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  • 1. Abramsson, Alexandra
    et al.
    Kurup, Sindhulakshmi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Yamada, Shuhei
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lindblom, Per
    Schallmeiner, Edith
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Ledin, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Bondjers, Göran
    Li, Jin-Ping
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lindahl, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Gerhardt, Holger
    Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development2007Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 21, nr 3, s. 316-331Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Distinct effects on heparan sulfate structure by different active site mutations in NDST-12003Ingår i: Biochemistry, Vol. 42, nr 7, s. 2110-2115Artikel i tidskrift (Refereegranskat)
  • 3.
    Busse, Marta
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Feta, Almir
    Presto, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Wilén, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Grønning, Mona
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kusche-Gullberg, Marion
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation2007Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 45, s. 32802-32810Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Dagälv, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Presto, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate biosynthesis: Characterization of an NDST1 splice variantManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparin biosynthesis2012Ingår i: Handbook of experimental pharmacology, ISSN 0171-2004, Vol. 207, s. 23-41Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ledin, Johan
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic developmentManuskript (Övrigt vetenskapligt)
  • 7.
    Carlsson, Pernilla
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Presto, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate biosynthesis: Characterization of an NDST1 splice variantManuskript (Övrigt vetenskapligt)
  • 8.
    Carlsson, Pernilla
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Presto, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lindahl, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains2008Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, nr 29, s. 20008-20014Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lack of both lethality and defective in vitro differentiation of embryonic stem cells N-deacetylase/N-sulfotransferase 1 and 2 causes early embryonicManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Åbrink, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 52, s. 44433-44440Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Åbrink, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Cell surface mast cell proteoglycans identified as heparin-substituted syndecan-2Manuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. 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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellen, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    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 bones2016Ingår i: Matrix Biology, ISSN 0945-053X, E-ISSN 1569-1802, Vol. 49, s. 82-92Artikel i tidskrift (Refereegranskat)
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans2017Ingår i: BIO-PROTOCOL, ISSN 2331-8325, Vol. 7, nr 15, artikel-id e2437Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 14.
    Dierker, Tabea
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Noborn, F.
    Gothenburg Univ, Dept Clin Chem & Transfus Med, Inst Biomed, Gothenburg, Sweden..
    Hinas, Andrea
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Identification of novel chondroitin sulfate sulfotransferases and proteoglycan core proteins in the nematode C. elegans2017Ingår i: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 98, nr 3, s. A4-A4Artikel i tidskrift (Övrigt vetenskapligt)
  • 15.
    Filipek-Gorniok, Beata
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Carlsson, Pernilla
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Haitina, Tatjana
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 3Artikel i tidskrift (Refereegranskat)
    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.

  • 16.
    Filipek-Gorniok, Beata
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi.
    Haitina, Tatjana
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Oliveira, Marta Bastos
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Hellgren, Charlotte
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development2013Ingår i: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, nr 8, s. 964-975Artikel i tidskrift (Refereegranskat)
    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.

  • 17. Forsberg, Erik
    et al.
    Pejler, Gunnar
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lunderius, Carolina
    Tomasini-Johansson, Bianca
    Kusche-Gullberg, Marion
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ledin, Johan
    Hellman, Lars
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Abnormal mast cells in mice deficient in a heparin-sythesizing enzyme1999Ingår i: Nature, Vol. 400, s. 773-776Artikel i tidskrift (Refereegranskat)
  • 18.
    Forsberg, Maud
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kundu, Soumi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Cancer och vaskulärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Dagälv, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Forsberg-Nilsson, Karin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Cancer och vaskulärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 14, s. 10853-10862Artikel i tidskrift (Refereegranskat)
    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.

  • 19.
    Frisk, Jun Mei Hu
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Melo, Fabio R.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Öhrvik, Helena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Pejler, Gunnar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.
    Mitogen-Activated Protein Kinase Signaling Regulates Proteoglycan Composition of Mast Cell Secretory Granules2018Ingår i: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 9, artikel-id 1670Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 20. Grobe, K
    et al.
    Ledin, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Forsberg, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Esko, Jeffrey D.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes.2002Ingår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1573, nr 3, s. 209-215Artikel i tidskrift (Refereegranskat)
    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.

  • 21. Grujic, Mirjana
    et al.
    Calounova, Gabriela
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Feyerabend, Thorsten
    Rodewald, Hans-Reimer
    Tchougounova, Elena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Pejler, Gunnar
    Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency2013Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 191, nr 7, s. 3931-3938Artikel i tidskrift (Refereegranskat)
    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.

  • 22. Holley, Rebecca J.
    et al.
    Deligny, Audrey
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Wei, Wei
    Watson, H. Angharad
    Ninonuevo, Milady R.
    Dagälv, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Leary, Julie A.
    Bigger, Brian W.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Merry, Catherine L. R.
    Mucopolysaccharidosis Type I, Unique Structure of Accumulated Heparan Sulfate and Increased N-Sulfotransferase Activity in Mice Lacking alpha-L-iduronidase2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 43, s. 37515-37524Artikel i tidskrift (Refereegranskat)
    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.

