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Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
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. , p. 55
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 870
Keywords [en]
Biology, mast cell, serine protease, tryptase, chymase, substrate specificity, phage display
Keywords [sv]
Biologi
National Category
Biological Sciences
Research subject
Molecular Immunology
Identifiers
URN: urn:nbn:se:uu:diva-3529ISBN: 91-554-5699-5 (print)OAI: oai:DiVA.org:uu-3529DiVA, id: diva2:163140
Public defence
2003-10-02, C10:305, Biomedicinskt center, Uppsala, 09:15
Opponent
Supervisors
Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved
List of papers
1. Rat mast cell protease 4 is a beta chymase with unusually stringent substrate recognition profile
Open this publication in new window or tab >>Rat mast cell protease 4 is a beta chymase with unusually stringent substrate recognition profile
2002 In: The Journal of Biological Chemistry, Vol. 277, no 21, p. 18579-18585Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90689 (URN)
Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved
2. Extended substrate specificity of rat mast cell protease 5, a rodent alpha chymase with elastase-like primary specificity
Open this publication in new window or tab >>Extended substrate specificity of rat mast cell protease 5, a rodent alpha chymase with elastase-like primary specificity
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 41, p. 39625-39631Article in journal (Refereed) Published
Abstract [en]

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Immunology
Identifiers
urn:nbn:se:uu:diva-90690 (URN)10.1074/jbc.M301512200 (DOI)12900423 (PubMedID)
Available from: 2003-09-04 Created: 2003-09-04 Last updated: 2017-12-14Bibliographically approved
3. 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
Open this publication in new window or tab >>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
Show others...
2000 In: Biochemistry, Vol. 39, no 42, p. 13068-13077Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90691 (URN)
Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved
4. Heparin antagonists are potent inhibitors of mast cell tryptase
Open this publication in new window or tab >>Heparin antagonists are potent inhibitors of mast cell tryptase
Show others...
2001 In: Biochemistry, Vol. 40, no 24, p. 7342-7349Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90692 (URN)
Available from: 2003-09-04 Created: 2003-09-04Bibliographically approved

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