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Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2–5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6–10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology. [ABSTRACT FROM AUTHOR]

Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells, and natural killer (NK) cells store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can account for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin-related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, intestinal parasite expulsion, killing of bacteria and fungi, induction, mobilization, or degradation of cytokines, and the degradation of connective tissue components. A very broad spectrum of primary cleavage specificities has also been observed, including chymase, tryptase, asp-ase, elastase, and met-ase specificities, which highlights the large flexibility in the active site of these proteases. Mast cells primarily express chymases and tryptases with chymotryptic or tryptic primary cleavage specificities, respectively. Neutrophils have several enzymes with chymase, elastase, and tryptase specificities. T cells and NK cells express between 5 and 14 different granzymes, depending on the species, and these enzymes have tryptase, asp-ase, chymase, and met-ase specificities. This review focuses on the appearance of these proteases during vertebrate evolution, their primary and extended cleavage specificities, and their potential in vivo substrates. The in vivo substrates and functions are a particular challenging issue because several of these enzymes have a relatively broad specificity and may therefore cleave a wide range of different substrates. [ABSTRACT FROM AUTHOR]

Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use. [ABSTRACT FROM PUBLISHER]

Human chymase (HC) constitutes a major granule protease in one of the two human mast cell (MC) types. The main biological role of this haematopoietic serine protease is probably not yet known, although it has been implicated in a large number of functions. Dogs, like humans, have only one chymase. This enzyme is closely related to its human homologue, and the MC subtypes of human and dog appear to be similar as well. Therefore, the functions of the dog chymase (DC) may closely reflect the functions of the HC. Moreover, dogs may serve as good models for studies of human MC functions and MC-related diseases. To reveal functional similarities and differences between the DC and HC, we have determined the extended cleavage specificity of the DC by substrate phage display. This method allows the simultaneous permutation of primed and unprimed substrate positions. The DC was found to have very similar preferences to its human counterpart for substrate positions P1, P3, P4 and P3′, whereas their preferences differ at positions P2, P1′ and P2′. Therefore, the HC and DC may have co-evolved with a substrate where positions P1, P3, P4 and P3′ are conserved between dogs and humans, whereas positions P2 and P1′ are not and P2′differs to a minor extent. The differences observed between these two enzymes suggest that results obtained from dog models cannot be directly extrapolated to human clinical settings but need to be evaluated carefully concerning potential differences in substrate preferences. [ABSTRACT FROM PUBLISHER]

Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2′. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2′ of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 → Gln and Lys192 → Met) and the combined double mutant revealed an altered specificity for P2′ amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2′ was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2′. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2′ amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes. [ABSTRACT FROM AUTHOR]

Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species. [ABSTRACT FROM AUTHOR]

Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are inflammatory cells primarily found in tissues in close contact with the external environment, such as the skin and the intestinal mucosa. They store large amounts of active components in cytoplasmic granules, ready for rapid release. The major protein content of these granules is proteases, which can account for up to 35 % of the total cellular protein. Depending on their primary cleavage specificity, they can generally be subdivided into chymases and tryptases. Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended chymotrypsin-like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic enzymes have been conserved, both in structure and extended cleavage specificity, for more than 200 million years, indicating major functions in MC-dependent physiological processes. We have also studied a third closely related protease, originating from the same chymase locus whose cleavage specificity is closely related to the apoptosis-inducing protease from cytotoxic T cells, granzyme B. The presence of both a chymase and granzyme B in all studied mammals indicates that these two proteases bordering the locus are the founding members of this locus. [ABSTRACT FROM AUTHOR]

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit β-chymase and a guinea pig α-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species. [ABSTRACT FROM AUTHOR]

Abstract: In rat and mouse the phylogenetic homologues of the human mast cell α-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate α-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell β-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1′ and P3′. The two enzymes differ clearly only in one position, the P2′ residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2′ of known substrates for the human chymase. Overall, these two rodent β-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases. [Copyright &y& Elsevier]

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna , granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species. • The analysis of the cleavage specificity of two Zebra mbuna granzyme A/K homologs is presented. • One of them is a classical tryptase similar to the mammalian counterparts. • The other enzyme is a tyrosine specific chymase. • GranzymeA/K members with chymotryptic specificity has only been observed in Cichlids. • This chymase may be an evolutionary adaptation to a lack of a classical mast cell chymase in fishes. [ABSTRACT FROM AUTHOR]

Abstract Serine proteases constitute the major protein content of mammalian mast cell granules and the selectivity for substrates by these proteases is of major importance for the role of mast cells in immunity. In order to address this subject, we present here the extended cleavage specificity of sheep mast cell protease-2 (MCP2), a chymotrypsin-type serine protease. Comparison of the extended specificity results to a panel of mammalian mast cell chymases show, in almost all aspects, the same cleavage characteristics. This includes preference for aromatic residues (Phe, Tyr, Trp) in the P1 position of substrates and a preference for aliphatic residues in most other substrate positions around the cleavage site. MCP2 also cleaved, albeit relatively low efficiency, after Leu in the P1 position. In contrast to the human, mouse, hamster and opossum chymases that show a relatively strong preference for negatively charged amino acids in the P2′position, the sheep MCP2, however, lacked that preference. Therefore, together with the rat chymase (rMCP1), sheep MCP2 can be grouped to a small subfamily of mammalian chymases that show fairly unspecific preference in the P2′position. In summary, the results here support the view of a strong evolutionary conservation of a potent chymotrypsin-type protease as a key feature of mammalian mast cells. Highlights • A chymotryptic enzyme is a key characteristic of mammalian mast cells. • The sheep MCP2 belongs to a small subfamily of chymases without preference for negative amino acids in the P2′position. • Serine proteases are the most abundant granule constituents of mammalian mast cells. • A number of potential physiological functions have been identified for these mast cell proteases. [ABSTRACT FROM AUTHOR]

Abstract: Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the β-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1′ distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2′ aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1′ and P2′ positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice. [Copyright &y& Elsevier]

Abstract: Serine proteases are major granule constituents of mast cells, neutrophils, T cells and NK cells. The genes encoding these proteases are arranged in different loci. The mast cell chymase locus e.g. comprises at least one α-chymase, one cathepsin G, and two granzyme genes in almost all mammalian species investigated. However, in the gray, short-tailed opossum (Monodelphis domestica) this locus contains only two genes. Phylogenetic analyses place one of them clearly with the α-chymases, whereas the other gene is equally related to cathepsin G and the granzymes. To study the function of opossum chymase, and to explore the evolutionary origin of mast cell chymases, we have analyzed the cleavage specificity of this enzyme. The protease was expressed in mammalian cells and the extended substrate specificity was determined using a randomized phage-displayed nonapeptide library. A strong preference for the aromatic amino acids Trp over Phe and Tyr in the P1 position was observed. This is in contrast to human chymase and mouse mast cell protease-4, which prefer Phe over Tyr and Trp in this position. However, in most other positions this enzyme shows amino acid preferences very similar to human chymase and mouse mast cell protease-4, i.e. aliphatic amino acids in positions P4, P3, P2 and P1′, and acidic amino acids (Glu and Asp) in the P2′ position. The overall specificity of MC chymase thereby seems to have been conserved over almost 200 million years of mammalian evolution, indicating a strong selective pressure in maintaining this specificity and an important role for these enzymes in mast cell biology. [Copyright &y& Elsevier]