cleavage specificity of mast cell chymases

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 429

Cleavage Specificity of Mast CellChymases

MATTIAS K. ANDERSSON

ISSN 1651-6214ISBN 978-91-554-7190-3urn:nbn:se:uu:diva-8714

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”Nu är det Din stund på jorden!”

Vilhelm Moberg

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 sub-strate selectivity. Molecular Immunology 2008; 45 (9): 2548-2558

II Andersson M.K., Karlson U. and Hellman L.

The extended cleavage specificity of the rodent �-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 chy-mase reveals a serine protease with well-defined substrate recog-nition 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 po-sition P2´ of substrates. Manuscript

Reprints were made with permission of the publisher.

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 �-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

Abbreviations

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

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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 pres-ence of invading microbes, an inflammatory response is initiated. The re-lease of inflammatory mediators leads to vasodilation and increased epithe-lial permeability, which allows an influx of leukocytes to the site of infec-tion. Mast cells (MC) are one of the important cell types in the early inflam-matory 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 initi-ate 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 invad-ing pathogens. Upon activation the B cells secrete antibodies towards ex-tracellular 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 reacti-vated memory cells (lymphocytes) that provide a fast response upon re-peated infections by the same microbes.

Unfortunately, sometimes the immunologic response is misdirected or re-acts too strongly, which may lead to tissue damage and disease. Autoimmu-nity 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 hyper-sensitivity 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, in-

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creased mucus production in the nose, sneezing and bronchial constriction. These symptoms affect the life quality of patients but are seldom lethal. Al-lergies 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. Al-though not granule stored, prostaglandins and leukotrienes are also produced and released during this early response. In addition, MCs also provide a de-layed response within hours of activation by de novo production and secre-tion 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 media-tors. These mediators will initiate an inflammatory response.

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The majority of all proteins stored in the secretory granules are serine prote-ases 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 increas-ing the understanding of chymases in MC biology, by determining the cleav-age 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 stain-ing 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 loca-tion, 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 tech-niques, the two MC subtypes can easily be distinguished by staining of hepa-rin, since it is present in CTMCs but not in MMCs.

Human MCs cannot be distinguished using the same immunohistochemi-cal 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 MMC-like 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 pro-portions. MCTC is the dominant subtype in skin, connective tissues and eso-phageal 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 para-sitic 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 in-volved 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 precur-sors and complete their maturation once they reach the peripheral tissues. The main cytokines that promote MC proliferation and differentiation are the

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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 de-pendent 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 spe-cific proteases mMCP-1 and mMCP-2 are in BMMC cultures, stimulated by TGF-�1 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, pro-teoglycans 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 decarboxy-lase. This biogenic amine act on four different histamine receptors, named H1-4. Upon binding these receptors, histamine causes constriction of bron-chial and intestinal smooth muscle, increases vascular permeability, secre-tion of gastric acid, and mediates neurotransmission. The H4 receptor is highly expressed in bone marrow and on leukocytes, which mediates chemo-taxis 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. Pro-teoglycans are large, negatively charged glycoproteins that are heavily gly-cosylated. They consist of a core protein with covalently linked and un-branched polysaccharide side chains called glycosaminoglycans (GAG). Two different proteoglycans are stored in MC granules, heparin and chon-droitin 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

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via a linker consisting of one xylose, two galactose residues and one glu-curonic 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 N-acetyl-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 proteogly-cans 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 CTMC Content

Rat Mouse Human (MCT) Rat Mouse Human

(MCTC) Proteoglycan

CS A, di B, E

CS A, E Heparin CS A, E

Heparin CS E

Heparin CS E

Heparin CS E

Histamine + + + ++ ++ + Prostaglandin PGD2

lo PGD2lo PGD2 PGD2 PGD2 PGD2

Leukotriene LTC4 LTC4 LTC4 LTC4lo - LTC4

lo Metalloprotease - - - CPA CPA CPA Serine protease Chymase

rMCP-8 fam. Chymase

Tryptase Chymase Tryptase

Chymase Tryptase

Chymase 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 prote-ases, 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 differ-ent subfamilies. The two main subfamilies expressed by MCs are the chy-mases 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 ex-press CPA, chymase and tryptase. Human MCT only express tryptase while

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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 C-terminal 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- pro-teoglycan 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 af-ter 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. How-ever, in vitro evidence also suggests that active monomers exist (37, 38).

Four groups of tryptase are found in humans, � (subtypes I and II), � (I, II and III), � (I and II) and � (I and II) (39). The �-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 pro-vide most tryptase activity in human MCs. While human tryptases are ex-pressed by all MCs, the rodent tryptases are restricted to CTMCs. Among the murine tryptases, mMCP-6 is most similar to human �-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 de-rived 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 fac-

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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-LT-receptors. 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, in-creases vascular permeability, and recruits neutrophils, monocytes and eosi-nophils to the site of inflammation.

Cytokines MCs also produce a number of cytokines, including tumour necrosis factor (TNF)-�, 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 IL-13 promotes IgE production by B-cells. The expression of these cytokines thus enhance the MC mediated immune response, e.g. towards parasites. IL-3, -4, -5, -13, TNF-� and GM-CSF induce chemotaxis, migration, activation and prolonged survival of human eosinophils and thereby enhance their anti-parasitic effects (43).

The multifunctional cytokine TNF-� is not only synthesized upon MC ac-tivation, but also stored as a preformed mediator in the secretory granules (44, 45). TNF-� is important in host defence against bacteria (46). It en-hances leukocyte recruitment by inducing endothelial expression of E-selectin (ELAM-1) and ICAM-1 and potentiates bactericidal and fungicidal activity of phagocytes (47-51). The in vivo role of MC derived TNF-� will be discussed in a later section.

In addition, MCs express chemokines including IL-8, eotaxin and mono-cyte chemotactic protein (MCP)-1 that are important for neutrophil, eosino-phil, 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 affin-ity receptors, Fc�RI, 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).

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MCs can also be activated by IgG antibodies, which bind to Fc�Rs on the MC surface. Fc�RI and III share the �-chain with Fc�RI and thus have com-mon intracellular signaling pathways. The high affinity receptor Fc�RI is expressed on human MCs (55), and cross-linking of these receptors lead to degranulation (56). The low affinity receptor Fc�RIII has been detected on murine CTMCs, whereas Fc�RI has not (57).

