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Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse - High specificity indicates high substrate selectivity
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Immunology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Immunology.
2008 (English)In: Molecular Immunology, ISSN 0161-5890, Vol. 45, no 9, 2548-2558 p.Article in journal (Refereed) Published
Abstract [en]

Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.

Place, publisher, year, edition, pages
2008. Vol. 45, no 9, 2548-2558 p.
Keyword [en]
Mast cell, Chymase, Mouse mast cell protease-1, Mouse mast cell protease-2, Cleavage specificity, Mucosal immunity, Intestinal permeability
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-97162DOI: 10.1016/j.molimm.2008.01.012ISI: 000255528400013PubMedID: 18313755OAI: oai:DiVA.org:uu-97162DiVA: diva2:171981
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2009-11-05Bibliographically 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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