MATTIASK.ANDERSSON CleavageSpecificityofMastCellChymases 429 DigitalComprehensiveSummariesofUppsalaDissertationsfromtheFacultyofScienceandTechnology

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(1)Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 429. Cleavage Specificity of Mast Cell Chymases MATTIAS K. ANDERSSON. ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008. ISSN 1651-6214 ISBN 978-91-554-7190-3 urn:nbn:se:uu:diva-8714.

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(152) List of papers. This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Andersson M.K., Pemberton A.D., Miller H.R.P. and Hellman L. Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse-High specificity indicates high substrate selectivity. Molecular Immunology 2008; 45 (9): 2548-2558. II. Andersson M.K., Karlson U. and Hellman L. The extended cleavage specificity of the rodent E-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase. Molecular Immunology 2008; 45 (3): 766-775. III. Andersson M.K., Enoksson M., Gallwitz M. and Hellman L. The extended cleavage specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile. Submitted manuscript. IV. Andersson M.K. and Hellman L. Synergetic interactions of Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2´ of substrates. Manuscript. Reprints were made with permission of the publisher..

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(154) Contents. Introduction.....................................................................................................9 General overview .......................................................................................9 Mast cells..................................................................................................10 Mast cell heterogeneity........................................................................11 Mast cell mediators..............................................................................12 Mast cell activation..............................................................................15 Mast cells in vivo .................................................................................17 Serine proteases........................................................................................19 Catalytic mechanism............................................................................20 Chymotrypsin-like serine proteases.....................................................20 Mast cell chymases ..............................................................................22 Present investigations....................................................................................31 Aim...........................................................................................................31 Results and discussion..............................................................................31 Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse-High substrate specificity indicates high substrate selectivity (Paper I)...............................................................31 The extended cleavage specificity of the rodent E-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase (Paper II) ........................................................................34 The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile (Paper III) ............................................................................................35 Synergetic interactions of Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2´ of substrates (Paper IV) .......................................................................36 Concluding remarks .................................................................................37 Sammanfattning på svenska..........................................................................40 Acknowledgements.......................................................................................45 References.....................................................................................................48.

(155) Abbreviations. Ang ASM BMMC CLP CPA CTMC DFP DPPI ECM GAG GM-CSF Graspase HC IL LPS LTC4 MC MCP-1 MCT MCTC MIP MMC mMCP MMP Ni-NTA PAF PGD2 rMCP rVC SCF TLR TNF-D. Angiotensin Airway smooth muscle Bone marrow derived mast cell Cecal ligation and puncture Carboxypeptidase A Connective tissue mast cell Diisopropyl fluorophosphate Dipeptidyl peptidase Extracellular matrix Glucosaminoglycan Granulocyte-macrophage colony-stimulating factor Granule-associated serine protease Human chymase Interleukin Lipopolysaccharide Leukotriene C4 Mast cell Monocyte chemotactic protein-1 Tryptase positive mast cell Tryptase and chymase positive mast cell Macrophage inflammatory protein Mucosal mast cell Mouse mast cell protease Matrix metalloproteinase Nickel-nitrilotriacetic acid Platelet activating factor Prostaglandin D2 Rat mast cell protease Rat vascular chymase Stem cell factor Toll-like receptor Tumour necrosis factor-D.

(156) Introduction. General overview We are constantly exposed to microorganisms that are a natural part of our environment. They are found everywhere; on the ground, in the air, in the food we eat, on our skin and in our intestine. In order to adapt to a life in such an environment, we and other vertebrates have developed the immune system. Anatomical and physiological barriers like the skin, mucosa, and the low pH of the stomach, are normally sufficient to stop unwanted microbes from entering our tissues. However, occasionally they manage to pass the barriers and we need to fight the infection. Upon tissue damage or the presence of invading microbes, an inflammatory response is initiated. The release of inflammatory mediators leads to vasodilation and increased epithelial permeability, which allows an influx of leukocytes to the site of infection. Mast cells (MC) are one of the important cell types in the early inflammatory response. Phagocytic cells like monocytes, macrophages and neutrophils are recruited to the area of inflammation. They can internalize (phagocytose) the microbes and kill them within their phagosomes. All these early components are part of the innate immune system. Simultaneously, dendritic cells internalize microbes and migrate to lymphoid tissues to initiate an adaptive immune response, which include B and T lymphocytes. The adaptive response is slower than the innate response, but also very efficient. The lymphocytes express antigen receptors and specifically target the invading pathogens. Upon activation the B cells secrete antibodies towards extracellular antigens and the T cells can either regulate the immune response or kill altered cells, e.g. virus infected cells or tumour cells. The immune response results in clearance of microbes and development of easily reactivated memory cells (lymphocytes) that provide a fast response upon repeated infections by the same microbes. Unfortunately, sometimes the immunologic response is misdirected or reacts too strongly, which may lead to tissue damage and disease. Autoimmunity is an example of such reactions, where the immune system attacks our own cells and tissues. This can lead to pathological conditions like multiple sclerosis, Crohn´s disease or rheumatoid arthritis. Other examples are hypersensitivity reactions like allergies and asthma, where we become sensitized against common antigens often derived from food, pollen, insect venoms, animal hair and drugs. Common symptoms of allergies are runny eyes, in9.

(157) creased mucus production in the nose, sneezing and bronchial constriction. These symptoms affect the life quality of patients but are seldom lethal. Allergies and asthma are common conditions in western countries and the prevalence have increased rapidly during the last decades. MCs are key players in these conditions.. Mast cells MCs are found primarily within the connective tissue and mucosal surfaces of the respiratory and gastrointestinal tracts, in the peritoneum and in the skin, often close to blood vessels and nerves. MCs have been described as the gatekeepers of the immune defence due to their tissue location at sites of microbial entry and their ability to immediately react against invading pathogens and recruit other immune cells by inducing local inflammation. Upon stimulation MCs can react instantly by releasing prestored mediators, including histamine, proteoglycans and neutral proteases. These mediators are stored in the abundant and characteristic cytoplasmic granules of MCs and their potent inflammatory effects are seen during allergic reactions. Although not granule stored, prostaglandins and leukotrienes are also produced and released during this early response. In addition, MCs also provide a delayed response within hours of activation by de novo production and secretion of cytokines. The prestored and the newly synthesized mediators initiate the inflammatory response (Fig. 1).. Figure 1. Upon activation, mast cells release prestored and newly produced mediators. These mediators will initiate an inflammatory response.. 10.