Toll-like receptor-mediated activation Toll-like receptors (TLR) are a family of receptors that directly binds to con-served structures of microbes, such as lipopolysaccharide (LPS), peptidogly-can 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 secre-tion or degranulation, depending on the stimulating agent (61).

Figure 2. Mast cells can be activated upon engagement of different receptors. Ab-breviations: 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 sur-face 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).

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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, �-chemokines macrophage inflamma-tory protein (MIP)-1� 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 lo-cated 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 es-sential for the development of MCs (72). This strain shows additional ab-normalities, 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 mortal-ity rate of only 25% (75). However, injection of anti-TNF-� antibodies di-rectly after CLP completely suppressed this protection, revealing a critical role for MC derived TNF-� in acute bacterial peritonitis. In a similar ex-periment the importance of TLR-4 was clearly documented. W/Wv-mice reconstituted with TLR-4 deficient MCs were associated with a higher mor-tality rate, and also a reduced recruitment of neutrophils and cytokine pro-duction compared to mice reconstituted with TLR-4 expressing BMMCs

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(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 CLP-model. 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, fur-ther 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 mecha-nism 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-� antibodies together with K. pneumo-niae in MC positive mice reduced the neutrophil influx by 70% (78). Im-paired 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 con-tribute 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 brasil-iensis, indicating different mechanisms for immunity against intestinal nematodes and helminths (83). Later experiments revealed a decrease of villous atrophy, neutrophil infiltration and TNF-�-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 docu-mented beneficial role towards snake and honeybee venoms (88).

Hence, MCs play an important role mainly in bacterial and parasitic infec-tions 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.

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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 prote-ases 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 ran-dom 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 sub-strate 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-Asp-Asp-Lys in its natural target trypsinogen.

Serine proteases are endopeptidases, which mean that they hydrolyze pep-tide 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´.

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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 sub-strate 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 sub-strate now form a tetrahedral intermediate. The donation of a proton from the catalytic His, leads to a collapse of the tetrahedral intermediate. An acyl-enzyme 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 se-quence is folded into two six-stranded �-barrels, with the active cleft be-tween the two �-barrels. The three amino acids of the catalytic triad are found in positions His57, Asp102 and Ser195, with His57 and Asp102 lo-cated on one side of the active cleft and Ser195 on the opposite side (chy-motrypsin 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 sub-classes, based on their primary cleavage specificity. The proteases have ei-ther 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

21

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 posi-tion 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 resi-dues (97).

Figure 4. The S1 pocket of chymotrypsin-like serine proteases with chymotrypsin-like, 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 stabi-lize the binding of a substrate. The backbone of enzyme residues 214-216 form an antiparallel �-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 non-substrate specific and does not discriminate between different substrates. Additional substrate binding pockets (beside the S1 pocket) also exist, pro-viding specific extended substrate interactions in chymotrypsin-like prote-

22

ases. In chymotrypsin, however, these interactions display little discrimina-tion.

The granule-associated serine proteases The granule-associated serine proteases (graspases) are a group of chy-motrypsin-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, neutro-phil 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 chymotrypsin-like 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). Addi-tionally, 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 sub-strates (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 specific-ity, 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

23

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 en-zymes, 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 chymotryp-sin-like cleavage specificity with the ability to bind the serine protease in-hibitor diisopropyl fluorophosphate (DFP) (115). The chymases are synthe-sized 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 �- and �-chymases Based on phylogenetic analyses the chymases can be divided into two sub-families, the �-chymases and the �-chymases (Fig. 5) (118). The �-chymases are found in all species investigated while the �-chymases appear to be present only in rodents.

The single human chymase (HC) is an �-chymase. This protease is ex-pressed in the human CTMC-like cells, MCTC. In rat and mouse a number of �-chymases are heterogeneously expressed in both CTMCs and MMCs (Ta-ble 2). In rat the �-chymase rMCP-5 and the �-chymase rMCP-1 are ex-pressed in CTMCs, while �-chymases rMCP-2, -3 and -4 are expressed in MMCs (24, 119). Mouse CTMCs express the �-chymase mMCP-5 and the �-chymase mMCP-4 whereas MMCs express the �-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 chy-mases 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 �-chymases rMCP-5 and mMCP-5 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

24

responsible for this change. In a recent study, the �-chymase hamster chy-mase-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 �-chymases. In addi-tion, the �-chymase in guinea pig was recently cloned and found to prefer Leu in the P1 position (127). The cleavage specificities of rodent �-chymases are thus displaying an astonishing diversity, compared to other species. A possible explanation is the addition of �-chymases in these spe-cies.

Figure 5. Dendogram showing the phylogenetic relationships of �- and �-chymases from different species. The amino acid sequences of mature proteases were used in the analysis, and the separation of �- and �-chymases are marked in the figure. Pic-ture 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 hu-mans. The presence of additional rodent �-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 �-chymases) in rat, mouse and human are most likely not the functional homologues. The functional homology be-tween chymases in primates and rodents are therefore not entirely clarified, which makes it difficult to directly extrapolate data from rodents to human

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MC biology. It seems that one or several of the rodent �-chymases probably have taken over the role of the �-chymase. Based on cellular location and biochemical properties, the CTMC �-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 func-tional homology between the CTMC chymases in rodents and humans (pa-per 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 possi-bly 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 inves-tigate 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 pre-ferred 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 ana-lyzed 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 N-terminal positions are targeted. In addition, different chromogenic or fluoro-genic 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 pre-sented above. Bastos et al. evaluated the P´ positions, however without con-sidering the N-terminal positions in the same reactions. In addition, this ex-

26

perimental setup held the P3´ position constant, potentially affecting the interactions of the S1´ and S2´ subsites of the HC. This study showed, how-ever, interesting indications of the important interactions of HC with the C-terminal 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 exam-ple 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 po-sitions 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 �-chymases ex-pressed in opossum, dog and rat and the �-chymase rMCP-4 as well as the data presented in this thesis ((125, 131, 132) and unpublished results Gall-witz 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 CTMC Serine protease

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 - - �-tryptase �-tryptase

rMCP-6 rMCP-7

mMCP-6 mMCP-7 mTMT

�-tryptasehi �-tryptasehi

rMCP-8 fam. 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 prote-ase; mMCP mouse mast cell protease; HC, human chymase; mTMT, mouse transmembrane tryptase.

