Tryptase from rat skin: purification and properties

Braganza, Vincent J.; Simmons, William H.

doi:10.1021/bi00234a023

Cited by 35 publications

(28 citation statements)

References 40 publications

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“…The insoluble diazotized collagen substrate Azocoll (>100 Mesh; Calbiochem, San Diego, Calif., USA) was used as a protein substrate [40]. Enzymes were activated with 2.5 m M L -cysteine by incubation for 10 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α<sub>1</sub>-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

Takai¹,

Kato²,

Ota³

et al. 2005

Int Arch Allergy Immunol

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Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcΕRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α₁-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α₁-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α₁-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.

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Section: Methodsmentioning

confidence: 99%

Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α<sub>1</sub>-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

Takai¹,

Kato²,

Ota³

et al. 2005

Int Arch Allergy Immunol

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“…Also, unlike the monomeric chymases and cathepsin G, enzymatically active tryptase is a tetramer. Heparin binds to and stabilizes the enzymatically active form of human tryptase (17), but surprisingly does neither to rat tryptase (18), even though both molecules colocalize to rat mast cell secretory granules. Mouse mast cell protease 7, another tryptase, by protein modeling, is predicted to bind to heparin through clustered His residues at acidic but not at neutral pH, this prediction being supported experimentally with recombinant mouse mast cell protease 7 precursor protein (19).…”

Section: Introductionmentioning

confidence: 99%

A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I.

Sakai

Ren²,

Schwartz³

1996

J. Clin. Invest.

144

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Tryptase is the major protein constituent of human mast cells, where it is stored within the secretory granules as a fully active tetramer. Two tryptase genes ( ␣ and ␤ ) are expressed by human mast cells at the level of mRNA and protein, each with a 30 amino acid leader sequence. Recombinant precursor forms of human ␣ -and ␤ -tryptase were produced in a baculovirus system, purified, and used to study their processing. Monomeric ␤ -protryptase first is shown to be intermolecularly autoprocessed to monomeric ␤ -pro'tryptase at acid pH in the presence of heparin by cleavage between Arg Ϫ 3 and Val Ϫ 2 in the leader peptide. The precursor of ␣ -tryptase has an Arg Ϫ 3 to Gln Ϫ 3 mutation that precludes autoprocessing. This may explain why ␣ -tryptase is not stored in secretory granules, but instead is constitutively secreted by mast cells and is the predominant form of tryptase found in blood in both healthy subjects and those with systemic mastocytosis under nonacute conditions. Second, the NH 2 -terminal activation dipeptide on ␤ -pro'tryptase is removed by dipeptidyl peptidase I at acid pH in the absence of heparin to yield an inactive monomeric form of tryptase. Conversion of the catalytic portion of ␤ -tryptase to the active homotetramer at acid pH requires heparin. Thus, ␤ -tryptase homotetramers probably account for active enzyme detected in vivo. Also, processing of tryptase to an active form should occur optimally only in cells that coexpress heparin proteoglycan, restricting this pathway to a mast cell lineage. ( J. Clin. Invest. 1996. 97: 988-995.)

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“…However, in neither case is formation of the tetramer dependent on heparin. Based on a multiple sequence alignment, we noted that recently cloned gerbil (22) and rat (23)(24)(25) MC tryptases also have the conserved residues that form the Trp-rich domain on the surface of mature mouse and human tryptases (4 -7, 21). Using site-directed mutagenesis, we now show that mMCP-7 requires this domain to form the enzymatically active tetramer.…”

mentioning

confidence: 99%

Formation of Enzymatically Active, Homotypic, and Heterotypic Tetramers of Mouse Mast Cell Tryptases

Huang¹,

Morales²,

Vagi³

et al. 2000

Journal of Biological Chemistry

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Mouse mast cell protease (mMCP) 6 and mMCP-7 are homologous tryptases stored in granules as macromolecular complexes with heparin and/or chondroitin sulfate E containing serglycin proteoglycans. When promMCP-7 and pseudozymogen forms of this tryptase and mMCP-6 were separately expressed in insect cells, all three recombinant proteins were secreted into the conditioned medium as properly folded, enzymatically inactive 33-kDa monomers. However, when their propeptides were removed, mMCP-6 and mMCP-7 became enzymatically active and spontaneously assumed an ϳ150-kDa tetramer structure. Heparin was not required for this structural change. When incubated at 37°C, recombinant mMCP-7 progressively lost its enzymatic activity in a time-dependent manner. Its N-linked glycans helped regulate the thermal stability of mMCP-7. However, the ability of this tryptase to form the enzymatically active tetramer was more dependent on a highly conserved Trp-rich domain on its surface. Although recombinant mMCP-6 and mMCP-7 preferred to form homotypic tetramers, these tryptases readily formed heterotypic tetramers in vitro. This latter finding indicates that the tetramer structural unit is a novel way the mast cell uses to assemble varied combinations of tryptases. Mouse mast cell (MC)1 protease (mMCP) 6 (1, 2) and mMCP-7 (3, 4) are homologous tryptases stored in abundance in their mature forms in the acidic secretory granules of numerous populations of MCs ionically bound to heparin and/or chondroitin sulfate E containing serglycin proteoglycans (1, 5, 6). Both tryptases are exocytosed when MCs are activated via their high affinity immunoglobulin (Ig) E receptors. mMCP-7 inhibits the formation of fibrin/platelet clots (7), whereas mMCP-6 induces the extravasation of neutrophils (8). Thus, the two tryptases often work in concert in the acute phases of MC-mediated inflammatory responses.A small amount of exocytosed mMCP-7 is retained in tissues for Ͼ1 h (5). However, most exocytosed mMCP-7 dissociates from its exocytosed protease/proteoglycan macromolecular complex in the extracellular matrix to allow the rapid diffusion of this tryptase away from the MC (5, 6). Because its proteoglycan-binding domain has more Lys and Arg residues than the corresponding domain in mMCP-7, exocytosed mMCP-6 cannot dissociate easily from its serglycin proteoglycan at neutral pH. Thus, due to its Ͼ10 million-dalton size, very little of the exocytosed mMCP-6/proteoglycan complex is able to enter the circulation.Pancreatic trypsin and most other serine proteases are enzymatically active in their ϳ30-kDa monomer states. Thus, it was a surprise when Schwartz and co-workers (9, 10) and then others (11-16) discovered that human MC tryptases purified from different tissues exist as tetramers. While it has been proposed that heparin is needed for human lung-and skinderived tryptases to form stable, enzymatically active tetramers (10, 14 -16), mMCP-7 is able to circulate in the blood of the V3 mastocytosis mouse for Ͼ1 h as an enzymatically active, homotypic tetrame...

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Tryptase from rat skin: purification and properties

Cited by 35 publications

References 40 publications

Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α<sub>1</sub>-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α<sub>1</sub>-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I.

Formation of Enzymatically Active, Homotypic, and Heterotypic Tetramers of Mouse Mast Cell Tryptases

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