| 1. |
- Fu, Zhirong, et al.
(author)
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Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity
- 2021
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:5
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Journal article (peer-reviewed)abstract
- Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.
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| 2. |
- Fu, Zhirong, et al.
(author)
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Extended cleavage specificity of sheep mast cell protease-2 : A classical chymase with preference to aromatic P1 substrate residues
- 2019
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In: Developmental and Comparative Immunology. - : ELSEVIER SCI LTD. - 0145-305X .- 1879-0089. ; 92, s. 160-169
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Journal article (peer-reviewed)abstract
- Serine proteases constitute the major protein content of mammalian mast cell granules and the selectivity for substrates by these proteases is of major importance for the role of mast cells in immunity. In order to address this subject, we present here the extended cleavage specificity of sheep mast cell protease-2 (MCP2), a chymotrypsin-type serine protease. Comparison of the extended specificity results to a panel of mammalian mast cell chymases show, in almost all aspects, the same cleavage characteristics. This includes preference for aromatic residues (Phe, Tyr, Trp) in the P1 position of substrates and a preference for aliphatic residues in most other substrate positions around the cleavage site. MCP2 also cleaved, albeit relatively low efficiency, after Leu in the P1 position. In contrast to the human, mouse, hamster and opossum chymases that show a relatively strong preference for negatively charged amino acids in the P2'position, the sheep MCP2, however, lacked that preference. Therefore, together with the rat chymase (rMCP1), sheep MCP2 can be grouped to a small subfamily of mammalian chymases that show fairly unspecific preference in the P2'position. In summary, the results here support the view of a strong evolutionary conservation of a potent chymotrypsin-type protease as a key feature of mammalian mast cells.
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| 3. |
- Fu, Zhirong, et al.
(author)
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Highly Selective Cleavage of Cytokines and Chemokines by the Human Mast Cell Chymase and Neutrophil Cathepsin G
- 2017
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In: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 198:4, s. 1474-1483
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Journal article (peer-reviewed)abstract
- Human mast cell chymase (HC) and human neutrophil cathepsin G (hCG) show relatively similar cleavage specificities: they both have chymotryptic activity but can also cleave efficiently after leucine. Their relatively broad specificity suggests that they may cleave almost any substrate if present in high enough concentrations or for a sufficiently long time. A number of potential substrates have been identified for these enzymes and, recently, these enzymes have also been implicated in regulating cytokine activity by cleaving numerous cytokines and chemokines. To obtain a better understanding of their selectivity for various potential in vivo substrates, we analyzed the cleavage of a panel of 51 active recombinant cytokines and chemokines. Surprisingly, our results showed a high selectivity of HC; only 4 of 51 of these proteins were substantially cleaved. hCG cleaved a few additional proteins, although this occurred after adding almost equimolar amounts of enzyme to target. The explanation for this wide difference in activity against peptides or other linear substrates compared with native proteins is most likely related to the reduced accessibility of the enzymes to potential cleavage sites in folded proteins. In this article, we present evidence that sites not exposed on the surface of the protein are not cleaved by the enzyme. Interestingly, both enzymes readily cleaved IL-18 and IL-33, two IL-1-related alarmins, as well as the cytokine IL-15, which is important for T cell and NK cell homeostasis. Cleavage of the alarmins by HC and hCG suggests a function in regulating excessive inflammation.
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| 4. |
- Fu, Zhirong, et al.
(author)
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Mast Cells and Basophils in the Defense against Ectoparasites : Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases
- 2021
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:23
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Journal article (peer-reviewed)abstract
- Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.
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| 5. |
- Pätsi, Jukka, et al.
(author)
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LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1.
- 2012
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2728. ; 1817:2, s. 312-318
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Journal article (peer-reviewed)abstract
- Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G > A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent Km for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A > G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G > A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G > A affecting the same extramembrane loop as the m.3376G > A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G > A transition in MTND1 is the pathogenic mutation and m.3865A > G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49 kD/PSST subunits. In all, m.3376G > A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.
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| 6. |
- Ramos-Ramirez, Patricia, et al.