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. Con-version of Ang I (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10)

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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 �- and �-chymases to convert Ang I. The human chymase was found to exclu-sively 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 �-chymases convert Ang I was later revised when the �-chymases mMCP-1 and rat vascular chymase (rVC) were shown to efficiently convert Ang I (Fig. 6) (136, 137). The �-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 op-posite 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 �-chymases in rat and mouse.

The HC actually converts Ang I with higher efficiency than the angio-tensin converting enzyme (ACE) and has been suggested to have an impor-tant 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 depend-ent 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 re-lease 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 recruit-ment (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 IL-8 homologue, MIP-2, and neutrophils at the injection site (149).

Furthermore, HC releases TGF-�1 from ECM and HC as well as rMCP-1 activate this cytokine (152, 153). Activated TGF-�1 act as a chemoattractant for monocytes, neutrophils and eosinophils (154, 155). IL-1� is, like TGF-�1, also secreted as an inactive precursor and needs to be activated extracel-lularly. HC has been shown to also activate this cytokine, which produces a highly potent inflammatory cytokine that, among other things, also can at-

28

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 secon-dary 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 ac-tivated and IL-6 and IL-13, which are degraded (158, 159).

Figure 6. Mast cell chymase cleavage sites in angiotensin I. Cleavage of the Phe8-His9 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 enzymati-cally 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 �-chymase and mMCP-4 (160-163). HC directly cleaves the MMP substrate procollagen and inacti-

29

vates tissue inhibitor of metalloproteinase-1 (TIMP-1) prolonging the half-life 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 fi-bronectin (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 in-hibits T-cell attachment to ASMs and epidermal growth factor-induced pro-liferation (170). Fibronectin degraded by HC may be involved in cell de-tachment 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-�1 has also been shown to enhance the produc-tion 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 endo-thelin 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

30

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 obser-vations, chymase is suggested to influence formation of atherosclerotic plaques.

Furthermore, HC is able to cleave the serine protease inhibitors C1-inhibitor and �(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 proin-flammatory effect has led to speculation that chymase is involved in dermati-tis and other inflammatory skin disorders. Chymase has further been sug-gested 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 per-meability regulation. Injection of HC in the skin of guinea pigs resulted in long lasting microvascular permeability and vascular leakage (191). Simi-larly, infusion of rat MMC protease rMCP-2 into the mesenteric artery in-creased 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 deposi-tion 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 acti-vate MMP-9 and were affected in the activation of MMP-2 as well. A re-duced 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 explana-tions 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.

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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 �- and �-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 �-chymase in the immu-nological responses towards intestinal nematodes. The mMCP-1-/- mice showed delayed expulsion of the intestinal nematode T. spiralis, and an in-creased 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 en-zyme 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.

32

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 nonam-ers, displayed on the surface of T7 phages. The library contains approxi-mately 5x107 different phage clones and the randomized peptides are ex-pressed 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 random-ized region of each phage clone is then determined.

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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-�1. Using the phage display system, the cleavage specificity of mMCP-1 was shown to display a high degree of se-lectivity in four out of the seven positions analyzed. Particularly strict pref-erences 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 indi-cated 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 Uni-ProtKB/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 accessi-ble 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 mem-branes, were identified as potential targets, as well as cell adhesion proteins belonging to the cadherin superfamily. Interestingly, the integrin �-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 perme-ability was also identified. Furthermore, a very interesting cleavage site was also found in the pro-domain of MMP-8, indicating a possible MMP-8 acti-vating 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 prote-olytic 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.

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The extended cleavage specificity of the rodent �-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase (Paper II) Based on phylogenetic studies, the chymases can be divided into two sub-populations, the �-chymases and the �-chymases (118). Most mammalian species investigated express a single �-chymase, except for rodents that also express members of the �-chymase subfamily. The rat and mouse �-chymases, rMCP-5 and mMCP-5 respectively, have lost their chymotrypsin-like cleavage specificity and instead become elastases (124, 125). The chy-mase activity in rat and mouse MC granules is therefore provided by the �-chymases. However, it is not known how the activity of the lost �-chymase is compensated for in rat and mouse. If the �-chymases have taken over this role, is it then by a single �-chymase or the combined effect of several mem-bers of the �-chymase subfamily? To address this question, we determined the extended cleavage specificity of the two �-chymases rMCP-1 and mMCP-4, which are the two main �-chymases expressed in rat and mouse CTMCs. This subpopulation of rodent MCs is most similar to the chymase positive MCTC in humans. rMCP-1 and mMCP-4 have also many properties in common. They are structural homologues and are expressed by CTMCs in two closely related species. Both enzymes are positively charged and readily bind heparin. Considering these facts the conclusion must be that they are similar enzymes that probably have the same function. In favor of such a model, both fibronectin and thrombin are degraded by these two enzymes (167, 168, 175, 176).

In order to evaluate the cleavage specificity we produced recombinant rMCP-1 and mMCP-4 in a mammalian expression system and utilized the previously described substrate phage display approach (Fig. 7) (131).

This analysis showed that the two �-chymases display very similar cleav-age specificities in the positions N-terminal of the cleaved bond. Both en-zymes display a clear preference for Phe in position P1 and aliphatic amino acids in positions P2-P4. P2 Leu, P3 Val and P4 Val are the single most fre-quently occurring residues in these positions. These results are in line with an earlier study, where the cleavage specificity for positions N-terminal of the cleaved bond was determined for rMCP-1 and HC, using chromogenic substrates (117). However, the C-terminal positions had previously never been determined for neither rMCP-1 nor mMCP-4, and an interesting differ-ence between the two �-chymases was here revealed. In position P2´ mMCP-4 but not rMCP-1 holds a striking preference for the acidic amino acids Asp and Glu. This difference becomes even more interesting when cleavage sites of natural targets for the human chymase are compared. Acidic amino acids are here commonly found in position P2´ and this prefer-ence seems to be the strongest preference found for this enzyme, apart from the primary preference for Phe in P1. The preference for negatively charged

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amino acids in the P2’ position hence seems important for substrate dis-crimination of the HC, and this preference is shared with mMCP-4. In con-clusion, mMCP-4 and rMCP-1 show similar cleavage specificities to HC in positions flanking the N-terminal side of the cleaved bond. On the C-terminal side, mMCP-4 but not rMCP-1 shows a strong preference for acidic amino acids, a preference that also seems to be shared by the HC. mMCP-4 shows great similarities in cleavage specificity to the human chymase, and these two chymases can therefore be considered each other’s direct func-tional homologues.

