3 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1 Higashi, Tsukuba 305-8566, Japan
Received on October 25, 2002; revised on June 30, 2003; accepted on June 30, 2003
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Abstract |
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Key words: AtFX/GER1 gene / coexpression / GDP-fucose synthesis / MUR1 gene / Saccharomyces cerevisiae
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Introduction |
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GDP-fucose is synthesized from GDP-mannose by a three-step reaction. The first step, oxidation of C-4 of mannose to keto group and reduction of C-6 of mannose to methyl residue, is catalyzed by GDP-mannose-4,6-dehydratase to yield GDP-4-keto-6-deoxymannose (Bonin et al., 1997). This enzyme requires the cofactor nicotinamide adenine dinucleotide phosphate (NADP+), which is reduced to NADPH during the reaction (Menon et al., 1999
). In the second step, epimerization occurs at the C-3 and C-5 position of 4-keto-6-deoxymannose. In the third step, ketone at the C-4 position of 4-keto-6-deoxymannose is reduced, yielding GDP-fucose (Tonetti et al., 1996
). The second and third steps are catalyzed by GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which requires NADPH as a cofactor.
These enzymes have been well characterized, and genes have been cloned in humans, plants, and bacteria. The gene for GDP-mannose-4,6-dehydratase was isolated in Arabidopsis thaliana as MUR1 (Bonin et al., 1997), and in human as GMD (Ohyama et al., 1998
; Sullivan et al., 1998
). The MUR1 protein and GMD protein were expressed in Escherichia coli, and GDP-mannose dehydratase activity was determined in vitro (Bonin et al., 1997
; Sullivan et al., 1998
). GDP-mannose-4,6-dehydratase in E. coli was isolated and its three-dimensional structure was determined by X-ray crystallography (Somoza et al., 2000
), indicating that the purified enzyme is stable and active. GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which has enzyme activities for the second and third reaction steps in a single polypeptide, has been cloned in humans and E. coli (Sullivan et al., 1998
; Tonetti et al., 1996
).
In vitro synthesis of fucosylated sugar chains using fucosyltransferase is still problematic because of the difficulty of economically producing GDP-fucose. We recently developed a system to produce GDP-fucose in vitro by introducing GDP-fucose synthesis genes in yeast cells. Another group reported a similar system using genes of E. coli (Mattila et al., 2000), but GDP-fucose productivity was unclear. Our system allows high production of GDP-fucose because yeast cells contain a large amount of the precursor, GDP-mannose. Furthermore, yeast cells do not consume GDP-fucose, so it is accumulated inside the cells as a final product.
We report the properties of the two enzymes in the yeast system and confirm their physical interactions. Because it is unclear whether these enzymes convert GDP-mannose to GDP-fucose in a cooperative fashion, we also aimed to clarify the interaction of the enzymes with each other, the effect of interaction on enzyme activity, and how the intermediate is transferred to the next enzyme.
Our results reveal that the presence of epimerase-reductase is a prerequisite for functional expression of dehydratase in synthesis of GDP-fucose, suggesting that interaction between the two enzymes plays a significant role in regulation of GDP-fucose synthesis.
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Results |
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Bonin et al. (1997) reported that MUR1 protein in MUR1-only expressing E. coli showed activity, which is contrary to our result in yeast cells. E. coli cells have inherent GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase enzyme encoded by wcaG gene, and the wcaG protein has high homology to AtFX/GER1 protein (Figure 1). Therefore the wcaG protein may function as a substitute for AtFX/GER1 protein. To test this hypothesis, we cloned wcaG gene from E. coli with Myc tag at the C-terminus and coexpressed it with MUR1 gene in yeast. The wcaG protein was expressed well in yeast cells (Figure 2B, lane 4, 6), and GDP-fucose synthesis activity was observed in assay of the cytoplasmic fraction of MUR1 and wcaG coexpressing cells (Figure 3f). Although expression level of wcaG protein in MUR1 and wcaG coexpressing yeast was higher than that of AtFX/GER1 protein (Figure 2B), GDP-fucose synthesis activity of MUR1 and wcaG coexpressing yeast was weaker than that of MUR1 and AtFX/GER1 coexpressing yeast (Figure 3e, 3f).
