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Kjellén, Lena
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Publications (10 of 65) Show all publications
Nakato, E., Liu, X., Eriksson, I., Yamamoto, M., Kinoshita-Toyoda, A., Toyoda, H., . . . Nakato, H. (2019). Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes. Glycobiology, 29(6), 479-489
Open this publication in new window or tab >>Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes
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2019 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 29, no 6, p. 479-489Article in journal (Refereed) Published
Abstract [en]

A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS INC, 2019
Keywords
cell culture, Drosophila, heparan sulfate, mutant
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-397309 (URN)10.1093/glycob/cwz020 (DOI)000493192100006 ()30869121 (PubMedID)
Funder
Vinnova
Available from: 2019-11-19 Created: 2019-11-19 Last updated: 2019-11-19Bibliographically approved
Noborn, F., Toledo, A. G., Nasir, W., Nilsson, J., Dierker, T., Kjellén, L. & Larson, G. (2018). Expanding the chondroitin glycoproteome of Caenorhabditis elegans. Journal of Biological Chemistry, 293(1), 379-389
Open this publication in new window or tab >>Expanding the chondroitin glycoproteome of Caenorhabditis elegans
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 1, p. 379-389Article in journal (Refereed) Published
Abstract [en]

Chondroitin sulfate proteoglycans (CSPGs) are important structural components of connective tissues in essentially all metazoan organisms. In vertebrates, CSPGs are involved also in more specialized processes such as neurogenesis and growth factor signaling. In invertebrates, however, knowledge of CSPGs core proteins and proteoglycan-related functions is relatively limited, even for Caenorhabditis elegans. This nematode produces large amounts of non-sulfated chondroitin in addition to low-sulfated chondroitin sulfate chains. So far, only nine core proteins (CPGs) have been identified, some of which have been shown to be involved in extracellular matrix formation. We recently introduced a protocol to characterize proteoglycan core proteins by identifying CS-glycopeptides with a combination of biochemical enrichment, enzymatic digestion, and nano-scale liquid chromatography MS/MS analysis. Here, we have used this protocol to map the chondroitin glycoproteome in C. elegans, resulting in the identification of 15 novel CPG proteins in addition to the nine previously established. Three of the newly identified CPGs displayed homology to vertebrate proteins. Bioinformatics analysis of the primary protein sequences revealed that the CPG proteins altogether contained 19 unique functional domains, including Kunitz and endostatin domains, suggesting direct involvement in protease inhibition and axonal migration, respectively. The analysis of the core protein domain organization revealed that all chondroitin attachment sites are located in unstructured regions. Our results suggest that CPGs display a much greater functional and structural heterogeneity than previously appreciated and indicate that specialized proteoglycan-mediated functions evolved early in metazoan evolution.

Keywords
Caenorhabditis elegans (C, elegans), chondroitin, glycosaminoglycan, mass spectrometry (MS), proteoglycan, proteoglycan structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341492 (URN)10.1074/jbc.M117.807800 (DOI)000419453200032 ()29138239 (PubMedID)
Available from: 2018-02-19 Created: 2018-02-19 Last updated: 2018-02-19Bibliographically approved
Frisk, J. M., Kjellén, L., Melo, F. R., Öhrvik, H. & Pejler, G. (2018). Mitogen-Activated Protein Kinase Signaling Regulates Proteoglycan Composition of Mast Cell Secretory Granules. Frontiers in Immunology, 9, Article ID 1670.
Open this publication in new window or tab >>Mitogen-Activated Protein Kinase Signaling Regulates Proteoglycan Composition of Mast Cell Secretory Granules
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2018 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 9, article id 1670Article in journal (Refereed) Published
Abstract [en]

Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2018
Keywords
mast cells, mitogen-activated protein kinase, MEK1/2, proteoglycans, heparin, chondroitin sulfate, tryptase, serglycin
National Category
Immunology Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-361689 (URN)10.3389/fimmu.2018.01670 (DOI)000439155000001 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Heart Lung FoundationSwedish Cancer Society
Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2018-10-03Bibliographically approved
Kjellén, L. & Lindahl, U. (2018). Specificity of glycosaminoglycan-protein interactions. Current opinion in structural biology, 50, 101-108
Open this publication in new window or tab >>Specificity of glycosaminoglycan-protein interactions
2018 (English)In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 50, p. 101-108Article in journal (Refereed) Published
Abstract [en]