(158) The majority of all proteins stored in the secretory granules are serine proteases belonging to the tryptase and chymase subfamilies. Due to their high abundance they are thought to be important for MC function. However their biological role is still not entirely clarified. This thesis will focus on increasing the understanding of chymases in MC biology, by determining the cleavage specificity of members of the chymase family.. Mast cell heterogeneity MCs are not a homogenous population of cells. Based on differences in the content of granules, at least two subpopulations of MCs with different staining properties have been identified. In rodents the two main populations are the connective tissue MCs (CTMC) and the mucosal MCs (MMC) (1). As their names indicate, they are found at different tissue locations. CTMCs are found in the skin and peritoneum and MMCs primarily beneath mucosal surfaces of the respiratory and gastrointestinal tracts. Apart from tissue location, they differ in terms of protease, proteoglycan and histamine content. CTMCs express tryptase, chymase, carboxypeptidase A (CPA), heparin and high amounts of histamine (Table 1). MMCs express chymase, chondroitin sulphate and low amounts of histamine. Using immunohistochemical techniques, the two MC subtypes can easily be distinguished by staining of heparin, since it is present in CTMCs but not in MMCs. Human MCs cannot be distinguished using the same immunohistochemical methods, since both subtypes express heparin (2). Instead the human MCs are subdivided based on their protease content and tissue distribution. The CTMC-like cells in humans are named MCTC due to their tryptase and chymase positive phenotype, however they also express CPA. The MMClike cells in humans are only tryptase positive and are therefore called MCT (3). MCTC and MCT are distributed in the same tissues, but in different proportions. MCTC is the dominant subtype in skin, connective tissues and esophageal submucosa, while the majority of MCs in lung are of the MCT-type. In the bowel, the distribution is more equal with a majority of MCT cells in bowel mucosa and more of MCTC cells in the submucosa (4). The functional difference between CTMCs and MMCs are not fully known, but they seem to have distinct roles. MMCs proliferate during parasitic infections and are thought to be involved in the expulsion of certain parasites (5). In contrast CTMCs change only minimally in numbers during infections. They are resident cells of the connective tissue and probably involved in connective tissue remodeling. MCs are derived from the haematopoietic stem cells in the bone marrow and are released into the circulation as CD34+/CD13+/c-kit+ precursors in humans or Thy-1lo c-kithi precursors in mice (6-8). They circulate as precursors and complete their maturation once they reach the peripheral tissues. The main cytokines that promote MC proliferation and differentiation are the 11.

(159) c-kit ligand, also known as stem cell factor (SCF), and interleukin (IL)-3 (9). The development of MCs into either CTMCs or MMCs is thought to be dependent on micro-environmental factors. Bone marrow derived MCs (BMMC) can in vitro be stimulated with different cytokines and differentiate into either MMC or CTMC like phenotypes. SCF and IL-3 stimulates mouse BMMCs to express the CTMC proteases mouse mast cell protease (mMCP)4, mMCP-5, mMCP-6 and CPA (10). Expression of the mouse MMC specific proteases mMCP-1 and mMCP-2 are in BMMC cultures, stimulated by TGF-E1 and IL-9 (11, 12).. Mast cell mediators MC granules store a number of important mediators that are released upon activation. Simultaneously both cell types also start a de novo production of other mediators (Table 1). The prestored mediators include histamine, proteoglycans and neutral proteases and the de novo produced mediators include cytokines, prostaglandins and leukotrienes. Granule stored mediators Histamine Histamine is a biogenic amine that is found in most tissues. However, high levels are found primarily in MCs and basophils (13, 14). Histamine is formed by decarboxylation of histidine by the enzyme histidine decarboxylase. This biogenic amine act on four different histamine receptors, named H1-4. Upon binding these receptors, histamine causes constriction of bronchial and intestinal smooth muscle, increases vascular permeability, secretion of gastric acid, and mediates neurotransmission. The H4 receptor is highly expressed in bone marrow and on leukocytes, which mediates chemotaxis of MCs (15). A mouse strain lacking histidine decarboxylase was shown to exhibit a decreased number of MCs and reduced granular content (16). Proteoglycans Another important constituent of MC granules are the proteoglycans. Proteoglycans are large, negatively charged glycoproteins that are heavily glycosylated. They consist of a core protein with covalently linked and unbranched polysaccharide side chains called glycosaminoglycans (GAG). Two different proteoglycans are stored in MC granules, heparin and chondroitin sulphate (17). In rodents, heparin is stored in CTMCs and chondroitin sulphate is found in MMCs, whereas human MCTC and MCT contain both heparin and chondroitin sulphate. The core protein found in both of these proteoglycans, serglycin, is a protein containing a centrally located sequence of repeated serines and glycines. The GAGs are attached to serine residues 12.

(160) via a linker consisting of one xylose, two galactose residues and one glucuronic acid. The GAGs consist of repetitive disaccharides, which in heparin are built up by glucuronic acid and N-acetyl-glucosamine. In chondroitin sulphate the core disaccharides consist instead of glucuronic acid and Nacetyl-galactosamine. Furthermore, the GAGs are modified in several steps (18). During the first modification step, the GAGs are sulfated, which adds negative charges to the proteoglycans. The negative charge of heparin and chondroitin sulphate is an important characteristic allowing the proteoglycans to function as a storage matrix for histamine and the positively charged proteases in the MC granules (19). The importance of heparin for storage of granule mediators has been demonstrated in mice with a targeted disruption of the heparin-sulfating enzyme N-deacetylase/N-sulfotransferase (NDST)-2 (20, 21). Table 1. Mediators found in rat, mouse and human mucosal and connective tissue mast cells. MMC1 Content Rat. Mouse. CS A, di B, E + PGD2lo LTC4 Chymase rMCP-8 fam.. CS A, E + PGD2lo LTC4 Chymase. Proteoglycan Histamine Prostaglandin Leukotriene Metalloprotease Serine protease. CTMC Human (MCT) Heparin CS A, E + PGD2 LTC4 -. Human (MCTC) Heparin Heparin Heparin CS E CS E CS E ++ ++ + PGD2 PGD2 PGD2 LTC4lo LTC4lo CPA CPA CPA Chymase Chymase Chymase Tryptase Tryptase Tryptase Rat. Mouse. Tryptase Data from (22-26) 1 Abbreviations: MMC, mucosal mast cell; CTMC, connective tissue mast cell; MCT, tryptase positive mast cell; MCTC, tryptase and chymase positive mast cell; CS, chondroitin sulphate; PGD2, prostaglandin D2; LTC4, leukotriene C4; CPA, carboxypeptidase A; rMCP-8 fam., rat mast cell protease-8 family. Neutral proteases The majority of proteins that are stored in the MC granules are neutral proteases, e.g. chymotrypsin-like or trypsin-like serine proteases or a MC specific carboxypeptidase A (CPA). Based on their primary cleavage specificity the serine proteases that are expressed by MCs have been classified into different subfamilies. The two main subfamilies expressed by MCs are the chymases and the tryptases, which have chymotrypsin-like and trypsin-like cleavage specificities, respectively. These serine proteases and CPA are stored as active enzymes in close connection to proteoglycans. However, due to the acidic environment within the granules, these enzymes are here unable to catalyze protein hydrolysis. They are called neutral proteases since they reach optimal activity at neutral pH. Rodent CTMCs and human MCTC express CPA, chymase and tryptase. Human MCT only express tryptase while 13.

