Characterization of Human γ-Tryptases, Novel Members of the Chromosome 16p Mast Cell Tryptase and Prostasin Gene Families

Mast cells (MCs) are highly specialized immune cells present in mammals and in lower organisms that predate the development of adaptive immunity. The strong evolutionary pressure to retain MCs for >500 million years suggests critical roles for these cells in our survival. In support of this conclusion, no human has been identified to date that lacks MCs, despite the adverse roles of MCs in systemic anaphylaxis and varied inflammatory disorders. MCs express numerous lineage-restricted neutral proteases, and four members of the chromosome 17A3.3 family of tryptases are preferentially expressed in mouse MCs. The anatomical location of MCs at host-environment interfaces has raised the possibility that some of these enzymes are evolutionally conserved because they are needed for combating infectious organisms. Here we review recent insights into the structure and function of MC tryptases in inflammation and host defense against bacteria and other infectious organisms. MC-restricted Granule Proteases and ProteoglycansA characteristic morphologic feature of mammalian mast cells (MCs) 3 is their electron-dense secretory granules, which contain serglycin proteoglycans (1, 2) bearing heparin (for review see Ref. 3) and chondroitin sulfates diB and E (4, 5). Heparin is the most negatively charged molecule in the body, and each rat and mouse peritoneal MC contains ϳ25 pg of this glycosaminoglycan, thereby explaining the avidity of MCs for cationic dyes (6). The repeating Ser-Gly sequence in serglycin where its glycosaminoglycans are attached cannot be cleaved by any known protease. This protease resistance feature is biologically relevant because the primary function of serglycin proteoglycans in MCs is to provide a scaffold for formation of macromolecular complexes with the cell's varied granule proteases. When properly folded, a positively charged face forms on the surface of each granule protease that allows it to bind tightly to the proteoglycan's negatively charged glycosaminoglycans (7-9).The proteases packaged in the secretory granules of mouse MCs include carboxypeptidase A3 (CPA3), granzyme B, cathepsin G, neuropsin, transmembrane tryptase/tryptase ␥/protease serine member S (Prss) 31, mouse MC protease (mMCP) 1-10, and mMCP-11/Prss34. Definitive evidence of the importance of heparin-containing serglycin proteoglycans in the packaging of specific cassettes of proteases in the MC's granules came with the characterization of N-deacetylase/Nsulfotransferase-2 (NDST-2)-null mice (10, 11). NDST-2 is essential for heparin biosynthesis and is preferentially expressed in MCs. Because of the loss of fully sulfated heparin, the MCs in the skin and peritoneal cavities of NDST-2-null mice store reduced amounts of mMCP-6 and almost no CPA3, mMCP-4, and mMCP-5, even though all four MC-restricted protease genes are transcribed at normal rates. The accumulated data led to the current view that serglycin proteoglycans are essential for the post-translational processing and granule accumulation of most, if not all, proteases stor...

Section: Mc-restricted Granule Proteases and Proteoglycansmentioning

confidence: 99%

Section: Mc-restricted Granule Proteases and Proteoglycansmentioning

confidence: 99%

Mast Cell-restricted Tryptases: Structure and Function in Inflammation and Pathogen Defense

McNeil

Adachi

Stevens

2007

“…Studies using recombinant human pro-␣-chymase suggest that DPPI can activate prochymase in vitro by removing the activation dipeptide (6). Tryptases also are a diverse group of structurally related proteases that include ␣, ␤, and ␥ isoforms in humans (12,13) and two or three tryptases (mMCP-6, mMCP-7, and transmembrane/␥) in mice (14). MMCP-7 is not expressed in C57BL/6 mice, and thus, its expression is strain-dependent (15).…”

mentioning

confidence: 99%

Dipeptidyl Peptidase I Is Essential for Activation of Mast Cell Chymases, but Not Tryptases, in Mice