Glycosaminoglycans (GAGs) interact with a variety of proteins with important functions in development and homeostasis. Most of these proteins bind to heparin in vitro, a highly sulfated GAG species, although heparan sulfate and/or chondroitin/dermatan sulfate are more frequent physiological ligands. Binding affinity and specificity are determined by charge distribution, mainly due to sulfate and carboxylate groups and by GAG chain conformation. Interactions may be nonspecific, essentially reflecting charge density or highly specific, dependent on rare GAG-structural features. Yet other GAG epitopes bind protein ligands with intermediate specificity and variable affinity. Studies of heparan sulfate biosynthesis point to stochastic but strictly regulated, cell-specific polymer modification. Together, these features allow for graded modulation of protein functional response.

Place, publisher, year, edition, pages
CURRENT BIOLOGY LTD, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-364480 (URN)10.1016/j.sbi.2017.12.011 (DOI)000443661300015 ()29455055 (PubMedID)
Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2018-10-30Bibliographically approved
Hu Frisk, J. M., Kjellén, L., Kaler, S. G., Pejler, G. & Öhrvik, H. (2017). Copper Regulates Maturation and Expression of an MITF: Tryptase Axis in Mast Cells. Journal of Immunology, 199(12), 4132-4141
Open this publication in new window or tab >>Copper Regulates Maturation and Expression of an MITF: Tryptase Axis in Mast Cells
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2017 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 199, no 12, p. 4132-4141Article in journal (Refereed) Published
Abstract [en]

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-340995 (URN)10.4049/jimmunol.1700786 (DOI)000417018100021 ()29127151 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Heart Lung FoundationTorsten Söderbergs stiftelseMagnus Bergvall FoundationÅke Wiberg FoundationNIH (National Institute of Health), Z01 HD008768.; Z01 HD008927
Available from: 2018-02-05 Created: 2018-02-05 Last updated: 2018-03-02Bibliographically approved
Xiong, A., Kundu, S., Forsberg, M., Xiong, Y., Bergström, T., Paavilainen, T., . . . Forsberg-Nilsson, K. (2017). Heparanase confers a growth advantage to differentiating murine embryonic stem cells, and enhances oligodendrocyte formation.. Matrix Biology, 62, 92-104
Open this publication in new window or tab >>Heparanase confers a growth advantage to differentiating murine embryonic stem cells, and enhances oligodendrocyte formation.
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2017 (English)In: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 62, p. 92-104Article in journal (Refereed) Published
Abstract [en]

Heparan sulfate proteoglycans (HSPGs), ubiquitous components of mammalian cells, play important roles in development and homeostasis. These molecules are located primarily on the cell surface and in the pericellular matrix, where they interact with a multitude of macromolecules, including many growth factors. Manipulation of the enzymes involved in biosynthesis and modification of HSPG structures alters the properties of stem cells. Here, we focus on the involvement of heparanase (HPSE), the sole endo-glucuronidase capable of cleaving of HS, in differentiation of embryonic stem cells into the cells of the neural lineage. Embryonic stem (ES) cells overexpressing HPSE (Hpse-Tg) proliferated more rapidly than WT ES cells in culture and formed larger teratomas in vivo. In addition, differentiating Hpse-Tg ES cells also had a higher growth rate, and overexpression of HPSE in NSPCs enhanced Erk and Akt phosphorylation. Employing a two-step, monolayer differentiation, we observed an increase in HPSE as wild-type (WT) ES cells differentiated into neural stem and progenitor cells followed by down-regulation of HPSE as these NSPCs differentiated into mature cells of the neural lineage. Furthermore, NSPCs overexpressing HPSE gave rise to more oligodendrocytes than WT cultures, with a concomitant reduction in the number of neurons. Our present findings emphasize the importance of HS, in neural differentiation and suggest that by regulating the availability of growth factors and, or other macromolecules, HPSE promotes differentiation into oligodendrocytes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-311793 (URN)10.1016/j.matbio.2016.11.007 (DOI)000411539900007 ()27890389 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilSwedish Childhood Cancer Foundation
Available from: 2017-01-02 Created: 2017-01-02 Last updated: 2018-04-09Bibliographically approved
Dierker, T., Noborn, F., Hinas, A. & Kjellén, L. (2017). Identification of novel chondroitin sulfate sulfotransferases and proteoglycan core proteins in the nematode C. elegans. Paper presented at Spring Meeting of the British-Society-for-Matrix-Biology (BSMB), APR 03-04, 2017, Univ Oxford, Oxford, ENGLAND. International journal of experimental pathology (Print), 98(3), A4-A4
Open this publication in new window or tab >>Identification of novel chondroitin sulfate sulfotransferases and proteoglycan core proteins in the nematode C. elegans
2017 (English)In: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 98, no 3, p. A4-A4Article in journal, Meeting abstract (Other academic) Published
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-345644 (URN)000408620000007 ()
Conference
Spring Meeting of the British-Society-for-Matrix-Biology (BSMB), APR 03-04, 2017, Univ Oxford, Oxford, ENGLAND
Available from: 2018-03-12 Created: 2018-03-12 Last updated: 2018-03-12Bibliographically approved
Dierker, T. & Kjellén, L. (2017). Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans. BIO-PROTOCOL, 7(15), Article ID e2437.
Open this publication in new window or tab >>Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans
2017 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 7, no 15, article id e2437Article in journal (Refereed) Published
Abstract [en]