(161) rodent MMCs express chymase and members of the rat mast cell protease (rMCP) -8 family. CPA is a Zn2+-dependent metalloprotease with exopeptidase activity. This means that it releases C-terminal amino acids of peptides, preferably at Cterminal aromatic or aliphatic amino acids (27, 28). The positively charged CPA is bound to heparin in the granules and consequently, the storage of CPA is impaired in mice lacking heparin (20). CPA has been suggested to act in synergy with chymase since CPA and chymase are associated with the same proteoglycan complexes, which are separated from the tryptase- proteoglycan complexes (29). CPA and chymase have furthermore been shown to act on mutual substrates, where chymase creates substrate with C-terminal aromatic residues, suitable for CPA cleavage (30, 31). The tryptases are trypsin-like serine proteases, which cleave substrates after the basic amino acids Arg and Lys. They are active as homotetramers, stabilized by heparin proteoglycan and other negatively charged polymers (32-35). If heparin is absent, the tetramers dissociate into inactive monomers (34). The interactions between the monomers are non-covalent, and they are linked in a way that directs the active sites towards each other (36). The monomers thus form a ring-shaped homotetramer with a central pore where the active sites face inwards. This structure restricts the entry of large protein substrates and also enhances the resistance towards many inhibitors. However, in vitro evidence also suggests that active monomers exist (37, 38). Four groups of tryptase are found in humans, D (subtypes I and II), E (I, II and III), J (I and II) and G (I and II) (39). The E-tryptases are found in MC granules as prestored active enzymes in complex with heparin. The three subtypes are very similar to each other and together they are thought to provide most tryptase activity in human MCs. While human tryptases are expressed by all MCs, the rodent tryptases are restricted to CTMCs. Among the murine tryptases, mMCP-6 is most similar to human E-tryptases both in primary amino acid sequence and cleavage specificity (40). mMCP-7 is similar to mMCP-6 and, like mMCP-6, also stored in CTMC granules. Tryptases have been attributed various functions and many of them are similar to chymases (39). The chymases will be discussed in detail in a later section. De novo synthesized mediators Eicosanoids Upon activation, MCs do not only degranulate, but also start to synthesize and release eicosanoids and cytokines. Eicosanoids are lipid mediators derived from arachidonic acids that are liberated from the cell membrane. The name eicosanoids refers to the 20 carbon atoms forming the backbone of these mediators. The arachidonic acid metabolites produced in MCs are prostaglandin D2 (PGD2), leukotriene C4 (LTC4) and platelet activating fac14.

(162) tor (PAF). PGD2 is synthesized through the cyclooxygenase pathway and is released within minutes after MC activation. PGD2 released from MCs leads to vasodilation and bronchoconstriction (41). LTC4 is synthesized via the lipoxygenase pathway and act on cys-LTreceptors. The interaction cause prolonged bronchoconstriction, enhanced vascular permeability and increased mucus production. These are effects commonly seen in asthmatic patients (25). PAF is derived from its precursor acyl-PAF by phospholipase A2 activity and acts as a potent proinflammatory agent. It induces vasodilation, increases vascular permeability, and recruits neutrophils, monocytes and eosinophils to the site of inflammation. Cytokines MCs also produce a number of cytokines, including tumour necrosis factor (TNF)-D, granulocyte-macrophage colony-stimulating factor (GM-CSF), SCF, IL-3, -4, -5, -6, -8, -10, -13, -14 and -16 (42). IL-4 is important for differentiation of TH-cells into the TH2 phenotype, and IL-4 together with IL13 promotes IgE production by B-cells. The expression of these cytokines thus enhance the MC mediated immune response, e.g. towards parasites. IL3, -4, -5, -13, TNF-D and GM-CSF induce chemotaxis, migration, activation and prolonged survival of human eosinophils and thereby enhance their antiparasitic effects (43). The multifunctional cytokine TNF-D is not only synthesized upon MC activation, but also stored as a preformed mediator in the secretory granules (44, 45). TNF-D is important in host defence against bacteria (46). It enhances leukocyte recruitment by inducing endothelial expression of Eselectin (ELAM-1) and ICAM-1 and potentiates bactericidal and fungicidal activity of phagocytes (47-51). The in vivo role of MC derived TNF-D will be discussed in a later section. In addition, MCs express chemokines including IL-8, eotaxin and monocyte chemotactic protein (MCP)-1 that are important for neutrophil, eosinophil, monocyte and T-cell chemotaxis.. Mast cell activation Fc receptor-mediated activation MCs can become activated by a variety of agents (Fig. 2). The most well studied pathway involves binding of IgE antibodies. IgE binds to high affinity receptors, FcHRI, which are found on the cell membrane of MCs. These receptors become cross linked upon IgE binding to multivalent antigens (or allergens) (52, 53). The cross linked receptors initiate intracellular signaling events and lead to degranulation of MCs which then causes the well known effects of hypersensitivity reactions (54). 15.

(163) MCs can also be activated by IgG antibodies, which bind to FcJRs on the MC surface. FcJRI and III share the J-chain with FcHRI and thus have common intracellular signaling pathways. The high affinity receptor FcJRI is expressed on human MCs (55), and cross-linking of these receptors lead to degranulation (56). The low affinity receptor FcJRIII has been detected on murine CTMCs, whereas FcJRI has not (57). Toll-like receptor-mediated activation Toll-like receptors (TLR) are a family of receptors that directly binds to conserved structures of microbes, such as lipopolysaccharide (LPS), peptidoglycan and double stranded RNA (58). A number of TLRs are expressed on MCs. TLR-2, -3, -4, -6, -7, -8 and -9 have been detected on rodent MCs (58, 59). Engagement of TLR-4 by LPS has been shown to induce secretion of inflammatory cytokines by MCs and the recruitment of neutrophils (60), while TLR-2 mediated activation of MCs can either lead to cytokine secretion or degranulation, depending on the stimulating agent (61).. Figure 2. Mast cells can be activated upon engagement of different receptors. Abbreviations: FcR, Fc receptor; TLR, toll-like receptor; CR, complement receptor; LPS, lipopolysaccharide; dsRNA double stranded RNA. Complement receptor-mediated activation Activation of the complement cascade results in the production of several low molecular weight cleavage products with MC activating properties. The anaphylotoxins C3a and C5a can interact with specific receptors on the surface of MCs and induce the release of histamine (62). In humans the C5a receptor, but no receptor for C3-fragments, are found on MCTC cells (63). However, in patients with systemic mastocytosis, receptors for both C5a and C3a are expressed (64). 16.

(164) Other mast cell activators In addition to the Fc, complement and Toll-like receptor ligands, a number of additional mediators have been found to activate MCs. For example, small peptides like substance P, endothelin, E-chemokines macrophage inflammatory protein (MIP)-1D and MCP-1 and degradation products of fibrinogen and fibronectin have all been shown to cause MC activation (65-69).. Mast cells in vivo MCs are well known for their pathological role in allergic reactions, but what is their beneficial role in our immune system? Since the MCs are located at the outer linings of the body, i.e. the skin and the mucosa of the GI tract and airways, they are among the first cells that come in contact with invading organisms. Immediately upon encounter with the microbe they rapidly release their inflammatory mediators. MCs have been conserved throughout evolution, which strongly suggests important functions for MCs in the immune system of the host (70, 71). However, the function of MCs is not yet fully understood. As a valuable tool to investigate the role of MCs in vivo, mouse strains lacking MCs have been studied. The most well studied model is the W/Wv-strain that is deficient in the c-kit receptor, which is essential for the development of MCs (72). This strain shows additional abnormalities, such as macrocytic anaemia, sterility and lack of the cells of Cajal and melanocytes (73). Another strain, called the W-sash (W-sh/W-sh) mice, has also been developed (74). This strain has a different mutation in the c-kit receptor, and is neither anaemic nor sterile and is proposed to be a better model for MC in vivo studies. However, up to this date most studies are based on the W/Wv-strain. These two MC deficient mice strains can be reconstituted with MCs from wild type mice by injection of bone marrow derived MCs (BMMC) to study the role of MCs in various disease models. Bacterial infections W/Wv-mice are more sensitive than wild type mice to many bacterial and parasitic infections (46). MC deficient mice subjected to cecal ligation and puncture (CLP), a model for acute bacterial peritonitis, resulted in 100% mortality of these mice, while MC reconstituted littermates showed a mortality rate of only 25% (75). However, injection of anti-TNF-D antibodies directly after CLP completely suppressed this protection, revealing a critical role for MC derived TNF-D in acute bacterial peritonitis. In a similar experiment the importance of TLR-4 was clearly documented. W/Wv-mice reconstituted with TLR-4 deficient MCs were associated with a higher mortality rate, and also a reduced recruitment of neutrophils and cytokine production compared to mice reconstituted with TLR-4 expressing BMMCs. 17.