  • 23.
    Holmborn, Katarina
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Dagälv, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Åbrink, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Divergent effects of heparan sulfate/heparin biosynthesis enzymes N-deacetylase/N-sulfotransferase 1 and 2 on Mast cell developmentManuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 24.
    Holmborn, Katarina
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Åbrink, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Impaired mast cell development in N-deacetylase/N-sulfotransferase deficient embryonic stem cellsManuskript (Övrigt vetenskapligt)
  • 25.
    Holmborn, Katarina
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ledin, Johan
    Smeds, Emanuel
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kusche-Gullberg, Marion
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is 6-O-sulfated but contains no N-sulfate groups2004Ingår i: The Journal of Biological Chemistry, Vol. 279, nr 41, s. 42355-58Artikel i tidskrift (Refereegranskat)
  • 26.
    Holmborn, Katarina
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ledin, Johan
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Smeds, Emanuel
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kusche-Gullberg, Marion
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is 6-O-sulfated but contains no N-sulfate groups.2004Ingår i: J Biol Chem, ISSN 0021-9258, Vol. 279, nr 41, s. 42355-8Artikel i tidskrift (Refereegranskat)
  • 27.
    Holmborn, Katarina
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Forsberg, Erik
    Kjellén, Lena
    Mice lacking N-deacetylase/N-sulfotransferase 1 and 2 are defective in early postimplantation developmentManuskript (Övrigt vetenskapligt)
  • 28.
    Hu Frisk, Jun Mei
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, S-75007 Uppsala, Sweden.
    Öhrvik, Helena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Copper Regulates Maturation and Expression of an MITF: Tryptase Axis in Mast Cells2017Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 199, nr 12, s. 4132-4141Artikel i tidskrift (Refereegranskat)
    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.

  • 29. Isidor, Bertrand
    et al.
    Pichon, Olivier
    Redon, Richard
    Day-Salvatore, Debra
    Hamel, Antoine
    Siwicka, Karolina A.
    Bitner-Glindzicz, Maria
    Heymann, Dominique
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    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 8q132010Ingår i: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 87, nr 1, s. 95-100Artikel i tidskrift (Refereegranskat)
    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.

  • 30. Jakobsson, L
    et al.
    Lundin, Lars
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Claesson-Welsh, L
    Heparan sulfate expression is critical in vascular developmentManuskript (Övrigt vetenskapligt)
  • 31.
    Jakobsson, Lars
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lundin, Lars
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Claesson-Welsh, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis2006Ingår i: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 10, nr 5, s. 625-634Artikel i tidskrift (Refereegranskat)
    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.

  • 32. Karlsen, Tine V
    et al.
    Iversen, Vegard V
    Forsberg, Erik
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Reed, Rolf K
    Gjerde, Eli-Anne B
    Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme.2004Ingår i: Am J Physiol Heart Circ Physiol, ISSN 0363-6135, Vol. 286, nr 3, s. H884-8Artikel i tidskrift (Refereegranskat)
  • 33. Kasza, Zsolt
    et al.
    Fredlund Fuchs, Peder
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Tamm, Christoffer
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Anna S.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    O'Callaghan, Paul
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heindryckx, Femke
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Larsson, Erik
    Le Jan, Sebastien
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Gerwins, Pär
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för radiologi.
    Kjellen, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 36, s. 25956-25963Artikel i tidskrift (Refereegranskat)
    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.

  • 34. Kawamura, Harukiyo
    et al.
    Li, Xiujuan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Goishi, Katsutoshi
    van Meeteren, Laurens A
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Jakobsson, Lars
    Cébe-Suarez, Stéphanie
    Shimizu, Akio
    Edholm, Dan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Ballmer-Hofer, Kurt
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Klagsbrun, Michael
    Claesson-Welsh, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization2008Ingår i: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 112, nr 9, s. 3638-49Artikel i tidskrift (Refereegranskat)
    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.

  • 35.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Glycobiology: Enzyme deficiencies deciphered2012Ingår i: Nature chemical biology, ISSN 1552-4469, Vol. 8, nr 2, s. 137-138Artikel i tidskrift (Refereegranskat)
    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.

  • 36.
    Kjellén, Lena
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Lindahl, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Specificity of glycosaminoglycan-protein interactions2018Ingår i: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 50, s. 101-108Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 37.
    Kreuger, Johan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Heparan Sulfate Biosynthesis: Regulation and Variability2012Ingår i: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 60, nr 12, s. 898-907Artikel i tidskrift (Refereegranskat)
    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)

  • 38.
    Kurup, S
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Abramsson, A
    Li, Jin-Ping
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lindahl, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Betsholtz, C
    Gerhardt, H
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulphate requirement in platelet-derived growth factor B-mediated pericyte recruitment2006Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 34, nr Pt 3, s. 454-455Artikel i tidskrift (Refereegranskat)
    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.

