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Hallgren, J., Hellman, L., Maurer, M., Nilsson, G. P., Wernersson, S., Åbrink, M. & Pejler, G. (2020). Novel aspects of mast cell and basophil function: Highlights from the 9th meeting of the European Mast Cell and Basophil Research Network (EMBRN)-A Marcus Wallenberg Symposium [Letter to the editor]. Allergy. European Journal of Allergy and Clinical Immunology, 75(3), 707-708
Open this publication in new window or tab >>Novel aspects of mast cell and basophil function: Highlights from the 9th meeting of the European Mast Cell and Basophil Research Network (EMBRN)-A Marcus Wallenberg Symposium
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2020 (English)In: Allergy. European Journal of Allergy and Clinical Immunology, ISSN 0105-4538, E-ISSN 1398-9995, Vol. 75, no 3, p. 707-708Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
John Wiley & Sons, 2020
National Category
Immunology in the medical area Respiratory Medicine and Allergy
Identifiers
urn:nbn:se:uu:diva-406838 (URN)10.1111/all.14065 (DOI)000518758900027 ()31557323 (PubMedID)
Funder
Swedish Cancer SocietySwedish Asthma and Allergy AssociationMarcus Wallenbergs Foundation for International Scientific CollaborationWenner-Gren FoundationsSwedish Research Council
Available from: 2020-03-12 Created: 2020-03-12 Last updated: 2020-04-07Bibliographically approved
Fu, Z., Akula, S., Thorpe, M. & Hellman, L. (2020). Potent and Broad but not Unselective Cleavage of Cytokines and Chemokines by Human Neutrophil Elastase and Proteinase 3. International Journal of Molecular Sciences, 21(2), Article ID 651.
Open this publication in new window or tab >>Potent and Broad but not Unselective Cleavage of Cytokines and Chemokines by Human Neutrophil Elastase and Proteinase 3
2020 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 21, no 2, article id 651Article in journal (Refereed) Published
Abstract [en]

In two recent studies we have shown that three of the most abundant human hematopoietic serine proteases-mast cell chymase, mast cell tryptase and neutrophil cathepsin G-show a highly selective cleavage of cytokines and chemokines with a strong preference for a few alarmins, including IL-18, TSLP and IL-33. To determine if this is a general pattern for many of the hematopoietic serine proteases we have analyzed the human neutrophil elastase (hNE) and human proteinase 3 (hPR-3) for their cleavage of a panel of 69 different human cytokines and chemokines. Our results showed that these two latter enzymes, in sharp contrast to the two previous, had a very potent and relatively unrestrictive cleavage on this panel of targets. Almost all of these proteins were cleaved and many of them were fully degraded. In light of the proteases abundance and their colocalization, it is likely that together they have a very potent degrading activity on almost any protein in the area of neutrophil activation and granule release, including both foreign bacterial or viral proteins as well as various self-proteins in the area of inflammation/infection. However, a few very interesting exceptions to this pattern were found indicating a high resistance to degradation of some cytokines and chemokines, including TNF-alpha, IL-5, M-CSF, Rantes, IL-8 and MCP-1. All of these are either important for monocyte-macrophage, neutrophil or eosinophil proliferation, recruitment and activation, suggesting that cytokines/chemokines and proteases may have coevolved to not block the recruitment of monocytes-macrophages, neutrophils and possibly eosinophils during an inflammatory response involving neutrophil activation.

Place, publisher, year, edition, pages
MDPI, 2020
Keywords
neutrophilic granulocyte, neutrophil elastase, proteinase 3, cytokine, chemokine
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-407515 (URN)10.3390/ijms21020651 (DOI)000515380000281 ()31963828 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2017-0022
Available from: 2020-03-25 Created: 2020-03-25 Last updated: 2020-03-25Bibliographically approved
Akula, S., Paivandy, A., Fu, Z., Thorpe, M., Pejler, G. & Hellman, L. (2020). Quantitative In-Depth Analysis of the Mouse Mast Cell Transcriptome Reveals Organ-Specific Mast Cell Heterogeneity. CELLS, 9(1), Article ID 211.
Open this publication in new window or tab >>Quantitative In-Depth Analysis of the Mouse Mast Cell Transcriptome Reveals Organ-Specific Mast Cell Heterogeneity
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2020 (English)In: CELLS, E-ISSN 2073-4409, Vol. 9, no 1, article id 211Article in journal (Refereed) Published
Abstract [en]