(165) (60). These results provide evidence for an important role of MCs in innate immune responses. MCs also express complement receptors, which are important in the CLPmodel. Mice lacking the complement component C3 exhibit a decrease in peritoneal MC degranulation and are more sensitive to CLP (76). Injections of C3 in this model enhanced MC degranulation and resistance to CLP, further suggesting an important role for MCs in bacterial infections. An important mediator of sepsis is the vasoconstrictor endothelin-1 and MCs express the endothelin-1 receptor ETA. MCs can enhance host survival in the CLP model, by ETA dependent activation (77). The suggested mechanism involves clearance of endothelin-1 levels due to chymase dependent degradation of the vasoconstrictor. More evidence for the role of MCs in bacterial infections was obtained by infecting W/Wv-mice with Klebsiella pneumoniae (78). The MC deficient mice had an almost 20-fold decrease in bacterial clearance rate and showed impaired neutrophil recruitment compared to wild type or MC reconstituted W/Wv-mice. Injection of anti-TNF-D antibodies together with K. pneumoniae in MC positive mice reduced the neutrophil influx by 70% (78). Impaired bacterial clearance is also observed in W-sh/W-sh mice when infected with Mycoplasma pneumoniae (79). MCs are also critical mediators in the vaccine-induced clearance of Helicobacter felis (80). Parasitic infections MCs are involved in the clearance of various parasites. After infection with the nematode Trichinella spiralis, W/Wv-mice expelled the nematodes slower than MC-reconstituted littermates. These results show that MCs contribute but are not essential for nematode expulsion (81, 82). The targeted deletion of the serine protease mMCP-1, expressed in MMCs, were shown to be involved in the expulsion of T. spiralis, but not of Nippostrongylus brasiliensis, indicating different mechanisms for immunity against intestinal nematodes and helminths (83). Later experiments revealed a decrease of villous atrophy, neutrophil infiltration and TNF-D-levels in mMCP-1-/- mice compared to mMCP-1+/+ mice (84). MCs are also involved in the immunity against Strongyloides ratti, of which expulsion was found to be slower in W/Wv-mice than the MC reconstituted animals (85, 86). Similar results were obtained also after infection with the protozoan Giardia lamblia (87). Apart from bacterial and parasitic infections, MCs have also a documented beneficial role towards snake and honeybee venoms (88). Hence, MCs play an important role mainly in bacterial and parasitic infections and serine proteases are demonstrably involved. The mechanisms for these beneficial effects of MCs are in many cases still unclear, which may open up for possible novel functions of the MC expressed serine proteases.. 18.

(166) Serine proteases Almost one third of all proteases in the human genome are serine proteases and they all share the same catalytic mechanism, involving a critical serine residue (89, 90). The catalytic triad, or charge relay system, of serine proteases is formed by an aspartic acid, a histidine and a serine residue. These amino acid residues are brought together in the correctly folded enzyme and provide a mechanism to cleave peptide bonds. To avoid hydrolysis of random peptide bonds causing peptide degradation, the enzyme and substrate have to interact in order to juxtapose the scissile bond close to the catalytic triad. The serine protease cleavage specificity is thus not determined by the catalytic triad, but of amino acid residues of the protease forming the substrate binding cleft. The cleavage specificity of different proteases may vary depending on their purpose. Digestive enzymes, like pancreatic elastase or trypsin have broad cleavage specificities, cleaving peptides after aliphatic or basic amino acids, respectively. Enterokinase on the other hand has stringent cleavage specificity and specifically recognizes the sequence Asp-Asp-AspAsp-Lys in its natural target trypsinogen. Serine proteases are endopeptidases, which mean that they hydrolyze peptide bonds within peptide chains. The amino acids N-terminal of the cleaved bond of a substrate are designated P1, P2, P3 etc., while on the C-terminal side they are called P1´, P2´, P3´ etc., and they interact with subsites of the enzyme accordingly named S1, S2, S1´ and S2´ (according to Schechter and Berger (91)). Therefore, the cleavage always occurs between the P1 and P1´ positions of the substrate (Fig. 3).. Figure 3. Definition of substrate and enzyme subsite interactions. Substrate amino acid residues are designated as position P1, P2, P3, …Pn, N-terminal of the cleaved bond and P1´, P2´, P3´, …Pn´, C-terminal of the cleaved bond, so that cleavage always occurs between positions P1 and P1´. The substrate amino acid residues interact with subsites of the enzyme accordingly numbered as S1, S2, S3, …Sn, and S1´, S2´, S3´, …Sn´.. 19.

(167) Catalytic mechanism Peptide bonds are very stable, but the catalytic triad of serine proteases (His, Asp, Ser) provides a refined mechanism of peptide bond hydrolysis. The rate of peptide bond hydrolysis by serine proteases is approximately 1010 times higher than the uncatalyzed reaction. The proteolytic processing is basically a two-step reaction, the acylation and deacylation of the enzyme (92). The acylation step is initialized by the His residue acting as a general base, which enhances the nucleophilicity of the Ser residue by forming a hydrogen bond to the Ser oxygen. The His residue is stabilized by forming a hydrogen bond to the Asp residue. The Ser can then attack the carbonyl carbon of the substrate scissile bond, which acquires a negative charge. This negatively charged oxyanion is stabilized by hydrogen bonding to amide nitrogens in a pocket of the enzyme, called the oxyanion hole. The enzyme and the substrate now form a tetrahedral intermediate. The donation of a proton from the catalytic His, leads to a collapse of the tetrahedral intermediate. An acylenzyme intermediate is formed and the C-terminal leaving group is released. The deacylation step is basically a recurrence of the acylation steps. This time, water attacks the carbonyl carbon of the acyl-enzyme, assisted by the His residue. A second tetrahedral intermediate is formed and subsequently collapses upon proton donation. The N-terminal acyl group is released and the catalytic Ser is restored again.. Chymotrypsin-like serine proteases Chymotrypsin-like serine proteases form one of the largest protease families, with more than 470 identifiers in the MEROPS database (93). The family name is derived from Chymotrypsin A in cattle (Bos taurus). Members of this gene family are involved in a number of physiological processes, like food digestion, reproduction, blood coagulation, fibrinolysis and immune responses. Chymotrypsin has 245 amino acid residues and the primary sequence is folded into two six-stranded E-barrels, with the active cleft between the two E-barrels. The three amino acids of the catalytic triad are found in positions His57, Asp102 and Ser195, with His57 and Asp102 located on one side of the active cleft and Ser195 on the opposite side (chymotrypsin numbering according to (94) which will be used throughout this thesis). The oxyanion hole, described above, is formed by the backbone amino (NH-) groups of Gly193 and Ser195. The majority of chymotrypsin-like proteases belong to one of three subclasses, based on their primary cleavage specificity. The proteases have either trypsin-like, chymotrypsin-like or elastase-like cleavage specificities, determined by the structure of the S1 pocket (Fig. 4). The S1 pocket is formed by residues 189–192, 214-216 and 224-228 (95), of which amino acids 189, 216 and 226 are of special interest. Amino acid 189 is located at 20.

