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.
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
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
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
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
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.
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
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.
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
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
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).
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.
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.
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