Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 870
Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases
BY ULRIKA KARLSON
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This thesis is based on the following articles, which are referred to in the text by their Roman numerals:
I. Karlson U., Pejler G., Fröman G., and Hellman L. “Rat mast cell protease 4 is a E-chymase with unusually stringent substrate recognition profile” The Journal of Biological Chemistry. (2002) 277:18579-85
II. Karlson U., Pejler G., Tomasini-Johansson B., and Hellman L. “Extended substrate specificity of rat mast cell protease 5, a rodent D-chymase with elastase-like primary specificity” Accepted for publication in The Journal of Biological Chemistry.
III. Hallgren J., Karlson U., Poorafshar M., Hellman L., and Pejler G. “Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.” Biochemistry. (2000) 39:13068-77.
IV. Hallgren J., Estrada S., Karlson U., Alving K., and Pejler G. “Heparin antagonists are potent inhibitors of mast cell tryptase.” Biochemistry. (2001) 40:7342-9.
Reprints were made with permission from the publishers. CONTENTS
ABBREVIATIONS...... 6 INTRODUCTION...... 7 GENERAL OVERVIEW ...... 7 MAST CELLS...... 8 Mast cell heterogeneity and granular content...... 8 Mast cell activation ...... 14 Mast cells in host defense ...... 15 SERINE PROTEASES...... 17 Catalytic mechanism ...... 18 Substrate binding and specificity of chymotrypsin-like serine proteases...... 18 Mast cell serine proteases...... 20 What is left to learn about mast cell serine proteases? ...... 28 SUMMARY OF PRESENT STUDIES...... 30 AIM...... 30 RESULTS AND DISCUSSION ...... 30 Paper I...... 30 Paper II...... 32 Paper III...... 34 Paper IV...... 35 CONCLUDING REMARKS ...... 36 ACKNOWLEDGEMENT...... 40 REFERENCES...... 42 ABBREVIATIONS aa amino acid(s) Ang angiotensin BMMC bone marrow-derived mast cells CPA carboxypeptidase A CS chondroitin sulfate CTMC connective tissue mast cells DFP diisopropyl fluorophosphate HC human chymase Ig immunoglobulin IL interleukin LPS lipopolysaccharide LT leukotriene MC mast cell MCs mast cells MMC mucosal mast cells mMCP mouse mast cell protease MMP matrix metallo-protease PG prostaglandin rMCP rat mast cell protease TGF-E transforming growth factor-E TLR Toll-like receptor TNF-D tumor necrosis factor-D INTRODUCTION
GENERAL OVERVIEW Every day we encounter bacteria, viruses and other potentially harmful microbes. The physical barriers, such as the skin and mucosal surfaces, prevent the bacteria from entering the body. However, microbes sometimes penetrate the physical barriers and then our immune system is activated. During an immune response, a battery of cells and effector molecules act to remove the invading microbes by various mechanisms. These mechanisms induce localized inflammatory responses that eliminate the infectious agent with minimal tissue damage to the host. Inflammation involves recruitment of immune cells, such as neutrophils, monocytes, lymphocytes and basophils to the tissue. The resident mast cells are thought to play an important role in the initiation and augmentation of the inflammatory process. Under certain conditions, the immune system can initiate an inappropriate response to a specific antigen, either by responding too vigorously, or by activating inappropriate cells. Allergy is an example of such misdirected actions of the immune system. The antigens in allergic reactions are called allergens. Examples of allergens are proteins from house dust mites, pollen, mold spores, and also fur, saliva and urine derived from different animals. The allergen induces an allergic response by activating mast cells, which react by releasing their pharmacologically potent mediators. The mediators are responsible for the symptoms seen in allergic individuals. Although the symptoms are most often not lethal, they do influence the quality of life of the affected person with manifestations such as sneezing, increased mucus production in the nose and runny eyes. Rodents can be used to investigate aspects of the mast cells and their role in allergy, inflammation and host defense. In the present study we have focused on rodent mast cell serine proteases, which are the major protein constituent of the mast cell granules. Serine proteases are enzymes that are released upon activation of the mast cell and due to their high abundance they are thought to have a central role in mast cell function. Much effort has also been devoted to studies of the biological targets of these proteases. However, there are still many important questions to be answered, and the
7 purpose of this thesis is to shed some light on the biochemical characteristics of these enzymes to increase the understanding of their function in mast cell biology.
MAST CELLS Mast cells (MCs) originate from hematopoietic stem cells located in the bone marrow. They circulate in the blood as precursors before homing to the tissue where they finally complete their maturation. Mature MCs are found scattered in the skin and mucosal layers, where they are dormant until activated by environmental stimuli. A general feature of MCs is the staining of their cytoplasmic granules with basic dyes. The acidic granules are storage vesicles for biologically active substances such as histamine, proteoglycans, cytokines and proteases. When the MC is activated, these stored mediators are released by a process called degranulation (Figure 1). Upon activation, the MC also initiates a de novo production of lipid mediators, e.g. leukotrienes and prostaglandins, as well as a cascade of cytokines, that are released by exocytosis minutes to hours after degranulation. The biological effects of these substances are vascular leakage, bronchoconstriction or intestinal hypermotility, increased mucus production and inflammation. These effects may be seen during allergic reactions, in which MCs are the main effector cells. Any allergic response must start with a sensitization phase, where the allergen induces B-lymphocytes to produce allergen-specific immunoglobulin E (IgE). IgE is secreted and binds with high affinity to FcHRI, located on the MC surface. Upon a second encounter with the allergen, the FcHRI-bound IgE molecules bind the allergen, and the MC starts an effector phase by releasing its mediators. In allergic asthma, the mediator release from MCs causes bronchoconstriction as well as mucus production and edema, which contribute to bronchial obstruction.
Mast cell heterogeneity and granular content Both rodent and human MCs display a high degree of heterogeneity, revealed most clearly by the content of their granules and their tissue location. Two main subtypes have been described in rodents, which are referred to as connective tissue MC (CTMC) and mucosal MC (MMC) (1). As indicated by their name, MMCs are located beneath mucosal surfaces, such as the lamina propria of the respiratory and gastrointestinal tract,
8 whereas CTMCs are found mainly in the skin and peritoneal cavity and can be relatively easily isolated from the peritoneal fluids of rodents (2).
Allergen
Mast cell
Minutes Minutes to hours Degranulation Secretion
Histamine Leukotrienes Cytokines Prostaglandins Proteases Chemokines Proteoglycans Cytokines
INFLAMMATION
Figure 1. The mast cell is activated by cross-linkage of its IgE, which stimulates degranulation and production of new mediators. Examples of mediators are histamine, proteases and cytokines.
The MMC and CTMCs differ with respect to proteoglycan content, amount of histamine and their granule proteases (Table I). Human MCs can be similarly subdivided into the two subtypes MCTC and MCT (Table I), depending on the granule proteases they contain. MCTC contains both tryptase and chymase, but MCT contains tryptase only (Table II, page 21). Unlike the rodent MC, the human MC subtypes cannot be distinguished by simple histochemical analysis, since both types contain heparin (3) and hence have the same staining properties. Instead, immunohistochemical techniques using anti-protease (chymase and tryptase) staining can be used
9 (4). In humans, most tissues seem to contain a mixture of the two MC types, with different relative numbers depending on organ and anatomical site (5). Studies on rodent MCs suggest that both MMCs and CTMCs are mature cells with distinct roles. For example, MMCs, but not CTMCs, increase in number during parasitic infections and are thought to contribute to the expulsion of certain parasites (reviewed in (6)).
Table I. Mediators of the two mast cell subtypes.
MMCa CTMC mouse rat human mouse rat human (MCT) (MCTC)
Proteoglycans CS A, di-B, CS A, E heparin heparin heparin heparin E CS A, E CS E CS E CS A, E
Histamine + + + ++ ++ +
Prostaglandin PGD2 low PGD2 low PGD2 PGD2 PGD2 PGD2
Leukotriene LTC4 LTC4 LTC4 LTC4 low - LTC4 low
Metallo- - - - CPA CPA CPA protease Data from (7-11) a Abbreviations; MMC mucosal mast cell; CTMC connective tissue mast cell; MCT -tryptase containing human mast cell; MCTC -tryptase and chymase containing human mast cell.