(168) the base of the S1 pocket, while amino acids 216 and 226 are positioned on the wall. Enzymes with chymotrypsin- or trypsin-like cleavage specificities usually contain Gly in positions 216 and 226, allowing large substrate side chains into the S1 binding pocket (96). In trypsin-like serine proteases Asp189 limits the primary specificity to positively charged, or basic, P1 side chains, Arg or Lys. In proteases with chymotrypsin-like substrate specificity Ser or other small amino acids in position 189 together with Gly216 and Gly226 create a preference for large hydrophobic amino acid side chains, as found in Phe, Tyr and Trp. Elastases have small amino acids like Ser in position 189, and larger, often non polar amino acids in positions 216 and 226, creating a more shallow S1 pocket. Therefore, elastases have a preference for cleaving after small aliphatic amino acids, like Ala and Val. However other structural elements distant from the cleavage site are also important as cleavage specificity determinants, since they stabilize substrate binding residues (97).. Figure 4. The S1 pocket of chymotrypsin-like serine proteases with chymotrypsinlike, trypsin-like or elastase-like primary cleavage specificity. The enzyme residues 189, 216 and 226 are of major importance as cleavage specificity determinants by restricting the S1 subsite for particular P1 side chains of substrates.. Chymotrypsin-like serine proteases also use extended interactions to stabilize the binding of a substrate. The backbone of enzyme residues 214-216 form an antiparallel E-sheet with the backbone of the P1-P3 residues of the substrate, by hydrogen bonding (98). Residues 214-216 are referred to as the polypeptide binding site and the interaction between the two backbones are important for efficient substrate hydrolysis. However, this interaction is nonsubstrate specific and does not discriminate between different substrates. Additional substrate binding pockets (beside the S1 pocket) also exist, providing specific extended substrate interactions in chymotrypsin-like prote21.

(169) ases. In chymotrypsin, however, these interactions display little discrimination. The granule-associated serine proteases The granule-associated serine proteases (graspases) are a group of chymotrypsin-like serine proteases stored in the granules of haematopoietic immune cells. They are closely related and are encoded from the same locus; the MC chymase locus located on chromosome 14 in human and mouse (99, 100). The enzymes encoded here are T-cell and NK-cell granzymes, neutrophil cathepsin G and the MC chymases, and they share important features. They have been found to lack a disulphide bond between Cys191 and Cys220 (101-103). This is a disulphide bond common to most chymotrypsinlike proteases, and the loss of it has created a new S3 pocket that interacts with substrate P3 side chains (104). The cleavage specificity of rMCP-1 and -2, which belong to the graspases, substantiate this observation. They cleave p-nitroanilide (pNA) substrates with optimal P3 side chains 100 times more efficient than with suboptimal P3 residues, while chymotrypsin show little or no difference of peptide hydrolysis with different P3 residues (104). Additionally, the graspases have three extra residues inserted between amino acids 39 and 40, which could interact with the P1´, P2´and P3´ residues of substrates. This loop may therefore create additional extended cleavage specificity interactions, compared to chymotrypsin. In graspases the side chain of amino acid 226 penetrates the bottom of the S1 pocket and directly interacts with the P1 side chain. This in contrast to other chymotrypsin-like proteases as discussed above. In NK-cell and T-cell granzyme B, amino acid 226Arg interacts with the P1 side chain creating a specificity for the negatively charged amino acid Asp in position P1 of substrates (105). When this enzyme is subjected to mutations in position 226, the cleavage specificity can be altered, which indicates the important role of amino acid 226 among the graspases. Exchanging Arg for a Gly in position 226 (Arg226Gly) gave an enzyme with chymotrypsin-like cleavage specificity, preferring aromatic amino acids in P1 (106), and an Arg226Glu mutant resulted in an enzyme with preference for basic amino acids (107). In human (but not mouse) neutrophil cathepsin G, residue 226 is a Glu resulting in a dual specificity of this enzyme accepting both aromatic and basic P1 side chains (108). The reason why human cathepsin G can cleave after aromatic amino acids is the fact that the side chain of 226Glu is directed slightly away from the P1 residue, allowing also aromatic side chains in the S1 pocket (109).. Mast cell chymases Serine proteases in MC granules are stored as active proteases. The low pH of the granules keeps the proteases inactive towards granule proteins and in 22.

(170) close contact with the proteoglycans for efficient packaging (110, 111). The proteases are also released in complex with heparin (112). The MC subpopulations have different tissue distributions and express different proteases. This suggests unique functions for each MC subtype, possibly due to interactions with site-specific substrates. The proteolytic activities of MC proteases include chymotrypsin-like and trypsin-like enzymes, referred to as chymases and tryptases, respectively. As described earlier, CTMCs in rodents and human MCTC express both chymases and tryptases, while rodent MMCs express chymases and human MCT express only tryptases. Additionally, a family of proteases with unknown cleavage specificity called the rMCP-8 family are expressed by mouse basophils and rat MMCs (24, 113, 114). Chymase was first described in 1959 as a MC enzyme with chymotrypsin-like cleavage specificity with the ability to bind the serine protease inhibitor diisopropyl fluorophosphate (DFP) (115). The chymases are synthesized as proproteins and activated intracellularly by the enzyme dipeptidyl peptidase (DPPI) (116). After degranulation the chymases become active in the neutral environment, and cleave peptides after aromatic amino acids with the order of preference Phe>Tyr>Trp (117). The D- and E-chymases Based on phylogenetic analyses the chymases can be divided into two subfamilies, the D-chymases and the E-chymases (Fig. 5) (118). The Dchymases are found in all species investigated while the E-chymases appear to be present only in rodents. The single human chymase (HC) is an D-chymase. This protease is expressed in the human CTMC-like cells, MCTC. In rat and mouse a number of E-chymases are heterogeneously expressed in both CTMCs and MMCs (Table 2). In rat the D-chymase rMCP-5 and the E-chymase rMCP-1 are expressed in CTMCs, while E-chymases rMCP-2, -3 and -4 are expressed in MMCs (24, 119). Mouse CTMCs express the D-chymase mMCP-5 and the E-chymase mMCP-4 whereas MMCs express the E-chymases mMCP-1 and -2 (120-122). Three-dimensional models of the four chymases in mouse revealed a stronger positive net charge on the surface of the CTMC chymases (123). CTMC chymases are also more tightly bound to heparin than MMC chymases are to chondroitin sulphate. The interaction between the protease and the proteoglycan is associated with limited diffusion and protection from inhibitors outside the cell. Based on sequence alignments, the two D-chymases rMCP-5 and mMCP5 are most similar to the HC (Fig. 5). However, rMCP-5 and mMCP-5 have lost their chymotrypsin-like cleavage specificity and gained elastase-like activity instead (124, 125). A Val residue in position 216 instead of a Gly, which is common in enzymes with chymotrypsin-like specificity, is partly 23.

