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Haematopoietic Serine Proteases: A Cleavage Specificity Analysis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Lars Hellman)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1144
Keyword [en]
Mast cell, cleavage specificity, phage display, chymase, serine protease, granzyme, fish protease
National Category
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-221891ISBN: 978-91-554-8945-8 (print)OAI: oai:DiVA.org:uu-221891DiVA: diva2:712000
Public defence
2014-06-04, C4:305, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-05-13 Created: 2014-04-07 Last updated: 2014-06-30Bibliographically approved
List of papers
1. Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
Open this publication in new window or tab >>Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 10, 2255-2267 p.Article in journal (Refereed) Published
Abstract [en]

Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-97165 (URN)10.1111/j.1742-4658.2010.07642.x (DOI)000277084600007 ()
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2014-09-24Bibliographically approved
2. Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis): an interesting animal model for the analysis of the function of the human mast cell chymase
Open this publication in new window or tab >>Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis): an interesting animal model for the analysis of the function of the human mast cell chymase
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2012 (English)In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 24, no 12, 771-782 p.Article in journal (Refereed) Published
Abstract [en]

Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use.

Keyword
animal model, chymase, cleavage specificity, human chymase, mast cell, macaque
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-190320 (URN)10.1093/intimm/dxs081 (DOI)000311903800004 ()
Available from: 2013-01-10 Created: 2013-01-07 Last updated: 2017-12-06Bibliographically approved
3. Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors
Open this publication in new window or tab >>Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, e65988- p.Article in journal (Refereed) Published
Abstract [en]

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr(4)-Ile(5) bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-208169 (URN)10.1371/journal.pone.0065988 (DOI)000322361200045 ()
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
4. Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
Open this publication in new window or tab >>Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
2016 (English)In: Developmental And Comparative Immunology, ISSN 0145-305X, Vol. 63, 84-95 p.Article in journal (Refereed) Published
Abstract [en]

Here we present the extended cleavage specificity of catfish granzyme-like I, previously identified in fish NK-like cells. This protease has been characterised using substrate phage display and further validated by using a panel of recombinant substrates. A strict preference for Met in the P1 (cleavage) position, indicating metase specificity was observed. A screening of potential in vivo substrates was performed based on the derived P5-P3' consensus: Arg-Val-Thr-Gly-Met(down arrow)Ser-Leu-Val. Channel catfish caspase 6 was one very interesting potential target identified. This site was present in an adjacent position to the classic caspase activation site (Asp179 in human caspase 6). Cleavage of this site (hence potential activation) by the catfish granzyme-like I could reveal a novel mechanism of caspase 6 activation. This poses an interesting idea that the role of granzyme-like proteases in the activation of caspase dependent apoptosis mechanisms has been conserved for over 400 million years.

Keyword
Fish; Serine protease; Cleavage specificity; Metase; NK cell; Caspase; Evolution
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-221554 (URN)10.1016/j.dci.2016.05.013 (DOI)000380623300010 ()27216028 (PubMedID)
Funder
Swedish Research Council, 621-2011-5007
Available from: 2014-04-09 Created: 2014-04-01 Last updated: 2016-11-23Bibliographically approved

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