Uridine Diphosphoglucose Dehydrogenase Regulates Proteoglycan Expression: cDNA Cloning and Antisense Study

Wegrowski, Yanusz; Perreau, Corinne; Bontemps, Yannick; Maquart, François‐Xavier

doi:10.1006/bbrc.1998.9262

Cited by 21 publications

(23 citation statements)

References 23 publications

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“…In addition, the sites of attachment of the photoaffinity label were more precisely defined by generating small peptide fragments of the labeled protein and separating the labeled peptides efficiently by one-step affinity chromatography. Third, the photolabeled peptide of the hUGDH identified is located within the proposed NAD ϩ -binding domain of many proteins (42)(43)(44)(45)(46)(47)(48), confirming that this sequence is expected to interact with NAD ϩ . It has been reported that the two substrates NAD ϩ and UDP-glucose bind in equimolar amounts rather than in a 2:1 ratio in favor of NAD ϩ over UDP-glucose in view of the sequential mechanism of the two-stage oxidation (3,40,54).…”

Section: Discussionmentioning

confidence: 68%

“…The conserved amino acids are uniformly distributed along the molecule. Interestingly, the upstream NAD ϩ -binding region (GXGXXG) is 100% identical in mammals and E. coli enzymes (42,43). The term "fold" was introduced by Rossmann (44 -47) to describe a nucleotide-binding domain found in families of oxidoreductases such as lactate dehydrogenase and flavodoxin.…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in the conserved glycine residues of the loop have been correlated with attenuation or elimination of enzyme activity (50 -52) and also with disease (53). The conserved structure suggests that UGDH may be part of a minimal genome for multicellular species and that the rigid basal structure is necessary for its activity (14,42). To our knowledge, the crystallographic structure of hUGDH has not been determined, and no studies by site-directed mutagenesis for the coenzyme-binding site(s) of the mammalian UGDHs have been reported yet.…”

Section: Discussionmentioning

confidence: 99%

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Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Huh

Yoon²,

Lee

et al. 2004

Journal of Biological Chemistry

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UDP-glucose dehydrogenase (UGDH) is (6, 7) show that the reaction of UGDH involves two successive oxidations to convert the 6Ј-hydroxyl of UDP-glucose to a carboxylate, together with the reduction of 2 molecules of NAD ϩ to NADH. In animals, it constitutes the unique pathway for glucuronate formation (8). Because glucuronate is a component of glycosaminoglycans (GAGs), the mutation inactivation of UGDH (sugarless) abolishes GAG assembly and, consequently, abolishes GAG-dependent growth factor signaling (9 -11). The primary structure of the mammalian enzyme was obtained from the protein sequence of bovine UGDH (12). The human gene was also recently cloned and assigned to chromosome 4p15.1 (13). It contains 12 exons, extends over 26 kb, and has one major transcription start site (14).GAG chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces. There is a growing body of information on the implication of GAGs in cell behavior, including signal transduction, cell proliferation, spreading, migration, and cancer growth and metastasis (15-17). GAG synthesis is influenced by cytokines and growth factors. Transforming growth factor-␤ is the most potent stimulator of proteoglycan and GAG synthesis, including that of hyaluronan. Its action, however, depends on the cell type (18). The synthesis of GAGs is also modulated by oxygen cell status. Hypoxic endothelial cells and lung fibroblasts enhanced the heparan sulfate/chondroitin sulfate ratio, which led to an increase of basic fibroblast growth factor reactivity on the cell surface (19,20). It was also shown that the level of intracellular UDP-glucuronate could influence GAG synthesis (8). Interestingly, the human UGDH was shown to be an early response gene after interleukin-1 treatment of ocular fibroblasts (21), as well as an early androgen response gene in breast cancer (22).Although a clone of human UGDH has been reported (13, 15), specific catalytic residues are not yet available. Therefore, further characterization of the active sites of human UGDH is needed to elucidate the physiological nature of the UGDH. In the present study, we have identified an NAD ϩ -binding site using photoaffinity labeling and cassette mutagenesis to gain a deeper insight into the structural basis of human UGDH. For this study, a 1509-base pair gene that encodes human UGDH has been chemically synthesized and expressed in Escherichia coli as a soluble protein. Identification of the nucleotide-binding sites of a variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert * This work was supported by Grant 03-PJ1-PG3-20900-0047 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (to S.-W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Huh

Yoon²,

Lee

et al. 2004

Journal of Biological Chemistry

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“…The cloned mammalian proteins from different species showed overall 97% identity. The human sequence has 27% identity with the Escherichia coli ortholog, with 100 and 60% identity of the NAD ϩ binding and the catalytic sites, respectively (8). Glycosaminoglycan chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces.…”

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confidence: 99%

Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression

Bontemps

Vuillermoz

Antonicelli

et al. 2003

Journal of Biological Chemistry

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“…21,22 The X-ray structure of S. pyogenes UGDH 14 suggests that the motif serves as a connection loop between a β-sheet (β1) and an α-helix (α1). The substitutions we made in the first two glycines of the motif provide evidence that a complex with NAD + is mediated by the glycines.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

Yoon¹,

Park²,

Park³

et al. 2009

Bulletin of the Korean Chemical Society

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Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the Km and Vmax for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

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Uridine Diphosphoglucose Dehydrogenase Regulates Proteoglycan Expression: cDNA Cloning and Antisense Study

Cited by 21 publications

References 23 publications

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

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