(171) responsible for this change. In a recent study, the D-chymase hamster chymase-2 was also shown to possess elastase-like cleavage specificity (126). In addition to position 216, amino acids 189 and 190 were also concluded to bring elastase-like cleavage specificity to the rodent D-chymases. In addition, the D-chymase in guinea pig was recently cloned and found to prefer Leu in the P1 position (127). The cleavage specificities of rodent Dchymases are thus displaying an astonishing diversity, compared to other species. A possible explanation is the addition of E-chymases in these species.. Figure 5. Dendogram showing the phylogenetic relationships of D- and E-chymases from different species. The amino acid sequences of mature proteases were used in the analysis, and the separation of D- and E-chymases are marked in the figure. Picture derived from Ulrika Karlson. The physiological function of chymase has not yet been fully clarified, which is most likely due to the differences seen between rodents and humans. The presence of additional rodent E-chymases, which have different cleavage specificities and heterogenic expression patterns, makes the rodents very different from humans. The situation is even more complicated, since the structural homologues (the D-chymases) in rat, mouse and human are most likely not the functional homologues. The functional homology between chymases in primates and rodents are therefore not entirely clarified, which makes it difficult to directly extrapolate data from rodents to human. 24.

(172) MC biology. It seems that one or several of the rodent E-chymases probably have taken over the role of the D-chymase. Based on cellular location and biochemical properties, the CTMC E-chymases rMCP-1 and mMCP-4 are likely to be the true functional homologues of HC (128). However, more detailed characterizations of these chymases are needed to establish the functional homology between the CTMC chymases in rodents and humans (paper II). As a consequence of this situation, it is difficult to determine the function of chymase since the most widely used model organisms, rat and mouse, are so different from humans. Similarities and differences can possibly be clarified by comparing the cleavage specificity and target substrates of the different chymases. Cleavage specificity of mast cell chymases Determining the cleavage specificity of MC chymases has been the objective of several studies. Powers et al. (117) used chromogenic substrates to investigate the cleavage specificity of the HC, rMCP-1, rMCP-2 and the dog chymase. All chymases had similar preferences for hydrophobic residues in the positions P1 to P4. Phe was the most favorable amino acid in position P1 and Val was preferred in P3 by all chymases. In position P2, the HC preferred Pro residues, while rMCP-1 and rMCP-2 had preferences for Leu. The HC and dog chymase equally preferred Phe or Met in the P4 position, where rMCP-1 and rMCP-2 preferred Met. The human chymase has also been analyzed for its cleavage specificity, using a combinatorial fluorogenic peptide library (129). The consensus sequence derived from this study was quite different from the study by Powers et al. (117). The consensus sequence from P4 to P1, Arg – Glu – Thr – Tyr, was furthermore identified in human albumin and found to be cleaved by the HC. These studies determined the extended cleavage specificity of positions N-terminal of the cleaved bond, without taking the C-terminal positions into consideration. In a study where non-cleavable peptide inhibitor libraries were used to study HC/substrate interactions, the C-terminal positions were evaluated (130). Interestingly, the P1´ and P2´ positions were found to preferably hold the negatively charged Glu and Asp, respectively, when the P3´ residue was held constant as an Arg. The positions flanking the scissile bond on the N-terminal side were found to hold (from P4 to P1) Ile – Glu – Pro – Phe. The methods used in these studies are however limited in several ways. When using chromogenic substrates and combinatorial libraries, only the Nterminal positions are targeted. In addition, different chromogenic or fluorogenic leaving groups are used, which are positioned on the C-terminal side of the cleaved bond and thus interacting with S´ subsites of the enzyme. These factors affect the results of the analyses, substantiated by the quite different results obtained in the two studies utilizing these strategies presented above. Bastos et al. evaluated the P´ positions, however without considering the N-terminal positions in the same reactions. In addition, this ex25.

(173) perimental setup held the P3´ position constant, potentially affecting the interactions of the S1´ and S2´ subsites of the HC. This study showed, however, interesting indications of the important interactions of HC with the Cterminal residues of substrates. Still, further characterization of the cleavage specificities is needed to evaluate these enzyme/substrate interactions in more detail. The limitations can be overcome by using different methods. One example of such a method is substrate phage display. This method is based on a library of random peptides, which are displayed on phages. The library can be screened for susceptible peptides, allowing simultaneous variation in positions on both sides of the cleaved bond, and without any foreign leaving groups. This strategy has been used to map the extended enzyme/substrate interactions of several mast cell chymases, including the D-chymases expressed in opossum, dog and rat and the E-chymase rMCP-4 as well as the data presented in this thesis ((125, 131, 132) and unpublished results Gallwitz et al.). These data will therefore be discussed in a later section. Table 2. Serine protease content of rat, mouse and human mast cells. MMC1 Serine protease. CTMC. Rat. Mouse. Human (MCT). Rat. Mouse. Human (MCTC). Chymase. rMCP-2 rMCP-3 rMCP-4. mMCP-1 mMCP-2. -. rMCP-1 rMCP-5. mMCP-4 mMCP-5. HC. Tryptase. -. -. D-tryptase E-tryptase. rMCP-6 rMCP-7. mMCP-6 mMCP-7 mTMT. D-tryptasehi E-tryptasehi. rMCP-8 rMCP-9 rMCP-10 Data from (22, 24, 133). 1 Abbreviations: MMC, mucosal mast cell; CTMC, connective tissue mast cell; MCT, tryptase positive mast cell; MCTC, tryptase and chymase positive mast cell; rMCP, rat mast cell protease; mMCP mouse mast cell protease; HC, human chymase; mTMT, mouse transmembrane tryptase. rMCP-8 fam.. Chymase substrates MC chymases have been shown to cleave many different substrates in vitro. However, which substrates that are important in vivo are in many cases not yet known. Most of the in vitro substrates and some in vivo effects that have been attributed to chymases will be presented here, and hopefully shed some light on the function of chymases. Angiotensin Angiotensin (Ang) is the most well studied substrate of the chymases. Conversion of Ang I (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10) 26.

