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PubMed1989

Identification of a tryptase-like enzyme in extracts of inflamed human gingiva by effector and gel-filtration studies.

Inflamed gingiva contain a serine proteinase which could not previously be identified on the basis of its substrate specificity and inhibitor response. Using the substrate ZAlaArgArgAFC at alkaline pH, the enzyme was shown to be extracted more efficiently in high salt buffer. Inclusion of NaCl in assays, however, caused progressive reduction of activity. There was also inhibition by CaCl2, MgCl2 and 2 mM TosLysCH2Cl but not by 2 mM TosPheCH2Cl. Heparin produced significant activation. In gel filtrations with 1.0 M NaCl, activity appeared in fractions corresponding to a molecular weight of about 135,000. These properties are all consistent with tryptase from human mast cells. The enzyme may participate in both the connective tissue destruction and the inflammatory and immunological processes of gingivitis and periodontitis.

Chromatography, Gel↗
PubMed1994

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1998

Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide.

The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.

Amino Acid Sequence↗
PubMed1988

Elevated histamine and tryptase levels in smokers' bronchoalveolar lavage fluid. Do lung mast cells contribute to smokers' emphysema?

Human lung mast cells have been reported recently to contain small amounts of the elastolytic protease present in the neutrophil and implicated in the pathogenesis of alveolar wall destruction in emphysema. Since mast cells are numerous within alveolar walls, release of inflammatory mediators (and possibly elastase) by cigarette smoking could contribute to alveolar injury in this disease. We therefore examined bronchoalveolar lavage (BAL) fluid for the mast cell granule constituents histamine and tryptase. The results, while not conclusive, supported the possibility that cigarette smoking increases secretion of histamine releasing activity by alveolar macrophages with subsequent degranulation of local mast cells. Mast cell discharge of inflammatory mediators (including neutrophil chemotactic factors and perhaps the elastolytic protease) could then participate in the destruction of alveolar walls.

Adult↗
PubMed2001

A novel murine tryptase involved in blastocyst hatching and outgrowth.

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.

Amino Acid Sequence↗
PubMed2005

Sulfated polysaccharides derived from dietary seaweeds increase the esterase activity of a lymphocyte tryptase, granzyme A.

Intake of sulfated polysaccharides, such as fucoidan or lambda-carrageenan extracted from seaweeds, has been shown to enhance immune responses, resulting in inhibition of tumor growth. However, little is known about the mechanisms by which these sulfated compounds mediate the enhancement. In the present study, we examined the effect of sulfated polysaccharides from seaweeds on esterase activity of a lymphocyte tryptase, granzyme A (GzmA), which is believed to induce the production of cytokines in a variety of cells. Inclusion of fucoidan (from Fucus vesiculosus) or lambda-carrageenan (from Gigartina aciculaire and Gigartina) in the reaction mixture increased the hydrolysis of Nalpha-benzyloxy-L-lysine thiobenzyl ester (BLT) by a recombinant rat GzmA in a concentration-dependent manner. Heparin, a sulfated polysaccharide from animal tissues, also increase the BLT hydrolysis, but the effect was less remarkable than those of the polysaccharides from the seaweeds. Hanes-Woolf analysis revealed that the enhancements in the presence of these sulfated compounds from the seaweeds were attributed to the increases in the affinity of the enzyme toward the substrate but not to those in the turnover rate. Chondroitin sulfate A, a sulfated polysaccharide found in animal and plant tissues, showed no positive effect on the hydrolysis. In the present paper, we propose that the enhancement of immune responses by intake of the sulfated polysaccharides from seaweeds can be partially accounted for by their direct effects on GzmA.

Animals↗
PubMed1992

Mast-cell phenotype in indolent forms of mastocytosis. Ultrastructural features, fluorescence detection of avidin binding, and immunofluorescent determination of chymase, tryptase, and carboxypeptidase.