Wolters

Pham²,

Muilenburg

et al. 2001

Self Cite

188

167

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI ؉/؉ and DPPI ؊/؊ mice. We find that DPPI ؊/؊ mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI ؊/؊ mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI ؊/؊ BMMCs is 25% that of DPPI ؉/؊ BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.Granule-associated serine proteases of cytotoxic lymphocytes, neutrophils, and mast cells (i.e. granzymes, elastase, cathepsin G, and chymases) are structurally related (1). They have a two-amino acid propeptide (also referred to as the "activation dipeptide") and an isoleucine at the NH 2 terminus of the activated enzyme (2). The activation dipeptide maintains the protease in an inactive state. After removal of the dipeptide, the new NH 2 -terminal isoleucine moves from its position on the surface of the protease to a region in the interior of the enzyme, where it interacts with an aspartate residue near the catalytic center (3). These movements are thought to change the enzyme substrate binding cleft and catalytic apparatus to a catalytically competent conformation.Initial studies investigating the activation of the granuleassociated serine proteases focused on the cysteine protease dipeptidyl peptidase I (DPPI), 1 also known as cathepsin C (2, 4 -8). DPPI was a logical candidate activator of these proteases because it is found in the same cells and is a promiscuous exopeptidase that hydrolyzes most NH 2 -terminal dipeptides (9). Studies using the DPPI inhibitor Gly-Phe-diazomethyl ketone reported that DPPI inhibition reduces granzyme A, neutrophil elastase, and cathepsin G activity in the cells (2). However, because the inhibitor is not entirely specific for DPPI and does not eliminate the activity of the proteases in question, the possibility remained that another protease was responsible for their activation. This possibility was tested in a DPPI knockout mouse (10). Cytotoxic lymphocytes of these animals produce normal amounts of granzymes A and B, but both enzymes are inactive and present in their pro-forms. These findings suggest that DPPI is the sole activator of granzymes A and B in vivo...

“…All populations of MCs examined to date store in their secretory granules various combinations of carboxypeptidase A, chymases, and tryptases ionically bound to serglycin proteoglycans that contain either heparin or highly sulfated chondroitin glycosaminoglycans. Human MCs express at least five distinct tryptases, and the cDNAs and genes have been isolated that encode human tryptases ␣, ␤I, ␤II, and ␤III, as well as a less homologous transmembrane tryptase (5)(6)(7)(8)(9)(10). Mapping and sequencing analysis of the region of human chromosome 16, where these genes reside, has revealed the presence of additional genes in the family (9,11), but it remains to be determined whether or not any of these genes encode enzymatically active proteases.…”

mentioning

confidence: 99%

Evaluation of the Substrate Specificity of Human Mast Cell Tryptase βI and Demonstration of Its Importance in Bacterial Infections of the Lung

Huang¹,

Sanctis²,

O’Brien³

et al. 2001

139

132

Human pulmonary mast cells (MCs) express tryptases ␣ and ␤I, and both granule serine proteases are exocytosed during inflammatory events. Recombinant forms of these tryptases were generated for the first time to evaluate their substrate specificities at the biochemical level and then to address their physiologic roles in pulmonary inflammation. Analysis of a tryptase-specific, phage display peptide library revealed that tryptase ␤I prefers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase ␤I was unable to activate cultured cells that express different types of protease-activated receptors, the numbers of neutrophils increased >100-fold when enzymatically active tryptase ␤I was instilled into the lungs of mice. In contrast, the numbers of lymphocytes and eosinophils in the airspaces did not change significantly. More important, the tryptase ␤I-treated mice exhibited normal airway responsiveness. Neutrophils did not extravasate into the lungs of tryptase ␣-treated mice. Thus, this is the first study to demonstrate that the two nearly identical human MC tryptases are functionally distinct in vivo. When MCdeficient W/W v mice were given enzymatically active tryptase ␤I or its inactive zymogen before pulmonary infection with Klebsiella pneumoniae, tryptase ␤I-treated W/W v mice had fewer viable bacteria in their lungs relative to zymogen-treated W/W v mice. Because neutrophils are required to combat bacterial infections, human tryptase ␤I plays a critical role in the antibacterial host defenses of the lung by recruiting neutrophils in a manner that does not alter airway reactivity.