(174) produces the potent vasoconstrictor Ang II by a single cleavage of the Phe8 – His9 bond. Early studies suggested a general difference in the ability of Dand E-chymases to convert Ang I. The human chymase was found to exclusively cleave the Phe8-His9 bond while rMCP-1 also cleaves the Tyr4-Ile5 bond and thereby degrades Ang I and II (118, 134, 135). The idea that only D-chymases convert Ang I was later revised when the E-chymases mMCP-1 and rat vascular chymase (rVC) were shown to efficiently convert Ang I (Fig. 6) (136, 137). The E-chymases hamster chymase-1 and mMCP-4 show activity against both sites but with a higher preference for the Phe8-His9 bond, leading primarily to conversion and a slow degradation of Ang I (138, 139). Ang I conversion is an example of the functional differences that can be found between various chymase members. rMCP-1 shows the complete opposite effect compared to HC, by destroying angiotensin, while mMCP-4 produces Ang II, but at a much slower rate than HC. HC is in this case more similar to the MMC chymase mMCP-1 than the CTMC E-chymases in rat and mouse. The HC actually converts Ang I with higher efficiency than the angiotensin converting enzyme (ACE) and has been suggested to have an important role in local vascular Ang II generation and blood pressure regulation (140-142). Chymase generated Ang II has in animal models been shown to be important in cardiovascular pathological states like vascular hyperplasia, aortic aneurysm, angiogenesis, myocardial infarction and cardiac fibrosis (143). The specific cleavage at the Phe8-His9 bond by HC is partly dependent on residue Lys40 of the enzyme (144). Cytokines Studies have shown that chymase has the potential to recruit neutrophils, eosinophils, macrophages, MCs and lymphocytes (145-150). This effect can at least partly be explained by activation or solubilization of cytokines and chemokines. HC has been shown to cleave membrane bound SCF and release it from cell membranes (151). Intradermal injection of HC in mice resulted in release of SCF from keratinocytes and increased number of MCs at the site of injection, suggesting an important role for SCF in MC recruitment (147). HC and mMCP-4 increase the expression of IL-8 and MIP-2 from eosinophils, respectively. IL-8 is a known neutrophil chemoattractant and intradermal injection of mMCP-4 increased the amount of the mouse IL8 homologue, MIP-2, and neutrophils at the injection site (149). Furthermore, HC releases TGF-E1 from ECM and HC as well as rMCP-1 activate this cytokine (152, 153). Activated TGF-E1 act as a chemoattractant for monocytes, neutrophils and eosinophils (154, 155). IL-1E is, like TGFE1, also secreted as an inactive precursor and needs to be activated extracellularly. HC has been shown to also activate this cytokine, which produces a highly potent inflammatory cytokine that, among other things, also can at27.

(175) tract macrophages and neutrophils (156). For several of these cytokines, the chemoattracting effect is only one of several proinflammatory functions. However, rMCP-1 and mMCP-4 show in this case obvious similar effects as HC. HC is also able to process the connective tissue-activating peptide (CTAP)-III into active chemokine neutrophil-activating peptide (NAP)-2 (157). It has also been suggested that the human chymase can act directly on monocytes and neutrophils as a chemoattractant, without activating a secondary attractant (145). This mechanism was proposed to be dependent of an active enzyme (chymase) that activates a still unknown chymase receptor by proteolytic cleavage. Additional cytokines that are processed by HC are proIL-18, which is activated and IL-6 and IL-13, which are degraded (158, 159).. Figure 6. Mast cell chymase cleavage sites in angiotensin I. Cleavage of the Phe8His9 bond converts angiotensin I into vasoactive angiotensin II. Cleavage of the Tyr4-Ile5 bond degrades angiotensin. Abbreviations: rMCP, rat mast cell protease; mMCP, mouse mast cell protease; HC, human chymase; rVC, rat vascular chymase.. Extracellular matrix components Both HC and rodent chymases have been attributed a role in extracellular matrix (ECM) remodeling, suggesting further similarities between the CTMC chymases. Various matrix metalloproteases (MMPs) are enzymatically activated. Collagenase (MMP-1) is activated by HC and rMCP-1, stromelysin (MMP-3) by rMCP-1 and -2, gelatinase A (MMP-2) is activated by mMCP-4 and gelatinase B (MMP-9) by dog D-chymase and mMCP-4 (160-163). HC directly cleaves the MMP substrate procollagen and inacti-. 28.

(176) vates tissue inhibitor of metalloproteinase-1 (TIMP-1) prolonging the halflife of MMPs (164, 165). The ECM protein fibronectin is cleaved by HC, rMCP-1 and mMCP-4, leading to disruption of cell adhesion to the ECM (128, 162, 166-168). mMCP-4-/- mice were further shown to be impaired in the turnover of fibronectin (128, 162). One consequence of chymase mediated fibronectin degradation is induction of apoptosis in neighboring smooth muscle cells (169). Chymase also affects airway smooth muscle (ASM) cells in several ways. Fibronectin in the pericellular matrix of ASMs is degraded and the cells increase their release of soluble CD44. Furthermore, chymase also inhibits T-cell attachment to ASMs and epidermal growth factor-induced proliferation (170). Fibronectin degraded by HC may be involved in cell detachment and apoptosis in human conjunctival epithelial cells (171). HC mediated degradation of fibronectin and occludin have also been proposed to decrease the barrier function of human corneal epithelial cells and to inhibit their migration (172). TGF-E1 has also been shown to enhance the production of ECM proteins, like collagen (22, 173), and as described above HC as well as rMCP1 activate this cytokine (152, 153). Vascular targets A role for chymase in regulation of coagulation has been documented. rMCP-1 and mMCP-4 inactivate thrombin and mMCP-4 also inactivates plasmin in a heparin dependent reaction (174-176). Heparin is suggested to attract thrombin and plasmin, bringing the substrates closer to the enzyme and thereby accelerates proteolysis. Other vascular substrates sensitive to chymase digestion are big endothelin and endothelin-1. Big endothelin is the precursor of endothelin-1, and is secreted by endothelial cells and airway epithelial cells along with mature endothelin-1. Endothelin-1 causes contraction of tracheal and vascular smooth muscle cells. HC was shown to cleave big endothelin at a single site creating a 31 amino acid long active variant, in contrast to the 21 amino acid long secreted endothelin-1 molecule, while rMCP-1 and rMCP-2 degrade the precursor (177). Endothelin-1 (1-21) has also been shown to be degraded by rMCP-1 and/or rMCP-5 in co-cultures of human aortic endothelial cells and rat peritoneal MCs (178). Mouse mMCP-4 and/or mMCP-5 have further been shown to contribute in limiting endothelin-1 induced toxicity upon intraperitoneal injection of endothelin-1 (77). Other vasoactive peptides degraded by chymases are bradykinin, kallidin and vasoactive intestinal protein (179, 180). Also serum albumin is cleaved by HC (129). Other substrates Chymase has been devoted a role in regulating lipoproteins. rMCP-1 was shown to degrade lipoproteins containing apolipoprotein (apo)A-I, apoA-II 29.