Immunoprecipitation analysis
Because enzyme activity was observed in cytoplasmic fractions of MUR1 and AtFX/GER1 or MUR1 and wcaG coexpressing cells and not in that of MUR1-only expressing cells, it is possible that MUR1 protein had some interaction with AtFX/GER1 protein or wcaG protein. To investigate such interactions, we performed immunoprecipitation analysis using anti-Myc antibody and Protein A Sepharose. Because AtFX/GER1 protein and wcaG protein have Myc tag at the C-terminus, MUR1 protein would be coprecipitated with them if such an interaction took place. As expected, western blotting showed a MUR1 protein band in MUR1 and AtFX/GER1 coexpressing cells and in MUR1 and wcaG coexpressing cells (Figure 4A, lane 4, 5). The amount of MUR1 protein in MUR1 and AtFX/GER1 coexpressing cells was very close to that in MUR1 and wcaG coexpressing cells (Figure 4A, lane 4, 5), even though productivity of wcaG protein was higher than that of AtFX/GER1 protein (Figure 4B, lane 4, 5). This result indicates that interaction between MUR1 protein and AtFX/GER1 protein is stronger than that between MUR1 protein and wcaG protein. MUR1 protein production was not observed in cytoplasmic fractions of cells expressing MUR1, AtFX/GER1, or wcaG only (Figure 4A, lanes 13).
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The amounts of MUR1 protein, AtFX/GER1 protein and wcaG protein were calculated by densitometric analysis of the western blot results in Figure 4. Based on the amounts of these proteins, the ratios of interacting MUR1 protein/AtFX/GER1 protein and MUR1 protein/wcaG protein were approximately 3:1 and 1:1, respectively. The free MUR1 protein was also calculated by compairing the amounts of MUR1 protein, AtFX/GER1 protein, and wcaG protein observed in western blot (Figure 2 and Figure 4), indicating that no free MUR1 protein is present in MUR1 and AtFX/GER1 or wcaG coexpressing cells.
Activity of GDP-fucose synthesis enzymes in vivo
To confirm the activity of GDP-fucose synthesis enzymes in yeast cell cytoplasm, we quantitated GDP-mannose and GDP-fucose in cell extracts (Shimma et al., 1997). Cells were grown in SD -leu and -ura medium until OD 1.0 at 30°C, and then pelleted. Formic acid (1 M) saturated with 1-butanol was added to the pellet, and cytoplasmic GDP-mannose and GDP-fucose were extracted. The extracts were subjected to Mono-Q column chromatography, and GDP-sugar fractions were isolated. These fractions were analyzed by HPLC, and quantities of GDP-mannose and GDP-fucose were determined by peak areas.
Only GDP-mannose was detected in wild-type yeast cells, and in transfectants expressing MUR1, AtFX/GER1, or wcaG gene only (Figure 5ad). The level of GDP-mannose was about 1.0 nmol per 2 x 108 cells (Table I). As expected, GDP-fucose and GDP-mannose were detected in MUR1 and AtFX/GER1, or MUR1 and wcaG coexpressing cells (Figure 5e, 5f), with quantitated levels of 3.5 nmol and 1.5 nmol per 2 x 108 cells, respectively (Table I). Levels of GDP-fucose and GDP-mannose in these coexpressing transfectants were respectively 3.5 times and 1.5 times higher than corresponding GDP-mannose levels in wild-type cells. In analogy to results of enzyme assay, intermediate peak was not observed and quantity of GDP-mannose remained the same in MUR1-only expressing cells. These results indicate that MUR1 protein in MUR1-only expressing cells showed no enzyme activity in vivo or in vitro, whereas when AtFX/GER1 or wcaG was present in the vicinity of MUR1 in the coexpressing cells, GDP-mannose was effectively converted to GDP-fucose both in vivo and in vitro.
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Discussion |
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MUR1 protein in MUR1-only expressing yeast cells did not show any dehydratase activity (Figure 3). The amount of MUR1 protein produced by MUR1 and AtFX/GER1 or MUR1 and wcaG coexpressing cells was six times higher than that in MUR1-only expressing cells (Figure 2A). It is plausible that in the MUR1-only expressing cells, degradation of MUR1 protein occurred through denaturation, resulting in instability and loss of activity (Figure 6). The results presented here suggest that the AtFX/GER1 and wcaG proteins may function to maintain MUR1 protein in active form by stabilizing its conformation. However, once MUR1 protein is in an active and stable form, further stabilization of conformation seems to be unnecessary (Figure 6). AtFX/GER1 protein disappeared when MUR1 and AtFX /GER1 coexpressing cells were cultivated for 60 h, but MUR1 protein still remained active (data not shown). This is consistent with the previously reported activity of purified MUR1 protein (Bonin et al., 1997).
