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  • 1. Abramsson, Alexandra
    et al.
    Kurup, Sindhulakshmi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Yamada, Shuhei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindblom, Per
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bondjers, Göran
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gerhardt, Holger
    Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development2007In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 21, no 3, p. 316-331Article in journal (Refereed)
    Abstract [en]

    During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor β (PDGFRβ) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.

  • 2.
    Bengtsson, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Distinct effects on heparan sulfate structure by different active site mutations in NDST-12003In: Biochemistry, Vol. 42, no 7, p. 2110-2115Article in journal (Refereed)
  • 3.
    Busse, Marta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Feta, Almir
    Presto, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wilén, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Grønning, Mona
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 45, p. 32802-32810Article in journal (Refereed)
    Abstract [en]

    The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.

  • 4.
    Carlsson, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dagälv, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Presto, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate biosynthesis: Characterization of an NDST1 splice variantManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    N-Deacetylase/N-sulfotransferases (NDSTs) are Golgi-located enzymes involved in the biosynthesis of heparan sulfate. They are bifunctional enzymes responsible for N-deacetylation of N-acetylglucosamine residues followed by N-sulfation of the generated free amino groups. In this paper we have identified and characterized a splice variant of NDST1 mRNA. The alternatively spliced mRNA transcript was shown to be present in varying amounts in different adult and embryonic mouse tissues. The protein resulting from translation of the spliced transcript (NDST1S) lacks the C-terminal half of fullength NDST and appears to be devoid of enzyme activity. As shown in HEK 293 cells overexpressing NDST1, a high expression of the splice variant resulted in reduced levels of NDST1. Unexpectedly, the level of N-sulfation was largely unaltered in heparan sulfate produced in NDST1S overexpressing cells while 6-O-sulfation was elevated and 2-O-sulfation was reduced. NDST1S shares the ability of NDST1 to interact with EXT2, one of the components of the heparan sulfate copolymerase. We speculate that NDST1S may alter the composition of the tentaive enzyme complex, the GAGosome, resulting in changes in the structure of heparan sulfate synthesized.

  • 5.
    Carlsson, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparin biosynthesis2012In: Handbook of experimental pharmacology, ISSN 0171-2004, Vol. 207, p. 23-41Article in journal (Refereed)
    Abstract [en]

    Heparin and heparan sulfate share the same polysaccharide backbone structure but differ in sulfation degree and expression pattern. Whereas heparan sulfate is found in virtually all cells of the human body, heparin expression is restricted to mast cells, where it has a function in storage of granular components such as histamine and mast cell specific proteases. Although differing in charge and sulfation pattern, current knowledge indicates that the same pathway is used for synthesis of heparin and heparan sulfate, with a large number of different enzymes taking part in the process. At present, little is known about how the individual enzymes are coordinated and how biosynthesis is regulated. These questions are addressed in this chapter together with a review of the basic enzymatic steps involved in initiation, elongation, and modification of the polysaccharides.

  • 6.
    Carlsson, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ledin, Johan
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic developmentManuscript (Other academic)
  • 7.
    Carlsson, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Presto, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate biosynthesis: Characterization of an NDST1 splice variantManuscript (Other academic)
  • 8.
    Carlsson, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Presto, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 29, p. 20008-20014Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.

