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Studies on the Role of UDP-Glucose Dehydrogenase in Polysaccharide Biosynthesis
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.

A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.

Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.

Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains.

Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 58
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1388
Keywords [en]
Biochemistry, UDP-glucose dehydrogenase, polysaccharide biosynthesis, glycosaminoglycan, A. thaliana, E. coli K5
Keywords [sv]
Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical and Physiological Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-4657ISBN: 91-554-6088-7 (print)OAI: oai:DiVA.org:uu-4657DiVA, id: diva2:165407
Public defence
2004-12-10, B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-11-17 Created: 2004-11-17Bibliographically approved
List of papers
1. cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney
Open this publication in new window or tab >>cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney
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1999 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 9, no 6, p. 595-600Article in journal (Refereed) Published
Abstract [en]

We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the first mammalian cDNA clone published. [After submission of the manuscript, a study appeared describing the molecular cloning and characterization of  the human and mouse UDP-glucose dehydrogenase genes(Spicer et al., 1998).] The enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronicacid, an essential precursor in glycosaminoglycan biosynthesis. The cDNA has an open reading frame of 1482 nucleotides coding for a 55 kDa protein. Expression of the enzyme in COS-7 cells showed a 3-fold increase inUDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acidswas shown not to be necessary for enzyme activity. Northernblots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a majortranscript size of 3.2 kilobases and a minor of 2.6 whereas mousetissues have a single 2.6 kilobase transcript. We have also developed a sensitive and direct assay using UDP-[14C]Glc as a substrate for detection of small amounts of UDPGDH activity. 

Keywords
UDP-Glc dehydrogenase, UDP-GlcA, glycosaminoglycan, proteoglycan, biosynthesis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92359 (URN)10336992 (PubMedID)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2018-11-13
2. Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide
Open this publication in new window or tab >>Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide
2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 374, no 3, p. 767-772Article in journal (Refereed) Published
Abstract [en]

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Keywords
bacterial capsule, Escherichia coli, glycosaminoglycan, K5 polysaccharide, UDP-glucose dehydrogenase, UDP-glucuronic acid
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92360 (URN)10.1042/BJ20030365 (DOI)000185546800021 ()12775214 (PubMedID)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2017-12-14Bibliographically approved
3. Glycosaminoglycan biosynthesis in UDP-glucose dehydrogenase overexpressing 293 cells
Open this publication in new window or tab >>Glycosaminoglycan biosynthesis in UDP-glucose dehydrogenase overexpressing 293 cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-92361 (URN)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2010-01-13Bibliographically approved
4. Increased expression of a UDP-glucose dehydrogenase gene in Arabidopsis thaliana results in altered cell wall composition and dwarfism
Open this publication in new window or tab >>Increased expression of a UDP-glucose dehydrogenase gene in Arabidopsis thaliana results in altered cell wall composition and dwarfism
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(English)Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-92362 (URN)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2011-06-20Bibliographically approved

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