The two types of MC share some features, for example the storage of biologically active components in their cytoplasmic granules. In addition, both types of cells initiate a de novo synthesis of some additional mediators, further amplifying the effects of the stored components.
De novo synthesized mediators Prostaglandins are a family of arachidonic acid derivatives that are released within minutes after mast cell degranulation. All prostaglandin (PG) synthesis starts with the release of arachidonic acid from the phospholipid bilayer into the cytoplasm. In MCs, the arachidonic acid is converted in subsequent steps into prostaglandin D2. Exocytosed PGD2 binds to receptors
10 on smooth muscle cells and acts as a vasodilator and bronchoconstrictor (12) and can also promote accumulation of inflammatory cells. Leukotrienes are another group of arachidonic acid metabolites, of which leukotriene C4 (LTC4) is the dominating molecule produced by MCs. Leukotrienes stimulate prolonged bronchoconstriction, enhance vascular permeability and promote increased mucus secretion (10), i.e. leukotrienes exert many of the detrimental effects seen in asthmatic airway obstruction. Mast cells also produce many cytokines, such as interleukins (IL) -1, -3, -4, -5, -6, -8, -13, -16, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-D (TNF-D) (13). IL-4, for example, is involved in polarization of immature T helper (TH) cells into the TH2 cell type, and IL-4, together with IL-13, promotes IgE synthesis. Thus, MCs produce cytokines that lead to an amplification of IgE-mediated immune responses, such as anti-parasite reactions. In addition, IL-5 stimulates eosinophil development and survival, further facilitating protective immune responses against parasites. In addition to being released as a newly synthesized cytokine, TNF-D can also be stored in the MC granules (14). This storage represents a very important initial source of this cytokine, before de novo synthesis has begun. TNF-D triggers endothelial cells to up-regulate adhesion molecules and to produce chemokines, facilitating recruitment of monocytes and neutrophils to sites of release.
Stored mediators Some mast cell mediators are preformed, i.e. they are synthesized and stored in the secretory granules until released by degranulation. One of the best- studied preformed substances is histamine, a biogenic amine produced by decarboxylation of histidine. Histamine acts by binding to receptors H1, H2, H3 or H4 (15, 16) expressed by various cell types. Binding of histamine to receptors on endothelial cells promotes vascular leakage. Histamine also causes constriction of bronchial and intestinal smooth muscle, thus contributing to bronchoconstriction. The function of histamine has been studied using histidine decarboxylase knockout mice, which are unable to synthesize histamine (17). It was shown that the storage of MC proteases and heparin was decreased, probably due to electrostatic imbalance in the MC granules. Further, these mice displayed impaired angiogenesis as well as impaired plasma extravasation. Interestingly, an augmented recruitment of
11 neutrophils was seen in the knockout mice that correlated with an improvement in bacterial clearance rate (18), suggesting that anti-histamines could be used in treatment of bacterial infections. The mast cell granules contain two major types of proteoglycans; heparin and chondroitin sulfate. In rodents, heparin is only present in CTMCs, whereas chondroitin sulfate is found in both types of MC (Table I). Proteoglycans consists of a protein core with covalently linked polysaccharide side chains known as glycosaminoglycans (GAG) (Figure 2).
GAG
Glucuronic acid
Galactose Link tetrasaccharide Galactose Xylose
O Serine residue CH2 Core protein CH
Core protein
GAG side chain
Figure 2. Schematic structure of serglycin proteoglycans. Proteoglycans consists of a core protein with attached glycosaminoglycans (GAG), which are unbranched polysaccharides.
The protein core in the mast cell granule proteoglycans is serglycin, which contains a repetitive serine-glycine sequence. The hydroxyl group of the serine residues is utilized to attach the GAG side chains. The side chains consist of unbranched repeated disaccharides, the identity of which differs between different types of GAG. For example, heparin contains glucuronic
12 acid and N-acetyl-glucosamine as disaccharide units, whereas chondroitin sulfate contains glucuronic acid and N-acetyl-galactosamine (19). The sugar residues on the GAG side chains undergo different types of modifications, e.g. sulfation (reviewed in (20)). The presence of carboxyl- or sulfate groups on the carbohydrate side chain makes proteoglycans negatively charged. Generally, heparin proteoglycan is more highly sulfated than chondroitin sulfate, and is consequently more negatively charged. The high net negative charge confers on the proteoglycans their characteristic properties that allow them to function as a storage matrix for the positively charged MC proteases and histamine (21). The importance of heparin proteoglycan was recently shown in mice with a targeted disruption of the heparin-sulfating enzyme, N- deacetylase/N-sulphotransferase-2, NDST-2 (22, 23). The CTMC displayed severe defects in storage of granula contents, such as histamine and proteases, as determined by transmission electron micrographs (22) and Western blots (22, 23). Mast cell granules also contain an abundance of neutral proteases that seem to be expressed exclusively by MCs. One of the neutral proteases is carboxypeptidase A (CPA), present only in CTMCs (both human and rodent). CPA is a Zn2+-dependent metalloprotease with exopeptidase activity, i.e. it degrades a protein target from the C-terminal side towards the N-terminal. CPA preferably cleaves targets that have C-terminal aromatic or aliphatic residues (24). One of the MC serine proteases, a chymase, as well as heparin proteoglycan seems to be required for efficient storage of CPA (22, 23, 25). Since chymase specifically cleaves after aromatic residues, it has been suggested that chymase and CPA may work in a consecutive manner, where chymase produces the substrates for CPA (26, 27). Another group of neutral proteases is the serine proteases. The proteolytic activities of the serine proteases include chymotrypsin- or trypsin-like specificity i.e. they cleave a substrate after hydrophobic or basic residues, respectively. In addition, a new group of proteases more related to T-cell granzyme B has been found in rat MC and mouse basophils (9, 28), the rMCP-8 family. However, the substrate specificity of these proteases is still unknown.
Mast cell activation Mast cells can be activated by a variety of immunological and non- immunological compounds. For instance, molecules that are produced by the
13 adaptive immune system, e.g. IgE and IgG, can activate mast cells (Figure 3). Interaction of a multivalent antigen (allergen) with specific IgE molecules that are attached to the mast cell via the high affinity receptor FcHRI (29, 30), brings the receptors in position to initiate intracellular signaling. This cross-linkage of IgE results in degranulation of the MC (31). Furthermore, murine MCs express IgG receptors (FcJR), which, upon aggregation by IgG coated antigens, stimulate degranulation. FcJRI and FcJRIII share the J-subunit with FcHRI and thus share some of the components in the subsequent intracellular signaling cascade (32).
C3b C3b Allergen/antigen Bb IgG IgE C5 C5a Receptor FcHRI U U FcJRIII U U C5aR U TLR U
Mast cell
Figure 3. Schematic drawing of some ways to activate mast cells.