  • 9.
    Dagälv, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lack of both lethality and defective in vitro differentiation of embryonic stem cells N-deacetylase/N-sulfotransferase 1 and 2 causes early embryonicManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    NDSTs (N-deacetylase/N-sulfotransferases) are enzymes responsible for N-sulfation during heparan sulfate and heparin biosynthesis. While lack of NDST2 results in defective mast cells and NDST1 deficiency causes neonatal death and lung, skeletal and brain defects, lack of both isoforms is not compatible with embryonic development. We here show that NDST1/2-/- embryos die before E6.5 and that embryos dissected out at E5.5 lack parts of the embryo/extraembryonic tissue. Consistent with their in vivo behavior, in vitro cultured NDST1/2 deficient embryos displayed impaired ability of inner cell mass proliferation. In addition, markers for all the three germ layers had a disturbed expression pattern in isolated NDST1/2 deficient embryonic stem (ES) cells. Characterization of heparan sulfate (HS) structure in control ES cells and in ES cells lacking NDST1, NDST2 or both NDST1 and NDST2 revealed big differences. As expected, control cells synthesized HS with the highest degree of sulfation closely followed by HS from NDST2-/- cells, which in turn was more sulfated than HS produced by NDST1-/- cells. HS from NDST1/2-/- cells was almost devoid of sulfate groups. Notably, lack of one NDST isoform did not result in increased expression of any of the others. While all cell types except the NDST1/2-/- cells produced HS with a higher degree of sulfation when allowed to differentiate for 8 days, HS from control cells was still more heavily sulfated than that produced by NDST2-/- cells followed by the HS of NDST1-/- cells. The increase in sulfation was paralleled by increased expression of NDST transcripts and could also be recorded as increased N-sulfotransferase activity of cell lysates. While NDST1/2 deficient ES cells were unable to differentiate into beating cardiomyocytes all NDST1-/- and control embryoid bodies had started to beat after 4 days of culture. Surprisingly, NDST2 deficiency resulted in delayed cardiomyocyte differentiation.

  • 10.
    Dagälv, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 52, p. 44433-44440Article in journal (Refereed)
    Abstract [en]

    Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (approximate to 30%) than in control MCs where approximate to 95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.

  • 11.
    Dagälv, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cell surface mast cell proteoglycans identified as heparin-substituted syndecan-2Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Connective tissue type mast cells isolated from the peritoneal cavity of mice and then cultured in vitro have been used to answer the question if one cell at a given time point can synthesize heparan sulfate chains with different structure. Characterization of cell surface proteoglycans made by the cells demonstrated that they were identical to syndecan-2, substituted with heparin chains. Ion exchange chromatography showed that the syndecan heparin chains behaved identically as heparin chains recovered from serglycin, inside the cells. This was also the case when mast cells from NDST2 deficient mice were studied. This time, syndecan-2 as well as serglycin derived polysaccharide chains had a lower but identical charge density. We conclude that mast cells only synthesize one kind of heparan sulfate/heparin chain at a time and that polysaccharide chains of identical structure will be found at the cell surface and inside the cell.

  • 12.
    Dierker, Tabea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Duisburg Essen, Ctr Med Biotechnol, Essen, Germany..
    Bachvarova, Velina
    Univ Duisburg Essen, Ctr Med Biotechnol, Essen, Germany..
    Krause, Yvonne
    Univ Duisburg Essen, Ctr Med Biotechnol, Essen, Germany..
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellen, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Seidler, Daniela G.
    Univ Munster, Inst Physiol Chem & Pathobiochem, D-48149 Munster, Germany..
    Vortkamp, Andrea
    Univ Duisburg Essen, Ctr Med Biotechnol, Essen, Germany..
    Altered heparan sulfate structure in Glce(-/-) mice leads to increased Hedgehog signaling in endochondral bones2016In: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 49, p. 82-92Article in journal (Refereed)
    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.

  • 13.
    Dierker, Tabea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Noborn, F.
    Gothenburg Univ, Dept Clin Chem & Transfus Med, Inst Biomed, Gothenburg, Sweden..
    Hinas, Andrea
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Identification of novel chondroitin sulfate sulfotransferases and proteoglycan core proteins in the nematode C. elegans2017In: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 98, no 3, p. A4-A4Article in journal (Other academic)
  • 14.
    Filipek-Gorniok, Beata
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Carlsson, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.

  • 15.
    Filipek-Gorniok, Beata
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Oliveira, Marta Bastos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hellgren, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development2013In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, no 8, p. 964-975Article in journal (Refereed)
    Abstract [en]

    Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.

  • 16. Forsberg, Erik
    et al.
    Pejler, Gunnar
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lunderius, Carolina
    Tomasini-Johansson, Bianca
    Kusche-Gullberg, Marion
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ledin, Johan
    Hellman, Lars
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Abnormal mast cells in mice deficient in a heparin-sythesizing enzyme1999In: Nature, Vol. 400, p. 773-776Article in journal (Refereed)
  • 17.
    Forsberg, Maud
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kundu, Soumi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dagälv, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Forsberg-Nilsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 14, p. 10853-10862Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.