This finding led to the speculation that the specificity for acidic P2´ resi-dues reflected the different Ang I converting abilities of rMCP-1, mMCP-4 and HC. Ang I is the most well studied substrate for chymases, and while rMCP-1 degrades this substrate, mMCP-4 and HC converts Ang I to Ang II. However, the results from paper IV indicate that other molecular mecha-nisms are involved.

The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile (Paper III) In human MCs only one chymase is expressed, the single human �-chymase. All mammals investigated express one �-chymase, except for ruminants where two very similar �-chymases are found (194-196). In contrast, rodents express an additional group of chymases called the �-chymases.

In paper II, we concluded that the �-chymase mMCP-4 is the functional homologue to the HC. This conclusion was based on the comparison of the cleavage specificity of mMCP-4, with the cleavage specificity of the HC and known HC substrates. However, the cleavage specificity of the HC had to that date only been investigated using chromogenic substrates, peptide li-braries or Ang I analogs, which either gives information concerning amino acid positions N-terminal or C-terminal of the cleaved bond. A more exten-sive analysis where amino acid residues are varied simultaneously both N-terminal and C-terminal of the cleaved bond has previously not been per-formed. In order to confirm the conclusion from paper II and to get a more complete picture of the extended cleavage specificity of the HC we wanted to investigate the HC using our substrate phage display approach.

Recombinant human �-chymase was produced in mammalian cells as de-scribed in paper II. Using the pure recombinant enzyme, we determined its cleavage specificity under conditions where all substrate positions, both N-terminal and C-terminal of the scissile bond, simultaneously were varied. Thus, after five rounds of selection the best combinations of amino acid resi-dues cleaved by HC were isolated and determined.

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The results reveal a high selectivity of peptide substrates cleaved by the HC. A high preference for the aromatic amino acids Phe and Tyr was ob-served for the P1 position. N-terminal of the cleaved bond aliphatic amino acids were preferred in all positions. In position P2 Ala, Val and Leu was equally represented. In the P3 position Val was the single most preferred amino acid and in P4, Gly was the most frequently occurring amino acid, closely followed by Leu and Val. C-terminal of the cleaved bond, the P1´ was the less restrictive position, showing preference for Ser. However, in position P2´, a strong preference for negatively charged amino acids was observed and P3´ was dominated by small aliphatic amino acids.

Compared to earlier studies, our results are in line with the view that ali-phatic amino acids often are seen N-terminal of the cleaved bond (117). In-terestingly, a Pro in position P2 has been suggested to be a determining fac-tor for conversion of Ang I and also preferred in other substrates (117, 129, 130, 186). We did not detect this preference, but rather an exclusion of Pro in this position. C-terminal of the cleaved bond, Ser is preferred in position P1´ and small aliphatic amino acids in P3´. The most striking finding was, however, the preference for acidic amino acids in the P2´ position. Structural studies have predicted this interaction (197-199), but we can here demon-strate the impact of this preference using a biochemical strategy. An earlier study, screening for HC inhibitors, suggested a preference for acidic amino acids in both P1´ and P2´ (130), but we can clearly show this preference to be specific for the P2´ position. Even though most substrate positions have been shown or predicted to fit well into each subsite pocket of the human chymase, the consensus we present have never been reported before.

As expected, the consensus sequence presented here is very similar to the cleavage specificity of the mouse CTMC �-chymase mMCP-4. Only one out of seven positions is not consistent, and this position (P1´) is the least selec-tive one for both enzymes. The homology in extended cleavage specificity between these two major connective tissue mast cell chymases could thus be confirmed.

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) Based on earlier studies presented in this thesis (paper I-III), the positions flanking the C-terminal side of the cleaved bond seem to be important in substrate discrimination by the MC chymases. One positions of special inter-est is the P2´ position, which seems to be of major importance for substrate recognition, when evaluating the known substrates of the HC. Both the HC and mMCP-4 here strongly prefers acidic amino acids,

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The molecular explanation for this preference has not been thoroughly in-vestigated, but molecular modeling studies based on the crystallized struc-ture of the human chymase have revealed three candidate residues. The amino acids Lys40, Arg143 and Lys192 are in the HC located in positions where they possibly can interact with P2´ residues of substrates. In addition to the HC and mMCP-4, we have also identified two other chymases, the opossum �-chymase and the rat �-chymase rMCP-5, to prefer acidic P2´ residues (125, 132). These four chymases hold Arg143 and Lys192 residues but varying amino acids in position 40. In contrast, four chymases that do not prefer acidic P2´ residues (the �-chymases rMCP1, mMCP-1 and rMCP-4 and the dog �-chymase (paper I and II, (131) and unpublished Gallwitz et al.)) do not possess Arg143 and Lys192 residues. However, Lys192 residues were present in three of those chymases. We therefore hypothesized that Arg143 alone or in cooperation with Lys192 mediate the preference for acidic amino acid residues in position P2´ of the human chymase. We de-cided to test our hypothesis by site-directed mutagenesis of the human chy-mase and evaluate the cleavage specificity of the mutants using our phage display strategy. We substituted Arg143 for Gln and Lys192 for Met, which are amino acids found in these positions in related chymases, lacking the specificity for acidic P2´. The HC Arg143Gln mutant, Lys192Met mutant and the Arg143Gln + Lys192Met double mutant were found to maintain the cleavage specificity of the wild type enzyme, with the exception for position P2´. The two single mutants showed a clear reduction in the P2´ preference for acidic amino acids and the double mutant totally abolished this prefer-ence. Of these data we could conclude that position 143 and 192 indeed are of major importance for determining the specificity for P2´ residues in the human chymase, and that synergetic effects of Arg143 and Lys192 are re-quired for obtaining a strong preference for acidic P2´ residues.

