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  • 1.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala University.
    The mast cell transcriptome and the evolution of granule proteins and Fc receptors2019Doktorsavhandling, 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.

    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
  • 2.
    Akula, Srinivas
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    The Appearance and Diversification of Receptors for IgM During Vertebrate Evolution2017Ingår i: IGM AND ITS RECEPTORS AND BINDING PROTEINS / [ed] Kubagawa, H Burrows, PD, SPRINGER-VERLAG BERLIN , 2017, s. 1-23Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Three different receptors that interact with the constant domains of IgM have been identified: the polymeric immunoglobulin (Ig) receptor (PIGR), the dual receptor for IgA/IgM (Fc alpha mu R) and the IgM receptor (Fc mu R). All of them are related in structure and located in the same chromosomal region in mammals. The functions of the PIGRs are to transport IgM and IgA into the intestinal lumen and to saliva and tears, whereas the Fc alpha mu Rs enhance uptake of immune complexes and antibody coated bacteria and viruses by B220+ B cells and phagocytes, as well as dampening the Ig response to thymus-independent antigens. The Fc mu Rs have broad-spectrum effects on B-cell development including effects on IgM homeostasis, B-cell survival, humoral immune responses and also in autoantibody formation. The PIGR is the first of these receptors to appear during vertebrate evolution and is found in bony fish and all tetrapods but not in cartilaginous fish. The Fc mu R is present in all extant mammalian lineages and also in the Chinese and American alligators, suggesting its appearance with early reptiles. Currently the Fc alpha mu R has only been found in mammals and is most likely the evolutionary youngest of the three receptors. In bony fish, the PIGR has either 2, 3, 4, 5 or 6 extracellular Ig-like domains, whereas in amphibians, reptiles and birds it has 4 domains, and 5 in all mammals. The increase in domain number from 4 to 5 in mammals has been proposed to enhance the interaction with IgA. Both the Fc alpha mu Rs and the Fc mu Rs contain only one Ig domain; the domain that confers Ig binding. In both of these receptors this domain shows the highest degree of sequence similarity to domain 1 of the PIGR. All Ig domains of these three receptors are V type domains, indicating they all have the same origin although they have diversified extensively in function during vertebrate evolution by changing expression patterns and cytoplasmic signaling motifs.

  • 3.
    Akula, Srinivas
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Mohammadamin, Sayran
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 5, s. e96903-Artikel i tidskrift (Refereegranskat)
    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.

  • 4.
    Akula, Srinivas
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Paivandy, Aida
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pjeler, Gunnar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    The mouse mast cell transcriptomeManuskript (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.

  • 5.
    Akula, Srinivas
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Boinapally, Vamsi
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 11, artikel-id e0143091Artikel i tidskrift (Refereegranskat)
    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.

  • 6.
    Fu, Zhirong
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Chahal, Gurdeep
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    de Garavilla, Lawrence
    GDL Pharmaceut Consulting & Contracting, Downingtown, PA 19335 USA.
    Kervinen, Jukka
    Tosoh Biosci LLC, 3604 Horizon Dr, King Of Prussia, PA 19406 USA.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Extended cleavage specificity of sheep mast cell protease-2: A classical chymase with preference to aromatic P1 substrate residues2019Ingår i: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 92, s. 160-169Artikel i tidskrift (Refereegranskat)
    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.

  • 7.
    Fu, Zhirong
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Chahal, Gurdeep
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    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 Stability2018Ingår i: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 9, artikel-id 2387Artikel i tidskrift (Refereegranskat)
    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.

  • 8.
    Fu, Zhirong
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 5, artikel-id e0154886Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus.

  • 9.
    Hellman, Lars T.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fu, Zhirong
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Tracing the Origins of IgE, Mast Cells, and Allergies by Studies of Wild Animals2017Ingår i: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, artikel-id 1749Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In most industrialized countries, allergies have increased in frequency quite dramatically during the past 50 years. Estimates show that 20-30% of the populations are affected. Allergies have thereby become one of the major medical challenges of the twenty-first century. Despite several theories including the hygiene hypothesis, there are still very few solid clues concerning the causes of this increase. To trace the origins of allergies, we have studied cells and molecules of importance for the development of IgE-mediated allergies, including the repertoire of immunoglobulin genes. These studies have shown that IgE and IgG most likely appeared by a gene duplication of IgY in an early mammal, possibly 220-300 million years ago. Receptors specific for IgE and IgG subsequently appeared in parallel with the increase in Ig isotypes from a subfamily of the recently identified Fc receptor-like molecules. Circulating IgE levels are generally very low in humans and laboratory rodents. However, when dogs and Scandinavian wolfs were analyzed, IgE levels were found to be 100-200 times higher compared to humans, indicating a generally much more active IgE synthesis in free-living animals, most likely connected to intestinal parasite infections. One of the major effector molecules released upon IgEmediated activation by mast cells are serine proteases. These proteases, which belong to the large family of hematopoietic serine proteases, are extremely abundant and can account for up to 35% of the total cellular protein. Recent studies show that several of these enzymes, including the chymases and tryptases, are old. Ancestors for these enzymes were most likely present in an early mammal more than 200 million years ago before the separation of the three extant mammalian lineages; monotremes, marsupials, and placental mammals. The aim is now to continue these studies of mast cell biology and IgE to obtain additional clues to their evolutionary conserved functions. A focus concerns why the humoral immune response involving IgE and mast cells have become so dysregulated in humans as well as several of our domestic companion animals.

  • 10.
    Thorpe, Michael
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells2016Ingår i: Developmental And Comparative Immunology, ISSN 0145-305X, Vol. 63, s. 84-95Artikel i tidskrift (Refereegranskat)
    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.

  • 11.
    Thorpe, Michael
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fu, Zhirong
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Albat, Emanuelle
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    de Garavilla, Lawrence
    GDL Pharmaceut Consulting & Contracting, Downingtown, PA USA.
    Kervinen, Jukka
    Tosoh Biosci LLC, Separat Business Unit, King Of Prussia, PA USA.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    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-52018Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, nr 12, artikel-id e0207826Artikel i tidskrift (Refereegranskat)
    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.

  • 12.
    Thorpe, Michael
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fu, Zhirong
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Chahal, Gurdeep
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Kervinen, Jukka
    Fraunhofer USA Ctr Mol Biotechnol, Newark, DE USA.
    de Garavilla, Lawrence
    GDL Pharmaceut Consulting & Contracting, Downingtown, PA USA.
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities2018Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, nr 4, artikel-id e0195077Artikel i tidskrift (Refereegranskat)
    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.

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