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Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
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.

Place, publisher, year, edition, pages
2010. Vol. 277, no 10, 2255-2267 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97165DOI: 10.1111/j.1742-4658.2010.07642.xISI: 000277084600007OAI: oai:DiVA.org:uu-97165DiVA: diva2:171984
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Cleavage Specificity of Mast Cell Chymases
Open this publication in new window or tab >>Cleavage Specificity of Mast Cell Chymases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 429
Keyword
Biology, Immunology, Mast cell, Serine protease, Chymase, Cleavage specificity, Phage display, Biologi
Identifiers
urn:nbn:se:uu:diva-8714 (URN)978-91-554-7190-3 (ISBN)
Public defence
2008-05-23, C10:305, BMC, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved
2. Haematopoietic Serine Proteases: A Cleavage Specificity Analysis
Open this publication in new window or tab >>Haematopoietic Serine Proteases: A Cleavage Specificity Analysis
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
Mast cell, cleavage specificity, phage display, chymase, serine protease, granzyme, fish protease
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-221891 (URN)978-91-554-8945-8 (ISBN)
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

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Thorpe, MichaelHellman, Lars

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