cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney

Thomas Lind, Elisabet Falk, Eva Hjertson, Marion Kusche-Gullberg and Kerstin Lidholt1

Department of Medical Biochemistry and Microbiology, University of Uppsala, The Biomedical Center, S-751 23 Uppsala, Sweden

Received on August 24, 1998; revised on October 20, 1998; accepted on October 26, 1998

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-glucuronic acid, 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 in UDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acids was shown not to be necessary for enzyme activity. Northern blots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a major transcript size of 3.2 kilobases and a minor of 2.6 whereas mouse tissues 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.

Key words: UDP-Glc dehydrogenase/UDP-GlcA/glycosaminoglycan/proteoglycan/biosynthesis

Introduction

UDP-Glucose dehydrogenase (UDPGDH) is an enzyme converting UDP-d-glucose (UDP-Glc) to UDP-d-glucuronic acid (UDP-GlcA) in the reaction: UDP-Glc + 2NAD+ + H2O -> UDP-GlcA + 2NADH + 2H+. In mammals, UDP-GlcA is one of the two sugar precursors needed in glycosaminoglycan polymerization and thereby is absolutely required for biosynthesis of heparin/heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronan (HA). UDP-GlcA is also a substrate for glucuronosylation of xenobiotics in the liver.

Recently, UDPGDH cDNA has been cloned in Drosophila melanogaster (Binari et al., 1997; Häcker et al., 1997; Haerry et al., 1997). Mutations in the gene encoding UDPGDH results in loss of heparan sulfate and wingless phenotype. This indicates the importance of glycosaminoglycans in embryonic development and the importance of UDPGDH in heparan sulfate biosynthesis. The predicted protein product of Drosophila UDPGDH is ~66% identical to the corresponding Caenorhabditis elegans, bovine, and soybean proteins, demonstrating that the protein is extremely conserved.

UDPGDH occurs in several bacterial species, and has been cloned from Streptococci pyogenes (hasB gene product) (Dougherty and van de Rijn, 1993), and E.coli K5 (kfiD gene product) (Petit et al., 1995). In bacteria, UDP-GlcA is a precursor for capsule polysaccharides such as HA from Streptococci (Dougherty and van de Rijn, 1993), the HS-like E.coli K5 polysaccharide (Petit et al., 1995) and the E.coli K4 polysaccharide with a carbohydrate backbone similar to CS (Rodriguez et al., 1988).

We have been studying polymerization reactions of glycosaminoglycan chains (Lidholt et al., 1988, 1989, 1992, 1994; Lind et al., 1993; Lidholt and Lindahl, 1992). As UDPGDH is an essential enzyme in the biosynthesis and no mammalian cDNA is published or expressed, we wanted to clone the mammalian UDPGDH to be able to study its regulatory functions.

We have now cloned and sequenced the UDPGDH from a bovine kidney cDNA library, with a human cDNA clone as a probe. The bovine kidney cDNA codes for a protein almost identical to bovine liver UDPGDH which was previously amino acid sequenced (Hempel et al., 1994). The difference is that the bovine kidney enzyme contains one additional internal and 25 additional C-terminal amino acids.

To be able to detect UDPGDH activity in small amounts, a sensitive and direct assay was developed where newly formed radiolabeled UDP-GlcA can be measured. UDPGDH was expressed in COS-7 cells and activity could be assayed both for the full length form of the protein and for a shorter form similar to the protein reported earlier (Hempel et al., 1994). Northern blot analysis of mouse and human mRNAs showed expression in most tissues, with highest expression in mouse liver.

