Mast Cell Proteases
Introduction
Mast cells (MCs) originate from the bone marrow, circulate in the blood as immature precursors, and then migrate into various tissues in which they undergo terminal differentiation under influence of local growth factors, in particular stem cell factor (SCF) (Gurish and Boyce, 2002). MCs are widely distributed throughout the body with a particular preponderance at sites close to the exterior, for example, mucosal surfaces and skin. This anatomic distribution is clearly in line with the notion of the MC as an important player in first‐line defense toward external insults. For various general aspects of MC biology, the reader is referred to excellent review articles (Galli 2005a, Galli 2005b, Metcalfe 1997).
When MCs mature, they acquire numerous electron dense cytoplasmic granules, which can be released following an appropriate stimulus. Out of such stimuli, cross‐linking of IgE molecules bound to the high‐affinity IgE receptor, FcεRI, by polyvalent antigen is the most well characterized (Blank and Rivera, 2004). It has also been known for many years that MCs can be activated in response to several other types of stimuli, including anaphylatoxins C5a and C3a, neuropeptides (e.g., substance P), endothelin 1 (ET‐1), and engagement of Toll‐like receptors or FcγRIII (Galli et al., 2005b).
MCs are undoubtedly most well known for their harmful effects in connection with immediate hypersensitivity reactions (Yu et al., 2006), but MCs are also implicated in various additional disorders such as multiple sclerosis (Secor et al., 2000), arthritis (Lee et al., 2002a), bullous pemphigoid (Chen et al., 2001), congestive heart failure (Hara et al., 2002), and squamous carcinoma (Coussens et al., 1999). Although reports describing harmful effects of MCs dominate the literature, it is important to emphasize that MCs can also be beneficial for their host by contributing to the innate immune defense toward certain pathogens, including bacteria (Echtenacher 1996, Malaviya 1996), parasites (Ha et al., 1983), and even toward snake venom poisoning (Metz et al., 2006). Much of this knowledge is based on experiments using a mouse strain that lacks MCs (the W/Wv strain), as a result of a mutation in the SCF receptor, c‐kit. By comparing the response to a certain stimulus, for example an experimentally induced disease, between wild‐type and MC‐deficient mice, it has been possible to specifically pinpoint an involvement of the MC in a variety of pathological settings. Importantly however, the exact mechanism by which MCs contribute to the various pathological conditions listed above has in many cases not been clarified.
When MCs degranulate, they release a number of preformed components to the exterior. MC activation may also cause de novo synthesis and release of additional compounds, including PGD2, PGE2, LTB4, and LTC4 as well as cytokines (Galli 2005a, Galli 2005b, Metcalfe 1997). Out of the preformed MC mediators, histamine is by far the most thoroughly characterized in terms of biological function. In addition, it has been known for a long time that MC granules contain proteoglycans (PGs), that is, protein “cores” to which glycosaminoglycan (GAG) side chains are attached, and it is now well established that MCs also contain preformed cytokines, for example, TNF‐α (Gordon et al., 1990). Finally, MC granules contain a number of MC‐specific proteases: tryptases, chymases, and MC carboxypeptidase A (MC‐CPA). Tryptases and chymases belong to the serine protease class, while MC‐CPA is a zinc‐dependent metalloprotease. The designation of these proteases relates to their substrate specificities, with tryptases having trypsin‐like cleavage specificities, while chymases have chymotrypsin‐like specificities. MC‐CPA has acquired its designation through its specificity for cleaving proteins/peptides from their C‐terminal end. The term “MC proteases” usually refers to the chymases, tryptases, and CPA that are specifically expressed by MCs and stored within MC granules. However, in addition to these MC‐specific proteases, MCs may contain and secrete other proteases, for example, matrix metalloproteases (MMPs) (Baram et al., 2001), cathepsin D, C, and E (Dragonetti 2000, Henningsson 2005, Wolters 2000). In this chapter, the term “MC proteases” will refer to the tryptases, chymases, and CPA that are present as releasable compounds within MC secretory granules.
Mature MCs contain conspicuously large amounts of stored proteases. For example, it has been calculated that human skin MCs contain altogether ∼16 μg of tryptase and chymase per 106 cells (Schwartz et al., 1987). Thus, MC degranulation will result in release of very large quantities of proteases and it is therefore not far fetched to assume that they may profoundly affect any process in which MCs are involved and in which MC degranulation is a component. Indeed, a number of potential functions for various MC proteases have been outlined during the years. Noteworthy however, only in a few cases has any suggested function of a MC protease been confirmed in vivo, for example, in experiments taking advantage of genetically modified animals. In this chapter, the aim has been to summarize current knowledge of the MC proteases, including novel insights into their biological function.
