Chymase Locus Research Articles

Two granzyme A/K homologs in Zebra mbuna have different specificities, one classical tryptase and one with chymase activity

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.

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway.

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.

Extended cleavage specificity of a Chinese alligator granzyme B homologue, a strict Glu-ase in contrast to the mammalian Asp-ases

Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates.

The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases.

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.

Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity.

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.

Extended Cleavage Specificity of the Rat Vascular Chymase, a Potential Blood Pressure Regulating Enzyme Expressed by Rat Vascular Smooth Muscle Cells

Serine proteases constitute the major protein content of the cytoplasmic granules of several hematopoietic cell lineages. These proteases are encoded from four different loci in mammals. One of these loci, the chymase locus, has in rats experienced a massive expansion in the number of functional genes. The human chymase locus encodes 4 proteases, whereas the corresponding locus in rats contains 28 such genes. One of these new genes has changed tissue specificity and has been found to be expressed primarily in vascular smooth muscle cells, and therefore been named rat vascular chymase (RVC). This β-chymase has been claimed to be a potent angiotensin-converting enzyme by cleaving angiotensin (Ang) I into Ang II and thereby having the potential to regulate blood pressure. To further characterize this enzyme, we have used substrate phage display and a panel of recombinant substrates to obtain a detailed quantitative view of its extended cleavage specificity. RVC was found to show a strong preference for Phe and Tyr in the P1 position, but also to accept Leu and Trp in this position. A strong preference for Ser or Arg in the P1’ position, just C-terminally of the cleavage site, and a preference for aliphatic amino acids in most other positions surrounding the cleavage site was also seen. Interesting also was a relatively strict preference for Gly in positions P3’ and P4’. RVC thereby shares similarity in its specificity to the mouse mucosal mast cell chymase mMCP-1, which efficiently converts Ang I to Ang II. This similarity adds support for the role of β-chymases as potent angiotensin converters in rodents, as their α-chymases, which have the capacity to efficiently convert Ang I into Ang II in other mammalian lineages, have become elastases. However, interestingly we found that RVC cleaved both after Arg2 and Phe8 in Ang I. Furthermore this cleavage was more than two hundred times less efficient than the consensus site obtained from the phage display analysis, indicating that RVC has a very low ability to cleave Ang I, raising serious doubts about its role in Ang I conversion.

Extended cleavage specificities of two mast cell chymase-related proteases and one granzyme B-like protease from the platypus, a monotreme.

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.

Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution.

Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus.

Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution.

Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

Extended substrate specificity of opossum chymase—Implications for the origin of mast cell chymases

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.

Expression profile of novel members of the rat mast cell protease (rMCP)-2 and (rMCP)-8 families, and functional analyses of mouse mast cell protease (mMCP)-8

Four hematopoietic serine proteases are common to the mast cell chymase locus of all analyzed mammals: alpha-chymase, cathepsin G, granzyme B, and granzyme C/H. Apart from these common genes, the mouse and rat loci hold additional granzyme-, beta-chymase-, and Mcpt8-like genes. To better understand the functional consequences of these additional enzymes and to be able to compare human and rodent immune functions, we have analyzed the expression of novel beta-chymase- and Mcpt8-like genes in the rat. Four novel genes, i.e., Mcpt2-rs2a, Mcpt2-rs2c, Mcpt8-rs1, and Mcpt8-rs4 were transcribed in tissues holding mucosal mast cells (MMC), where also the classical MMC protease Mcpt2 was expressed. We also found transcripts of rat vascular chymase (rVch) in some of these tissues. RVch is a beta-chymase that converts angiotensin I, like the human chymase. Rat MMC may therefore have similar angiotensin-converting properties as chymase-positive human mast cells, although these are mostly regarded the counterpart of rat connective tissue mast cells. The human mast cells that are considered the counterpart of rat MMC express, however, only tryptase, whereas rat MMC express various proteases, but no tryptase. We further studied the proteolytic activity of mMCP-8 as a first representative for the Mcpt8-subfamily. Based on sequence comparison and molecular modeling, mMCP-8 may prefer aspartic acid in substrate P1 position. However, we could not detect hydrolysis of chromogenic substrates or phage-displayed random nonapeptides despite numerous trials. On the other hand, we have obtained evidence that the function of the Mcpt8-like proteases depends on proteolytic activity. Namely, the expression of the only Mcpt8-family member with a mutation in the catalytic triad, Mcpt8-rs3, was strongly reduced. Thus, the substrate specificity of mMCP-8 may be too narrow to be detected with the employed methods, or the enzyme may require a substrate conformation that is not provided by the analyzed peptides.

Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution

The acidic granules of natural killer (NK) cells, T cells, mast cells, and neutrophils store large amounts of serine proteases. Functionally, these proteases are involved, e.g., in the induction of apoptosis, the recruitment of inflammatory cells, and the remodeling of extra-cellular matrix. Among the granule proteases are the phylogenetically related mast cell chymases, neutrophil cathepsin G, and T-cell granzymes (Gzm B to H and Gzm N), which share the characteristic absence of a Cys(191)-Cys(220) bridge. The genes of these proteases are clustered in one locus, the mast cell chymase locus, in all previously investigated mammals. In this paper, we present a detailed analysis of the chymase locus in cattle (Bos taurus) and opossum (Monodelphis domestica). The gained information delineates the evolution of the chymase locus over more than 200 million years. Surprisingly, the cattle chymase locus contains two alpha-chymase and two cathepsin G genes where all other studied chymase loci have single genes. Moreover, the cattle locus holds at least four genes for duodenases, which are not found in other chymase loci. Interestingly, duodenases seem to have digestive rather than immune functions. In opossum, on the other hand, only two chymase locus-related genes have been identified. These two genes are not arranged in one locus, but appear to have been separated by a marsupial-specific chromosomal rearrangement. Phylogenetic analyses place one of the opossum genes firmly with mast cell alpha-chymases, which indicates that the alpha-chymase had already evolved as a separate, clearly identifiable gene before the separation of marsupials and placental mammals. In contrast, the second gene in opossum is positioned phylogenetically between granzymes, cathepsin G, and the duodenases. These genes, therefore, probably evolved as separate subfamilies after the separation of placental mammals from marsupials. In platypus, only one chymase locus-like sequence could be identified. This previously published "granzyme" does not cluster clearly with any of the chymase locus gene families, but shares the absence of the Cys(191)-Cys(220) bridge with the other chymase locus proteases. These findings indicate that all chymase locus genes are derived from a single ancestor that was present more than 200 million years ago.

Rapid lineage-specific diversification of the mast cell chymase locus during mammalian evolution

Serine proteases constitute the major protein granule content of cells of several hematopoietic cell lineages. A subgroup of these proteases, including the mast cell chymases, neutrophil cathepsin G, and T cell granzymes B to F and N, are in all investigated mammals encoded in one locus, the chymase locus. It is interesting to note that this locus has diversified greatly during the last 95 Myr of mammalian evolution. This divergence is exemplified by the presence of Mcpt8-related genes and multiple beta-chymases in the mouse and rat, which lack direct counterparts in primates and in seven functional granzyme genes in the mouse where the human locus has only two. To study the expansion of the locus during rodent evolution and to better understand the evolutionary origin of beta-chymases and the Mcpt8-family, we have performed a detailed analysis of the chymase locus of four mammalian species, i.e., human, dog, mouse, and rat. As a result, we report here a second chymase-like gene in dog, Cma2, which clusters with beta-chymases in phylogenetic analyses. This finding supports a duplication of the common ancestor for alpha- and beta-chymases before the major radiation of placental mammals, and a loss of the ancestral beta-chymase gene sometime during primate evolution. Moreover, we show that in the rat, the Mcpt8-family diversified relatively recently together with sequences related to the beta-chymase Mcpt2. Eight novel genes were identified in the duplication region, four of which are predicted to be functional. Duplications of rat granzyme B- and C-like sequences occurred seemingly independently within a similar time frame, but did not give rise to functional genes. Due to the duplications in rat and deletions in the carnivore/primate lineage, the rat chymase locus is approximately 15 and 9 times larger than its counterparts in dog and human, respectively. These findings illustrate the importance of gene duplications in conferring rapid changes in mammalian genomes.

Granzymes: a family of lymphocyte granule serine proteases.

Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.