Results

UDP-Glucose dehydrogenase (UDPGDH) from bovine liver was previously found to be composed of 468 amino acid units and have a molecular weight of ~52,000, as deduced from amino acid sequencing of the purified enzyme (Hempel et al., 1994). When this sequence was compared with DNA sequences translated into all six reading frames (tblastn search in GenBank), a human EST (Expressed Sequence Tag) clone was found. This clone was obtained from the National Center for Biotechnology Information (NCBI) and sequenced. The cDNA insert had an open reading frame that coded for a polypeptide (180 aa) that was similar to the C-terminal part of the bovine protein. The main difference was that the human protein had 25 additional amino acid residues in the C-terminal, when compared with the bovine protein. The human EST clone was used as a probe to screen a bovine kidney cDNA library to obtain full-length UDPGDH. Four different cDNA clones were obtained, which were identical in the 3[prime]-end but differed in the length of the 5[prime]-untranslated sequence. As the bovine cDNA was inserted into a [lambda]gt10 vector with EcoRI restriction sites and the cDNA had an internal cleavage site for EcoRI (Figure 1), two cDNA pieces of the insert (approximately 1.5 and 1.3 kb) were isolated and sequenced. The larger fragment contained the coding sequence but lacked the C-terminal part of the protein. The shorter fragment contained the C-terminal, and in addition a large part of the 3[prime]- noncoding sequence. The two combined sequences contained an open reading frame, encoding bovine UDPGDH (Figure 1). This cDNA sequence revealed one additional internal amino acid, Leu 157, and 25 additional amino acids at the C-terminus compared with the protein sequence that was found earlier. Of the latter residues, 23 are identical to the human and mouse sequences (Figure 2A). The bovine kidney UDPGDH thus has 494 amino acids and a calculated molecular weight of ~55,000.


Figure 1. Nucleotide and amino acid sequence of bovine kidney UDPGDH. The box indicates the internal EcoRI cleavage site. EBI/GenBank accession number is AF095792.


Figure 2. Alignment of UDP-glucose dehydrogenase sequences. (A) Deduced amino acid sequence of bovine kidney UDPGDH and alignment with human, mouse, Drosophila melanogaster, and Arabidopsis thaliana homologues. Dashes indicate amino acid identity, gaps show absence of a particular amino acid, and the numbers are the position of the amino acids in the corresponding protein. The arrow highlights the Leu 157 (boldface), and the additional 25 C-terminal amino acids (boldface) are underlined. (B) Alignment of partial amino acid sequences of bovine liver UDPGDH (Hempel et al., 1994), bovine kidney UDPGDH (this study), short form construct of UDPGDH, and long form construct of UDPGDH. In boldface are the unrelated amino acids from the constructs (see Materials and methods). The numbers indicate the position of the amino acids.

A human and a mouse multiple tissue Northern blot was analyzed with a 295 bp cDNA probe from the coding region of the UDPGDH (see Materials and methods). It shows expression in all tissues in both human and mouse, with markedly low and high expression in skeletal muscle and mouse liver, respectively (Figure 5). The expression of UDPGDH mRNA showed no correlation to the amount of control G3PDH. In the human blot, transcripts of 2.6 and 3.2 kilobases exist whereas in the mouse there seems to be only one single mRNA of 2.6 kilobases.

To be able to detect low amount of UDPGDH activity a sensitive and direct method was developed. The assay measures the formation of UDP-[14C]GlcA from UDP-[14C]Glc. Cell lysate was incubated with 14C-labeled UDP-Glc and NAD+ as substrates, and subsequently applied to an anion exchange column to which the newly formed UDP-[14C]GlcA bound. The column was eluted using a salt gradient and the elution position of UDP-GlcA (Figure 3) could be identified by comparison with a standard UDP-[14C]GlcA. The amount of eluted UDP-[14C]GlcA was then detected by scintillation counting.


Figure 3. UDPGDH activity assay. Cell lysate was incubated with UDP-[14C]Glc and after incubation, newly-formed UDP-[14C]GlcA was separated from nonutilized UDP-[14C]Glc on anion exchange chromatography (MonoQ) as described in Materials and methods. Arrows indicate elution positions of the UDP-sugars.

