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Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
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2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 6, e65988- p.Article in journal (Refereed) Published
Abstract [en]

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr(4)-Ile(5) bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.

Place, publisher, year, edition, pages
2013. Vol. 8, no 6, e65988- p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-208169DOI: 10.1371/journal.pone.0065988ISI: 000322361200045OAI: diva2:651178
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2014-05-13Bibliographically approved
In thesis
1. Haematopoietic Serine Proteases: A Cleavage Specificity Analysis
Open this publication in new window or tab >>Haematopoietic Serine Proteases: A Cleavage Specificity Analysis
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions.

In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates.  The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity.

As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors.  Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions.

The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development.

Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr.

Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 63 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1144
Mast cell, cleavage specificity, phage display, chymase, serine protease, granzyme, fish protease
National Category
urn:nbn:se:uu:diva-221891 (URN)978-91-554-8945-8 (ISBN)
Public defence
2014-06-04, C4:305, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2014-05-13 Created: 2014-04-07 Last updated: 2014-06-30Bibliographically approved

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