Immunoprecipitation experiments showed that MUR1 protein interacts with AtFX/GER1 protein and wcaG protein (Figure 4). This interaction is necessary to maintain the active and stable form of MUR1 protein. The amount of AtFX/GER1 protein needed to coprecipitate MUR1 protein was less than the amount of wcaG protein needed (Figure 4A, lane 5, 6; 4B, lane 5, 6). This suggests that MUR1 protein interacts more strongly with AtFX /GER1 protein than with wcaG protein. In vitro experiments also support this hypothesis. GDP-fucose synthesis activity of MUR1 and AtFX/GER1 coexpressing cytoplasmic fraction was higher than that of MUR1 and wcaG coexpressing fraction, even though amount of AtFX/GER1 protein was less than that of wcaG protein (Figures 2 and 3).
The interaction between dehydratase and epimerase-reductase may have a significant role in vivo. The intermediate GDP-4-keto-6-deoxymannose is unstable, as reported earlier. If these two GDP-fucose synthesis enzymes existed separately, the intermediate would be broken down. However, if the dehydratase and epimerase-reductase form a complex, the intermediate can be quickly transferred from the former to the latter, and efficient synthesis of GDP-fucose is possible (Figure 6). Thus GDP-fucose synthesis activity of MUR1 and AtFX/GER1 coexpressing cytoplasmic fraction was higher than that of MUR1 and wcaG coexpressing cytoplasmic fraction due to strong interaction between MUR1 protein and AtFX/GER1 protein. In addition, this dehydratase and epimerase-reductase interaction may also have another role in controlling the amount of GDP-mannose and GDP-fucose in vivo. GDP-mannose-4,6-dehydratase activity is inhibited by GDP-fucose (Koizumi et al., 2000). However, GDP-mannose-4,6-dehydratase activity is not inhibited in cells where only GDP-mannose-4,6-dehydratase is active due to the absence of both GDP-4-keto-6-deoxymannose-3,5-epimerase-6-reductase and GDP-fucose, resulting no accumulation of GDP-mannose. The lack of GDP-mannose causes inhibition of N-linked oligosaccharide synthesis, then yielding cell death, because GDP-mannose is essential for the synthesis of N-linked encoplasmic reticulum core oligosaccharide. Therefore the GDP-mannose-4,6-dehydratase activity without GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase will be toxic for yeast cells. This may be the other reason for the absence of MUR1 enzyme activity in MUR1-only expressing cells.
The methods for chemical or biological synthesis of GDP-fucose have been reported (Adelhorst and Whitesides, 1993; Ichikawa et al., 1992
; Yamamoto et al., 1984
). Synthesizing GDP-fucose using these methods was expensive, because it required expensive starting materials or enzymes. Recently, Koizumi et al. (2000)
reported a method for GDP-fucose synthesis using bacteria. This method is capable of producing large amounts of GDP-fucose but still has some problems. First, for production of GDP-fucose using their system, three E. coli gene transfectants are needed, E. coli's GDP-mannose synthesis enzymes, GDP-mannose-4,6-dehydratase (gmd), and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (wcaG), along with Corynebacterium ammoniagenes for production of GDP-fucose. Furthermore, they used detergent and organic solvents to transfer intermediates from transfectant to transfectant. Therefore new cells are required for each reaction. Second, feeding the starting materials, GMP and mannose, were essential. Finally, because E. coli's GDP-mannose-4,6-dehydrase activity is strongly suppressed by low concentration of GDP-fucose, a low yield of GDP-fucose resulted from using the gmd transfectant and wcaG transfectant simultaneously (Koizumi et al., 2000
). Therefore they had to add the wcaG transfectants after accumulation of the intermediate GDP-4-keto-6-deoxymannose product. Thus their method is complicated and requires monitoring the accumulation of GDP-4-keto-6-deoxymannose.
Compare to Koizumi et al. (2000) method, our system is simpler and easier. Our method requires the culture of MUR1 and AtFX/GER1 coexpressing yeast only, without the starting materials or the control of intermediate accumulation. Therefore it is easy to scale up the production level. Mattila et al. (2000)
also have reported a similar GDP-fucose production system, but they failed to show the amount of GDP-fucose. They used the E. coli gmd gene (the same gene used in the method of Koizumi et al.) instead of MUR1 gene. Because GDP-mannose-4,6-dehydratase encoded by the gmd gene is inhibited its activity by low concentration of GDP-fucose, enough GDP-fucose may be not accumulated in their system.