Based on these results other related chymases are predicted to share the P2´ specificity for acidic amino acid residues. Among these are the �-chymases: macaca and baboon chymase, sheep mast cell protease, and the rodent mMCP-5 and hamster chymase-2. Also the �-chymases rMCP-3, hamster chymase-1 and gerbil chymase-1 hold these positions intact and have a predicted acidic P2´ specificity.

Concluding remarks This thesis has focused on characterizing the extended cleavage specificity of members of the chymase family of enzymes. The studies are based on phage display methodology, which has several advances compared to other methods. The phage library provides a fast and convenient way to screen millions of substrates in a single reaction. In the phage library used by us, 50 millions unique nonamers are displayed on phages. Considering that a poten-

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tial aromatic P1 residue can be randomly placed in nine different positions of each substrate, the total number of cleavage sites screened in each reaction is very high. While other commonly used methods, are limited to investigate the positions N-terminal of the cleaved bond, the phage display approach further expands the range to also include the C-terminal positions. Determi-nations of cleavage specificities have historically therefore focused on the N-terminal residues, and the C-terminal positions have been neglected. Finally, the phage display methodology can detect subsite interdependence, i.e. if a particular amino acid in one position affects the preference for residues in other positions.

The characterization of the cleavage specificity of mast cell chymases has in this study provided insight into the importance of the C-terminal positions in substrate recognition. Three of the chymases studied in this thesis showed strict preferences for amino acids in C-terminal positions. The most obvious differences in cleavage specificity between the chymases are accordingly found in the C-terminal positions. The preference for acidic P2´ residues seems to be conserved among the �-chymases or �-chymase homologues, which are expressed by CTMCs. In contrast, the MMC expressed chymases mMCP-1 and rMCP-4 have strict preferences for the hydrophilic P1´ resi-dues Ser and Arg and aliphatic amino acids in P2´. The CTMC and MMC chymases, however, share similar preferences for hydrophobic, preferably aliphatic, amino acids N-terminal of the cleaved bond. Relatively small dif-ferences in cleavage specificity are thus seen among the chymases expressed in either CTMCs or MMCs, compared to the differences seen between the chymases expressed by these two MC subpopulations. These features sub-stantiate the idea that the different subsets of serine proteases expressed by the MMCs or CTMCs target different substrates and thereby provide differ-ent functions to the host.

Important information regarding the functional relationships between chymases in rodents and human has also been obtained. The conclusion that the mouse �-chymase mMCP-4 is the functional homologue of the human �-chymase is interesting in several ways. The function of �-chymases is most likely very important, considering the wide expression in mammalian spe-cies and that an ancestral �-chymase gene has been found in the American opossum. This specificity can thereby be traced back to the last common ancestor of marsupials and placental mammals, which has been dated to approximately 185 million years ago (196). In mouse, this important func-tion has probably been preserved by the �-chymase mMCP-4, when the �-chymase (mMCP-5) changed its primary specificity from chymotrypsin-like to elastase-like. This finding can potentially be very important when investi-gating the function of the human �-chymase. The rodent specific expression of �-chymases has made it difficult to extrapolate data from our most com-monly used animal models, rat and mouse. By clarifying the functional rela-tions between the chymases expressed in these species, more can be learned

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from these models. An mMCP-4 knockout has been developed and can po-tentially provide highly interesting data about the function of the HC (128).

Phage display provides a powerful tool to determine substrate specificity of enzymes, in this case MC chymases. This is however only the first step to understand the physiological role of these enzymes. As the next step their in vivo targets have to be identified. In paper I, an in silico screening for poten-tial substrates of mMCP-1 was performed. The candidate substrates may have theoretically very interesting effects, but taking the step from database searches to identify an in vivo target might be difficult. The substrate must be available at the same tissue locations as the enzyme, and the specific tar-get sequence must be accessible for the enzyme. Identifying novel targets would most likely require a screening of a substantial amount of candidates. However, when searching for potential substrates that can explain an ob-served effect of the enzyme, this list can be drastically reduced. In addition, when testing in vitro cleavage of candidate targets, the potential effect of proteoglycans should be tested. CTMC chymases are released in complex with heparin, and the heparin might either repel or attract potential sub-strates.

In conclusion, the results presented in this thesis have provided new in-sights of the importance of the primed positions in substrate recognition by mast cell chymases. The preferences for amino acids in these positions pro-vide evidence for different functions of the rodent �-chymases expressed in either CTMCs or MMCs. Important information rearding functional similari-ties between the rodent �-chymases and �-chymases expressed in other spe-cies was also obtained by the identification of virtually identical cleavage specificities of the mouse �-chymase mMCP-4 and the human �-chymase. The molecular mechanisms responsible for the preference for acidic amino acids in position P2´, shared by these two enzymes were also identified. Many of the uncertainties regarding mast cell chymases have been clarified and the information concerning the extended cleavage specificity of these chymases is an important contribution to the determination of their function.

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Sammanfattning på svenska

Vi lever i en miljö där vi ständigt exponeras för mikroorganismer såsom bakterier, virus och parasiter. För att överleva i en sådan miljö har ett för-svarssystem som kallas immunförsvaret utvecklats. Immunförsvaret skyddar oss från infektioner som om de inte stoppas snabbt kan leda till allvariga och dödliga sjukdomar. Immunförsvaret består av en mängd olika celltyper och vävnader som finns spridda i kroppen. Även våra barriärer mot omvärlden, t.ex. huden och slemhinnorna i mag-tarmsystemet och luftvägarna, räknas ibland hit eftersom de i de flesta fall förhindrar att mikroorganismerna kom-mer in i kroppen. När de, trots barriärerna, ändå kommer in i kroppen, akti-veras immunförsvaret. Den del av immunförsvaret som kallas det medfödda immunförsvaret reagerar först, och bland dessa celler hittar vi bl.a. mastcel-lerna. Gemensamt för cellerna som hör till det medfödda immunförsvaret är att de har receptorer på sin yta som binder till proteiner som är vanliga på mikroorganismerna men som inte finns på våra kroppsegna celler. När dessa receptorer binder till mikroorganismerna aktiveras cellerna, vilket kommer leda till en inflammatorisk reaktion. Cellerna släpper då ut substanser som leder till att vävnaden svullnar och blir röd och smärtkänslig. Andra substan-ser som samtidigt släpps ut, leder till att fler immunceller lockas till inflam-mationshärden. En del av cellerna i det medfödda immunförsvaret kallas för fagocyter, vilka har förmågan att kunna ”äta upp” mikroorganismer. Andra celler kan utsöndra giftiga substanser som dödar mikroorganismerna.