In order to express UDPGDH, two different constructs were made using the internal EcoRI restriction site. A short form UDPGDH-S (see Materials and methods), containing most of the coding region but lacking the last 23 amino acids and a long form UDPGDH-L, encoding the whole protein (Figure 2B). The two constructs code for a His/FLAG UDPGDH fusion protein. Transient expression in COS-7 cells clearly shows that both constructs introduce an overexpression of UDPGDH activity compared to control cells, transfected with vector alone. The long form construct induced a 3-fold increase in UDPGDH activity and the short form doubled the activity compared to control cells (Table I). Western blot analysis (Figure 4) of cell extracts shows that a FLAG-fusion protein of the correct size (~57,000) is made with both constructs, although the signal is stronger for the long construct. No signal is generated in the control cells transfected with the vector plasmid alone. This clearly shows that the last 23 amino acids are not essential for enzyme activity and that the His/FLAG epitope seems not to interfere with enzyme activity.


Figure 4. Western blot of FLAG-tagged UDPGDH. Cell lysate (20 µg protein) from transfected COS-7 cells, (lane 1) empty vector, (lane 2) UDPGDH-S, (lane 3) UDPGDH-L were separated by SDS-PAGE and transferred to PVDF-membrane as described in Materials and methods. FLAG-tagged UDPGDH was detected with anti-FLAG M2 monoclonal antibody and visualized by ECL Western blot detection system.


Figure 5. Distribution of UDP-glucose dehydrogenase mRNA in human and mouse tissues. Two multiple tissue northern (MTN) blots, one mouse (A) and one human (B) were hybridized with 32P-labeled probes (see Materials and methods) recognizing a 295 bp sequence, unique for the dehydrogenase (the filter was washed in 2× SSC, 0.05% SDS at 55°C). As a control the same filters were hybridized with human G3PDH (C and D) and washed (in 0.1× SSC, 0.1% SDS at 50°C).

Table I. Overexpression of UDPGDH in COS-7 cells
Construct UDPGDH activity(cpm/µg protein/min)
Exp.1a
pcDNA3 0.42 ± 0.045
UDPGDH-L 1.5 ± 0.085
Exp. 2a
pcDNA3 1.9 ± 0.025
UDPGDH-S 2.9 ± 0.075
UDPGDH-L 4.2 ± 0.005
COS-7 cells were transfected with vector alone (pcDNA3), UDPGDH-S, or UDPGDH-L and cell lysate was prepared as described under Materials and methods. UDPGDH activity was assayed by detecting formed UDP-[14C]GlcA. The data represents means ± SD of duplicate samples.
apH of the lysis buffer in Exp. 1 was 7.2 whereas in Exp. 2 it was 8.5.

Discussion

In mammals, the formation of UDP-GlcA is an important regulatory event since it is one of the precursors in the biosynthesis of glycosaminoglycan chains. The kinetics of the enzyme have been investigated in several studies (Ordman and Kirkwood, 1977; Franzen et al., 1983; Jaenicke et al., 1986; Dickinson, 1988; Hempel et al., 1994) and bovine liver UDPGDH has been purified and amino acid sequenced; however, the enzyme has not previously been cloned from any mammalian source. The bovine UDPGDH cDNA clone that we have sequenced is in accord with the previously published amino acid sequence (Hempel et al., 1994). The differences (one additional internal amino acid residue and 25 additional amino acids at the C-terminus (also confirmed by the human and mouse sequences)) could be due to errors in the earlier amino acid sequencing, or to the existence of a truncated form of the protein. The expression of the full length form in COS-7 cells demonstrated that an active form of the protein was produced. A cDNA coding for a shorter form of the protein more similar to the amino acid sequence published by Hempel et al. also showed activity. These results show that the last 23 C-terminal amino acids are not necessary for dehydrogenase activity and that the FLAG-tag does not inactivate the enzyme. Comparing UDPGDH from such different species as bovine and the plant Arabidopsis thaliana reveals a highly conserved protein with 60% identity at the amino acid level (Lidholt, 1997). However, the C-terminus is less conserved, which is in agreement with our finding that the catalytic activity is not dependent on the C-terminal amino acids. It should also be noted that we have cloned and sequenced the cDNA from a kidney library whereas the published amino acid sequence is derived from a liver enzyme.