Section snippets
Expression of MC Proteases
The specific expression patterns for the MC proteases vary considerably between species and also among different MC subclasses found within a certain species (Table 1). Moreover, the repertoire of expressed MC proteases is also a function of the degree of cellular differentiation and can be modified following, for example, the course of a disease. The phylogenetic relationships between the various MC tryptases and chymases are indicated in Figure 1, Figure 2, respectively. As indicated in Fig. 1
The chymase locus
The human MC α‐chymase gene (CMA1) is located on chromosome 14q11.2 at one end of a small cluster of four genes, covering ∼130 kb (Caughey et al., 1993b). This cluster contains the neutrophil‐specific cathepsin G gene (CTSG) 65 kb upstream of CMA1 and the T‐cell‐specific granzyme H and B genes (GZMH and GZMB) located 30 and 55 kb upstream of CTSG (Table 2; Fig. 3A). The transcriptional orientations of these genes are the same, and their overall genetic organization is conserved in rodents, dog,
MCs in evolution
MCs or MC‐like cells have been observed in essentially all vertebrates, as well as in some invertebrates, for example, clams (Ulrich and Boon, 2001) and tunicates (Cavalcante et al., 2002). The widespread presence of MCs indicates that they have indeed played an important beneficial role, also in early evolution. However, most research on MC‐derived proteases has been done in mammals. Therefore, it is still an open question whether MC proteases have an ancient origin or if they are restricted
Protein Organization and Processing
The MC proteases are all synthesized as preproenzymes, that is, with an N‐terminal signal peptide directing them into the ER lumen. Cleavage of the signal peptide then results in generation of proforms of the respective proteases, and subsequent cleavage of the propetide results in formation of mature enzyme. In no case has a proform of any MC protease been isolated from natural sources to allow N‐terminal sequencing and determination of the exact site for cleavage of the signal peptide in vivo
Chymase
The crystal structures have been solved both for rMCP‐2 (Remington et al., 1988) and human chymase (McGrath 1997, Pereira 1999; Fig. 4A). In addition, the structure of human prochymase was solved (Reiling et al., 2003). The three‐dimensional structure of human MC chymase revealed a high degree of similarity with pancreatic chymotrypsin, rMCP‐2, and cathepsin G. A striking feature of human chymase, but not of rMCP‐2, is the presence of a large number of basic amino acid residues (Lys/Arg) in two
Cleavage Specificity
The key to understanding the function of any protease is to identify its in vivo substrate(s). Important clues to this issue may come from determining the fine cleavage specificity of a protease, and then search for matching peptide sequences in potential target proteins/peptides. Information regarding the exact cleavage specificity of a protease can also be useful when constructing inhibitors of the protease. In the following, cleavage sites are designated by using the nomenclature of
Storage
Based on the colocalization of MC proteases with anionic heparin/CS PGs within granules, it was early suggested that these compounds are interacting (Schwartz and Austen, 1980). Evidence for such an interaction between PGs and MC proteases came when it was shown that degranulation of BMMCs caused release of complexes of PGs and serine proteases (Serafin et al., 1986) as well as MC‐CPA/PG complexes (Serafin et al., 1987). High concentrations of NaCl disrupted these complexes, demonstrating that
Substrates for MC Proteases
Given that the MC proteases, with certain exceptions, have a relatively broad cleavage specificity (Section 7), it would be expected that a large number of proteins/peptides could be potential substrates. Indeed, a multitude of proteins and peptides are known to be cleaved by MC proteases (Table 5, Table 6, Table 7). Many of these have been identified through incubation of the MC protease with the protein/peptide in a purified system. In other cases, substrates have been identified in cell
In Vivo Function
A multitude of potential biological functions have been ascribed to the various MC proteases (Table 9, Table 10). These functions have been inferred by different approaches. In many cases, the protease has been added to a cell culture system followed by recording of a response. In other approaches, purified protease has been administered into an experimental animal, followed by assessment of, for example, inflammatory parameters. MC protease functions have also been indicated by the correlation
Synthetic inhibitors
As a consequence of the multitude of pathological settings in which MC proteases have been implicated, much effort has been invested in generating potent and selective MC protease inhibitors. Some of these inhibitors are peptide based, as exemplified by the potent chymase inhibitor Z‐Ile‐Glu‐Pro‐Phe‐COOMe (Bastos et al., 1995). However, due to a requirement for orally available compounds with favorable pharmacokinetics, recently developed MC protease inhibitors are mainly nonpeptide based and
Summary and Future Perspectives
As is evident from this chapter, past research on various MC proteases has gathered large amounts of information regarding their structure, processing mechanisms, expression profiles, cleavage specificity, and potential in vivo substrates and functions. However, the critical questions remain in large parts unanswered, that is, what is their true biological function? Since the MC proteases are released during various inflammatory conditions, it is likely that they at least under certain
Acknowledgments
We are grateful to Stefan Knight for generating the images used in Fig. 4. The authors of this chapter receive support from the Swedish Research Council, The Swedish Cancer Foundation, King Gustaf V's 80th anniversary Fund, the Mizutani Foundation for Glycoscience, the Vårdal Foundation and Formas.
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