Den andra delen av immunförsvaret kallas för det adaptiva immunförsva-ret. Bland cellerna i denna del hittar vi T- och B-lymfocyterna. Dessa celler har alla unika receptorer på sina cellytor. Det innebär att alla lymfocyter kan binda till och aktiveras av olika delar av enskilda mikroorganismer. Vid en infektion aktiveras därför bara en bråkdel av alla lymfocyter, men dessa för-ökar sig snabbt och bildar snart en stor koloni av likadana celler. När B-cellerna aktiveras börjar de producera och utsöndra antikroppar. Antikroppar är Y-formade lösliga proteiner som kan binda till samma ytstruktur på mik-roorganismerna som B-cellerna de producerats av. När antikroppar binder till mikroorganismerna synliggörs de för det övriga immunförsvaret (bl.a. fagocyterna), som effektivare kan bekämpa inkräktaren. B-cellerna och anti-kropparna är speciellt viktiga för att bekämpa bakterier. T-cellerna är där-emot specialiserade på att bekämpa virusinfektioner. De cirkulerar i kroppen och känner av om våra egna celler är infekterade av virus. Om de stöter på

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en virusinfekterad cell har T-cellerna verktygen att döda denna cell, så att infektionen inte sprids vidare.

Immunförsvaret är med andra ord ett väldigt raffinerat och komplicerat system som skyddar oss mot allvarliga infektioner. Hela systemet bygger på ett fungerande samspel mellan de olika cellerna och vävnaderna och att de inflammatoriska reaktionerna kan regleras på ett mycket välkontrollerat sätt. Om inte detta fungerar riskerar vi livsfarliga autoimmuna reaktioner, d.v.s. en situation då kroppens immunceller felaktigt börjar bekämpa kroppens egna celler, eller kronisk inflammation. Sådana reaktioner kan leda till all-varliga sjukdomar som t.ex. multipel skleros (MS) reumatoid artrit (RA) eller typ 1 diabetes. Även överkänslighetsreaktioner som allergier och astma är exempel på sjukdomar där regleringen av immunförsvaret inte fungerar som det ska. I dessa fall är det mastceller som blir aktiverade mot vanliga och ofarliga substanser, som t.ex. födoämnen, pollen eller pälsdjur. Dessa överkänslighetsreaktioner har ökat kraftigt i västvärlden under de senaste årtiondena och skapar stora besvär för de som drabbas. Idag vet vi mindre om vilka positiva effekter mastcellerna har än deras roll i dessa sjukdomstill-stånd. Det är därför viktigt att forska om mastcellernas funktion.

Mastcellerna finns utspridda utmed kroppens ytor mot omvärlden, främst i huden och bukhålan samt i slemhinnorna i tarmen och luftvägarna. De är alltså perfekt placerade för att snabbt kunna möta inkräktande mikroorga-nismer. När mastcellerna blir aktiverade släpper de omedelbart ut inflamma-toriska mediatorer som är lagrade i korn, s.k. granula, inuti cellerna, vilket bl.a. leder till rekrytering av fler immunceller. På grund av dessa egenskaper har mastcellerna liknats vid kroppens vaktposter som håller utkik efter infek-tioner och kan skaffa förstärkning när det behövs.

Mastcellerna aktiveras oftast genom att antikroppar av IgE-typ, som sitter bundna på cellernas yta, binder till proteiner på mikroorganismer eller aller-gen (de ofarliga substanserna vi kan bli allergiska mot). Beroende på inne-hållet i kornen och var mastcellerna hittas i kroppen kan man i mus och råtta dela in mastcellerna i bindvävsmastceller och slemhinnemastceller. I männi-ska ser mastcellerna lite annorlunda ut, men de som mest liknar bindvävs-mastceller kallas MCTC och de som liknar slemhinnemastceller kallas MCT.

Mediatorerna som lagras i mastcellernas korn är bl.a. histamin som ger många av de allergiska symptomen och heparin som är viktigt för lagringen av mediatorerna. Det finns även gott om s.k. serinproteaser i dessa korn. Proteaser är proteiner som klyver andra proteiner och serinproteaser är en grupp av proteaser som är beroende av aminosyran serin för att kunna genomföra klyvningen. Den här avhandlingen syftar till att öka kunskapen kring vissa av dessa proteaser eftersom vi vet ganska lite om deras funktion.

Det finns i mastcellerna två huvudsakliga underfamiljer av serinproteaser; kymaser och tryptaser. Kymaserna, som är den underfamiljen som jag har arbetat med, klyver proteiner efter stora vattenavvisande aminosyror medan tryptaser klyver proteiner efter positivt laddade aminosyror. Proteiner är

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långa pärlband av sammanfogade aminosyror som är veckade på ett bestämt sätt för att kunna utföra sina speciella funktioner. I människa finns det endast ett kymas i mastceller, och det kallas för det humana �-kymaset. Detta ky-mas hittar vi bara i de bindvävsmastcell-lika cellerna i människa, MCTC. I gnagare, som mus och råtta, finns det förutom ett �-kymas även en grupp liknande proteaser som kallas för �-kymas. Dessa �-kymas finns i både bindvävsmastceller och slemhinnemastceller. Hos gnagarna har �-kymasen förändrats så att de klyver efter andra aminosyror än �-kymasen i andra ar-ter. Vi tror därför att �-kymasen i gnagare inte har samma funktion som det humana �-kymaset. Istället har gnagarna �-kymas, som inte har förändrats på samma sätt, och antagligen kan utföra samma funktion som det humana �-kymaset. Idag vet vi inte med säkerhet vad kymaserna har för huvudsaklig funktion när de släpps ut ur mastcellerna. Anledningen är delvis beroende på skillnaderna i vilka kymas som finns i människor jämfört med mus och råtta. Dessa gnagare är de viktigaste modelldjuren vi använder oss av inom den biologiska forskningen, och så länge vi inte med säkerhet vet vilket eller vilka kymas som har samma funktion som det humana �-kymaset är det svårt att undersöka dess funktion.

I den här avhandlingen presenteras karaktäriseringar av både �-kymas och �-kymas, för att reda ut skillnader och likheter mellan olika kymaser i människa, mus och råtta. Jag har analyserat den förlängda klyvningsspecifi-citeten för dessa proteas, vilket betyder att jag har undersökt vilka aminosy-rasekvenser som de olika kymasen helst klyver. Till min hjälp har jag använt en metod som heter ”phage display” som med ett svenskt ord skulle kunna kallas fag-presentation. Fager är små virus som bara infekterar bakterier. På ytan av dessa fager har vi modifierat dem att presentera, eller visa upp, svan-sar av slumpvis sammansatta aminosyrasekvenser (peptider). Vi har ett helt bibliotek med olika slumpvis sammansatta peptider, vilket sammanlagt inne-håller ungefär 50 miljoner unika sekvenser. Genom att binda upp dessa fags-vansar på en fast yta, kan vi tillsätta kymaset vi vill analysera och se vilka peptider proteaset helst klyver. Det är viktigt att veta att varje fag bara visar upp en specifik sekvens, så vi vet vilka sekvenser som klyvts. När proteaset har klyvt en fagsvans lossnar fagen från den fasta ytan och genom att samla upp dessa och tillsätta dem till bakterier, produceras nya fager med hela fagsvansar igen. De nya fagerna binds därefter upp på nytt och kymaset får än en gång välja bland de svansar som finns på fagerna och klyva de som passar bäst för proteaset. Genom ett antal sådana anrikningar av klyvnings-bara peptider kan vi sedan se vilka peptider som varje kymas helst klyver.

När vi analyserade klyvningsspecificiteten för det humana �-kymaset och de två �-kymasen rMCP-1 från råtta och mMCP-4 från mus, fann vi att de hade väldigt lika klyvningsspecificitet. Speciellt mMCP-4 och det humana �-kymaset valde mycket likartade peptider vilket tyder på att musens mMCP-4 har samma funktion som den humana �-kymaset. Dessa kymas är

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även lika varandra på många andra sätt och finns i motsvarande bindvävs-mastceller i varje art, d.v.s. de finns i samma vävnader i kroppen.

När kymasen klyver peptider krävs det att peptiderna kan binda till det ställe på proteaset där klyvningen kan äga rum. Denna plats kallas för den peptidbindande regionen. Varje aminosyra i det pärlband som bygger upp en peptid interagerar med en specifik plats på proteaset. När vi analyserade det humana �-kymaset och de två �-kymasen rMCP-1 och mMCP-4 fann vi att det humana �-kymaset och mMCP-4, men inte rMCP-1 föredrog negativt laddade aminosyror i en position två aminosyror bort från stället där peptid-kedjan klyvs (position P2´). När vi undersökte de ställen i proteiner där vi vet att det humana �-kymaset kan klyva, fann vi att detta kymas för det mes-ta väljer just negativt laddade aminosyror i denna position. Denna preferens verkar därför extra viktig när det humana �-kymaset och mus �-kymaset mMCP-4 väljer sina peptider som ska klyvas. Vi ville därför undersöka vilka aminosyror i det humana �-kymaset som bestämmer att detta kymas väljer negativt laddade aminosyror i position P2´. Genom att jämföra utseendet på olika kymas som har denna preferens med andra kymas som inte har den-samma, hittade vi två aminosyror som skulle kunna leda till denna preferens. Det var aminosyrorna arginin143 och lysin192. Arginin och lysin är två po-sitivt laddade aminosyror som kan attrahera negativt laddade aminosyror och siffrorna 143 och 192 anger var i kymasproteinet som aminosyrorna finns. För att undersöka detta förändrade vi aminosyrasekvensen för det humana �-kymaset och ersatte arginin143 och lysin192 med andra aminosyror. Resul-tatet visade att båda aminosyrorna bidrog till att locka negativt laddade ami-nosyror i position P2´ hos peptider, till det humana �-kymaset, och att både arginin143 och lysin192 behövdes för att få ett starkt urval av sådana pepti-der.

Vi har även undersökt ett �-kymas som finns i slemhinnemastceller i mus, för att se hur annorlunda �-kymasen som finns i bindvävsmastceller är från dem som finns i slemhinnemastceller. Kymaset som vi undersökte var mMCP-1, som i möss har visat sig ha en effekt i immunförsvaret mot vissa parasiter. Klyvningsspecificiteten som mMCP-1 hade, visade sig vara väl-digt specifik i fyra positioner. Två av dessa skiljde sig väldigt mycket från dem som väljs ut av kymas som finns i bindvävsmastceller. Detta tyder på att �-kymaserna som finns i olika mastceller också har olika funktioner. Vi sökte också i en proteindatabas som innehåller alla proteiner som finns i mus efter proteiner som innehåller aminosyrasekvenser som liknar de vi såg att mMCP-1 gärna klöv. Vi hittade då ett antal olika proteiner som mMCP-1 skulle kunna klyva och som kan förklara hur detta kymas bidrar till immun-försvaret mot parasiter.

Resultaten som presenteras i den här avhandlingen visar att �-kymaset i mus, mMCP-4, klyver i princip identiska peptider som det humana �-kymaset och att de med största sannolikhet har samma funktion. Genom att studera mMCP-4 kan vi därmed lära oss väldigt mycket om det humana �-

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kymaset. Vi visar även att �-kymaser som uttrycks i olika mastceller verkar ha olika funktioner.

Genom att leta i databaser som innehåller de proteiner som finns i en or-ganism efter proteinsekvenser som liknar de som kymaserna föredrar att klyva, kan våra resultat framöver också leda till att vi kan hitta nya klyv-ningsbara proteiner, eller substrat, för olika mastcell-kymas. Det kan leda till att vi hittar nya funktioner till kymasen eller kan förklara funktioner som redan är kända. Detta kommer att ge oss en förbättrad bild av mastcellens funktion i vårt immunförsvar.

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Acknowledgements

Arbetet med den här avhandlingen under de senaste fem åren har varit en händelserik och lärorik tid, och den hade inte varit möjlig utan hjälp av en mängd människor som funnits i min närhet. Jag är skyldig er alla ett stort tack.