On murine MCs, the low affinity receptor for IgG, FcJRIII, is the IgG receptor responsible for degranulation as assessed by studies on FcJRIII–/– mice (33). This receptor can be triggered by immune complexes, such as antibody coated microbes, and thereby induce MC degranulation (34). To determine the contribution of the different receptors in anaphylaxis, the ability to induce systemic anaphylaxis has been tested on mice devoid of FcHRI or the FcJ-chain (devoid of all receptors with the common J-chain, i.e. FcHRI, FcJRI/III) (35). The FcJ–/– mice were severely deficient in the ability to express systemic anaphylactic responses. In contrast, the FcHRI- deficient mice had only slightly reduced responses compared to control, and the mortality rate was still very high. This finding was also in correlation with the level of mast cell degranulation, which was reduced but still substantial in the FcHRI-deficient mice. Thus, the FcJRIII contributes
14 substantially to MC degranulation in vivo. However, human mast cells seem to lack expression of FcJRIII (36) making MC activation independent of FcJRIII in humans. The innate immune system is the first line of defense against infection. The mechanism by which microbes induce host innate immune responses is complex. Toll-like receptors (TLR), for instance, are membrane bound receptors that are capable of binding various bacterial products, such as conserved patterns on microorganisms, including lipopolysaccharides (LPS), lipoproteins, peptidoglycans, bacterial DNA and yeast zymosan (reviewed in (37)). Binding of bacterial products on TLR present on MCs ultimately lead to expression and release of cytokines by the MC (38) or in some cases, degranulation of the MC (39) (Figure 3). MCs can also be activated via complement receptors by the complement products C3a, C5a (anaphylotoxins) and other C3-derived fragments, which are also products of activated innate immune responses (40). Human mast cells display differential expression of the C5a receptor (C5aR) (41), but do not express the receptors for C3 derived fragments, such as CR1, CR2, CR3 and CR4 (41). However, bone marrow MCs from patients with systemic mastocytosis had high levels of CR1, CR4 as well as C5aR (42), thus indicating a mechanism for up-regulation of these receptors. In addition, mouse bone marrow-cultured MCs could be triggered to up-regulate CR3 upon stimulation with LPS (43), further supporting a role for complement receptors in activation of MCs e.g. during bacterial infections.
Mast cells in host defense After the identification of the mast cell by Paul Ehrlich in the 1870s, the MC physiological function has to a large extent remained a mystery. The MC role in the pathophysiology of allergic diseases has been recognized for a long time, but what about its role in normal host defense? Considering that the MC has been preserved throughout evolution, it must possess qualities that are valuable to the host. For instance, MCs have two properties that make them perfect as initiators of immune responses. Firstly, their location at the outermost surfaces of the body, such as the skin and mucosal layers, makes them ideally placed to be the first immune cells to encounter microbial attack. Secondly, MCs store their pre-synthesized mediators in their granules, ready to be released at the first encounter with the microbe.
15 As stated earlier, these mediators have the ability to instantly trigger a strong inflammatory response, suggesting a role in host defense. Studies on mast cell-deficient mice (44) (e.g. W/Wv-mice, defective in the c-kit intracellular signaling) have been used to assess the contribution of MCs in host defense. Reversal of the W/Wv phenotype can be done by reconstitution of bone marrow-derived mast cells (BMMC) from normal littermates, which also ensures that the observed phenotype is dependent on the absence of MCs. Using this model, MCs have been documented to have a protective role in acute bacterial peritonitis, as shown by 100% mortality of W/Wv mice compared to 25% in MC-reconstituted W/Wv control mice (45). MCs are also important in the defense against Klebsiella pneumoniae, where the MC deficient mice had an impaired bacterial clearance rate (46). By subjecting mice to acute bacterial peritonitis, the importance of Toll-like receptor (TLR) expression on MCs was ascertained. The W/Wv-mice, which were reconstituted with TLR4-deficient BMMC, displayed increased mortality as well as a defective neutrophil recruitment compared to controls (38). TLR on mast cells can therefore be a link to MC involvement in innate immunity to bacteria. MCs have also been shown to participate in the expulsion of certain helminthes, such as Trichinella spiralis (47) and Strongyloides ratti (47-49). However, in some cases of helminth infection, such as Nippostrongulus brasiliensis in rat, MCs seem to promote the infection rather than having a beneficial role (6, 50). An additional function of MCs was revealed by studies on mice with a targeted deletion of NDST-2, affecting CTMC granula storage. These mice exhibited defects in fibronectin turnover, suggesting a role for MC in extracellular matrix remodeling (51). The redundant activities of the MC granula components make it difficult to document the contribution of a single mediator. However, the targeted disruption of mMCP-1, a serine protease expressed only in MMC, caused delayed expulsion of the parasite T. spiralis, confirming a specific role of this protease in defense against nematodes (52). Serine proteases are the most abundant protein mediators in the MC granules, and their structure, substrate specificity and their role in host defense as well as in pathogenesis of allergic diseases will be further discussed in the next section of this thesis.
16 SERINE PROTEASES Proteases are ordered into families by their catalytic-site residues, and also often by common sequence motifs around the catalytic residues. The term “serine protease” indicates the importance of a serine residue in the catalytic mechanism (53). The catalytic charge-relay system in serine proteases consists of an aspartic acid, a histidine and a serine residue, henceforth referred to as the catalytic triad. These residues are spread throughout the polypeptide chain, but are brought together and stabilized in the correctly folded enzyme. The catalytic triad does not determine the substrate specificity, but only provides a mechanism by which to cleave the substrate. Proteolytic enzymes also require unique structural elements to permit interaction with and binding of the substrate, and to coordinate the substrate in a position where cleavage can occur. To avoid random proteolysis and thus target protein degradation, many of the members of the serine protease family exhibit limited proteolysis, which means that they have restricted specificity. Serine proteases with limited specificity are required in many important biological processes where zymogen activation is necessary, including complement activation, blood coagulation and fibrinolysis. Serine proteases are endopeptidases, which means that they are capable of cleavage within a polypeptide chain. In the cleaved substrate, the amino acid residues extending toward the N-terminal from the cleavage site are called P1, P2, P3 etc., while the residues in the C-terminal direction are called P1’, P2’ etc (54) (Figure 4).
Protease Figure 4. Target sequence P4P3 P2 P1 P1’ P2’ definition. The Pn,.., P2, P1, P1’, P2’, .., Pn’ defines the positions surrounding the actual + Ala Val - NH3 Ala Phe Gly Asp COO cleavage site. The cleavage occurs between the P1 and P1’ Target amino acid sequence residues.
17 Catalytic mechanism Proteolytic processing, i.e. hydrolysis of peptide bonds, is initiated in serine proteases by creating a reactive serine. The catalytic triad, which in addition to the serine, consists of a histidine and an aspartic acid residue, forms a charge relay system (55). The histidine residue acts as a general base and enhances the nucleophilicity of the serine residue. This charge relay system stabilizes a proton transfer during enzyme catalysis. Substrate hydrolysis starts with a nucleophilic attack by the serine residue on the substrate carbonyl carbon atom adjacent to the scissile bond, creating a substrate/enzyme intermediate complex. This intermediate falls apart when a proton is transferred to the leaving group of the substrate, i.e. the C-terminal portion of the protein substrate, resulting in its liberation from the complex. The N-terminal portion of the protein substrate, still covalently attached to the enzyme, is released in a deacylation reaction that involves a nucleophilic attack by a water molecule (55).
Substrate binding and specificity of chymotrypsin-like serine proteases Virtually all chymotrypsin-like serine proteases share the common feature of having a P1 specificity that is limited to a few related amino acids. Depending on their substrate specificity, the majority of these enzymes fall into one of three subclasses; the elastase, the chymotrypsin and the trypsin subclass. These three subclasses have specificity for small hydrophobic, large hydrophobic and basic residues, respectively. The substrate pocket that binds the P1 residue is shaped to be highly complementary to a few specific residues, e.g. trypsin has an acidic residue at the base of its substrate pocket, thus allowing positively charged P1 residues to bind (56). The overall substrate-binding cavity is formed by two main loops consisting of residues 182-195 (chymotrypsin numbering according to (57) will be used throughout this thesis) and 214-228 (58). The backbone of the substrate P4-P2 residues forms a short anti-parallel E-sheet to the backbone of the enzyme residues 214-216, the non-specific substrate-binding loop (Figure 5). This interaction is suggested to direct the substrate to a position where the scissile bond is exposed to the reactive serine, and cleavage can then occur (58). However, structural elements distant from the substrate binding site are also important for substrate specificity, since they confer a stable conformation on the
18 substrate-binding residues (58). Amino acids 216 and 226 are usually a Gly residue in members of the trypsin and chymotrypsin family, as other residues restrict the admittance of substrates with larger P1 residues. Consequently, elastase does contain 216 and 226 residues with side-chains of considerable size, hence creating a shallower cavity shaped to accommodate small hydrophobic residues. Moreover, the NK-cell protease Met-ase (later designated granzyme M) also has larger residues at positions 216 and 226, which confers the peculiar methionine (and similar sized residues) specificity on this enzyme (59). This was established by producing the Met- ase mutant Ser216Gly, which could accommodate large aromatic amino acids, reflecting the importance of this residue in providing the Met-ase specificity.
