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The extended cleavage specificity of the rodent β-chymases rMCP-1 and mMCP-4 reveal major fumctional similarities to the human mast cell chymase
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.
2008 (English)In: Molecular Immunology, ISSN 0161-5890, Vol. 45, no 3, 766-775 p.Article in journal (Refereed) Published
Abstract [en]

In rat and mouse the phylogenetic homologues of the human mast cell alpha-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate alpha-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell beta-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1' and P3'. The two enzymes differ clearly only in one position, the P2' residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2' of known substrates for the human chymase. Overall, these two rodent beta-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.

Place, publisher, year, edition, pages
2008. Vol. 45, no 3, 766-775 p.
Keyword [en]
Mast cell, Chymase, Rat mast cell protease-1, Mouse mast cell protease-4, Cleavage specificity
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-97163DOI: 10.1016/j.molimm.2007.06.360ISI: 000251350900020PubMedID: 17681377OAI: oai:DiVA.org:uu-97163DiVA: diva2:171982
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2010-01-28Bibliographically 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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