The synthesis of the UDP-sugars is thought to occur in the cytosol in mammalian cells while the polymerization of the proteoglycan chains occurs in the Golgi. Active transport of both UDP-Glc (Persat et al., 1984) and UDP-GlcA (Nuwayhid et al., 1986) into the Golgi lumen has been demonstrated. The amount of available UDP-GlcA can be rate limiting in the chain elongation reaction either directly in the Golgi lumen if the UDPGDH exists there, or indirectly via the UDP-GlcA transporters. In earlier studies of heparin biosynthesis (Lidholt and Lindahl, 1992) we have shown that the GlcA transfer reaction has a ~100-fold higher Km for the UDP-GlcA than for the UDP-GlcNAc in the corresponding GlcNAc transfer. This finding indicates that the amounts of UDP-GlcA are rate limiting, and that the biosynthesis may be regulated by the amount of UDP-GlcA available. The biosynthesis of hyaluronan is thought to take place at the cell membrane (Prehm, 1984) where the glycosyltransferases can be in direct contact with the cytosol and thereby be directly regulated by the pools of UDP-sugars.

UDP-GlcA is also an important precursor for UDP-xylose (UDP-Xyl) through the reaction, UDP-GlcA -> UDP-Xyl + CO2, catalyzed by UDP-GlcA decarboxylase. Xylose is the first sugar unit added to the protein core in CS and HS proteoglycan biosynthesis and UDP-Xyl is the nucleotide sugar used for initiation of the polysaccharide chains. The pool of UDP-Xyl is dependent on the amount of UDP-GlcA. Xylose transfer has been shown to utilize both exogenous UDP-Xyl transported into the ER lumen and UDP-Xyl generated from UDP-GlcA within the lumen (Kearns et al., 1993).

UDPGDH occurs in several bacterial species, such as Streptococci (hasB gene product), which synthesizes hyaluronan (Dougherty and van de Rijn, 1993), and E.coli K5 (kfiD gene product), which produces a capsule polysaccharide with the same structure as the heparin/heparan sulfate precursor polymer (Petit et al., 1995). In both these bacteria, the UDPGDH-coding genes are located directly downstream from the polysaccharide synthase genes hasA and kfiC, respectively. This arrangement suggests that UDPGDH is closely connected with polysaccharide biosynthesis. The organization of the UDPGDH gene, its regulation in the mammalian cell and the role the enzyme plays in glycosaminoglycan biosynthesis have been subject of a recent study (Spicer et al., 1998) that appeared after this article was submitted.

Materials and methods

cDNA library screening

A [lambda]gt10 bovine kidney 5[prime]-stretch plus library (Clontech), and host strain C600 Hfl was used to screen for the bovine UDPGDH. The screening procedure was done according to the manufacturer's recommendations (#PT1010-1, version #PR47377). The cDNA probe used for screening was derived from an EST (Expressed Sequence Tag) clone from Soares human placenta Nb2HP library, GenBank ID: R28477 IMAGE (Integrated Molecular Analysis of Genome Expression) Consortium, in vector pT7T3D (Pharmacia Biotech) with a modified polylinker, restriction sites NotI and EcoRI.

DNA sequencing and analysis

The human placenta EST clone was sequenced in the vector described above. The insert from the bovine [lambda]gt10 clone was subcloned into a plasmid vector pUC19.

The nucleotide sequence of the isolated cDNA was determined by repeated sequencing of both strands of alkaline-denatured plasmid DNA using the Cy5 AutoRead Sequencing Kit (Pharmacia Biotech) with Cy5-labeled Universal and Reverse Primers. Internal oligonucleotide primers were made and recognized sequences about 300 base pairs apart. The nucleotide sequences were labeled using Cy5-dATP labeling mix and the sequence reactions were performed using T7 DNA polymerase (Pharmacia Biotech). The sequence was read on an ALFexpress system (Pharmacia Biotech) and analyzed using DNA-Star (DNASTAR Inc.) on a Macintosh computer. The nucleotide and protein sequences were compared with other database sequences using BLAST search, NCBI (National Center for Biotechnology Information) on Internet address: http://www.ncbi.nlm.nih.gov/