Commercially available GDP-mannose is produced by yeast at a price about 30 times lower than that of GDP-fucose. In this study, we showed that the amount of GDP-fucose was 3.5 times larger than that of GDP-mannose using MUR1 and AtFX/GER1 coexpressing yeast. The result indicates that the efficient production of GDP-fucose is possible using MUR1 and AtFX/GER1 coexpressing yeast system. We believe that our GDP-fucose synthesis method using MUR1 and AtFX/GER1 coexpressing yeast is more suitable than any previous reported GDP-fucose production methods.
In conclusion, we successfully cloned AtFX/GER1 gene from A. thaliana cDNA library and coexpressed it with A. thaliana MUR1 gene in yeast. AtFX/GER1 protein in combination with MUR1 protein not only showed GDP-fucose synthesis ability but also formed a complex with MUR1 protein and stabilized its activity. These results show that the yeast expression system is useful for analysis of cofactors or interaction with a protein of foreign origin that does not exist in yeast.
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Materials and methods |
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MUR1 gene was amplified by PCR with primers (5'-GTCGAATTCATGGCGTCAGAGAACAAC-3' and 5'-GAACTCGAGAGGTTGCTGCTTAGCATC-3') that incorporated EcoRI and XhoI sites. AtFX/GER1 gene was amplified by PCR with primers (5'-ATTGGTACCAT-GTCTGACAAATCTGCCAAAATCTTCGTC-3' and 5'-TTAGTCGACGATATCTCGGTTGCAAACATTC-TTCAAATACCAATCATAAG-3') that incorporated KpnI and EcoRV sites. For amplification of the genes the cDNA library of A. thaliana (Arabidopsis QUICK-Clone cDNA, Clontech, Palo Alto, CA) was used as the template DNA. WcaG gene was amplified by PCR with primers (5'-AAGGTACCATGAGTAAACAACGAGTTTTTATTGCTGG-TC-3' and 5'-TTGTCGACTTACCCGGGCCGAAAGC-GGTCTTGATTCTCAAGGAACC-3') that incorporated KpnI, SmaI, and SalI sites. Genomic DNA of E. coli was used for amplification of wcaG gene, and the amplified genes were ligated into pCR2.1.
After confirming the nucleotide sequence by sequencing, these genes were introduced into expression vectors. MUR1 gene was cut by EcoRI and XhoI restriction enzymes and introduced into EcoRIXhoI sites of the expression vector YEp352GAP, which contains the GAPDH promoter (Kainuma et al., 1999). A triple influenza hemagglutinin epitope (HA) gene fragment was excised from HAp316 vector with NruI and Ecl136II and inserted into PvuII site of YEp352GAP that contains the MUR1 gene; this plasmid was designated YEp-MUR1HA. Gene of triple c-Myc epitope tag (Myc) was amplified by PCR with primers (5'-GGTGAACAAAAGTTGATTTCTGAAGAAGAT-3' and 5'-CTAGAGGTTCAAGTCTTCTTCTGAGATTAA-3'). This PCR product was introduced directly to EcoRV site of AtFX/GER1 gene and SmaI site of wcaG gene. Myc and AtFX/GER1 fusion gene (AtFXMyc) was cut by KpnI and XhoI enzymes and inserted into KpnISalI site of YEp352GAP-II of which the multicloning site was exchanged for EcoRISalI oligomer of pUC vector. The gene fragment containing a GAPDH promoter, AtFXMyc, and a GAPDH terminator was cut by BamHI, and this fragment was inserted to BamHI site of pYO325 (Sikorski and Hieter, 1989
). This plasmid was designated pYO-AtFXMyc. The same method was followed to construct the plasmid pYO-wcaGMyc. WcaGMyc gene was cut by KpnI and SalI and inserted into YEp352GAP-II. A GAPDH promoter, wcaGMyc, and a GAPDH terminator fragment were obtained by BamHI digestion and introduced in pYO325. This plasmid was designated pYO-wcaGMyc. Expression vector of AtFX/GER1 protein without Myc tag (pYO-AtFX) was constructed similarly to the construction of pYO-AtFXMyc, except that insertion of Myc tag epitope was not performed.
