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cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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1999 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 9, no 6, 595-600 p.Article 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. 

Place, publisher, year, edition, pages
1999. Vol. 9, no 6, 595-600 p.
Keyword [en]
UDP-Glc dehydrogenase, UDP-GlcA, glycosaminoglycan, proteoglycan, biosynthesis
Identifiers
URN: urn:nbn:se:uu:diva-92359PubMedID: 10336992OAI: oai:DiVA.org:uu-92359DiVA: diva2:165403
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2011-06-16Bibliographically approved
In thesis
1. Studies on the Role of UDP-Glucose Dehydrogenase in Polysaccharide Biosynthesis
Open this publication in new window or tab >>Studies on the Role of UDP-Glucose Dehydrogenase in Polysaccharide Biosynthesis
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. 58 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1388
Keyword
Biochemistry, UDP-glucose dehydrogenase, polysaccharide biosynthesis, glycosaminoglycan, A. thaliana, E. coli K5, 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:nbn:se:uu:diva-4657 (URN)91-554-6088-7 (ISBN)
Public defence
2004-12-10, B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-11-17 Created: 2004-11-17Bibliographically approved

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