Human skin tryptase: Kinetic characterization of its spontaneous inactivation

Schechter, Norman M.; Eng, Grace Y.; McCaslin, Darrell R.

doi:10.1021/bi00061a020

Cited by 47 publications

(71 citation statements)

References 36 publications

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“…In each instance, Ͼ80% of the expressed mutant bound to a heparinSepharose column (data not shown). Thus, the conversion of Trp 14 or Trp 206 to Leu did not cause an extensive alteration in the overall three-dimensional structure of mMCP-7.…”

Section: Identification Of Two Trp Residues In Mmcp-7 Required For Itmentioning

confidence: 88%

“…Site-directed mutagenesis was then performed with the Altered Sites ® II in vitro mutagenesis system (Promega), according to the manufacturer's instructions. The mutagenic oligonucleotide 5Ј-AA-CAAGTGGCCCTTGCAGGTGAGCCTG-3Ј (corresponding to nucleotides 112-138 of our mMCP-7 cDNA) was used to convert Trp 14 to Leu, whereas the mutagenic oligonucleotide 5Ј-ACCTTGCTGCAGGCAG-GCGTGGTC-3Ј (corresponding to nucleotides 696 -720 of our mMCP-7 cDNA) was used to convert Trp 206 to Leu. These Trp residues were mutated to Leu residues rather than to Asp, Glu, Arg, or Lys residues in order to maintain the general hydrophobic nature of the Trp-rich domain on the surface of folded mMCP-7.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, it was a surprise when Schwartz and co-workers (9, 10) and then others (11)(12)(13)(14)(15)(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 tetramer free of proteoglycan (5).…”

mentioning

confidence: 99%

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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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Section: Identification Of Two Trp Residues In Mmcp-7 Required For Itmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…The conformational changes in the tetramer are followed by reversible dissociation into inactive monomers. The monomers slowly decay in an irreversible fashion to non-reassociating species (30). Like TAFIa, tryptase apparently does not have a natural inhibitor in vivo; therefore, the spontaneous decay of tryptase activity may serve to limit its activity following release from secretory granules (29).…”

Section: Discussionmentioning

confidence: 99%

Roles of Thermal Instability and Proteolytic Cleavage in Regulation of Activated Thrombin-activable Fibrinolysis Inhibitor

Boffa

Bell

Stevens

et al. 2000

Journal of Biological Chemistry

111

177

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We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin-and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.The balance between the activities of the coagulation and fibrinolytic cascades is essential to prevent excessive blood loss upon injury to the vasculature while maintaining the blood in a fluid state at sites distant from the injury. The recent identification of thrombin-activable fibrinolysis inhibitor (TAFI 1 (1)) (also known as plasma procarboxypeptidase B (2) and carboxypeptidase U (3)) has illuminated a novel molecular linkage between these cascades. The production of thrombin by the coagulation cascade results in the generation of the active form of TAFI (TAFIa) that down-regulates fibrinolysis by removal of the carboxyl-terminal lysine residues from partially degraded fibrin that are important for the development of positive feedback in the fibrinolytic cascade (4).The M r ϳ60,000 TAFI zymogen is cleaved after Arg-92 by thrombin to yield an activation peptide and a M r ϳ35,000 enzyme (TAFIa) that possesses basic carboxypeptidase activity (1, 2). The catalytic efficiency of thrombin activation of TAFI is enhanced 1250-fold in the presence of the cofactor thrombomodulin (5). The enzymatic activity of TAFIa is intrinsically unstable, decaying with a half-life of about 8 -9 min at body temperature in the absence of additional proteolytic cleavage (6). Previous studies from our laboratory have revealed that the instability of TAFIa activity is highly sensitive to temperature; the half-life of TAFIa is increased approximately 15-fold by decreasing from 37 to 25°C the temperature at which the enzym...

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“…Changes in tertiary structure also occur with this type of inactivation, because mAbs against β-tryptase were made that recognize conformational epitopes on active tetramers that are not available on inactive monomers [67]. A kinetic analysis of β-tryptase decay suggested active tetramers first became inactive tetramers (reversible by adding heparin), and then inactive monomers (irreversible) [68,69]. Evidence for active monomers was first reported by Addington et al [70], this conclusion was based upon analyzing monomers that had been loaded onto a gel-filtration column equilibrated with 10 mM Mes buffer, pH 6.1, containing 10 % glycerol, 1M NaCl and 0.01% NaN 3 .…”

Section: Tetramer ↔ Monomermentioning

confidence: 99%

Active monomers of human β-tryptase have expanded substrate specificities

Fukuoka

Schwartz

2007

International Immunopharmacology

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Abstractβ-Tryptase, a product of the TPSAB1 and TPSB2 genes, is a trypsin-like serine protease that is a major and selective component of the secretory granules of all human mast cells, accounting for as much as 25% of cell protein. Once mast cells are activated, β-tryptase is released along with histamine and heparin proteoglycan. β-Tryptase is a unique enzyme with a homotetrameric structure in which active sites face into the central cavity of the four monomers, stabilized by heparin-proteoglycan. This structure makes β-tryptase resistant to most biological inhibitors of serine proteases. Without stabilization, at neutral pH β-tryptase converts to inactive monomers. Tryptase levels are elevated in bronchoalveolar lavage (BAL) fluid obtained from atopic asthmatics and in serum during systemic anaphylactic shock. Several synthetic small molecular weight β-tryptase inhibitors reduced Aginduced airway hypersensitivity in animals, suggesting β-tryptase is involved in the pathogenesis of airway inflammation. Although the major biologic substrate(s) of β-tryptase remain ambiguous, the protease can digest several proteins of potential biologic importance, including fibrinogen, fibronectin, pro-urokinase, pro-matrix metalloprotease-3 (proMMP-3), protease activated receptor-2 (PAR2) and complement component C3. Recently, monomers of β-tryptase with enzymatic activity have been detected in vitro. Here we discuss how β-tryptase monomers with enzymatic activity were identified as well as their potential role in vivo.

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Human skin tryptase: Kinetic characterization of its spontaneous inactivation

Cited by 47 publications

References 36 publications

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

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

Roles of Thermal Instability and Proteolytic Cleavage in Regulation of Activated Thrombin-activable Fibrinolysis Inhibitor

Active monomers of human β-tryptase have expanded substrate specificities

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