Preparation of cytoplasmic fractions
The yeast W303 was transformed with expression plasmids YEp352GAP, pYO325, YEp-MUR1HA, pYO-AtFXMyc, pYO-wcaGMyc, and pYO-AtFX and grown in 200 ml of SD -leu and -ura medium for 12 h at 30°C. Cells were collected by centrifugation at 3000 x g for 5 min, washed with 1% KCl, and resuspended in 5 ml 100 mM TrisHCl (pH 7.5), 2 mM dithiothreitol (DTT), protease inhibitor (1 tablet of Complete/50 ml, Roche, Mannheim, Germany; 1% aprotinin, Sigma, St. Louis, MO). Glass beads (0.450.5 mm) were added to half the cell suspension volume and homogenized by vortex mixer for 1 min; this was repeated three times with cooling. Homogenates were filtrated by G1 glass filter and centrifuged at 10,000 x g for 20 min. Supernatant was collected and centrifuged at 100,000 x g for 1 h. The supernatant was collected as cytoplasmic fraction, and (NH4)2SO4 (516 mg/ml) was added. After 1 h at 4°C, the sample was centrifuged at 10,000 x g for 20 min, and the pellet was dissolved in water. The resulting sample was desalted by Fast Desalting Column HR 10/10 (Amersham Pharmacia Biotech AB, Little Chalfont, UK) with 20 mM TrisHCl (pH 7.5) containing 0.5 mM DTT and protease inhibitors. This desalted sample was used as the cytoplasmic fraction.
Western blot analysis
Protein concentration was determined by BCA protein assay reagent. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) was performed using 100 µg protein from the cytoplasmic fraction. Proteins were then transferred to polyvinylidene fluoride membrane filter using electroblotter (0.4 A, 1 h). After incubation of the membrane filter for 1 h in 3% skim milk (Difco, Detroit, MI), 100 mM TrisHCl (pH 7.5), and 500 mM NaCl (blocking buffer), it was transferred to 10 ml of anti-HA monoclonal antibody (16B12, Convance, Berkeley, CA) or anti-Myc monoclonal antibody (9E10, Convance) at a dilution of 1:1000 in blocking buffer with 0.05% Tween 20. The membrane filter was incubated for 1 h at room temperature; washed thre times with 100 mM TrisHCl (pH 7.5), 500 mM NaCl, and 0.5% Tween 20 (wash buffer) for a total of 30 min; and then incubated for 1 h with anti-mouse IgG alkaline phosphatase conjugate (ICN Pharmaceuticals, Inc., Aurora, OH) at a dilution of 1:1000. The membrane filter was again washed three times and incubated with 10 ml of 100 mM TrisHCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2 containing nitro blue tetrazolium chloride and X-phosphate. Coloring reaction was stopped by washing the membrane filter for 5 min with 50 ml of 10 mM TrisHCl (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA).
Assay of GDP-fucose synthesis activity and GDP-mannose-4,6-dehydratase activity
GDP-fucose synthesis activity was assayed in 50 µl of 20 mM TrisHCl (pH 7.5), 10 mM EDTA, 5 mM NADPH, and 1 mM GDP-mannose for 1 h at 37°C. Each cytoplasmic fraction containing 400 µg protein was used for the standard assay. The reaction was stopped by boiling for 3 min, followed by centrifugation for 1 min at 15,000 rpm. Supernatants were filtered by Ultrafree C3LGC to remove proteins having molecular weight >10 kDa. Samples were analyzed by HPLC using Wakosil 5C18-200 (4.6 x 250 mm) column with 0.5 M KH2PO4 as running buffer at a flow rate of 1.0 ml/min (Tonetti et al., 1996). GDP-mannose and GDP-fucose were detected by absorbance at 254 nm. GDP-mannose-4,6-dehydratase activity was assayed in 50 µl of 20 mM TrisHCl, pH 7.5, 10 mM EDTA, 1 mM GDP-mannose for 1 h at 37°C. After the reaction was completed, the intermediate product was reduced by adding 1 µmol of NaBH4 and incubating for 90 min at 37°C (Tonetti et al., 1996
).
Immunoprecipitation
A cytoplasmic fraction containing 500 µg protein was preincubated with Protein A Sepharose (Amersham Pharmacia Biotech AB) in 10 µl suspension of water overnight at 4°C, followed by centrifugation for 1 min at 15,000 rpm. Two microliters of anti-Myc monoclonal antibody was added to the supernatant and incubated for 1 h at 4°C, then 10 µl of Protein A Sepharose was added and incubated for 3 h at 4°C. Protein A Sepharose was pelleted by centrifugation for 1 min at 15,000 rpm and washed three times with the wash buffer used in western blot analysis. SDSPAGE sample buffer (20 µl) was added, and the proteins analyzed by western blot analysis as described.
