Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Human beta-tryptase is a ring-like tetramer with active sites facing a central pore. 952 29

We have previously shown that fibroblast and keratinocyte supernatants up-regulate expression of mast cell characteristics in the human immature mast cell line HMC-1. This effect could not be induced in HMC-1 cells by the well-known mast cell growth factor stem cell factor (SCF), probably due to mutations of the SCF receptor c-Kit in these cells. Here we report the effects of several known fibroblast- and keratinocyte-derived growth factors, namely nerve growth factor (NGF), basic fibroblast growth factor, platelet-derived growth factor and transforming growth factor-beta, on mast cell differentiation, using HMC-1 cells as a model. NGF, at 0.1-50 ng/ml concentrations, caused a marked, dose-dependent up-regulation of tryptase, Fc epsilon RI and histamine within 10 days of culture, associated with an enhanced expression of mRNA for Fc epsilon RI and mast cell tryptase. On restriction analysis, only mast cell beta-tryptase, but not alpha-tryptase, could be demonstrated. Furthermore, the high-affinity NGF receptor (TrkA) was found at both the transcriptional and protein levels, while expression of the low-affinity NGF receptor was detectable at the mRNA level only. None of the other growth factors caused a significant alteration of the mast cell markers studied when added to HMC-1 cells at concentrations known to be biologically active in other culture systems. Immature human mast cells are thus induced to assume a more mature phenotype in vitro in response to NGF, most probably via stimulation of the high-affinity NGF receptor expressed on these cells. Besides SCF, NGF should therefore be considered as an additional mast cell growth factor that contributes to human mast cell maturation at tissue sites.
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PMID:Effects of nerve growth factor (NGF) and other fibroblast-derived growth factors on immature human mast cells (HMC-1). 977 35

Mast cell tryptase purified from human adult skin (AS), adult lung (AL) and newborn foreskin (NS) with a monoclonal antitryptase B2 immunoaffinity Sepharose column was further fractionated by HPLC using a Mono-S cation exchange column at pH 6.5. Tryptases exhibited two clearly separated major fractions, both of which also revealed at least two overlapping peaks. Native tryptase molecules from skin consisted of two diffuse protein bands in SDS-PAGE at about 31 and 35 kDa, whereas those from lung usually exhibited a predominant diffuse band at about 29 kDa. The forms of tryptases separated by Mono-S HPLC gave a different banding pattern in SDS-PAGE. Tryptase from NS exhibited chromatographic peaks that each showed Mr values approximately 1-3 kDa higher than those of tryptase from AS. By gel filtration, the Mr values for native major fractions of tryptases derived from AS and AL were 178 kDa and 141 kDa, respectively. After carbohydrate removal by glycanase, the observed differences in Mr values in SDS-PAGE reduced to two similar sharp bands of Mr approximately 28 kDa and 30 kDa for all tryptase preparations. AS and AL tryptases and their subfractions exhibited similar enzyme kinetic values and similar immunoreactivities in a tryptase immunoassay. Inactivation rates at physiologic ionic strength were similar for both AL and AS tryptases. The results show the enzymatic and antigenic similarity between lung and skin tryptases, and suggest that tryptase is stored mainly as beta-tryptase in human mast cells. Tryptase immunoassay measures similarly both lung and skin tryptases and, thus, this assay is suitable for detection of mast cell activation, in contrast to assays for other proteinases of mast cells, e.g. chymase, cathepsin G and carboxypeptidase, that are present in MC(TC) cells mainly in skin only.
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PMID:Identification and characterization of multiple forms of tryptase from human mast cells. 1019 93

Tryptases alpha and beta/II were expressed in insect cells to try to ascertain why human mast cells express these two nearly identical granule proteases. In contrast to that proposed by others, residue -3 in the propeptide did not appear to be essential for the three-dimensional folding, post-translational modification, and/or activation of this family of serine proteases. Both recombinant tryptases were functional and bound the active-site inhibitor diisopropyl fluorophosphate. However, they differed in their ability to cleave varied trypsin-susceptible chromogenic substrates. Structural modeling analyses revealed that tryptase alpha differs from tryptase beta/II in that it possesses an Asp, rather than a Gly, in one of the loops that form its substrate-binding cleft. A site-directed mutagenesis approach was therefore carried out to determine the importance of this residue. Because the D215G derivative of tryptase alpha exhibited potent enzymatic activity against fibrinogen and other tryptase beta/II-susceptible substrates, Asp215 dominantly restricts the substrate specificity of tryptase alpha. These data indicate for the first time that tryptases alpha and beta/II are functionally different human proteases. Moreover, the variation of just a single amino acid in the substrate-binding cleft of a tryptase can have profound consequences in the regulation of its enzymatic activity and/or substrate preference.
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PMID:Human tryptases alpha and beta/II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the substrate-binding cleft. 1039 6

