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The extended substrate cleavage specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile
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.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2009 (English)In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 21, no 1, 95-104 p.Article in journal (Refereed) Published
Abstract [en]

The human chymase (HC) is a major granule constituent of mast cells (MCs) residing in the connective tissue and the sub-mucosa. Although many potential substrates have been described for this important MC enzyme, its full range of in vivo substrates has most likely not yet been identified. A major step toward a better understanding of the function of the HC is therefore to determine its extended cleavage specificity. Using a phage-displayed random nonapeptide library, we show that the HC has a rather stringent substrate recognition profile. Only aromatic amino acids (aa) are accepted in position P1, with a   strong preference for Tyr and Phe over Trp. Aliphatic aa are preferred in positions P2 to P4 N-terminal of the cleaved bond. In the P1' position C-terminal of the cleaved bond, Ser is clearly over-represented and acidic aa Asp and Glu are strongly preferred in the P2' position. In P3', the small aliphatic aa Ala, Val and Gly were frequently observed. The consensus sequence, from P4 to P3': Gly/Leu/Val-Val/Ala/Leu-Ala/Val/Leu-Tyr/Phe-Ser-Asp/Glu-Ala/Val/Gly,   provides an instrument for the identification of novel in vivo substrates for the HC. Interestingly, a very similar cleavage specificity was recently reported for the major chymase in mouse connective tissue mast cells (CTMCs), the beta-chymase mouse mast cell   protease-4, suggesting functional homology between these two enzymes. This indicates that a rather stringent chymotryptic substrate recognition profile has been evolutionary conserved for the dominant CTMC chymase in mammals.

Place, publisher, year, edition, pages
2009. Vol. 21, no 1, 95-104 p.
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-97164DOI: 10.1093/intimm/dxn128ISI: 000261898300009OAI: oai:DiVA.org:uu-97164DiVA: diva2:171983
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2010-08-09Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 429
Biology, Immunology, Mast cell, Serine protease, Chymase, Cleavage specificity, Phage display, Biologi
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
Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved

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