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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
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.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
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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.

Place, publisher, year, edition, pages
2012. Vol. 24, no 12, 771-782 p.
Keyword [en]
animal model, chymase, cleavage specificity, human chymase, mast cell, macaque
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-190320DOI: 10.1093/intimm/dxs081ISI: 000311903800004OAI: oai:DiVA.org:uu-190320DiVA: diva2:585895
Available from: 2013-01-10 Created: 2013-01-07 Last updated: 2014-09-24Bibliographically approved
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1144
Mast cell, cleavage specificity, phage display, chymase, serine protease, granzyme, fish protease
National Category
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)
Available from: 2014-05-13 Created: 2014-04-07 Last updated: 2014-06-30Bibliographically approved

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