(author)
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House Dust Mite and Cat Dander Extract Induce Asthma-Like Histopathology with an Increase of Mucosal Mast Cells in a Guinea Pig Model
- 2023
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In: Journal of Immunology Research. - : Hindawi Publishing Corporation. - 2314-8861 .- 2314-7156. ; 2023
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Journal article (peer-reviewed)abstract
- Background. Asthma is a chronic inflammatory disease with structural changes in the lungs defined as airway remodelling. Mast cell responses are important in asthma as they, upon activation, release mediators inducing bronchoconstriction, inflammatory cell recruitment, and often remodelling of the airways. As guinea pigs exhibit anatomical, physiological, and pharmacological features resembling human airways, including mast cell distribution and mediator release, we evaluated the effect of extracts from two common allergens, house dust mite (HDM) and cat dander (CDE), on histopathological changes and the composition of tryptase- and chymase-positive mast cells in the guinea pig lungs. Methods. Guinea pigs were exposed intranasally to HDM or CDE for 4, 8, and 12 weeks, and airway histology was examined at each time point. Hematoxylin and eosin, Picro-Sirius Red, and Periodic Acid-Schiff staining were performed to evaluate airway inflammation, collagen deposition, and mucus-producing cells. In addition, Astra blue and immunostaining against tryptase and chymase were used to visualize mast cells. Results. Repetitive administration of HDM or CDE led to the accumulation of inflammatory cells into the proximal and distal airways as well as increased airway smooth muscle mass. HDM exposure caused subepithelial collagen deposition and mucus cell hyperplasia at all three time points, whereas CDE exposure only caused these effects at 8 and 12 weeks. Both HDM and CDE induced a substantial increase in mast cells after 8 and 12 weeks of challenges. This increase was primarily due to mast cells expressing tryptase, but not chymase, thus indicating mucosal mast cells. Conclusions. We here show that exposure to HDM and CDE elicits asthma-like histopathology in guinea pigs with infiltration of inflammatory cells, airway remodelling, and accumulation of primarily mucosal mast cells. The results together encourage the use of HDM and CDE allergens for the stimulation of a clinically relevant asthma model in guinea pigs.
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| 7. |
- Thorpe, Michael, et al.
(author)
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Extended cleavage specificities of mast cell proteases 1 and 2 from golden hamster : Classical chymase and an elastolytic protease comparable to rat and mouse MCP-5
- 2018
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In: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:12
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Journal article (peer-reviewed)abstract
- Serine proteases constitute the major protein content of mast cell secretory granules. Here we present the extended cleavage specificity of two such proteases from the golden hamster, Mesocricetus auratus. Analysis by phage display technique showed that one of them (HAM1) is a classical chymase with a specificity similar to the human mast cell chymase. However, in contrast to the human chymase, it does not seem to have a particular preference for any of the three aromatic amino acids, Phe, Tyr and Trp, in the P1 position of substrates. HAM1 also efficiently cleaved after Leu similarly to human and many other mast cell chymases. We observed only a 3-fold lower cleavage activity on Leu compared to substrates with P1 aromatic amino acids. Chymotryptic enzymes seem to be characteristic for connective tissue mast cells in mammalian species from opossums to humans, which indicates a very central role of these enzymes in mast cell biology. HAM1 also seems to have the strongest preference for negatively charged amino acids in the P2 position of all mast cell chymases so far characterized. The second hamster chymase, HAM2, is an elastolytic in its activity, similarly to the alpha-chymases in rats and mice (rMCP-5 and mMCP-5, respectively). The presence of an alpha-chymase that developed elastase activity thereby seems to be a relatively early modification of the alpha-chymase within the rodent branch of the mammalian evolutionary tree.
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| 8. |
- Thorpe, Michael, et al.
(author)
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Extended cleavage specificity of human neutrophil cathepsin G : A low activity protease with dual chymase and tryptase-type specificities
- 2018
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:4
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Journal article (peer-reviewed)abstract
- Human neutrophils express at least four active serine proteases, cathepsin G, N-elastase, proteinase 3 and neutrophil serine protease 4 (NSP4). They have all been extensively studied due to their importance in neutrophil biology and immunity. However, their extended cleavage specificities have never been determined in detail. Here we present a detailed cleavage specificity analysis of human cathepsin G (hCG). The specificity was determined by phage display analysis and the importance of individual amino acids in and around the cleavage site was then validated using novel recombinant substrates. To provide a broader context to this serine protease, a comparison was made to the related mast cell protease, human chymase (HC). hCG showed similar characteristics to HC including both the primary and extended specificities. As expected, Phe, Tyr, Trp and Leu were preferred in the P1 position. In addition, both proteases showed a preference for negatively charged amino acids in the P2 A position of substrates and a preference for aliphatic amino acids both upstream and downstream of the cleavage site. However, overall the catalytic activity of hCG was similar to 10-fold lower than HC. hCG has previously been reported to have a dual specificity consisting of chymase and tryptase-type activities. In our analysis, tryptase activity against substrates with Lys in P1 cleavage position was indeed only 2-fold less efficient as compared to optimal chymase substrates supporting strong dual-type specificity. We hope the information presented here on extended cleavage specificities of hCG and HC will assist in the search for novel in vivo substrates for these proteases as well as aid in the efforts to better understand the role of hCG in immunity and bacterial defence.
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| 9. |
- Thorpe, Michael, et al.
(author)
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Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis) : an interesting animal model for the analysis of the function of the human mast cell chymase
- 2012
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In: International Immunology. - : Oxford University Press (OUP). - 0953-8178 .- 1460-2377. ; 24:12, s. 771-782
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Journal article (peer-reviewed)abstract
- Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use.
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| 10. |
- Zhongwei, Yuan, et al.
(author)
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Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases : Two Highly Specific Leu-Ases
- 2019
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 20:24
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Journal article (peer-reviewed)abstract
- Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit beta-chymase and a guinea pig alpha-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species.
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