Lasse, min handledare, tack för att du guidat mig in och igenom immunolo-gins och mastcell-proteasernas fascinerande men ibland något förvirrande värld, och även för ditt brinnande intresse för evolution som smittat av sig. Jag vill även önska dig lycka till i din fortsatta forskning om mastcell-proteaser, evolution och terapeutiska vaccin.

Jenny, vi har följts åt under de här åren vilket har varit ett sant nöje. Tack för din positiva energi och smittande skratt samt alla påminnelser om tidiga möten. Du har många bra egenskaper jag försöker ta efter varje dag, speciellt din målmedvetenhet. Maike, vi har också haft många år tillsammans på ICM, och du har varit en stor inspirationskälla för det vetenskapliga och kritiska tänkandet. Lycka till med din nya karriär. I also wish you, Sayran, and your family, all the best and good luck with your work on Fc receptors.

Jag vill även passa på att tacka de gamla doktoranderna och en postdoc, Ulrika, Anna, Camilla L, Lotta, Molly och Sara, som fanns på plats när jag först kom till gruppen och som skapade en glad och välkomnande atmo-sfär. Särskilt tack till Ulrika som lade grunden för projektet jag arbetat med och visade stort tålamod när jag var ny. Nu ser jag skillnad på filter och skyddspapper!!

AveMaria, Parvin och Siv, ni var klipporna på immunologilabbet och höll både labbet och mitt humör på topp. Tack för alla frågor som ni svarat på, jag har lärt mig mycket av er.

Sandra, jag är glad att du fattade beslutet att flytta din forskargrupp till oss här på BMC. Du har tillfört ett kliniskt tänkande till immunologin, som har varit intressant och spännande att få ta del av. Sofia, tack för att du slutat sparka mig på smalbenen. Allvarligt talat, jag är imponerad av självsäkerhe-ten du har i det du gör. Nästa gång är det din tur, kör hårt! Kajsa, jag upp-skattar din raka attityd och charm, jag tror att det kommer att leda dig till en karriär som går spikrakt uppåt. Även tack för gott samarbete på den sista kursen och att du lärt mig vikten av att låsa dörrar! Cissi, du dök upp hos oss med din glada och avslappnade attityd när jag hade en lite tyngre period och behövde det som mest. Det är dessutom trevligt att höra lite öschötska på

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labbet. Maria A, du hann bara göra ett kort gästspel på BMC innan du dis-puterade, men det räckte för att introducerade Twinings överlägsna Earl Grey. Det är både jag och te-producenten glada för.

Jag vill även passa på att tacka mina projektstudenter Mattias E, Maria F, Lena, Per H. och Camilla P. Ni har alla bidragit med viktiga delar till mina projekt. Lycka till i framtiden! Lycka till framöver vill jag även önska de nuvarande projektstudenterna Yue, Erik och Mia och vår gästforskare Anders. Dessutom, tack för trevliga middagar med spridda immunologer i PAFIBB-sällskapet och trevliga luncher med Erika.

Jag vill rikta ett varmt tack till alla medarbetare på ICM. Alla seniora forskare, speciellt Diarmaid Hughes för uppmuntrande samtal när det be-hövts. Alla doktorander/postdocs som har skapat en trevlig arbetsatmosfär, roliga ölklubbar och fester. På MCB, speciellt tack till Per som var min för-sta mentor på ICM, och på senare tid för diskussioner om vetenskapen och framtiden. Niklas, för trevliga sena luncher med Al Bundy. ICMs Magnum look-a-like Pontus. Nicole, Greta, Christoph och Ben, det har varit ett nöje att dela korridor med er. Lycka till i andra änden av BMC! Alla er på Mikro särskilt: Marie, för många roliga konsertminnen, Disa, Keso-Jonas, Tobbe och Patricia för att ni bildar den trevligaste gruppen på ICM (utanför immu-nologiprogrammet). Tack för alla era trevliga sammankomster jag fått vara med på. Klas, för att du delar med dig av din nattsvarta humor. Helene, du är en kämpe, envishet lönar sig. Ema, stort lycka till med familjen och karri-ären i Makedonien. Ett stort lycka till i fortsättningen även till er övriga (Cia, Erik, Johan R, Fredrik, Shiying, Hava, Pernilla, Bhupi, Sonchita, Henrik T, Emma, Johan A, Karin och gamlingarna Magnus och B-Mathias (ni är så många..))! På struktur vill jag särskilt tacka mina goda vänner, Daniel som hjälpt mig med omslaget och Henrik I som sponsrar min pokerkarriär.

Självklart vill jag även tacka den fasta staben på ICM; Administratörerna (framför allt Sigrid) som alltid tar sig tid, Christer som fixar allt det prak-tiska, Akiko som ser till att datorerna fungerar samt Ulrika, Eva och Ewa för teknisk assistans och material. Tack för att ni skapar förutsättningarna att bedriva forskning på ICM.

Jag vill tacka alla mina vänner utanför institutionen (Håkan, Dieter, Emil, Daniel, Henrik I, Knocksson, Itti, Maria A, Katja, Peter K, Hen-rik A och Fredrik B). Tack för alla Roskilderesor, pokerkvällar, fotbolls- och hockeymatcher med en öl i handen m.m. Ni har alla indirekt bidragit till denna avhandling genom att störa mitt vetenskapliga fokus och tillåtit mig att slappna av vissa kvällar och helger. Fortsätt med det!

Jag har enormt mycket att tacka min familj för. Mina kära föräldrar Alve och Gunilla, ni har alltid ställt upp för mig och stöttat mig i mina val. Ni är fantastiska och det är skönt att veta att ni finns där. Syster Maria med sambo Peter och busfröna Charlie, Clara, Amanda och Johanna. Det är alltid

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lika roligt att träffa er och det blir alldeles för sällan. Vår traditionella vecka på Öland är en höjdpunkt varje år.

Slutligen vill jag tacka den person som varit helt ovärderlig under denna tid. Min kära Linda. Jag är fantastiskt glad att du stått ut med mig den senas-te tiden och att du stöttat mig och finns här för mig. Nu är jag också klar och vi har alla möjligheter att forma vår framtid, hur vi vill och var vi vill. Det är dags att ta tillvara på vår tid på jorden! Jag är glad att vi gör det tillsammans.

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cleavage specificity of mast cell chymases

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