  • 18. Grobe, K
    et al.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forsberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Esko, Jeffrey D.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes.2002In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1573, no 3, p. 209-215Article in journal (Refereed)
    Abstract [en]

    Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.

  • 19. Grujic, Mirjana
    et al.
    Calounova, Gabriela
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Feyerabend, Thorsten
    Rodewald, Hans-Reimer
    Tchougounova, Elena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pejler, Gunnar
    Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency2013In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 191, no 7, p. 3931-3938Article in journal (Refereed)
    Abstract [en]

    Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

  • 20. Holley, Rebecca J.
    et al.
    Deligny, Audrey
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wei, Wei
    Watson, H. Angharad
    Ninonuevo, Milady R.
    Dagälv, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Leary, Julie A.
    Bigger, Brian W.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Merry, Catherine L. R.
    Mucopolysaccharidosis Type I, Unique Structure of Accumulated Heparan Sulfate and Increased N-Sulfotransferase Activity in Mice Lacking alpha-L-iduronidase2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 43, p. 37515-37524Article in journal (Refereed)
    Abstract [en]

    Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from alpha-L-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.

  • 21.
    Holmborn, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dagälv, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Divergent effects of heparan sulfate/heparin biosynthesis enzymes N-deacetylase/N-sulfotransferase 1 and 2 on Mast cell developmentManuscript (preprint) (Other academic)
    Abstract [en]

    Deficiency of the heparan sulfate (HS) biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed HS biosynthesis in all organs, while lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, cultured terminally differentiated MCs from adult mice, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. While MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, NDST1 deficiency results in the formation of MCs which compared to wild type MCs contain increased levels of proteases specific for the connective tissue type MCs as well as increased amounts and degree of sulfation of heparin. A model of the GAGosome, a tentative Golgi enzyme complex, is used to explain the results.

  • 22.
    Holmborn, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Impaired mast cell development in N-deacetylase/N-sulfotransferase deficient embryonic stem cellsManuscript (Other academic)
  • 23.
    Holmborn, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ledin, Johan
    Smeds, Emanuel
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is 6-O-sulfated but contains no N-sulfate groups2004In: The Journal of Biological Chemistry, Vol. 279, no 41, p. 42355-58Article in journal (Refereed)
  • 24.
    Holmborn, Katarina
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ledin, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Smeds, Emanuel
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is 6-O-sulfated but contains no N-sulfate groups.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 41, p. 42355-8Article in journal (Refereed)
  • 25.
    Holmborn, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ringvall, Maria
    Forsberg, Erik
    Kjellén, Lena
    Mice lacking N-deacetylase/N-sulfotransferase 1 and 2 are defective in early postimplantation developmentManuscript (Other academic)
  • 26.
    Hu Frisk, Jun Mei
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kaler, Stephen G
    Eunice Kennedy Shriver Natl Inst Child Hlth & Hum, Sect Translat Neurosci, Mol Med Branch, NIH, Bethesda, MD 20892 USA.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, S-75007 Uppsala, Sweden.
    Öhrvik, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Copper Regulates Maturation and Expression of an MITF: Tryptase Axis in Mast Cells2017In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 199, no 12, p. 4132-4141Article in journal (Refereed)
    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.

  • 27. Isidor, Bertrand
    et al.
    Pichon, Olivier
    Redon, Richard
    Day-Salvatore, Debra
    Hamel, Antoine
    Siwicka, Karolina A.
    Bitner-Glindzicz, Maria
    Heymann, Dominique
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kraus, Cornelia
    Leroy, Jules G.
    Mortier, Geert R.
    Rauch, Anita
    Verloes, Alain
    David, Albert
    Le Caignec, Cedric
    Mesomelia-Synostoses Syndrome Results from Deletion of SULF1 and SLCO5A1 Genes at 8q132010In: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 87, no 1, p. 95-100Article in journal (Refereed)
    Abstract [en]

