uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Glycosaminoglycan Biosynthesis in Zebrafish
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders.

This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1143
Keyword [en]
Heparan sulfate, chondroitin/dermatan sulfate, biosynthesis, development, N-deacetylase N-sulfotransferase, glycosyltransferases, morpholino, CRISPR-Cas9
National Category
Biochemistry and Molecular Biology Developmental Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-264269ISBN: 978-91-554-9368-4 (print)OAI: oai:DiVA.org:uu-264269DiVA: diva2:859704
Public defence
2015-11-27, C8:305, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2015-11-05 Created: 2015-10-08 Last updated: 2015-11-10
List of papers
1. The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation
Open this publication in new window or tab >>The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation
Show others...
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3Article in journal (Refereed) Published
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.

National Category
Evolutionary Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-251802 (URN)10.1371/journal.pone.0119040 (DOI)000351277500060 ()25767878 (PubMedID)
Available from: 2015-04-28 Created: 2015-04-24 Last updated: 2017-12-04Bibliographically approved
2. Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
Open this publication in new window or tab >>Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
Show others...
2013 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, no 8, 964-975 p.Article in journal (Refereed) Published
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.

Keyword
chondroitin sulfate, polymerase, CSGALNACT, CHSY, CHPF, zebrafish
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204834 (URN)10.1002/dvdy.23981 (DOI)000321843400008 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2017-12-06Bibliographically approved
3. On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
Open this publication in new window or tab >>On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
Show others...
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 40, 33905-33916 p.Article in journal (Refereed) Published
Abstract [en]

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Keyword
Protein Linkage Region, Molecular-Cloning, Hspg Synthesis, Cell Polarity, Growth-Plate, Expression, Ext2, Proteoglycans, Glypican, Xylose
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-185209 (URN)10.1074/jbc.M112.401646 (DOI)000309602100071 ()
Note

De två första författarna delar förstaförfattarskapet.

De två sista författarna delar sistaförfattarskapet.

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2017-12-07Bibliographically approved
4. Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis
Open this publication in new window or tab >>Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-258588 (URN)
Available from: 2015-07-27 Created: 2015-07-16 Last updated: 2015-11-10

Open Access in DiVA

fulltext(1167 kB)327 downloads
File information
File name FULLTEXT01.pdfFile size 1167 kBChecksum SHA-512
7fb2cac2b34e7050001a801a6449e0d13d8c0c4b87b212cf60ea9cce90078015852f49bfd62bd39525a960d8245a6f510777c59b28f04c7b5b1c938273a805d2
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Filipek-Górniok, Beata

Search in DiVA

By author/editor
Filipek-Górniok, Beata
By organisation
Department of Medical Biochemistry and Microbiology
Biochemistry and Molecular BiologyDevelopmental BiologyMedical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
Total: 327 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1127 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf