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Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2000 In: Biochemistry, Vol. 39, no 42, 13068-13077 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2000. Vol. 39, no 42, 13068-13077 p.
Identifiers
URN: urn:nbn:se:uu:diva-90691OAI: oai:DiVA.org:uu-90691DiVA: diva2:163138
Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved
In thesis
1. Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases
Open this publication in new window or tab >>Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.

The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 55 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 870
Keyword
Biology, mast cell, serine protease, tryptase, chymase, substrate specificity, phage display, Biologi
National Category
Biological Sciences
Research subject
Molecular Immunology
Identifiers
urn:nbn:se:uu:diva-3529 (URN)91-554-5699-5 (ISBN)
Public defence
2003-10-02, C10:305, Biomedicinskt center, Uppsala, 09:15
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Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved

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