Mast cells (MCs) are primarily resident hematopoietic tissue cells that are localized at external and internal surfaces of the body where they act in the first line of defense. MCs are found in all studied vertebrates and have also been identified in tunicates, an early chordate. To obtain a detailed insight into the biology of MCs, here we analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR-based transcriptomics. We show that MCs at different tissue locations differ substantially in their levels of transcripts coding for the most abundant MC granule proteins, even within the connective tissue type, or mucosal MC niches. We also demonstrate that transcript levels for the major granule proteins, including the various MC-restricted proteases and the heparin core protein, can be several orders of magnitude higher than those coding for various surface receptors and enzymes involved in protease activation, as well as enzymes involved in the synthesis of heparin, histamine, leukotrienes, and prostaglandins. Interestingly, our analyses revealed an almost complete absence in MCs of transcripts coding for cytokines at baseline conditions, indicating that cytokines are primarily produced by activated MCs. Bone marrow-derived MCs (BMMCs) are often used as equivalents of tissue MCs. Here, we show that these cells differ substantially from tissue MCs with regard to their transcriptome. Notably, they showed a transcriptome indicative of relatively immature cells, both with respect to the expression of granule proteases and of various enzymes involved in the processing/synthesis of granule compounds, indicating that care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. Furthermore, the latter finding indicates that the development of fully mature tissue-resident MCs requires a cytokine milieu beyond what is needed for in vitro differentiation of BMMCs. Altogether, this study provides a comprehensive quantitative view of the transcriptome profile of MCs resident at different tissue locations that builds nicely on previous studies of both the mouse and human transcriptome, and form a solid base for future evolutionary studies of the role of MCs in vertebrate immunity.

Place, publisher, year, edition, pages
MDPI, 2020
Keywords
transcriptome, mRNA, mast cell, tryptase, chymase, serine protease, Fc epsilon RI, heparin, histamine
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-407608 (URN)10.3390/cells9010211 (DOI)000515398200211 ()31947690 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2017-0022
Available from: 2020-04-01 Created: 2020-04-01 Last updated: 2020-04-01Bibliographically approved
Liu, J., Fu, Z., Hellman, L. & Svärd, S. G. (2019). Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins. Molecular and biochemical parasitology (Print), 227, 29-38
Open this publication in new window or tab >>Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins
2019 (English)In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 227, p. 29-38Article in journal (Refereed) Published
Abstract [en]

Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery.

Keywords
Cysteine protease, Defensins, Diarrhea, Immunoglobulins, Parasite, Phage display
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-372982 (URN)10.1016/j.molbiopara.2018.10.004 (DOI)000457660900006 ()30458129 (PubMedID)
Funder
Swedish Research Council, 2017-02918
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-03-05Bibliographically approved
Zhongwei, Y., Akula, S., Fu, Z., de Garavilla, L., Kervinen, J., Thorpe, M. & Hellman, L. (2019). Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases: Two Highly Specific Leu-Ases. International Journal of Molecular Sciences, 20(24), Article ID 6340.
Open this publication in new window or tab >>Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases: Two Highly Specific Leu-Ases
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2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 24, article id 6340Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
mast cells, chymase, tryptase, cleavage specificity, Leu-ase, phage display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-404709 (URN)10.3390/ijms20246340 (DOI)000506840100233 ()31888202 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW2017.0022
Available from: 2020-02-26 Created: 2020-02-26 Last updated: 2020-02-26Bibliographically approved
Fu, Z., Akula, S., Thorpe, M., Chahal, G., de Garavilla, L., Kervinen, J. & Hellman, L. (2019). Extended cleavage specificity of sheep mast cell protease-2: A classical chymase with preference to aromatic P1 substrate residues. Developmental and Comparative Immunology, 92, 160-169
Open this publication in new window or tab >>Extended cleavage specificity of sheep mast cell protease-2: A classical chymase with preference to aromatic P1 substrate residues
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2019 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 92, p. 160-169Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCI LTD, 2019
Keywords
Mast cell, Chymase, Sheep, Cleavage specificity, Animal model
National Category
Immunology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-378627 (URN)10.1016/j.dci.2018.11.019 (DOI)000458596200017 ()30481523 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-11Bibliographically approved
Fu, Z., Akula, S., Thorpe, M. & Hellman, L. (2019). Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity. International Journal of Molecular Sciences, 20(20), Article ID 5147.
Open this publication in new window or tab >>Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity
2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 20, article id 5147Article in journal (Refereed) Published
Abstract [en]

Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and the environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is tryptase. To look deeper into the question of their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b, and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting regulatory functions may involve controlling excessive TH2-mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15, and IL-21, indicating a potent negative feedback loop on TH2 immunity.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
mast cell, tryptase, chymase, serine protease, human chymase, cleavage specificity, cytokine, chemokine, TH2
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-400005 (URN)10.3390/ijms20205147 (DOI)000498822800179 ()31627390 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2017-0022
Available from: 2019-12-19 Created: 2019-12-19 Last updated: 2019-12-19Bibliographically approved
Thorpe, M., Fu, Z., Albat, E., Akula, S., de Garavilla, L., Kervinen, J. & Hellman, L. (2018). 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. PLoS ONE, 13(12), Article ID e0207826.
Open this publication in new window or tab >>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
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 12, article id e0207826Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2018
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-373000 (URN)10.1371/journal.pone.0207826 (DOI)000452307600021 ()30521603 (PubMedID)
Funder
Swedish Research Council, 621-2011-5007
Available from: 2019-01-14 Created: 2019-01-14 Last updated: 2019-01-14Bibliographically approved
Thorpe, M., Fu, Z., Chahal, G., Akula, S., Kervinen, J., de Garavilla, L. & Hellman, L. (2018). Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities. PLoS ONE, 13(4), Article ID e0195077.
Open this publication in new window or tab >>Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 4, article id e0195077Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-353202 (URN)10.1371/journal.pone.0195077 (DOI)000430026900017 ()29652924 (PubMedID)
Funder
Swedish Research Council, 621-2011-5007
Available from: 2018-06-13 Created: 2018-06-13 Last updated: 2018-06-13Bibliographically approved
Fu, Z., Thorpe, M., Akula, S., Chahal, G. & Hellman, L. (2018). Extended Cleavage Specificity of Human Neutrophil Elastase, Human Proteinase 3, and Their Distant Ortholog Clawed Frog PR3-Three Elastases With Similar Primary but Different Extended Specificities and Stability. Frontiers in Immunology, 9, Article ID 2387.
Open this publication in new window or tab >>Extended Cleavage Specificity of Human Neutrophil Elastase, Human Proteinase 3, and Their Distant Ortholog Clawed Frog PR3-Three Elastases With Similar Primary but Different Extended Specificities and Stability
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2018 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 9, article id 2387Article in journal (Refereed) Published
Abstract [en]

Serine proteases are major granule constituents of several of the human hematopoietic cell lineages. Four proteolytically active such proteases have been identified in human neutrophils: cathepsin G (hCG), N-elastase (hNE), proteinase 3 (hPR-3), and neutrophil serine protease 4 (hNSP-4). Here we present the extended cleavage specificity of two of the most potent and most abundant of these enzymes, hNE and hPR-3. Their extended specificities were determined by phage display and by the analysis of a panel of chromogenic and recombinant substrates. hNE is an elastase with a relatively broad specificity showing a preference for regions containing several aliphatic amino acids. The protease shows self-cleaving activity, which results in the loss of activity during storage even at +4 degrees C. Here we also present the extended cleavage specificity of hPR-3. Compared with hNE, it shows considerably lower proteolytic activity. However, it is very stable, shows no self-cleaving activity and is actually more active in the presence of SDS, possibly by enhancing the accessibility of the target substrate. This enables specific analysis of hPR-3 activity even in the presence of all the other neutrophil enzymes with addition of 1% SDS. Neutrophils are the most abundant white blood cell in humans and one of the key players in our innate immune defense. The neutrophil serine proteases are very important for the function of the neutrophils and therefore also interesting from an evolutionary perspective. In order to study the origin and functional conservation of these neutrophil proteases we have identified and cloned an amphibian ortholog, Xenopus PR-3 (xPR-3). This enzyme was found to have a specificity very similar to hPR-3 but did not show the high stability in the presence of SDS. The presence of an elastase in Xenopus closely related to hPR-3 indicates a relatively early appearance of these enzymes during vertebrate evolution.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2018
Keywords
neutrophilic granulocyte, serine protease, hematopoiesis, proteinase 3, N-elastase, amphibian, neutropenia, phage display
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-368440 (URN)10.3389/fimmu.2018.02387 (DOI)000447372500001 ()
Funder
Swedish Research Council, 621-2011-5007
Available from: 2018-12-10 Created: 2018-12-10 Last updated: 2018-12-10Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1459-3815

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