    Mesomelia-synostoses syndrome (MSS) or mesomelic dysplasia with acral synostoses Verloes-David-Pfeiffer type is a rare autosomal-dominant disorder characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations. So far, five patients in four unrelated families have been reported worldwide with MMS. By using whole-genome oligonucleotide array CGH, we have identified an interstitial deletion at 8q13 in all patients. The deletions vary from 582 Kb to 738 Kb in size, but invariably encompass only two genes: SULF1, encoding the heparan sulfate 6-O-endosulfatase 1, and SLCO5A1, encoding the solute carrier organic anion transporter family member 5A1. SULF1 acts as a regulator of numerous growth factors in skeletal embryonic development whereas the function of SLCO5A1 is yet unknown. Breakpoint sequence analyses performed in two families showed nonrecurrent deletions. Real-time quantitative RT-PCR analysis showed the highest levels of SULF1 transcripts in human osteoblasts and cartilage whereas SLCO5A1 was highly expressed in human fetal and adult brain and heart. Our results strongly suggest that haploinsufficiency of SULF1 contributes to this mesomelic chondrodysplasia, highlighting the critical role of endosulfatase in human skeletal development. Codeletion of SULF1 and SLCO5A1-which does not result from a low-copy repeats (LCRs)-mediated recombination event in at least two families-was found in all patients, so we suggest that haploinsufficiency of SULF1 combined with haploinsufficiency of SLCO5A1 (or the altered expression of a neighboring gene through position effect) could be necessary in the pathogenesis of MSS.

  • 28. Jakobsson, L
    et al.
    Lundin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, L
    Heparan sulfate expression is critical in vascular developmentManuscript (Other academic)
  • 29.
    Jakobsson, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lundin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis2006In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 10, no 5, p. 625-634Article in journal (Refereed)
    Abstract [en]

    Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

  • 30. Karlsen, Tine V
    et al.
    Iversen, Vegard V
    Forsberg, Erik
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reed, Rolf K
    Gjerde, Eli-Anne B
    Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme.2004In: Am J Physiol Heart Circ Physiol, ISSN 0363-6135, Vol. 286, no 3, p. H884-8Article in journal (Refereed)
  • 31. Kasza, Zsolt
    et al.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tamm, Christoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Anna S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heindryckx, Femke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Erik
    Le Jan, Sebastien
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Kjellen, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 36, p. 25956-25963Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

  • 32. Kawamura, Harukiyo
    et al.
    Li, Xiujuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Goishi, Katsutoshi
    van Meeteren, Laurens A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jakobsson, Lars
    Cébe-Suarez, Stéphanie
    Shimizu, Akio
    Edholm, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ballmer-Hofer, Kurt
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Klagsbrun, Michael
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization2008In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 112, no 9, p. 3638-49Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor (VEGF)-A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165-containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.

  • 33.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Glycobiology: Enzyme deficiencies deciphered2012In: Nature chemical biology, ISSN 1552-4469, Vol. 8, no 2, p. 137-138Article in journal (Refereed)
    Abstract [en]

    Mucopolysaccharidoses are inherited disorders in which inactivation of lysosomal enzymes results in accumulation of glycosaminoglycans within cells, causing tissue and organ dysfunction. A method to determine the unique end structures of the accumulated glycosaminoglycans offers a new way for diagnosis.

  • 34.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heparan Sulfate Biosynthesis: Regulation and Variability2012In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 60, no 12, p. 898-907Article in journal (Refereed)
    Abstract [en]

    Nearly all vertebrate cells have been shown to express heparan sulfate proteoglycans (HSPGs) at the cell surface. The HSPGs bind to many secreted signaling proteins, including numerous growth factors, cytokines, and morphogens, to affect their tissue distribution and signaling. The heparan sulfate (HS) chains may have variable length and may differ with regard to both degree and pattern of sulfation. As the sulfation pattern of HS chains in most cases will determine if an interaction with a potential ligand will take place, as well as the affinity of the interaction, a key to understanding the function of HSPGs is to clarify how HS biosynthesis is regulated in different biological contexts. This review provides an introduction to the current understanding of HS biosynthesis and its regulation, and identifies research areas where more knowledge is needed to better understand how the HS biosynthetic machinery works. (J Histochem Cytochem 60:898-907, 2012)

  • 35.
    Kurup, S
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Abramsson, A
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Betsholtz, C
    Gerhardt, H
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulphate requirement in platelet-derived growth factor B-mediated pericyte recruitment2006In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 34, no Pt 3, p. 454-455Article in journal (Refereed)
    Abstract [en]

    HS (heparan sulphate) plays a key role in angiogenesis, by interacting with growth factors required in the process. it has been proposed that HS controls the diffusion, and thus the availability, of platelet-derived growth factor B that is needed for pericyte recruitment around newly formed capillaries. The present paper summarizes our studies on the importance of HS structure in this regulatory process.