(177) and apoB (30, 181, 182). The HC can similarly degrade apoA-I, A-II, apoE and phospholipid transfer protein (181, 183, 184). As a result of these observations, chymase is suggested to influence formation of atherosclerotic plaques. Furthermore, HC is able to cleave the serine protease inhibitors C1inhibitor and D(2)-macroglobulin, neurotensin, birch and human profilin and to cause autolysis (cleave HC) (28, 141, 185-187). In vivo role of chymase Mast cell chymases have been implicated in a vast number of biological processes and diseases in vivo (188). A selection of those is presented here. As described in an earlier section, chymases have been shown to recruit neutrophils, eosinophils and monocytes in vivo. (148-150, 189). This proinflammatory effect has led to speculation that chymase is involved in dermatitis and other inflammatory skin disorders. Chymase has further been suggested to be involved in cardiac and vascular diseases. For example, the mMCP5-/- model showed reduced ischemia-reperfusion injury of skeletal muscles (190). Other studies have revealed the importance of chymase in epithelial permeability regulation. Injection of HC in the skin of guinea pigs resulted in long lasting microvascular permeability and vascular leakage (191). Similarly, infusion of rat MMC protease rMCP-2 into the mesenteric artery increased the epithelial permeability and translocation of rMCP-2 and Evans blue-labeled human serum albumin into the jejunal lumen within minutes after infusion (192, 193). The mouse homologue to rMCP-2, mMCP-1, has been detected in the gut lumen during expulsion of the nematode T. spiralis, which indicates a similar mechanism for this chymase (5). In a later study, it was shown that the expulsion of T. spiralis was delayed and that the deposition of muscle larvae was increased in a mouse strain lacking mMCP-1 (83). Taken together, the function of rMCP-2 and mMCP-1 seem identical and the increased permeability may be crucial to allow translocation of immune cells and antibodies to the intestinal lumen. Another chymase knockout model has also been studied. The mMCP-4-/strain revealed that mMCP-4 is responsible for most chymotryptic activity in peritoneum and ear tissue (128). The mMCP-4 deficient mice could not activate MMP-9 and were affected in the activation of MMP-2 as well. A reduced ability to process fibronectin was also observed (162). Thus, chymases have been implicated in many biological processes and many in vitro substrates have been identified. However, detailed explanations of which targets that are involved are in most cases not established. Therefore further studies are needed in order to explain the true biological function(s) of MC chymases.. 30.

(178) Present investigations. Aim The aim of this study was to increase the knowledge about MC specific chymases in placental mammals. By characterizing the cleavage specificity of chymases expressed in mouse, rat and human, information regarding functional relationships of D- and E-chymases between and within these species, can be obtained. The cleavage specificity of these chymases may also be used as a valuable tool to identify novel natural substrates, possibly providing important knowledge of the in vivo function of chymases.. Results and discussion Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse-High substrate specificity indicates high substrate selectivity (Paper I) Mouse MMCs express two chymases, mMCP-1 and mMCP-2, but only mMCP-1 has been shown to provide proteolytic activity to these cells (120). MMCs are found mainly as intraepithelial cells in the gastrointestinal tract and are thought to provide a defense mechanism against gastrointestinal nematodes. Infections by these organisms are accordingly associated with intraepithelial MC hyperplasia in the host. An mMCP-1 knockout model has provided evidence for the in vivo relevance of this E-chymase in the immunological responses towards intestinal nematodes. The mMCP-1-/- mice showed delayed expulsion of the intestinal nematode T. spiralis, and an increased deposition of muscle larvae in the host (83). Furthermore, during these infections mMCP-1 appear in high amounts in the jejunal lumen and in the circulation. Despite the clear evidence for the important role of mMCP-1 in intestinal immune responses against nematodes, the function of this enzyme has not been established. Since mMCP-1 is found to cross epithelial and endothelial layers, a suggested role for this chymase is to increase epithelial and endothelial permeability. Although no such substrates have been identified, the suggested target is indirect or direct degradation of tight junction proteins.. 31.

(179) In this study, we performed a detailed analysis of the extended cleavage specificity of mMCP-1, by utilizing a substrate phage display approach (Fig. 7). We used a T7 phage library consisting of randomly synthesized nonamers, displayed on the surface of T7 phages. The library contains approximately 5x107 different phage clones and the randomized peptides are expressed at the C-terminus of the capsid protein of the phages with an affinity His6-tag at the C-terminal end. Each phage displays one specific peptide. The phages are immobilized on nickel-nitrilotriacetic acid (Ni-NTA) beads and after washing; the phage particles are subjected to the protease. Phages that express a random peptide that is sensitive to protease hydrolysis will be released from the matrix and become available for amplification in E. coli bacteria. The newly formed sublibrary is immobilized on fresh Ni-NTA beads and subjected to a second round of selection. After several selection rounds, individual phage clones are isolated and the sequences encoding the randomized regions are determined.. Figure 7. The principle of substrate phage display. A library of phages expressing a random nonamer followed by an affinity His6-tag (1), is immobilized on Ni-NTA beads (2). Protease is added and phages expressing a random peptide susceptible to protease cleavage are released (3). Released phages are collected and amplified in E. coli bacteria (4). The sublibrary is immobilized on fresh Ni-NTA beads (5) to start a new selection round (6). After the last of several rounds of selection (7), phages are plated on E. coli bacteria and individual phage clones are isolated (8). The randomized region of each phage clone is then determined.. 32.

(180) To obtain pure protease for the analysis of the extended cleavage specificity, mMCP-1 was purified from MMC-like bone marrow-derived mast cells cultured in the presence of TGF-E1. Using the phage display system, the cleavage specificity of mMCP-1 was shown to display a high degree of selectivity in four out of the seven positions analyzed. Particularly strict preferences were detected for Phe over Tyr in the P1 position and Ser in position P1´. mMCP-1 also showed high selectivity for large hydrophobic amino acids Trp, Phe and Leu in position P2 and the aliphatic amino acids Leu, Val and Ala in position P2´. Also in position P3 and P4, aliphatic amino acids were clearly overrepresented and in P3´ mMCP-1 seem to be more tolerant, allowing a variety of amino acids like Arg, Ser, Gly and Ala. Based on these results, the potentially best substrate for mMCP-1 is from P4 to P3´: Val/Pro-Val-Leu/Phe/Trp-Phe-Ser-Leu-Xaa, where the P1 position is indicated in bold letters. The strict preferences in position P2 to P2´ indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The UniProtKB/Swiss-Prot database was screened for potential suitable substrates with the following amino acids allowed in position P2 to P2´: P2 (Leu/Phe/Trp), P1 (Phe), P1´ (Ser/Arg) and P2´ (Leu/Val/Ala). This led to the identification of 113 hypothetical mouse substrates, potentially accessible for mMCP-1. A selection of 17 out of the 113 proteins found, are cell adhesion proteins, extracellular matrix proteins and a matrix metalloprotease (MMP), which might be of interest to explain the permeability increasing effects of mMCP-1. Laminin chains, building up vascular basement membranes, were identified as potential targets, as well as cell adhesion proteins belonging to the cadherin superfamily. Interestingly, the integrin D-7 chain, which has been shown to attach MMC to basement membranes was also identified, raising the possibility that mMCP-1 can modify MC attachment to basement membranes. Another potential target, the atrial natriuretic peptide clearance (ANP-C) receptor, with a documented effect on vascular permeability was also identified. Furthermore, a very interesting cleavage site was also found in the pro-domain of MMP-8, indicating a possible MMP-8 activating mechanism of mMCP-1. Several unsuccessful attempts were also made to determine the cleavage specificity of the second serine protease expressed by mouse MMCs, mMCP-2. Earlier trials have also revealed difficulties in detecting proteolytic activity of this enzyme. The most likely explanation is that mMCP-2 is proteolytically inactive, due to a two amino acid deletion in the region close to the specificity-conferring triplet. An alternative explanation would be that mMCP-2 has an extremely strict substrate recognition profile. However, screening of millions of unique substrates would most likely detect even the most strict substrate specificity, making this explanation less likely.. 33.

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