SDS-polyacrylamide gel electrophoresis and immunoblotting

Cell lysate was analyzed by denaturing (SDS) 10%-polyacrylamide gel electrophoresis on a Bio-Rad MiniProtean unit according to the manufacturer's description. Separated protein was transferred to a PVDF membrane (Millipore) in a Bio-Rad Trans-Blot semi-dry electroblot system. Transfer buffer was 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) pH 11 with 5% MeOH under transfer conditions 6 V for 40 min. Western blot of FLAG-tagged UDPGDH was done using the anti-FLAG M2 antibody (Kodak) and made according to the Hybond ECL manual (Amersham Pharmacia Biotech) using PBS-0.1% Tween 20 as buffer and 15% bovine serum as blocking agent. The ECL signal was recorded on a Bio-Rad G525 phosphoimaging device.

Northern blot hybridization

Two Multiple Tissue Northern Blots (MTN) from Clontech, prepared with mRNA from human and mouse tissues, were hybridized with a 32P-probe prepared from cDNA of bovine UDPGDH. The probe was produced as follows: cDNA of bovine UDPGDH was cleaved with BglII to yield a 295 bp fragment corresponding to nt 144-438 in Figure 1, which was electrophoresed on 1% agarose gel. The fragment was purified with a Wizard column (Promega), denatured, and labeled with [[alpha]32P]-dCTP using Ready-to-go DNA labeling beads (Pharmacia Biotech). Unincorporated [[alpha]32P]-dCTP was removed with a Microspin S-300 HR column (Pharmacia Biotech). The blots were hybridized with the cDNA-probe at 60°C, Expresshyb solution (Clontech) and then washed in 2× SSC (SSC is 150 mM NaCl, 15 mM sodium citrate buffer, pH 7), 0.05% SDS at 55°C. The same membranes were hybridized with human G3PDH (glyceraldehyde-3-phosphate dehydrogenase, Clontech) cDNA at 68°C and then washed in 0.1× SSC, 0.1% SDS at 50°C.

Construction of expression plasmids

Excising of UDPGDH cDNA from the lambda phage with the restriction enzyme EcoRI yields two fragments of 1537 and 1334 base pairs. The 1537 base pair fragment contains the start M (nt 127 in Figure 1) and most of the coding region. To facilitate subcloning, a PCR (UlTma, Perkin Elmer) fragment was ligated at the unique NsiI restriction site (nt 253 in Figure 1) which introduces a BamHI restriction site at the start methionine. This was done with the PCR primers forward 5[prime]-CCGGATCCGTTTGAAATTAAGAAGAT and reverse 5[prime]-CCATGCATTGATTCTTGATTC with the 1547 base pair fragment as template (anneal 55°C 30 sec, 72°C 30 sec, 94°C 60 sec and 25 cycles). The PCR product was cleaved with BamHI and NsiI and ligated to the NsiI-cleaved 1547 base pair fragment. Restriction sites BamHI and EcoRI were used to subclone the modified fragment into the expression vector pcDNA3 (Invitrogen) which introduced a His/FLAG (MGGSHHHHHHDYKDDDDK-) tag in the N-terminal end and exchanged the 23 last amino acids to 14 unrelated amino acids in the C-terminal (-LQISITLAAARACI). This construct is named UDPGDH-S (short). The 1334 bp fragment was added to the first construct, UDPGDH-S, via an EcoRI restriction site which reintroduces the native 3[prime]-end, resulting in the full length version of the UDPGDH. This construct is named UDPGDH-L (long). The new 5[prime]-end was sequenced to ascertain that no errors were introduced by the PCR product.