  • 36.
    Le Jan, Sébastien
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hayashi, Makoto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kasza, Zsolt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bishop, Joseph R
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Lars
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esko, Jeffrey D
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis2012In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 32, no 5, p. 1255-1263Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis.

    METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, TGFβ, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and platelet-derived growth factor B signal transduction.

    CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

  • 37. Ledin, Johan
    et al.
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wilén, Maria
    Thuveson, Maria
    Kusche-Gullberg, Marion
    Forsberg, Erik
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    In vivo regulation of liver heparan sulfate biosynthesis in NDST1 and NDST2 deficient miceArticle in journal (Refereed)
  • 38.
    Ledin, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ringvall, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thuveson, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wilén, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forsberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Enzymatically active N-deacetylase/N-sulfotransferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 47, p. 35727-35734Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST ( glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HSN-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1(-/-) mice was drastically lowered, liver HS from wild-type mice, from NDST1(-/-), NDST2(-/-), and NDST1(-/-), NDST2(-/-) mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1(-/-) NDST2(-/-) embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1(-/-), and NDST1(-/-) embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximate to 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1(-/-) embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

  • 39.
    Ledin, Johan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Staatz, William
    Li, Jin-Ping
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Götte, Martin
    Selleck, Scott
    Kjellén, Lena
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate structure in mice with genetically modified heparan sulfate production.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 41, p. 42732-41Article in journal (Refereed)
  • 40.
    Li, Jin-Ping
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hagner-McWhirther, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Palgi, Jaan
    Jalkanen, Markku
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Biosynthesis of heparin / heparan sulfate: cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 44, p. 28158-28163Article in journal (Refereed)
    Abstract [en]

    Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-p. (1994) J. Biol. Chem. 269, 26953-26958). The present report describes the molecular cloning and functional expression of the lung enzyme. The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da. N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence. The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells. Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of 3H2O from a polysaccharide substrate containing C5-3H-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO3)n substrate resulted in conversion of approximately 20% of the GlcUA units into IdceA residues. Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant approximately 9-kilobase (kb) mRNA and a minor approximately 5-kb species. Mouse mastocytoma cells showed only the approximately 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.

  • 41.
    Lindahl, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pathophysiology of heparan sulphate: many diseases, few drugs2013In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 273, no 6, p. 555-571Article, review/survey (Refereed)
    Abstract [en]

    Heparan sulphate (HS) polysaccharides are covalently attached to the core proteins of various proteoglycans at cell surfaces and in the extracellular matrix. They are composed of alternating units of hexuronic acid and glucosamine, with sulphate substituents in complex and variable yet cell-specific patterns. Whereas HS is produced by virtually all cells in the body, heparin, a highly sulphated HS variant, is confined to connective-tissue-type mast cells. The polysaccharides interact with a multitude of proteins, mainly through ionic binding, and thereby control key processes in development and homoeostasis. Similar interactions also implicate HS in various pathophysiological settings, including cancer, amyloid diseases, infectious diseases, inflammatory conditions and some developmental disorders. Prospects for the development of HS-based drugs, which are still largely unrealized, are discussed.

  • 42.
    Lindahl, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lidholt, K
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    More to "heparin" than anticoagulation1994In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 75, no 1, p. 1-32Article in journal (Refereed)
  • 43.
    Menard, Julien A.
    et al.
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Christianson, Helena C.
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Kucharzewska, Paulina
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Bourseau-Guilmain, Erika
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Svensson, Katrin J.
    Harvard Med Sch, Dana Farber Canc Inst, Boston, MA USA.;Harvard Med Sch, Dept Cell Biol, Boston, MA USA..
    Lindqvist, Eva
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Chandran, Vineesh Indira
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Welinder, Charlotte
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden.;Lund Univ, Ctr Excellence Biol & Med Mass Spectrometry CEBMM, Biomed Ctr D13, Lund, Sweden..
    Bengzon, Johan
    Lund Univ, Lund Stem Cell Ctr, Lund, Sweden.;Lund Univ, Sect Neurosurg, Dept Clin Sci, Lund, Sweden..
    Johansson, Maria C.
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden..
    Belting, Mattias
    Lund Univ, Sect Oncol & Pathol, Dept Clin Sci, Barngatan 2B, SE-22185 Lund, Sweden.;Skane Univ Hosp, Lund, Sweden..
    Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis2016In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, no 16, p. 4828-4840Article in journal (Refereed)
    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.

