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Extended substrate specificity of opossum chymase: Implications for the origin of mast cell chymases
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.
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 7, 2116-2125 p.Article in journal (Refereed) Published
Abstract [en]

Serine proteases are major granule constituents of mast cells, neutrophils, T cells and NK cells. The genes encoding these proteases are arranged in different loci. The mast cell chymase locus e.g. comprises at least one alpha-chymase, one cathepsin G, and two granzyme genes in almost all mammalian species investigated. However, in the gray, short-tailed opossum (Monodelphis domestica) this locus contains only two genes. Phylogenetic analyses place one of them clearly with the alpha-chymases, whereas the other gene is equally related to cathepsin G and the granzymes. To study the function of opossum chymase, and to explore the evolutionary origin of mast cell chymases, we have analyzed the cleavage specificity of this enzyme. The protease was expressed in mammalian cells and the extended substrate specificity was determined using a randomized phage-displayed nonapeptide library. A strong preference for the aromatic amino acids Trp over Phe and Tyr in the P1 position was observed. This is in contrast to human chymase and mouse mast cell protease-4, which prefer Phe over Tyr and Trp in this position. However, in most other positions this enzyme shows amino acid preferences very similar to human chymase and mouse mast cell protease-4, i.e. aliphatic amino acids in positions P4, P3, P2 and P1', and acidic amino acids (Glu and Asp) in the P2' position. The overall specificity of MC chymase thereby seems to have been conserved over almost 200 million years of mammalian evolution, indicating a strong selective pressure in maintaining this specificity and an important role for these enzymes in mast cell biology.

Place, publisher, year, edition, pages
2008. Vol. 45, no 7, 2116-2125 p.
Keyword [en]
Mast cell, Chymase, Cleavage specificity, Evolution, Marsupial
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-97131DOI: 10.1016/j.molimm.2007.10.015ISI: 000254260500031PubMedID: 18022236OAI: oai:DiVA.org:uu-97131DiVA: diva2:171934
Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2009-11-13Bibliographically approved
In thesis
1. Hematopoietic Serine Proteases from the Mast Cell Chymase and Tryptase Loci - a Functional and Evolutionary Analysis
Open this publication in new window or tab >>Hematopoietic Serine Proteases from the Mast Cell Chymase and Tryptase Loci - a Functional and Evolutionary Analysis
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish.

Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.

To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.

Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 427
Keyword
Cell and molecular biology, immune system, mast cell, basophil, mast cell chymase locus, multigene tryptase locus, serine protease, evolution, Cell- och molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8676 (URN)978-91-554-7179-8 (ISBN)
Public defence
2008-05-16, C10:305, BMC, Husargatan 3, Box 596, 75124 Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2011-07-08Bibliographically approved

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