed against mast cell tryptase (AA1) and the eosinophil cationic protein (EG2). Eleven symptomatic atopic asthmatics treated with beta 2-agonists alone and six normal subjects were studied. Over a period of 2 wk prior to bronchoscopy, patients recorded asthma symptom scores, bronchodilator usage, and twice-daily peak expiratory flow. Five days before bronchoscopy, methacholine responsiveness was assessed. Two biopsies were taken from the subcarinae, one of which was processed into araldite for immunostaining by the streptavidin biotin immunoperoxidase method and the other into Spurr resin for electron microscopy. The number of AA1 staining mast cells present in the bronchial mucosa was not significantly different in the epithelium or submucosa between the asthmatic and the normal subjects. However, in the biopsies from asthmatics, there were significantly greater numbers of EG2-staining eosinophils in the epithelium (median, 1.2/mm versus zero; p less than 0.005) and in the submucosa (median, 50/mm2 versus 1/mm2; p less than 0.001). Electron microscopy showed morphologic features of mast cell and eosinophil degranulation in the asthmatics. No correlation could be established between mast cell or eosinophil numbers and indices of disease activity of PC20 methacholine, which points to the complexity of mechanisms responsible for the symptoms and the airway hyperresponsiveness of asthma.
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PMID:Quantitation of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry. 202 38

The susceptibility of connective tissue elements to degradation by human mast cells was explored using purified mast cell tryptase and sonicated mast cell preparations. The R-22 strain of smooth muscle cells from rat heart was used for preparation in vitro of a labelled anchored matrix. Digestion of 11.9 +/- 1.2% (n = 5) of this matrix was observed after overnight incubation with the mast cell sonicates. Pretreatment of the sonicate with a tryptase inhibitor TLCK reduced the digestion by 42%. Digestion of 12 +/- 1% (n = 4) of the matrix was observed with purified tryptase. The susceptible substrate within this anchored insoluble matrix resided in the glycoprotein compartment as defined by enzymatic characterization of the residual matrix. Mast cells may play a role in mediating connective tissue degradation through the release of proteases specifically synthesized by this cell.
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PMID:The mast cell as an effector of connective tissue degradation: a study of matrix susceptibility to human mast cells. 222 42

An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.
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PMID:Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase. 225 Jan 89

Tryptase was previously shown to undergo rapid inactivation under physiological conditions unless stabilized by the presence of heparin. The current study shows that increasing the concentration of free tryptase enhances the preservation of enzymic activity, consistent with dissociation of the tetramer, rather than autodegradation, as the mechanism of inactivation. Heparin glycosaminoglycan fragments of Mr greater than 5700 are necessary for complete stabilization of tryptase activity. This stabilizing effect depends upon negative charge density rather than carbohydrate composition. Thus, keratan sulphate or hyaluronic acid were no better than physiological buffer alone; chondroitin monosulphates and heparan sulphate each prolonged the t1/2 about 20-fold over buffer alone; chondroitin sulphate E prolonged the t1/2 69-fold; and dextran sulphate and heparin provided complete stabilization of tryptase activity for 120 min. Poly-D-glutamic acid prolonged the t1/2 55-fold. In each case the loss of tryptase activity followed apparent first-order kinetics. Increasing the NaCl concentration from 0.01 M to 1.0 M increased the stability of free tryptase. In contrast, increasing the NaCl concentration in the presence of stabilizing polysaccharides decreased the stability of tryptase until dissociation of tryptase from each polysaccharide presumably occurred; thereafter tryptase stability increased as did that of free tryptase. The effect of salt concentration on heparin-stabilized tryptase activity (as opposed to stability) was also evaluated. The mast cell proteoglycans heparin and chondroitin sulphate E, by virtue of containing the naturally occurring glycosaminoglycans of highest negative charge density, may play a major role in the regulation of mast cell tryptase activity in vivo.
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PMID:Regulation of human mast cell tryptase. Effects of enzyme concentration, ionic strength and the structure and negative charge density of polysaccharides. 244 72

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.
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PMID:Activation of latent rheumatoid synovial collagenase by human mast cell tryptase. 245 61

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.
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PMID:Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret. 249 55

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.
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PMID:Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation. 250 77

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.
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PMID:Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut. 264 38


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