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Glycosaminoglycan Biosynthesis in Zebrafish

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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1143 Glycosaminoglycan Biosynthesis in Zebrafish BEATA FILIPEK-GÓRNIOK ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9368-4 UPPSALA urn:nbn:se:uu:diva-264269 2015 Dissertation presented at Uppsala University to be publicly examined in C8:305, BMC, Husargatan 3, Uppsala, Friday, 27 November 2015 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Associate Professor Kay Grobe (Institute for Physiological Chemistry and Pathobiochemistry, University of Münster). Abstract Filipek-Górniok, B. 2015. Glycosaminoglycan Biosynthesis in Zebrafish. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1143. 54 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9368-4. 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. Keywords: Heparan sulfate, chondroitin/dermatan sulfate, biosynthesis, development, N- deacetylase N-sulfotransferase, glycosyltransferases, morpholino, CRISPR-Cas9 Beata Filipek-Górniok, Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, SE-75123 Uppsala, Sweden. © Beata Filipek-Górniok 2015 ISSN 1651-6206 ISBN 978-91-554-9368-4 urn:nbn:se:uu:diva-264269 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264269) Nothing that comes to easy has any significance. Jacek Hugo-Bader, journalist and reporter Supervisors: Lena Kjellén, Professor Department of Medical Biochemistry and Microbiology Uppsala University Uppsala, Sweden Johan Ledin, Researcher Department of Organismal Biology Uppsala University Uppsala, Sweden Faculty opponent: Kay Grobe, Associate Professor Institute for Physiological Chemistry and Pathobiochemistry University of Münster Münster, Germany Examining Committee: Dan Larhammar, Professor Department of Neuroscience, Pharmacology Uppsala University Uppsala, Sweden Hiroshi Nakato, Associate Professor Department of Genetics, Cell Biology and Development University of Minnesota Minnesota, US Sally Stringer, Honorary Lecturer/Editorial team leader Cardiovascular Division University of Manchester/HealthCare21 Communications Ltd, Manchester, UK Chairperson: Jin-ping Li, Professor Department of Medical Biochemistry and Microbiology Uppsala University Uppsala, Sweden Cover: Adult female zebrafish. Drawing by Beata Filipek-Górniok, 2015. List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Filipek-Górniok B., Carlsson P., Haitina T., Habicher J., Ledin J., Kjellén L. (2015) The NDST gene family in zebrafish: role of NDST1b in pharyngeal arch formation. PLoS One, 10(3):e0119040. doi: 10.1371/journal.pone.0119040. II Filipek-Górniok B., Holmborn K., Haitina T., Habicher J., Oliveira M.B., Hellgren C., Eriksson I., Kjellén L., Kreuger J., Ledin J. (2013) Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development. Developmental Dynamics, 242(8):964-75. doi: 10.1002/dvdy.23981. III Holmborn K., Habicher J., Kasza Z., Eriksson A.S., Filipek- Górniok B., Gopal S., Couchman J.R., Ahlberg P.E., Wiweger M., Spillmann D., Kreuger J., Ledin J. (2012) On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis. The Journal of biological chemistry, 287(40):33905-16. IV Habicher J., Filipek-Górniok B., Varshney G., Ahlberg P.E., Burgess S., Kjellén L., Ledin J. (2015) Large-scale generation of zebrafish mutants with defective glycosaminoglycan biosynthesis. Manuscript. Reprints were made with permission from the respective publishers. Additional Publications I Fisher S., Filipek-Górniok B., Ledin J. (2011) Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling. BMC Developmental Biology, 11:53. doi: 10.1186/1471-213X-11-53. II Dagälv A., Lundequist A., Filipek-Górniok B., Dierker T., Eriksson I., Kjellén L. (2015) Heparan sulfate structure: methods to study N- sulfation and NDST action. Methods in molecular biology, 1229:189- 200. doi: 10.1007/978-1-4939-1714-3_17. Contents Introduction ...................................................................................................13 Background ...................................................................................................14 Proteoglycans and glycosaminoglycans ...................................................14 Heparan sulfate proteoglycans.............................................................15 Chondroitin/dermatan sulfate proteoglycans .......................................16 Heparan sulfate biosynthesis................................................................16 Chondroitin/dermatan sulfate biosynthesis..........................................19 …and one to rule them all: PAPS.............................................................21 Regulation of the glycosaminoglycans biosynthesis – a puzzle not yet solved. ............................................................................................22 Glycosaminoglycans in biological processes.......................................23 Zebrafish as a model system.....................................................................28 Zebrafish in glycosaminoglycan research............................................30 Method considerations..............................................................................32 Overexpression studies ........................................................................32 Rise and fall of the morpholino technology.........................................32 Selected methods for targeted genome editing in zebrafish ................33 Present investigation .....................................................................................36 Aim ...........................................................................................................36 Paper I ..................................................................................................36 Paper II.................................................................................................37 Paper III ...............................................................................................38 Paper IV ...............................................................................................39 Concluding remarks and future perspectives ................................................41 Populärvetenskaplig sammanfattning ...........................................................43 Acknowledgments.........................................................................................44 References .....................................................................................................46 Abbreviations ADP Adenosine diphosphate APS Adenosine 5’-phosphosulfate ATP Adenosine triphosphate b3gat3 Glucuronyltransferase I BMP Bone morphogenetic protein C4ST Chondroitin sulfate 4-O-sulfotransferase C6ST Chondroitin sulfate 6-O-sulfotransferase 1 CS/DS2ST Chondroitin sulfate/dermatan sulfate 2-O-sulfotransferase CHPF/CHSY2 Chondroitin polymerization factor CHPF2 Chondroitin polymerization factor 2 CHSY1 Chondroitin synthase 1 CHSY3 Chondroitin synthase 3 CS Chondroitin sulfate CSGALNACT, GalNAcT Chondroitin sulfate N-acetyl galactosaminyl transferase DS Dermatan sulfate EXTL Exostosin-like
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