Gly 216 Tyr 215 Ser 214 HO O N N N O H O H
O Figure 5. Hydrogen bonds between the backbone of the substrate residues P1-P3 and the enzyme residues 214- R H O 2 H O 216. Chymotrypsin-like enzymes interact with the substrate in the form N N N of an anti-parallel E-sheet, formed by the residues 214-216, thereby O R3 R1 providing a binding platform for the substrate. P3 P2 P1
Most chymotrypsin-like proteases have a disulphide bond that is located in the substrate-binding region, bridging Cys 191 to Cys 220. However, a subclass of hematopoietic granula-associated proteases e.g. the T-cell granzymes, neutrophil cathepsin G and the MC chymases, lack this disulphide bond (60-62). The deletion of the disulphide bond creates a new pocket that can specifically bind P3 side chains, thus creating extended interactions with the substrate (63). This is also reflected in the substrate specificity; the granula proteases can cleave a substrate 100 times faster if
19 the substrate has a superior P3 side chain, whereas chymotrypsin has little or no preference for different P3 side chains (63). In addition, the granula serine proteases have an extra loop inserted between amino acids 39-40 that could interact with the substrate P1’ and P2’ residues, thus expanding the substrate specificity to also involve residues at the C-terminal side of the scissile bond. As with other members holding the chymotrypsin-fold, the P1 substrate pocket of granula serine proteases is formed by residues 188-192 and 221- 226. The side chain of residue 226 is oriented to penetrate the bottom of the pocket, and hence interacts directly with the P1 side chain. In granzyme B, which has specificity for P1 Asp, Arg 226 at the bottom of the substrate pocket interacts with the negatively charged Asp chain of the substrate (64). Substitution of Arg 226 in granzyme B with Gly gave preference for aromatic amino acids, similar to chymases (65). Moreover, the granzyme B Arg226Glu mutant gained preference for basic amino acids, further suggesting a pivotal role of residue 226 in determining primary substrate specificity of granula proteases (66). Interestingly, human, but not mouse, cathepsin G has a Glu residue at position 226, which confers it with a dual specificity for both aromatic and basic amino acids (67). The specificity for aromatic side chains arises due to the position of the Glu carboxyl group, which is locked in a position directed slightly away from the substrate P1 (68).
Mast cell serine proteases The MC serine proteases are stored in the granules as fully mature enzymes, ready to be released upon activation of the MC. They catalyze the hydrolysis of peptide and ester bonds at neutral pH. Consequently, the low pH in the secretory granules (69) prevents the stored proteases from degrading any granula proteins. In addition, the proteases are rendered positively charged at a low pH and thus remain tightly bound to the negatively charged serglycine proteoglycan side chains, e.g. heparin (70). This interaction reduces the electrostatic repulsion, allowing dense packaging of the proteases in the granules. Upon activation of the MC, the proteoglycan-bound proteases are released as a macromolecular complex (71). The different MC subtypes express different serine proteases (Table II). The tissue-specific expression suggests that the proteases have different substrate specificities since the substrates they encounter may have localized expression (further discussed in paper I). In addition, the type and amount of
20 inhibitors that are present at different locations may vary, suggesting that the proteases may be different in their resistance to inhibitors.
Table II. Serine protease content of the different mast cell subtypes.
MMCa CTMC mouse rat human mouse rat human (MCT) (MCTC)
PROTEASES chymase mMCP-1 rMCP-2 mMCP-4 rMCP-1 HC b mMCP-2 rMCP-3 mMCP-5 rMCP-5 mMCP-9 ? rMCP-4
tryptase D,E-tryp mMCP-6 rMCP-6 D,E-tryphigh mMCP-7 rMCP-7 TM-tryp granzyme B rMCP-8 –like rMCP-9 rMCP-10 Data from (7, 9, 72). a Abbreviations; MMC mucosal mast cell; CTMC connective tissue mast cell; MCT -tryptase containing human mast cell; MCTC -tryptase and chymase containing human mast cell; mMCP mouse mast cell protease; rMCP rat mast cell protease; HC human chymase; tryp tryptase; TM-tryp transmembrane tryptase; bWas shown in Paper II to possess elastase rather than chymase specificity, although it falls into the chymase branch in the phylogenetic tree
The majority of the MC proteases react with diisopropyl fluorophosphate, DFP, a low molecular weight inhibitor that covalently binds the catalytic serine residue. The proteolytic activities of the serine proteases include chymotrypsin- or trypsin-like specificities, and these enzymes are therefore referred to as chymases and tryptases, respectively. In addition, a new group of proteases more related to T-cell granzyme B, the rMCP-8 family, has been found in rat MMC and mouse basophils (Table II) (9, 28, 73). However, the substrate specificity of these proteases is still unknown, despite several attempts by our group to characterize substrate specificity using chromogenic substrates and substrate phage display (unpublished data). Notably, members of the rMCP-8 family have a similar amino acid
21 residue composition in their active site as human D-tryptase, which was recently shown to be inactive towards peptide substrates due to steric hindrance in the substrate binding cleft (74, 75).
Chymases The name chymase was first used in the 1960s to denote a group of enzymes expressed in the secretory granules of MC, which was similar to pancreatic chymotrypsin. The target specificity of chymases is chymotrypsin-like; protein targets are cleaved on the carbonyl side of aromatic residues, with the order of preference Phe!Tyr!Trp (76). Chymases are synthesized as proenzymes that are activated by removal of the acidic two-residue propeptide. Activation occurs intracellularly by the enzyme dipeptidyl peptidase I (77), which also removes N-terminal propeptides of several members of the granzyme family (78). Five of the ten rat MC serine proteases, rMCP-1, -2, -3, -4, -5 and five of the nine mouse MC serine proteases, mMCP-1, -2, -4, -5, -9, are thought to be chymases (Table II). In rodents, chymases are heterogeneously expressed by both MMC and CTMC, whereas expression of the single human chymase (HC) is restricted to CTMC-like cells. Structural modeling of the mouse and rat MC chymases revealed that areas with higher charge density were present on chymases expressed by CTMC, but were absent on MMC chymases (79). As a result, the CTMC chymases are more tightly bound to the heparin matrix, affecting diffusion and resistance to inhibitors after release into the extracellular space. A phylogenetic analysis reveals that chymases form two distinct subgroups, D and E chymases (Figure 6) (80). There seems to be a single D- chymase present in every species investigated, but E-chymases have only been found in rodents. Early functional studies revealed a difference in substrate selectivity of D- and E-chymases regarding angiotensin I (Ang I) conversion into angiotensin II (Ang II) (81). The vasoconstrictor, Ang II, is formed by a single cleavage of the Ang I peptide, Asp1-Arg2-Val3-Tyr4-Ile5- His6-Pro7-Phe8-His9-Leu10, at the Phe8-His9 bond. D-chymases were shown to exclusively cleave the Phe8-His9 bond to form Ang II, whereas E-chymases (rMCP-1 and rMCP-2) cleave both the Tyr4-Ile5 and the Phe8-His9 bonds, thereby degrading Ang II (80). However, recent studies on E-chymases reveal heterogeneity within this group. Rat vascular chymase and mMCP-1 display almost exclusively Ang I converting activity (82, 83). Furthermore,
22 mMCP-4 and hamster chymase-1 hydrolysed both sites, but with a small preference for the Phe8-His9 bond, having both converting and destroying activities (84, 85). In conclusion, Ang I degradation is not a mandatory quality of E-chymases, and, as discussed in Paper II, Ang II generation is not a general feature of D-chymases.
rMCP-4 1000 rMCP-3 rMCP-1 mMCP-4 1000 hamster chymase-1 E gerbil chymase-1 rMCP-2 977 rat vascular chymase 804 mMCP-1 mMCP-9
hamster chymase-2 rodent gerbil chymase-2 1000 rMCP-5 D mMCP-5 1000 sheep mast cell protease dog chymase 909 macaca chymase 905 baboon chymase 1000 human chymase
bovine chymotrypsin 0.05
Figure 6. Phylogenetic relationships of chymases. The amino acid sequences of the mature proteases (i.e. without signal sequence or propeptide) were aligned using Clustal W. The symbols D and E marks the branches for D- and E-chymases, respectively.
The substrates and biological function of chymases (Figure 7) have been addressed in several studies. In vitro, human chymase has been demonstrated to activate the potent inflammatory cytokine IL-1E (86) and to cleave membrane bound SCF resulting in its release from cells (87). The release of
23 SCF was shown in a later experiment to induce MC migration, resulting in an accumulation of MC at sites with chymase injections (88). Furthermore, human chymase releases latent TGF-E1 from extracellular matrix complexes (89). This was also confirmed in experiments where rat mast cells expressing rMCP-1, could activate TGF-E1 (90). Activated TGF-E1 can promote production of extracellular matrix proteins (91) and it has been shown to be chemotactic for monocytes, neutrophils and eosinophils (92, 93). Several studies, both in vivo and in vitro, indicated that human chymase is a potent recruiter of inflammatory cells (88, 94-97). Although activation of cytokines (above) has been implicated, the actual substrate responsible for this effect has yet to be identified.
Extracellular Coagulation Inflammation matrix remodeling and fibrinolysis SCF release fibronectin degradation proIL-1} activation MMP activation thrombin inactivation TGF} activation TGF} activation plasmin inactivation
in vitro and ex vivo
ChymaseChymase knockout Delayed parasite in vivo expulsion
Inflammation Permeability recruitment of increased vascular and inflammatory cells epithelial permeability
Figure 7. The diverse roles of mast cell chymases.
A role for chymase in tissue remodeling is suggested by the ability of chymase to activate pro-MMPs (matrix metalloproteases) such as collagenase (MMP-1) (98), stromelysin (MMP-3) (99) and gelatinase B (MMP-9) (100). Moreover, chymase might also prolong the half-life of
24 MMPs through degradation of the MMP inhibitor, TIMP-1 (101). HC has also been shown to directly degrade type 1 procollagen (102). Further, HC and the CTMC chymases, mMCP-4 and rMCP-1, have the ability to cleave fibronectin, an extracellular matrix protein (51, 103, 104). Cleavage of fibronectin disrupts cell adhesion to extracellular matrix (ECM), and this disruption can lead to a loss of viability of cells residing in ECM (104). In vitro studies have indicated a role for rodent chymases in regulating coagulation (105, 106). Thrombin is inactivated by the CTMC chymases, mMCP-4 and rMCP-1, and, in addition, plasmin is inactivated by mMCP-4. Cleavage of these substrates is strongly dependent on heparin proteoglycan, which attracts both plasmin and thrombin and hence enables the contact with the heparin bound chymase (106). In vivo evidence demonstrates that human chymase has effects on epithelial permeability in organs such as the skin. When injecting HC into the skin of guinea pigs, vascular leakage is observed as a result of increased permeability (107). A similar effect has also been obtained with the rat MMC protease, rMCP-2, where infusion of rMCP-2 into the vasculature of rat jejunum resulted in increased macromolecular leakage into the intestinal lumen (108, 109). Further, the mouse homologue mMCP-1 was translocated into the lumen during parasitic infections, suggesting that a similar effect is obtained with this protease (6). An interesting observation was made when mMCP-1 was subjected to targeted deletion. When challenging mMCP-1-/- mice with the helminth parasite, Trichinella spiralis, a delayed expulsion of the parasite was observed (52), suggesting that this serine protease is an important contributor to MC inflammatory responses in the intestine. The observed effect in the knockout could be due to a more intact intestinal epithelial membrane, diminishing rapid outflow of immune cells and anti- worm antibodies into the intestinal lumen.
Tryptases Tryptases are a family of serine proteases with trypsin-like substrate specificity, i.e. they cleave a substrate at the carboxyl side of arginine and lysine residues (110). All known tryptases are initially translated as zymogens, with an approximately 10 aa long N-terminal propeptide. Processing of the zymogen occurs before storage in the granules, but the enzyme responsible for this action has yet to be identified. However, some results suggest that tryptase might be partially activated by dipeptidyl peptidase I (77, 111). Several isoenzymes are present both in human (D, EIa,
25 EIb, EII, EIII, and transmembrane/J-tryptase I and II) and rodents (mMCP-6, -7 and transmembrane tryptase) (112, 113). The E-tryptases are highly homologous with 98-99% identity at the protein level, whereas the D- tryptase display a91% identity with the E-tryptases (112). D-tryptase is not stored in the secretory granules, but is instead constitutively secreted by MC and can be found circulating in blood (111, 114). The structure of D-tryptase was recently solved, and due to a single amino acid change in the active site, Gly216 in E-tryptases to an Asp in D-tryptase, this enzyme displays very low proteolytic activity (74, 75). Therefore, in humans, the tryptase activity in MC can be attributed to E-tryptases. Tryptases are active in the form of a non-covalently linked homotetramer. Structural analysis revealed that the monomers are positioned in the corners of a quadrant, with the active sites facing a central pore (115) (Figure 8). The central pore, similar to the hole of a doughnut, restricts the access of macromolecular substrates to the active sites. This confers tryptase with distinct substrate specificity properties, such as preference for low molecular weight peptide substrates and high resistance towards large protease inhibitors. Each of the tryptase monomers interacts with its neighboring monomers via two intrinsically different interfaces (116) One of the interfaces has a small surface area and is stabilized mainly by hydrophobic interactions (116), whereas the other has a large surface area and is stabilized by hydrogen bonds and ionic interactions as well as hydrophobic moieties. The glycosaminoglycans present in the MC stabilize the weaker interface by binding positively-charged residues on the protease surface and thereby bridging the monomers, resulting in a strengthened interaction and a decrease in the electrostatic repulsion between the monomers (116). As discussed earlier, tryptases are resistant to most endogenous protease inhibitors present in plasma and the extracellular space due to the restricted entrance of inhibitors into the central cavity where the active sites are located. Therefore, regulation of the activity of human tryptase in vivo must mostly depend on dissociation of the active tetramer into inactive monomers. Some proteins that form tight complexes with heparin compete with tryptase for the binding of heparin and have thus been shown to inhibit tryptase in vitro (117, 118) and in vivo (119).
26 Heparin proteoglycan D A
Tryptase monomer
C B
Figure 8. Tryptase tetramers are stabilized by heparin. Four tryptase monomers are arranged as a quadrant with their active sites facing a central hole. The monomers are bridged by proteoglycans, e.g. heparin, which increase the total strength of the connection between the monomers A-B and C-D.
Numerous substrates are cleaved by tryptase in vitro, several of which possess matrix-remodeling and anti-coagulant activities, e.g. activation of MMPs and uPA, the plasminogen activator (120-123), and degradation of fibronectin (122) and fibrinogen (124, 125). Furthermore, tryptase stimulates proliferation of fibroblasts (126), smooth muscle cells (127) and epithelial cells (128, 129). These mitogenic effects might, in the long run, contribute to the thickening of airway walls and lead to increased airway responsiveness, as seen in asthmatic patients. Tryptase also possess pro-inflammatory activities, displayed as recruitment of inflammatory cells (130), most likely through stimulation of production and release of the chemokine IL-8, a potent attractant of granulocytes (128, 129, 131). The stimulation of IL-8 production might be mediated by activation of the proteinase-activated receptor-2 (132), which has a potential cleavage site for tryptase (133). The pro-inflammatory effect has also been tested in animal models (134-136), where recruitment of
27 neutrophils by tryptase was demonstrated to be important for combating bacterial infections (135). Further, the demonstration that MC had an essential role in recruiting neutrophils during bacterial peritonitis in mice, not solely dependent on TNF-D, again suggests that tryptase may contribute to host defense against bacterial infections (46). In allergic asthma, aerosolized tryptase causes bronchoconstriction as suggested by in vivo and ex vivo studies on experimental animals (137, 138) and an ex vivo study on human lungs (139). Furthermore, several studies have demonstrated that tryptase inhibitors blocked allergen-induced bronchoconstriction, thus verifying involvement of tryptase in bronchoconstriction (140-142). The biological substrate(s) that mediates this effect is not fully elucidated, but the bronchodilator VIP, vasoactive intestinal peptide, is degraded by tryptase and may thus contribute to the bronchoconstriction (143). Another way by which tryptase is suggested to cause bronchoconstriction is via activation of resident mast cells (144). In summary, tryptase inhibition may have a positive effect on asthmatic patients, by reducing bronchoconstriction, infiltration of inflammatory cells and the mitogenic effect on smooth muscle cells.
What is left to learn about mast cell serine proteases? Mast cells are key players in airway pathologies, such as allergic asthma, but have also been shown to be involved in host defense against bacteria and parasitic worms. The MC proteases are major protein components of mast cell granules and, consequently, should contribute to MC function. Much of the current knowledge of function of mast cell proteases must be revised as new members are discovered that might possess subtle but important changes in activities and hence have another biological function. Studies of cleavage specificities of the proteases can be a way of characterizing new biological substrates that may be involved in immunological and pathological responses. The species variations in MC protease functions must also be clarified in order to be able to extrapolate results obtained in animal models to humans. As stated earlier, MC serine proteases are important in host defense, but they may also contribute to airway obstruction, inflammation and bronchoconstriction in asthmatic patients. Further characterization of MC proteases may thus contribute to our knowledge of their structure and activity and, as a result, ways to inactivate them.
28 29 SUMMARY OF PRESENT STUDIES
AIM The aim of this thesis was to help clarify, by biochemical studies, the specific roles of individual MC serine proteases in the biological activity of the mast cell. Several of these proteases were thus characterized regarding heparin-dependence, catalytic activity, susceptibility to inhibitors and substrate specificity.
RESULTS AND DISCUSSION
E-chymases can differ with respect to extended substrate specificity as assessed by substrate phage display (Paper I) Chymases are a group of serine proteases with chymotrypsin-like substrate specificity. Phylogenetic analysis reveals that chymases form two distinct subgroups, D and E chymases. In the E-chymase subfamily, only rodent chymases are represented. The E-chymases are heterogeneously expressed, rMCP-1 only by CTMC and rMCP-2 by MMC. One of the other E- chymases, rMCP-4, is also expressed by MMC (145). So, considering these facts, an intriguing question arises. Why do rat and mouse express not only one, but several E-chymases? One hypothesis is that they perform discrete tasks. If this is so, one can predict that they also have different target specificity. The substrate specificity of rMCP-1 and rMCP-2 is quite well studied (76, 105, 146); they both prefer small aliphatic P2 and P3 residues together with Phe or Tyr in the P1 position. rMCP-2 has generally lower activity than rMCP-1 towards chromogenic substrates, and also cleaves substrates with charged P2 residues very poorly (76). However, one of the chymases, rMCP-4, has never been studied at the protein level. The aim was therefore to do a biochemical analysis of rMCP-4, and compare the results with those of the other E-chymases. In this paper as well as in Paper II, a phage display system was used to determine the substrate specificity of the investigated proteases. The phage display strategy enables millions of peptide substrates to be screened in a
30 single reaction. The peptide library was constructed as a fusion protein between the phage capsid protein and a randomized nine residues long peptide. Each phage will therefore carry one specific peptide, displayed at its surface. The selection of phages sensible to cleavage by the protease involves immobilization of the phage, followed by its release by proteolytic cleavage.
Agarose
Ni2+ Agarose Protease T7
Ni2+
Add nickel- 2. Immobilize Add agarose the phages protease Agarose
Agarose Randomized region His6-tag Ni2+ XXXXXXXXX HHHHHH. 1.Produce a library of 3.Cleave the Ni2+ C-terminal of the phage peptides, displayed at susceptible capsid protein the surface of T7 peptides phages T7 T7 T7
Amplify 4. Collect the Remove the phages selected uncleaved Agarose in bacteria phages phages Agarose
Ni2+ Ni2+ T7 After several rounds of selection, T7 isolate individual phage DNA and sequence randomized region
Figure 9. Phage display substrate analysis. (1) Phages displaying random peptides fused to their capsid protein are (2) immobilized on an affinity support. The phages are treated with the protease (3) and uncleaved phages, still attached to the support, are removed. The cleaved phages (4) are amplified in order to start a new round of selection, or their variable sequence is determined by DNA sequencing.
To generate the peptide library, oligomeric nucleotides were inserted in the gene of the phage capsid protein. The oligonucleotide construct contained a randomized region encoding nine amino acids, followed by a six histidine long affinity tag. After packaging of recombinant phage DNA into phage
31 particles in vitro, the phages were amplified to express the fusion protein at the surface (Fig. 9, step 1). The His-tag allowed the phages to bind to an immobilized phase, in this case a Ni2+-agarose resin (Fig. 9, step 2). The protease to be analyzed is then added, and phages expressing peptides sensitive to proteolytic cleavage are released from the solid phase (Fig. 9, step 3). The released phages are then recovered (Fig. 9, step 4), amplified in bacteria to again express substrate peptides. A new selection process can be started using the phage sub-library carrying peptides that were cleaved in the first selection. The selection process is repeated several times to extract phages highly susceptible to the analyzed protease. To assess how rMCP-4 differs from other chymases in its substrate specificity, we produced recombinant rMCP-4 protein, free from any contaminating mast cell proteases. The extended substrate specificity was studied in more detail using phage displayed peptide substrates (see above). Surprisingly, rMCP-4 was shown to have a very stringent specificity, with very little variation in the P4-P1 positions. Further, two subsequent aromatic aa were present in the P2 and P1 positions. This characteristic is not a regular quality of chymases, and this adds to the hypothesis that the E- chymases are not redundant proteases. Moreover, the specificity for P1’ and P2’ residues has never been fully exploited for E-chymases, but we were able to show that rMCP-4 displayed specificity also for P2’ residues. The aim was also to see if we could extract any potential targets from the protein database using the substrate specificity data. We found substrates belonging to three different categories; the blood clotting and fibrinolytic pathway (protein C and coagulation factor V, plasminogen activator inhibitor-1), immunological receptor proteins (TGF-E receptor type III, FcJRIII) and a protein involved in matrix remodeling (procollagen C- proteinase enhancer). All these potential substrates have qualities that, upon cleavage, could contribute to the biological activity and overall effect of the MC granula components. However, until tested in vivo, we can still not be sure that the true substrate is among those found in the database.