  • 44. Nguyen, Thao K. N.
    et al.
    Tran, Vy M.
    Sorna, Venkataswamy
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kojima, Akinori
    Koketsu, Mamoru
    Loganathan, Duraikkannu
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dorsky, Richard I.
    Chien, Chi-Bin
    Kuberan, Balagurunathan
    Dimerized Glycosaminoglycan Chains Increase FGF Signaling during Zebrafish Development2013In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 8, no 5, p. 939-948Article in journal (Refereed)
    Abstract [en]

    Proteoglycans (PGs) modulate numerous signaling pathways during development through binding of their glycosaminoglycan (GAG) side chains to various signaling molecules, including fibroblast growth factors (FGFs). A majority of PGs possess two or more GAG side chains, suggesting that GAG multivalency is imperative for biological functions in vivo. However, only a few studies have examined the biological significance of GAG multivalency. In this report, we utilized a library of bis- and tris-xylosides that produce two and three GAG chains on the same scaffold, respectively, thus mimicking PGs, to examine the importance of GAG valency and chain type in regulating FGF/FGFR interactions in vivo in zebrafish. A number of bis- and tris-xylosides, but not mono-xylosides, caused an elongation phenotype upon their injection into embryos. In situ hybridization showed that elongated embryos have elevated expression of the FGF target gene mkp3 but unchanged expression of reporters for other pathways, indicating that FGF/FGFR signaling was specifically hyperactivated. In support of this observation, elongation can be reversed by the tyrosine kinase inhibitor SU5402, mRNA for the FGFR antagonist sprouty4, or FGF8 morpholino. Endogenous GAGs seem to be unaffected after xyloside treatment, suggesting that this is a gain-of-function phenotype. Furthermore, expression of a multivalent but not a monovalent GAG containing syndecan-1 proteoglycan recapitulates the elongation phenotype observed with the bivalent xylosides. On the basis of these in vivo findings, we propose a new model for GAG/FGF/FGFR interactions in which dimerized GAG chains can activate FGF-mediated signal transduction pathways.

  • 45.
    Noborn, Fredrik
    et al.
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Toledo, Alejandro Gomez
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Nasir, Waqas
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Nilsson, Jonas
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Dierker, Tabea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala, Sweden..
    Larson, Goran
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Expanding the chondroitin glycoproteome of Caenorhabditis elegans2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 1, p. 379-389Article in journal (Refereed)
    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.

  • 46. Noborn, Fredrik
    et al.
    Toledo, Alejandro Gomez
    Sihlbom, Carina
    Lengqvist, Johan
    Fries, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellen, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nilsson, Jonas
    Larson, Goran
    Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans2015In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 1, p. 41-49Article in journal (Refereed)
    Abstract [en]

    Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CSlyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

  • 47. Pegeot, Mathieu
    et al.
    Sadir, Rabia
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Simorre, Jean-Pierre
    Gans, Pierre
    Lortat-Jacob, Hugues
    Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture C-13-glucose metabolic labeling2015In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 25, no 2, p. 151-156Article in journal (Refereed)
    Abstract [en]

    Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a H-1-C-13 2D NMR-based approach, directly performed on HS isolated from C-13-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development.

  • 48.
    Presto, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Carlsson, Pernilla
    Spillmann, Dorothe
    Kjellén, Lena
    Heparan sulfate biosynthesis: PAPS regulates NDST-mediated formation of N-sulfated domainsManuscript (Other academic)
  • 49.
    Presto, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thuveson, Maria
    Busse, Marta
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfationManuscript (Other academic)
  • 50.
    Presto, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thuveson, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Carlsson, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Busse, Marta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wilén, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 12, p. 4751-4756Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

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