  • 39.
    Le Jan, Sébastien
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Hayashi, Makoto
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Cancer och vaskulärbiologi.
    Kasza, Zsolt
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bishop, Joseph R
    Weibrecht, Irene
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Heldin, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Lars
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Esko, Jeffrey D
    Claesson-Welsh, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Cancer och vaskulärbiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis2012Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 32, nr 5, s. 1255-1263Artikel i tidskrift (Refereegranskat)
    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.

  • 40. Ledin, Johan
    et al.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Wilén, Maria
    Thuveson, Maria
    Kusche-Gullberg, Marion
    Forsberg, Erik
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    In vivo regulation of liver heparan sulfate biosynthesis in NDST1 and NDST2 deficient miceArtikel i tidskrift (Refereegranskat)
  • 41.
    Ledin, Johan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Thuveson, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Wilén, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kusche-Gullberg, Marion
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Forsberg, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Enzymatically active N-deacetylase/N-sulfotransferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation2006Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 47, s. 35727-35734Artikel i tidskrift (Refereegranskat)
    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.

  • 42.
    Ledin, Johan
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Staatz, William
    Li, Jin-Ping
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Götte, Martin
    Selleck, Scott
    Kjellén, Lena
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Spillmann, Dorothe
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Heparan sulfate structure in mice with genetically modified heparan sulfate production.2004Ingår i: J Biol Chem, ISSN 0021-9258, Vol. 279, nr 41, s. 42732-41Artikel i tidskrift (Refereegranskat)
  • 43.
    Li, Jin-Ping
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hagner-McWhirther, Åsa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Palgi, Jaan
    Jalkanen, Markku
    Lindahl, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Biosynthesis of heparin / heparan sulfate: cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung1997Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, nr 44, s. 28158-28163Artikel i tidskrift (Refereegranskat)
    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.

  • 44.
    Lindahl, Ulf
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Pathophysiology of heparan sulphate: many diseases, few drugs2013Ingår i: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 273, nr 6, s. 555-571Artikel, forskningsöversikt (Refereegranskat)
    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.

  • 45.
    Lindahl, Ulf
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lidholt, K
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    More to "heparin" than anticoagulation1994Ingår i: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 75, nr 1, s. 1-32Artikel i tidskrift (Refereegranskat)
  • 46.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, 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 Endocytosis2016Ingår i: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, nr 16, s. 4828-4840Artikel i tidskrift (Refereegranskat)
    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.

  • 47.
    Nakato, Eriko
    et al.
    Univ Minnesota, Dept Genet Cell Biol & Dev, 6-160 Jackson Hall,321 Church St SE, Minneapolis, MN 55455 USA.
    Liu, Xin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Yamamoto, Maki
    Ritsumeikan Univ, Fac Pharmaceut Sci, 1-1-1 Nojihigashi, Kusatsu, Shiga 5258577, Japan.
    Kinoshita-Toyoda, Akiko
    Ritsumeikan Univ, Fac Pharmaceut Sci, 1-1-1 Nojihigashi, Kusatsu, Shiga 5258577, Japan.
    Toyoda, Hidenao
    Ritsumeikan Univ, Fac Pharmaceut Sci, 1-1-1 Nojihigashi, Kusatsu, Shiga 5258577, Japan.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Li, Jin-Ping
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Nakato, Hiroshi
    Univ Minnesota, Dept Genet Cell Biol & Dev, 6-160 Jackson Hall,321 Church St SE, Minneapolis, MN 55455 USA.
    Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes2019Ingår i: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 29, nr 6, s. 479-489Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 48. Nguyen, Thao K. N.
    et al.
    Tran, Vy M.
    Sorna, Venkataswamy
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kojima, Akinori
    Koketsu, Mamoru
    Loganathan, Duraikkannu
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Dorsky, Richard I.
    Chien, Chi-Bin
    Kuberan, Balagurunathan
    Dimerized Glycosaminoglycan Chains Increase FGF Signaling during Zebrafish Development2013Ingår i: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 8, nr 5, s. 939-948Artikel i tidskrift (Refereegranskat)
    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.

  • 49.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala, Sweden..
    Larson, Goran
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, SE-41345 Gothenburg, Sweden..
    Expanding the chondroitin glycoproteome of Caenorhabditis elegans2018Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, nr 1, s. 379-389Artikel i tidskrift (Refereegranskat)
    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.

  • 50. Noborn, Fredrik
    et al.
    Toledo, Alejandro Gomez
    Sihlbom, Carina
    Lengqvist, Johan
    Fries, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellen, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Nilsson, Jonas
    Larson, Goran
    Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans2015Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, nr 1, s. 41-49Artikel i tidskrift (Refereegranskat)
    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.

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