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Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
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2015 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 10, no 3, e0121957Article in journal (Refereed) Published
Abstract [en]

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

Place, publisher, year, edition, pages
2015. Vol. 10, no 3, e0121957
National Category
Evolutionary Biology
URN: urn:nbn:se:uu:diva-252231DOI: 10.1371/journal.pone.0121957ISI: 000352083900161PubMedID: 25793894OAI: diva2:809943
Available from: 2015-05-05 Created: 2015-05-04 Last updated: 2015-10-01Bibliographically approved
In thesis
1. Glycosaminoglycan Biosynthesis and Function in Zebrafish Development: Sugars Shaping Skeletons
Open this publication in new window or tab >>Glycosaminoglycan Biosynthesis and Function in Zebrafish Development: Sugars Shaping Skeletons
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) proteoglycans are glycosylated proteins with important roles in animal development and homeostasis. HS and CS/DS are long, linear glycosaminoglycan (GAG) polysaccharides and attached to a core protein they form proteoglycans. GAGs on proteoglycans are often modified by sulfate groups and mainly found in the extracellular matrix or associated to the cell membrane. They interact with different proteins, for example signaling molecules, and influence developmental processes. Cells in cartilage produce a functionally specialized dense extracellular matrix, full of proteoglycans. Using the zebrafish as a model to study GAG biosynthesis we discovered that HS production is prioritized over CS/DS production, if the availability of link structures is restricted. We also found that the effects of removing HS and CS/DS biosynthetic enzymes in zebrafish larvae typically differ from what could be hypothesized solely from knowledge of the activity of each enzyme. These findings indicated a highly complex regulation of GAG biosynthesis and we thus proceeded to identify novel GAG biosynthetic enzymes in zebrafish and characterized their expression during early development. Notably, strong expression of CS/DS glycosyltransferases was found in cartilage structures, correlating with a drastic increase of CS/DS synthesis after two days of development, and high CS/DS deposition in cartilage. Finally, to understand how different GAG biosynthetic enzymes affect zebrafish development, we decided to use the CRISPR/Cas9 technology to generate new loss of function alleles for enzymes in HS and CS/DS biosynthesis. Some mutants show disturbed larval development or adult morphology, but we found many mutants to develop into adults without major morphological abnormalities, suggesting a high redundancy for GAG biosynthetic enzymes. Many GAG glycosyltransferases and modification enzymes have multiple isoforms, suggesting that a combination of mutations in one individual will become necessary to study the loss of specific modifications. To conclude, the zebrafish model gives new insights into the GAG machinery and the CRSIPR/Cas9 technology allows for swift production of new loss of function zebrafish lines with defective GAG biosynthesis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 65 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1266
National Category
Natural Sciences Biological Sciences Developmental Biology Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-259079 (URN)978-91-554-9282-3 (ISBN)
Public defence
2015-09-11, Friessalen, Norbyvägen 14, Uppsala, 09:15 (English)
Available from: 2015-08-20 Created: 2015-07-27 Last updated: 2015-10-01

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Habicher, JudithHaitina, TatjanaEriksson, IngerHolmborn, KatarinaDierker, TabeaAhlberg, Per E.Ledin, Johan
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