An imposter among chymases -the rat D-chymase rMCP-5 has elastase-like substrate specificity (Paper II) Chymases can be subdivided into two groups, D- and E-chymases (Figure 6, page 23) (80). This subdivision is based on phylogenetic data only, not on
32 any information about the biological activity. Much effort has been devoted to study the function and catalytic activities of the E-chymases (61, 76, 83, 85, 105, 147, 148), but the rodent D-chymases have so far been neglected. In the D-chymase group we find the well-studied human chymase clustered together with the other primate D-chymases and also the dog and sheep chymase. The rodent D-chymases form a separate group within the D- chymases. A previous study has shown that an exchange of a single residue in the active site of human chymase, Gly 216 to Val, could dramatically alter its substrate specificity (149). All rodent D-chymases contain a Val in this position, but how this affected substrate specificity for these proteases was not known. To answer the question if rodent D-chymases are the functional homologues of the human chymase, we decided to produce and characterize recombinant rMCP-5, the rat D-chymase. Analysis of the primary specificity with chromogenic substrates demonstrated that rMCP-5 did not display chymotrypsin-like substrate specificity, and could therefore not be regarded as a chymase. Instead the activity was elastase-like, with preference for small aliphatic amino acids such as Val, Ala and Ile. When rMCP-5 was subjected to substrate phage display analysis (see Paper I for details) the extended substrate specificity was shown to be P4-(Gly, Pro, Val), P3-(Leu, Val, Glu), P2-(Val, Leu, Thr), P1-(Val, Ile, Ala), P1’-(Xaa) and P2’-(Glu, Leu, Asp). This result confirms that rMCP-5 is an elastase, and that it has preference for small hydrophobic aa in several of the positions on the N- terminal side of the scissile bond. However, the most interesting finding is the very high specificity for residues with acidic side chains i.e. Asp or Glu, in the P2’ position. Due to a loss of a disulfide bridge in the granzyme/chymase family of serine proteases, a new cavity has been formed that can accommodate aa on the C-terminal side of the scissile bond. The phage display substrate analysis clearly demonstrates that these proteases, with rMCP-5 as a representative, have gained specificity for certain C- terminal residues. Furthermore, in previous studies of HC, the acidic nature of the P2’ residue has been implicated to be of importance for HC’s Ang I converting properties (150). However, although rMCP-5 displays high specificity for P2’ acidic residues, it is highly unlikely that rMCP-5 has any Ang I-converting properties, since the specificity for P1 aromatic residues is lost. As rodents possess an D-chymase with altered specificity, which protease(s) in rodents fulfill the tasks equivalent to that of human chymase?
33 Many have suggested that the E-chymases are redundant or “atypical” chymases. However, recent studies on E-chymases demonstrated that they share some characteristics with human chymase, such as the ability to release TGFE (89, 90) and to increase epithelial permeability (107, 147). An additional mutual characteristic of human chymase and E-chymases is fibronectin degradation (51, 103, 104), as well as the newly discovered Ang I-converting properties by some of the E-chymases, qualities that have previously been confined to the D-chymases. To summarize, the present study strongly suggests that E-chymases in many ways are the functional homologues of human chymase, as the D- chymases in rodents have lost their chymotryptic activity and instead gained elastase-like specificity.
Heparin proteoglycan regulates the activity of mouse tryptase mMCP-6 (Paper III) Tryptases are very unusual enzymes for one major reason; they are active in the form of a homotetramer with the active sites facing a central pore, and are therefore resistant to most endogenous protease inhibitors. In mouse, tryptase expression is confined to connective tissue mast cells, where efficient storage in vivo is dependent on proteoglycans (22, 23). Moreover, proteoglycans seem to have a pivotal role in stabilizing the tryptase tetramer, as seen in several studies on human E tryptase (151, 152). However, some findings regarding the mouse tryptase mMCP-6 (78% identity to human tryptase E) indicated that this tryptase might be active in the absence of proteoglycans (134). Therefore, two major questions were raised; what conditions are needed for tetramer formation of mouse tryptase mMCP-6 and is heparin proteoglycan required for tetramer formation? To obtain pure mMCP-6 protein, this protease was produced as a recombinant protein in mammalian cells. To generate a protein that could easily be purified, mMCP-6 was produced as an inactive zymogen, with a
His6 purification tag followed by an enterokinase site replacing the natural N-terminal propeptide. Recombinant mMCP-6 was purified on Ni2+-agarose from cell culture media. The mature protease was subsequently obtained by enterokinase digestion, which removes the purification tag by cleavage at the enterokinase site. Analysis by SDS-PAGE confirmed that mMCP-6 was pure and that removal of the purification tag was successful.
34 The formation of active enzyme was studied under several different conditions; in the absence or presence of heparin, and at various pH. Addition of heparin was demonstrated to be mandatory to gain catalytic activity. A low pH ( 6.5) during incubation with heparin was also essential for enzymatic activity. To determine whether heparin facilitated any tetramerization, mMCP-6 was subjected to size-exclusion chromatography. As suspected, mMCP-6 tetramers were formed only in the presence of heparin, and at a low pH. The necessity of having a low pH during tetramerization is most likely a reflection of the heparin-binding properties of mMCP-6; binding of mMCP-6 to heparin-Sepharose could only be accomplished at a low pH. A supplementary study on pH and enzyme stability confirmed that inactivation, i.e. dissociation of the active tetramer into inactive monomers, was greatly increased at higher, i.e. physiological, pH. The pivotal role of heparin for mMCP-6 activity was also studied in vivo. mMCP-6 activated with or without heparin was injected into the peritoneum of mice, and the recruitment of inflammatory cells was measured. In the mice injected with mMCP-6 only, or in the PBS and heparin control, no increase of peritoneal lavage cells could be detected, whereas mice injected with mMCP-6 plus added heparin showed a marked recruitment of neutrophils. Thus, catalytically active mMCP-6 is needed to attain a pro- inflammatory activity, and heparin is a requirement to obtain catalytically active mMCP-6.
Heparin antagonists are potent inhibitors of mast cell tryptase (Paper IV) The recognition of tryptase as an agent with pro-inflammatory and broncho- contractile effects has boosted the search for tryptase inhibitors. One such inhibitor, APC-366, has already been tested in vivo on allergic sheep (140), pigs (153), and humans (141), where it has been shown to suppress the late phase response in asthmatics. Active site inhibitors (such as APC-366) for tryptase are generally very small, since tetrameric tryptase is resistant to most large inhibitors due to steric hindrance. Small inhibitors with a good selectivity profile, i.e. those that preferably inhibit exclusively the actual target are quite difficult to produce. APC-366, for example, also displays some inhibitory activity towards thrombin. The construction of a selective tryptase inhibitor may therefore pose a challenge. However, some proteins
35 that form tight complexes with heparin compete with tryptase for the binding of heparin and have thus been shown to inhibit tryptase. Examples of such proteins are lactoferrin (119) and myeloperoxidase (118). The assumption that heparin binding proteins, such as heparin antagonists, could be a way to inhibit tryptase, was therefore further exploited in this study. The polycationic heparin-antagonists, protamine and Polybrene were both shown to be good inhibitors of human tryptase. The efficacy of the heparin antagonists was also compared to APC-366, the active site inhibitor. The rate of inhibition was shown to be fastest with protamine, but the IC50-value was lower for Polybrene, indicating that less inhibitor is required to obtain full inhibition. Inhibition with APC-366 was both slower and less effective than that of the heparin antagonists, with 4 h required to obtain full inhibition. Surprisingly, APC-366 also inhibited rMCP-1, the rat CTMC chymase, albeit with a higher Ki value. Inhibition of tryptase by heparin antagonists should, due to the nature of inhibition, display noncompetitive kinetics. Unexpectedly, the arginine rich protein protamine displayed competitive kinetics, indicative of active site binding. When subjected to size exclusion chromatography, the protamine- treated tryptase eluted as a monomer, verifying that the mechanism of inhibition involves destabilization of the tetramer. In addition, no degradation products of protamin were detected on SDS-PAGE, which excludes the theory that protamine is a substrate for tryptase. Thus, the positively charged protamine may interact both with heparin and with some negatively charged residues around the active site, thereby blocking substrate binding. Taken together, heparin antagonists are potent inhibitors of tryptase, with a potential to serve as suppressors of asthmatic symptoms.
