<p>Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. </p><p>In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an <i>in vivo</i> substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.</p>

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.

Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics. The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential in vivo substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme. Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference. Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.

Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions. In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates.  The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity. As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors.  Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions. The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development. Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr. Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man) 5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5 mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS-PAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced KM, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns.

Human mast-cell chymase (EC 3.4.21.39) is a chymotrypsin-like serine protease that is stored in and released from mast-cell granules. This enzyme has been expressed in Pichia pastoris by homologous recombination of the cDNA coding for the mature active chymase into the Pichia genome. Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor. Methanol induction resulted in the secretion of active chymase into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days. Active enzyme was purified from the culture media with a 22 % yield of activity by a simple two-step procedure involving hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.

No description available.

Dialkylsilanediols are a novel class of non-hydrolyzable analogues of the tetrahedral intermediate of amide hydrolysis, shown to be good inhibitors of HIV-1 protease, angiotensin converting enzyme (ACE), and thermolysin. An impediment to utilization of these silanediol structures, however, has been the methods for their assembly. This research describes the reductive lithiation of hydridosilanes and alkoxysilanes, and the use of the resulting silyl anions to develop efficient methods to synthesize silanediol precursors. In the first part of research, lithiation of hydridosilanes was studied. As part of this study, a simple 1H NMR method was developed for monitoring and analyzing the progress of lithiation. In addition, this method was converted to a titration for silyllithium reagents using BHT as an internal standard. Silanediols 107 and 177 are analogues of a potent chymase inhibitor, NK-3201 (82). In the second part, diphenylsilanes 108 and 170, precursors to silanediols 107 and 177, were synthesized using addition of silyllithium to sulfinimine 113 as a key step. In the third part, lithiation of alkoxysilanes was studied. (Si,O)-Dianions, generated from lithiation of silane alcohol 175 or 2,2-diphenyl-1-oxa-2-silacyclopentane (225), were reacted with a wide variety of electrophiles to give potentially useful silicon-containing building blocks. Addition of the (Si,O)-dianion 284 to sulfinimines gave silanediol inhibitor precursors with full control of stereochemistry. In the last part, a new method featuring 1,1-diphenyl-2-azaallyllithium chemistry were utilized to synthesize a series of protected α-amino silanes 323, 329 - 331. / Chemistry

<p>Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. </p><p>Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.</p><p>To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.</p><p>Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an <i>in vitro</i> purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.</p>