Transient expression of UDPGDH in COS-7 cells

COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/nutrient mix F12 (Life Technologies, Inc.) supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% (v/v) heat inactivated (56°C, 30 min) fetal calf serum, at 37°C and 7.5% CO2. For each transfection, 70% confluent cells in a 175 cm2 flask were trypsinized and washed with PBS supplemented with 2 mM MgCl2 in 10 mM Hepes (N-[2-hydroxyethyl)piperazine-N[prime]-[2-ethanesulfonic acid]), pH 7.2. The cells were resuspended in 500 µl of washing buffer, and 30 µg of plasmid cDNA was added together with 50 µg of fish sperm DNA (Boehringer) as carrier. Electrotransfection was carried out in a 0.4 cm cuvette (BTX) with settings 360 V and 500 µF (Gene pulser II, Electroporation system, Bio-Rad). After transfection the cells were resuspended in medium containing 2% DMSO and transferred to 10 cm culture dishes at room temperature for 20 min before incubation at 37°C for 72 h.

UDPGDH activity assay

Cells washed in PBS were scraped off the plate in lysis buffer (50 mM Tris-HCl pH 8.5, 1% Triton X-100, 150 mM NaCl, and leupeptin 1 µM), gently rocked for 1 h at 4°C and centrifuged for 5 min at 12,000 × g. An aliquot of supernatant containing 70 µg of solubilized protein was added to assay buffer containing 20 mM Tris-HCl pH 8.5, 0.5 mM NAD, 0.5 mM UDP-Glc, 0.12 µCi UDP-[14C] d-glucose (287 mCi/mmol, Amersham), and 1 mM AMP-PNP (5[prime]-adenylylimidodiphosphate; Sigma) in a total volume of 25 µl. After 20 min incubation at 30°C, the reaction was stopped by transferring the sample to -20°C. Newly formed UDP-[14C]GlcA was detected by anion exchange chromatography on a MonoQ column using an HPLC system (both, Pharmacia Biotech); 25 µl of sample was diluted in 0.5 ml of 50 mM NaCl in 50 mM acetate buffer pH 4, applied to the column and washed with 10 ml of the same buffer. Labeled UDP-[14C]GlcA was eluted with a salt gradient, 50-300 mM NaCl at a flow rate of 0.5 ml/min for 80 min. Fractions of 1 ml were collected, and the radioactivity was detected by scintillation counting (Beckman, LS 6000 IC).

Acknowledgments

This work was supported by Grants 10440 and 10155 from the Swedish Medical Research Council, by Polysackaridforskning AB (UPPSALA, Sweden), Magnus Bergvalls stiftelse and stiftelsen Lars Hiertas minne.

Abbreviations

GlcNAc, N-acetyl-d-glucosamine; GlcA, d-glucuronic acid; Xyl, d-xylose; UDPGDH, UDP-glucose dehydrogenase; CS, chondroitin sulfate; HA, hyaluronan; HS, heparan sulfate; EST, expressed sequence tag.

References

Binari ,R.C., Staveley,B.E., Johnson,W.A., Godavarti,R., Sasisekharan,R. and Manoukian,A.S. (1997) Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development, 124, 623-2632.

Dickinson ,F.M. (1988) Studies on the unusual behaviour of bovine liver UDP-glucose dehydrogenase in assays at acid and neutral pH and on the presence of tightly bound nucleotide material in purified preparations of this enzyme. Biochem. J., 255, 775-780. MEDLINE Abstract

Dougherty ,B.A. and van de Rijn,I. (1993) Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A Streptococci. Demonstration of UDP-glucose dehydrogenase activity. J. Biol. Chem., 268, 7118-7124. MEDLINE Abstract

Franzen ,J.S., Marchetti,P.S., Lockhart,A.H. and Feingold,D.S. (1983) Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase. Biochim. Biophys. Acta, 746, 146-153. MEDLINE Abstract

Haerry ,T.E., Heslip,T.R., Marsh,J.L. and O'Connor,M.B. (1997) Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila. Development, 124, 3055-3064. MEDLINE Abstract

Häcker ,U., Lin,X. and Perrimon,N. (1997) The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development, 124, 3565-3573. MEDLINE Abstract

Hempel ,J., Perozich,J., Romovacek,H., Hinich,A., Kuo,I. and Feingold,D.S. (1994) UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases. Prot. Sci., 3, 1074-1080.