CONCLUDING REMARKS The in vitro analysis of the substrate specificity of a protease can be performed in several ways; by a limited number of synthetic substrates, by positional scanning using synthetic combinatorial libraries, or by the substrate phage display method described in this thesis. Synthetic substrates have the advantage of easy determinations of kinetic constants such as kcat and KM, thus allowing easy comparisons of substrate hydrolysis by different enzymes. On the other hand, making a full substrate analysis using this
36 method is very time consuming, and the synthetic substrate analysis has hence evolved to a combinatorial library technique (133, 154-157). This technique utilizes substrates with the general structure Ac-X-X-X-P1-AMC, where AMC is a fluorochrome. In each setting, one of the positions is defined with one of the twenty amino acids, while the remaining positions are randomized, thus creating a library of compounds. This method will allow easy screening of virtually any protease, providing full information about substrate specificity on the N-terminal side of the scissile bond. Human chymase has recently been subjected to positional scanning (158), a study which generated both new targets and inhibitors. However, the positional scanning technique neither resolves any P1’ or P2’ specificity, nor does it provide any information regarding subsite interdependence, i.e. if a particular amino acid in one position affects amino acid composition of another subsite. On the contrary, the phage display substrate technique allows rapid determination of full substrate specificity on both sides of the scissile bond, and information concerning subsite interdependence can be obtained. However, one of the most crucial steps in interpreting phage display data is the alignment of substrate sequences, which can sometimes be complicated. Therefore, in some cases, positional scanning and phage display have been used together as complementary analyses (156, 159). The biochemical characterization of mast cell proteases is a first step towards understanding their contribution to MC function. One of the questions addressed in this study is the target specificity of rodent MC chymases. We have shown that the rat protease rMCP-4 differs from other chymases in its substrate recognition (Paper I), and that the predicted chymase rMCP-5 is actually not a chymase but rather has elastase-like specificity (Paper II). However, there is still a gap between the substrate specificity and the biological target that needs to be bridged. A predicted target for rMCP-4 worthy of note due to its potential as a modulator of the immune response, is the low affinity receptor for IgG, FcJRIII (Paper I). Many cell types express this receptor, e.g. mast cells, which can degranulate upon stimulation with IgG/antigen immune complexes. The cleavage site of FcJRIII is located in the extracellular part of the receptor, close to the transmembrane region. rMCP-4 could thus liberate FcJRIII from the cell surface, resulting in decreased activation through this receptor. In addition, the released receptor may function as a “decoy”, blocking the binding of immune complexes to cell-bound receptors.
37 A search in the Swissprot database using substrate phage display data for rMCP-5 (Paper II), revealed several potential substrates for this enzyme (unpublished data). Examples of retrieved candidate targets are members of the cadherin family. Cadherins are cell adhesion molecules that play an important role in holding adjacent cells together, e.g. in various epithelia. Each cadherin protein contains several cadherin domains, and in most of these domains there seems to be a potential cleavage site for rMCP-5. Therefore, there is a possibility that rMCP-5, despite its difference in substrate specificity, still generates similar biological effects as human chymase, i.e. an increase in epithelial permeability. However, as stated earlier, any potential substrate must be tested for protease sensitivity, before it can be classified as a substrate. The overall biological effect of a protease is not only dependent on its substrate specificity. The resistance to inhibitors and the half-life of an enzyme determines the magnitude of its proteolytic actions. The mast cell tryptases are very resistant to endogenous inhibitors, but instead they do have another Achilles' heel; they are continuously inactivated at physiologic pH due to dissociation into monomers (Paper III, (152)). An acidic pH, on the other hand, was shown to both activate and stabilize tryptases, most likely by increasing its heparin binding properties (Paper III, (152)). As the pH is lowered locally at sites of inflammation, a much greater overall effect by mMCP-6 and similar enzymes can be anticipated during inflammatory conditions. The chance that tryptases are released at sites of inflammation is very likely, since many of the MC mediators trigger inflammatory responses, including mMCP-6 itself (Paper III, (134)). In many autoimmune diseases MC also seem to accumulate at sites of inflammation. Sometimes local inflammations are beneficial, for example during host defense reactions against various pathogens, but sometimes suppression of an inflammatory response is desired. As the mast cell tryptases contribute to inflammation, e.g. by recruiting immune cells, they are potential therapeutic targets. In paper IV the heparin antagonists protamine and Polybrene were shown to be efficient inhibitors of both the mouse and human tryptases. The ongoing research in this field has generated many types of inhibitors (reviewed in (160)). Although few compounds have reached the clinic, several molecules, including the heparin antagonists, are promising candidates as a treatment for asthma and other allergic and inflammatory disorders.
38 39 ACKNOWLEDGEMENT
A great adventure… …never ending? That’s what I thought in the beginning of my PhD studies. But now I’ve finally (to my own surprise) reached the “grand finale”. Some people have helped me tremendously during this journey, and I especially would like to thank…
…first and foremost, Lasse, my supervisor, for guidance, suggestions and a minimal of criticism. Thank you for your support and faith in me! I really doubt that I would have started any PhD studies if you had not given me the chance to join your mast cell-, basophil-, Ig-evolution-, zinc-finger-, vaccine-group. PS. If I ever want to have fresh input to any new project, I’ll give you a call, because I know you’ll be able to provide me with enough research ideas to last a lifetime.
…Gunnar P, my “co-supervisor”, for scientific discussions, paper revisions and help whenever I got stuck in my laboratory work. I’m also very grateful that you “adopted” me into your group meetings, which have expanded my “protease horizon” as well as stimulated me to participate in scientific discussions.
…Pilis, for having all the answers. Always.
…the girls in the lab; Maryam and Carolina, for being the best teachers ever. Maryam, for telling me how things could be done, and Carolina, for telling me how things should be done; but still both of you always allowed me to do things my way. The combination of you two resulted in the most complete supervisor I could ever hope for. Maria, for having all the answers on laboratory work, Molly, for being so fun and unique (snus being one thing), Lotta, because you are such a talkative and dance- loving “shejka-loss” person, Jeannette, for being exceptionally rich in exams (skeppar-, dykar-, jägar-, doktor-…etc) and in principles (Robbie-bubbles, I’ll say no more), Anna, because you have always positive things to say about my presentations and everything else, (although I don’t always deserve it, it’s still nice to hear), Parvin, for always being so joyful and helpful, Camilla, for your help with my never-ending “fill in forms” anxiety, and for your vivid personality, Maike, for being so good at everything you do that it is a privilege to work with and discuss things with you, Sara, for having the courage to continue the path as a researcher at the university, I wish I could be like you. The new members Jenny and Mattias, as well as project students in the lab, Kaisa, Klas, and Sofia, for creating a good lab
40 atmosphere with scientific discussions and less scientific discussions (party, party). I wish you good luck, Mattias, in continuing the work on mast cell proteases.
…the Gunnar P group: Jenny, I’m very thankful that you decided to work on mMCP-6, Frida, Anders, Elena, Ignacio, Mirjana and, of course, Magnus. Thanks for all your help and fresh ideas in laboratory work, and for including me in your cake-eating Friday sessions.
…the Cod group; no one mentioned, no one forgotten. Thanks for all help with laboratory work and computer support.
…Frida and Otti that introduced me to the immunology course-labs and “real” immunology, and Erika, Anders and Johan L, for being fun lunch dates and interesting discussion partners.
…Gunnar F, for providing me with the most important tool of my research; the library of phage displayed substrates.
…the helpful staff at ICM and IMBIM; especially Sigrid, Christer, Eva, Tony and Ylva.
...Alan McWhirter for his linguistic revision (this section was not included, however, so don’t blame him for all the mistakes here).
…my friends outside the lab (however, some still belong to the family of research- fanatics*): Jessica*, Aneta*, Fredrik, Mattias*, Stefan, Anna, Åsa, Mathias, Maria, Linda, Jonas, Kicki and my Jönköping-dwelling friends Agneta, Mia, and Sofi. Thank you all for making my spare-time more fun.
…my parents, Inger and Morgan, för att ni alltid har ställt upp för mig och låtit mig göra mina egna val vad gäller studier och annat, samt för den djuplodade språkgranskning av den här avhandlingen som pappa gjorde, and my brother, Stefan, för att du alltid hör av dig (trots att jag är väldigt dålig på att ringa tillbaks) och alltid tar dig tid att träffa mig när jag kommer hem och hälsar på.
…and last, but certainly not least, Thomas, my fiancé, “sambo” and soul mate. Thank you for always being there for me. I love you.
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55 Acta Universitatis Upsaliensis Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology Editor: The Dean of the Faculty of Science and Technology
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