The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs are needed. Such methods have previously been optimized for other species such as mice and zebrafish. After modifying existing purification protocols, we could recently show that the nematodes also produce chondroitin sulfate, in addition to heparan sulfate, thus challenging the view that only non-sulfated chondroitin was synthesized by C. elegans. We here present our protocol adapted for C. elegans. Since the purification strategy involves separation of non-sulfated and sulfated GAGs, it may also be useful for other applications where this approach could be advantageous.

Keywords
Glycosaminoglycans, Caenorhabditis elegans, Proteoglycans, Ion exchange chromatography, Sulfation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-389483 (URN)10.21769/BioProtoc.2437 (DOI)000457836100019 ()
Funder
Swedish Research Council
Available from: 2019-07-16 Created: 2019-07-16 Last updated: 2019-07-16Bibliographically approved
Dierker, T., Bachvarova, V., Krause, Y., Li, J.-P., Kjellen, L., Seidler, D. G. & Vortkamp, A. (2016). Altered heparan sulfate structure in Glce(-/-) mice leads to increased Hedgehog signaling in endochondral bones. Matrix Biology, 49, 82-92
Open this publication in new window or tab >>Altered heparan sulfate structure in Glce(-/-) mice leads to increased Hedgehog signaling in endochondral bones
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2016 (English)In: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 49, p. 82-92Article in journal (Refereed) Published
Abstract [en]

One of the key regulators of endochondral ossification is Indian hedgehog (Ihh), which acts as a long-range morphogen in the developing skeletal elements. Previous studies have shown that the distribution and signaling activity of Ihh is regulated by the concentration of the extracellular glycosaminoglycan heparan sulfate (HS). An essential step during biosynthesis of HS is the epimerization of D-glucuronic to L-iduronic acid by the enzyme glucuronyl C5-epimerase (Hsepi or Glce). Here we have investigated chondrocyte differentiation in Glce deficient mice and found increased regions of proliferating chondrocytes accompanied by a delayed onset of hypertrophic differentiation. In addition, we observed increased expression levels of the Ihh target genes Patched1 (Ptch1) and Parathyroid hormone related peptide (Pthrp; Parathyroid hormone like hormone (Pthlh)) indicating elevated Ihh signaling. We further show that Ihh binds with reduced affinity to HS isolated from Glce(-/-) mice. Together our results strongly indicate that not only the level, but also the structure of HS is critical in regulating the distribution and signaling activity of Ihh in chondrocytes.

Keywords
Indian hedgehog, Ihh, Chondrocyte differentiation, Heparan sulfate, Epimerization, Glce, Endochondral ossification
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-283810 (URN)10.1016/j.matbio.2015.06.004 (DOI)000371944000005 ()26116392 (PubMedID)
Funder
Swedish Research Council, K2012-67X-21128-01-4
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-11-30Bibliographically approved
Menard, J. A., Christianson, H. C., Kucharzewska, P., Bourseau-Guilmain, E., Svensson, K. J., Lindqvist, E., . . . Belting, M. (2016). Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis. Cancer Research, 76(16), 4828-4840
Open this publication in new window or tab >>Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis
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2016 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, no 16, p. 4828-4840Article in journal (Refereed) Published
Abstract [en]

Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo. On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-304214 (URN)10.1158/0008-5472.CAN-15-2831 (DOI)000382297700024 ()27199348 (PubMedID)
Available from: 2016-11-21 Created: 2016-10-03 Last updated: 2017-11-29Bibliographically approved
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