Jaenicke ,R., Rudolph,R. and Feingold,D.S. (1986) Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme. Biochemistry, 25, 7283-7287. MEDLINE Abstract

Kearns ,A.E., Vertel,B.M. and Schwartz,N.B. (1993) Topography of glycosylation and UDP-xylose production. J. Biol. Chem., 268, 11097-11104. MEDLINE Abstract

Lidholt ,K. (1997) Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria. Biochem. Soc. Trans., 25, 866-870. MEDLINE Abstract

Lidholt ,K. and Lindahl,U. (1992) Biosynthesis of heparin. The d-glucuronosyl- and N-acetyl-d-glucosaminyltransferase reactions and their relation to polymer modification. Biochem. J., 287, 21-29. MEDLINE Abstract

Lidholt ,K., Riesenfeld,J., Jacobsson,K.-G., Feingold,D.S. and Lindahl,U. (1988) Biosynthesis of heparin: modulation of polysaccharide chain length in a cell-free system. Biochem. J., 254, 571-578. MEDLINE Abstract

Lidholt ,K., Kjellén,L. and Lindahl,U. (1989) Biosynthesis of heparin: relationship between the polymerization and sulphation processes. Biochem. J., 261, 999-1007. MEDLINE Abstract

Lidholt ,K., Weinke,J.L., Kiser,C.S., Lugemwa,F.N., Bame,K.J., Cheifetz,S., Massagué,J., Lindahl,U. and Esko,J.D. (1992) A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc. Natl. Acad. Sci. USA, 89, 2267-2271. MEDLINE Abstract

Lidholt ,K., Fjelstad,M., Jann,K. and Lindahl,U. (1994) Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E.coli K5 capsular polysaccharides. Carbohydr. Res., 255, 87-101. MEDLINE Abstract

Lind ,T., Lindahl,U. and Lidholt,K. (1993) Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the d-glucuronosyl- and the N-acetyl-d-glucosaminyltransferase reactions. J. Biol. Chem., 268, 20705-20708. MEDLINE Abstract

Nuwayhid ,N., Glaser,J.H., Johnson,J.C., Conrad,H.E., Hauser,S.C. and Hirschberg,C.B. (1986) Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum. J. Biol. Chem., 261, 12936-12941. MEDLINE Abstract

Ordman ,A.B. and Kirkwood,S. (1977) UDP-glucose dehydrogenase. Kinetics and their mechanistic implications. Biochim. Biophys. Acta, 481, 25-32. MEDLINE Abstract

Persat ,F., Azzar,G., Martel,M.B. and Got,R. (1984) Evidence for coupling between transport of UDP-glucose and its synthesis by membrane-bound pyrophosphorylase in Golgi apparatus of cat liver. Biochim. Biophys. Acta, 769, 377-380. MEDLINE Abstract

Petit ,C., Rigg,G., Pazzani,C., Smith,A., Sieberth,V., Stevens,M., Boulnois,G., Jann,K. and Roberts,I.S. (1995) Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for biosynthesis of the K5 polysaccharide. Mol. Microbiol., 17, 611-620. MEDLINE Abstract

Prehm ,P. (1984) Hyaluronate is synthesized at plasma membranes. Biochem. J., 220, 597-600. MEDLINE Abstract

Rodriguez ,M.-L., Jann,B. and Jann,K. (1988) Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5: K4: H4, a fructose-containing polysaccharide with a chondroitin backbone. Eur. J. Biochem., 177, 117-124. MEDLINE Abstract

Spicer ,A.P., Kaback,L.A., Smith,T.J. and Seldin,M.F. (1998) Molecular cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes. J. Biol. Chem., 273, 25117-25124. MEDLINE Abstract


1To whom correspondence should be addressed at: Department of Medical Biochemistry and Microbiology, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden


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