Nerve growth factor-beta (NGF) is known as a growth factor for human basophils and murine mast cells and has recently been shown to also up-regulate mast cell characteristics in human leukaemic mast cells. We have examined here the effect of NGF on the differentiation of normal human mast cells from cord blood progenitors during culture with stem cell factor (SCF), NGF alone or in combination, or fibroblast supernatants. All these supplements induced mast cell immunoreactivity against tryptase, c-Kit and FcepsilonRIalpha, but none of the cells reacted against the basophil specific antibody 2D7 before or during culture. Intracellular tryptase activity increased as well, with maximal levels on combined culture with SCF and NGF. On reverse transcription-polymerase chain reaction (RT-PCR), cells lacked tryptase and chymase and expressed low levels of FcepsilonRI and c-Kit mRNA prior to culture, with marked up-regulation of FcepsilonRI and c-Kit, and with de novo expression of mast-cell specific alpha- and beta-tryptase by week 3, and of chymase by week 5. Only the TrkA and not the p75 NGF receptor was detected at m-RNA and protein level, and only the TrkA NGF receptor was up-regulated during NGF-driven culture. These findings show therefore that, like SCF, NGF is another growth factor that can induce and regulate human mast-cell development and differentiation.
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PMID:Nerve growth factor-beta induces mast-cell marker expression during in vitro culture of human umbilical cord blood cells. 1071 72

Total tryptase levels of 20 ng/mL or higher in a baseline serum sample when the ratio of total to beta-tryptase is 20 or greater strongly suggest underlying systemic mastocytosis. Whether these criteria prove to be more sensitive than a bone marrow biopsy will require further study. Although the absolute level of total tryptase does not predict disease severity, it may provide a practical method for assessing the efficacy of therapeutic interventions designed to reduce the mast cell burden.
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PMID:Serum tryptase and the laboratory diagnosis of systemic mastocytosis. 1090 44

alpha- and beta-tryptase genes encode serine proteases that are abundantly expressed by mast cells. Under physiologic conditions other myeloid cells are virtually tryptase negative. However, tryptases are also expressed in several myeloid leukemia cell lines. In this study, serum total tryptase levels were determined in 150 patients with acute leukemias (de novo acute myeloid leukemia [AML], n = 108; secondary AML, n = 25; acute lymphoid leukemia [ALL], n = 17) by fluoroenzyme immunoassay. In healthy subjects (n = 30), tryptase levels ranged between 2.0 and 12.6 ng/mL. Elevated tryptase levels (> 15) were detected in 42 (39%) of 108 patients with de novo AML and in 11 (44%) of 25 patients with secondary AML. No elevated tryptase levels were found in patients with ALL. In de novo AML, elevated tryptase levels were frequently detected in patients with French-American-British classification M0 (6 of 9), M2 (9 of 14), M3 (4 of 6), and M4eo (7 of 7), and less frequently in M1 (7 of 20), M4 (6 of 26), M5 (2 of 18), M6 (0 of 5), or M7 (1 of 3). The highest tryptase levels were found in M4eo. Immunohistochemical staining of bone marrow sections with anti-tryptase antibody as well as immunoelectron microscopy revealed tryptase expression in the cytoplasm of myeloblasts. As assessed by Northern blotting and reverse transcriptase-polymerase chain reaction, AML cells expressed alpha-tryptase messenger RNA (mRNA) but little or no beta-tryptase mRNA. In AML patients with elevated serum tryptase before chemotherapy, who entered complete remission, tryptase levels returned to normal or near normal values. Blast cell persistence or regrowth was associated with a persistently elevated level or recurrent increase of tryptase. Together, tryptase is expressed in myeloblasts in a group of AML and may serve as a useful disease-related marker.
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PMID:Expression of mast cell tryptase by myeloblasts in a group of patients with acute myeloid leukemia. 1156 8

Tryptases are serine proteases primarily expressed in mast cells. Normal blood basophils express only trace amounts of the enzyme. However, recent immunohistochemical studies have raised the possibility that neoplastic basophils express significant amounts of tryptase. In this study, tryptase expression was analyzed in normal and neoplastic basophils by immunoelectron microscopy using antitryptase monoclonal antibody G3. Basophils were obtained from patients with chronic myeloid leukemia (CML), idiopathic myelofibrosis (IMF), and myelodysplastic syndrome (MDS), and from healthy donors. Tryptase-immunoreactive material was detected in cytoplasmic granules of basophils in CML, IMF, and MDS. By contrast, normal basophils did not contain significant amounts of tryptase by immunoelectron microscopy. As assessed by reverse transcription-polymerase chain reaction, neoplastic basophils contained messenger RNA (mRNA) for alpha-tryptase, but no beta-tryptase mRNA. In summary, these data provide evidence that neoplastic basophils in CML, IMF, and MDS can express detectable amounts of tryptase. Therefore, tryptase should not be regarded as specific for mast cells when neoplastic myeloid cells are analyzed.
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PMID:Detection of tryptase in cytoplasmic granules of basophils in patients with chronic myeloid leukemia and other myeloid neoplasms. 1158 60

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.
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PMID:Tryptase's potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions. 1179 23

Four 11-residue peptides based on the Bowman-Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human beta-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.
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PMID:Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides. 1190 11


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