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The mast cell transcriptome and the evolution of granule proteins and Fc receptors
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala University. (Molecular Immunology)ORCID-id: 0000-0001-6628-1640
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Protection against disease-causing pathogens, known as immunity, involves numerous cells organs, tissues and their products. To able to understand the biology of immune cells (hematopoietic cells) and their role in an immune system, we have used several different methods, including transcriptome analyses, bioinformatics, production of recombinant proteins and analyses of some of them, focusing on the granule proteases by substrate phage display.

Hematopoietic cells express surface receptors interacting with the constant region of immunoglobulins (Igs) known as Fc receptors (FcRs). These receptors play major roles in the immune system, including enhancing phagocytosis, activating antibody dependent cellular cytotoxicity and cell activation. A detailed bioinformatics analysis of FcRs reveals that the poly-Ig receptors (PIGR), FcR-like molecules and common signalling γ chain all appeared very early with the appearance of the bony fishes, and thereby represent the first major evolutionary step in FcR evolution. The FcμR, FcαμR, FcγR and FcεR receptors most likely appeared in reptiles or early mammals, representing the second major step in FcR evolution.

Cells of several of the hematopoietic cell lineages contain large numbers of cytoplasmic granules, and serine proteases constitute the major protein content of these granules. In mammals, these proteases are encoded from four different loci: the chymase, the met-ase, the granzyme (A/K) and the mast cell tryptase loci. The granzyme (A/K) locus was the first to appear and came with the cartilaginous fishes. This locus is also the most conserved of the three. The second most conserved locus is the met-ase locus, which is found in bony fishes. The chymase locus appeared relatively late, and we find the first traces in frogs, indicating it appeared in early tetrapods.

To study the early events in the diversification of these hematopoietic serine proteases we have analyzed key characteristics of a protease expressed by an NK-like cell in the channel catfish, catfish granzyme–like I. We have used phage display and further validated the results using a panel of recombinant substrates. This protease showed a strict preference for Met at the P1 (cleavage) position, which indicates met-ase specificity. From the screening of potential in vivo substrates, we found an interesting potential target caspase 6, which indicates that caspase-dependent apoptosis mechanisms have been conserved from fishes to mammals.

A larger quantitative transcriptome analysis of purified mouse peritoneal mast cells, cultured mast cells (BMMCs), and mast cells isolated from mouse ear and lung tissue identified the major tissue specific transcripts in these mast cells as the granule proteases. Mast cell specific receptors and processing enzymes were expressed at approximately 2 orders of magnitude lower levels. The levels of a few proteases were quite different at various anatomical sites between in vivo and cultured BMMCs. These studies have given us a new insights into mast cells in different tissues, as well as key evolutionary aspects concerning the origins of a number of granule proteases and FcRs.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2019. , s. 55
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1803
Nyckelord [en]
Mast cell, Fc receptors, Granule serine protease, Evolution and transcriptome.
Nationell ämneskategori
Biologiska vetenskaper Immunologi
Forskningsämne
Immunologi
Identifikatorer
URN: urn:nbn:se:uu:diva-381377ISBN: 978-91-513-0645-2 (tryckt)OAI: oai:DiVA.org:uu-381377DiVA, id: diva2:1304279
Disputation
2019-06-05, C8:301, BMC, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-05-13 Skapad: 2019-04-12 Senast uppdaterad: 2019-06-17
Delarbeten
1. Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution
Öppna denna publikation i ny flik eller fönster >>Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution
2014 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 5, s. e96903-Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR gamma chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.

Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi
Identifikatorer
urn:nbn:se:uu:diva-228481 (URN)10.1371/journal.pone.0096903 (DOI)000336838000078 ()
Tillgänglig från: 2014-07-15 Skapad: 2014-07-15 Senast uppdaterad: 2019-04-12Bibliografiskt granskad
2. Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution
Öppna denna publikation i ny flik eller fönster >>Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution
2015 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 11, artikel-id e0143091Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
urn:nbn:se:uu:diva-271027 (URN)10.1371/journal.pone.0143091 (DOI)000365070700134 ()26569620 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 621-2011-5007
Tillgänglig från: 2016-01-05 Skapad: 2016-01-05 Senast uppdaterad: 2019-04-12Bibliografiskt granskad
3. Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
Öppna denna publikation i ny flik eller fönster >>Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
2016 (Engelska)Ingår i: Developmental And Comparative Immunology, ISSN 0145-305X, Vol. 63, s. 84-95Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Here we present the extended cleavage specificity of catfish granzyme-like I, previously identified in fish NK-like cells. This protease has been characterised using substrate phage display and further validated by using a panel of recombinant substrates. A strict preference for Met in the P1 (cleavage) position, indicating metase specificity was observed. A screening of potential in vivo substrates was performed based on the derived P5-P3' consensus: Arg-Val-Thr-Gly-Met(down arrow)Ser-Leu-Val. Channel catfish caspase 6 was one very interesting potential target identified. This site was present in an adjacent position to the classic caspase activation site (Asp179 in human caspase 6). Cleavage of this site (hence potential activation) by the catfish granzyme-like I could reveal a novel mechanism of caspase 6 activation. This poses an interesting idea that the role of granzyme-like proteases in the activation of caspase dependent apoptosis mechanisms has been conserved for over 400 million years.

Nyckelord
Fish; Serine protease; Cleavage specificity; Metase; NK cell; Caspase; Evolution
Nationell ämneskategori
Immunologi
Identifikatorer
urn:nbn:se:uu:diva-221554 (URN)10.1016/j.dci.2016.05.013 (DOI)000380623300010 ()27216028 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 621-2011-5007
Tillgänglig från: 2014-04-09 Skapad: 2014-04-01 Senast uppdaterad: 2019-04-12Bibliografiskt granskad
4. The mouse mast cell transcriptome
Öppna denna publikation i ny flik eller fönster >>The mouse mast cell transcriptome
Visa övriga...
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

Mast cells (MCs) are highly specialized tissue resident cells that are often found at the interphase between body and environment such as the skin, lung and intestinal mucosa. To obtain a more detailed picture of the biology of MCs we have analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR based transcriptomics.  The results show that MCs at different tissue locations can differ quite substantially in transcript levels of several of the most abundant granule proteins even if they belong to the same basic MC type, i.e connective tissue or mucosal MCs. We can also see that transcript levels for the major granule proteins, like the various proteases and the heparin core protein can be several orders of magnitude higher than the surface receptors.  This also applies for the processing enzymes involved in activation of the proteases and in the synthesis of heparin and histamine. Interestingly also is the almost complete absence of transcripts for cytokines in the MC populations of the various organs, indicating that cytokines only are produced by activated MCs. Bone marrow derived MCs are often used as equivalents of tissue MCs.  We here show that these cells differ substantially in their transcriptome from tissue MCs. They show a transcriptome of relatively immature cells both with respect to the granule components and to the processing enzymes indicating that care should be taken when transferring findings from these cells to the in vivo function of tissue resident MCs.  This latter finding also give clear indication for that additional cytokines are needed, in addition to the stem cell factor (SCF), for the development into fully mature tissue MCs.

Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
Immunologi
Identifikatorer
urn:nbn:se:uu:diva-381501 (URN)
Tillgänglig från: 2019-04-11 Skapad: 2019-04-11 Senast uppdaterad: 2019-04-15Bibliografiskt granskad

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