The factor(s) that causes excessive mast cell (MC) proliferation in indolent forms of mastocytosis is not known, nor is it known whether that proliferation is a regulated clonal expansion or merely a non-neoplastic hyperplasia. Human MCs display phenotypes that depend on the microenvironment. Thus, if the phenotype of MCs in mastocytosis lesions is determined to be abnormal for that tissue site (and therefore the MCs are refractory to microenvironmental signals) then a clonal process would be suggested. The authors determined the phenotypes of MCs from the lesional skin of 17 patients with indolent mastocytosis and the bone marrows of 9 patients. They compared them with the phenotypes of MCs from the lesional skin of 8 patients with various dermatitides, the skin of 2 patients with idiopathic anaphylaxis, and the breast skin of 15 control patients. MCs from all the skin specimens showed the characteristic skin MC phenotype, with predominantly scroll-poor granules by ultrastructure and containing tryptase and chymase by immunofluorescence detection (the MCTC immunophenotype). The skin MCs of each patient bound avidin and contained carboxypeptidase by immunofluorescence detection. MCs from the bone marrow of patients with indolent mastocytosis, the source of progenitors, also showed the scroll-poor and MCTC phenotypes. These findings do not support an unregulated clonal expansion in indolent forms of mastocytosis. They are consistent with a non-neoplastic hyperplasia or possibly a clonal process in which MCs remain responsive to microenvironmental regulation.

Avidin↗
PubMed1988

Tryptase and chymase: comparison of extraction and release in two dog mastocytoma lines.

Mast cell secretory granules contain unique tryptic and chymotryptic serine proteases that differ between species and tissues. Direct comparison of these proteases in single-cell types has been hindered by the difficulty of obtaining adequate numbers of pure mast cells. In this study, we were able to compare tryptic and chymotryptic enzyme activity in cells of presumed monoclonal origin, using two stable lines ('BR' and 'G') of dog mastocytomas. The gel-filtration profiles, inhibitor susceptibilities and substrate preferences of tryptic and chymotryptic mastocytoma protease activities established their close resemblance to the tryptases and chymases of human and rodent mast cells. Striking heterogeneity was observed in the amounts and solubilities of the tryptic and chymotryptic activity in the two different mastocytoma cell lines. Incubation of cells from both lines with calcium ionophore A23187 caused non-cytotoxic release of protease activity. In contrast to chymase from rat connective tissue mast cells, protease activity that was insoluble after extraction at low ionic strength became soluble following ionophore-stimulated release. Neither tryptic nor chymotryptic activity was activated during degranulation, suggesting the absence of inactive precursors. Cells of the 'BR' line released both tryptic and chymotryptic activity in parallel with the granule marker histamine; cells of the 'G' line released a much smaller proportion of tryptic activity than of either chymotryptic activity or histamine. These differences in release of granule constituents from cells of common origin could be explained by developmental variations in the production of performed mediators or by differential regulation of preformed mediator release. We conclude that the differences in protease content, solubility and release in these mastocytoma lines are useful in evaluating the potential pathophysiological significance of the contribution of proteases to mast cell heterogeneity.

Animals↗
PubMed1986

Mast cells in human keloid, small intestine, and lung by an immunoperoxidase technique using a murine monoclonal antibody against tryptase.

A murine monoclonal antibody (G5) against human lung mast cell tryptase was used for selective staining of human mast cells by an indirect immunoperoxidase method. Human tissues (keloid, small bowel, lung) were fixed in either Carnoy's fluid or neutral buffered formalin. In all three tissues the number and location of G5-stained mast cells corresponded closely with metachromatic toluidine blue-stained mast cells, although the immunospecific technique appeared to be more sensitive. In lung the average concentration of G5-positive mast cells after Carnoy's fixation was 15,695/cu mm of subepithelial tissue in bronchi and bronchioles and 26,580/cu mm of alveolar wall, in small bowel was 20,958/cu mm of mucosa and 8576/cu mm of submucosa, and in keloid was 3068/cu mm. Formalin fixation significantly reduced concentrations of G5-positive mast cells in all tissues except keloid.

Animals↗
PubMed2004

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities.

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.

Animals↗