Extraction of GDP-sugars from yeast cells
Yeast cell culture (10 ml) was cultivated until OD600 1.0 (2 x 108 cells) and harvested. Two milliliters of ice-cold 1 M formic acid saturated with 1-butanol were added to the cells and incubated for 30 min at 0°C (Shimma et al., 1997). The supernatant was collected and dried by lyophilization. The dried sample was dissolved in 200 µl water, and the nucleotide diphosphate sugar fraction was isolated by Mono-Q column chromatography using SMART System (Amersham Pharmacia Biotech AB). The Mono-Q column was equilibrated with 10 mM KH2PO4 at a flow rate of 100 µl/min. After 5 min of sample injection, the ratio of 0.5 M KH2PO4 was increased linearly up to 100% for 20 min. Isolated nucleotide diphosphate sugar fractions were analyzed by HPLC as described previously.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: jigami.yoshi{at}aist.go.jp
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Abbreviations |
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References |
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Bonin, C.P. and Reiter, W.D. (2000) A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis. Plant J., 21, 445454.[CrossRef][ISI][Medline]
Bonin, C.P., Potter, I., Vanzin, G.F., and Reiter, W.D. (1997) The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose. Proc. Natl Acad. Sci. USA, 94, 20852090.
Ichikawa, Y., Lim, Y.-C., Dumas, D.P., Shen, G.-J., Garcia-Junceda, E., Willams, M.A., Bayer, R., Ketcham, C., Walker, L.E., Paulson, J.C., and Wong, C.-H. (1992) Chemical-enzymatic synthesis and conformational analysis of sialyl Lewis x and derivatives. J. Am. Chem. Soc., 114, 92839298.[ISI]
Kainuma, M., Ishida, N., Yoko-o, T., Yoshioka, S., Takeuchi, M., Kawakita, M., and Jigami, Y. (1999) Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae. Glycobiology, 9, 133141.
Koizumi, S., Endo, T., Tabata, K., Nagano, H., Ohnishi, J., and Ozaki, A. (2000) Large-scale production of GDP-fucose and Lewis x by bacterial coupling. J. Ind. Microbiol. Biotechnol., 25, 213217.[CrossRef][ISI]
Lowe, J.B., Stoolman, L.M., Nair, R.P., Larsen, R.D., Berhend, T.L., and Marks, R.M. (1990) ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell, 63, 475484.[ISI][Medline]
Mattila, P., Rabina, J., Hortling, S., Helin, J., and Renkonen, R. (2000) Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae. Glycobiology, 10, 10411047.
Menon, S., Stahl, M., Kumar, R., Xu, G.Y., and Sullivan, F. (1999) Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli. J. Biol. Chem., 274, 2674326750.
Ohyama, C., Smith, P.L., Angata, K., Fukuda, M.N., Lowe, J.B., and Fukuda, M. (1998) Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells. J. Biol. Chem., 273, 1458214587.
Phillips, M.L., Nudelman, E., Gaeta, F.C., Perez, M., Singhal, A.K., Hakomori, S., and Paulson, J.C. (1990) ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le x. Science, 250, 11301132.[ISI][Medline]
Polley, M.J., Phillips, M.L., Wayner, E., Nudelman, E., Singhal, A.K., Hakomori, S., and Paulson, J.C. (1991) CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewis x. Proc. Natl Acad. Sci. USA, 88, 62246228.[Abstract]
Shimma, Y., Nishikawa, A., bin Kassim, B., Eto, A., and Jigami, Y. (1997) A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae. Mol. Gen. Genet., 256, 469480.[CrossRef][ISI][Medline]
Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122, 1927.
Somoza, J.R., Menon, S., Schmidt, H., Joseph-McCarthy, D., Dessen, A., Stahl, M.L., Somers, W.S., and Sullivan, F.X. (2000) Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose. Structure Fold Des., 8, 123135.[Medline]
Sullivan, F.X., Kumar, R., Kriz, R., Stahl, M., Xu, G.Y., Rouse, J., Chang, X.J., Boodhoo, A., Potvin, B., and Cumming, D.A. (1998) Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J. Biol. Chem., 273, 81938202.
Tonetti, M., Sturla, L., Bisso, A., Benatti, U., and De Flora, A. (1996) Synthesis of GDP-L-fucose by the human FX protein. J. Biol. Chem., 271, 2727427279.
Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science, 250, 11321135.[ISI][Medline]
Yamamoto, K., Maruyama, T., Kumagai, H., Tochikura, T., Seno, T., and Yamaguchi, H. (1984) Preparation of GDP-L-fucose by using microbial enzymes. Agric. Biol. Chem., 48, 823824.[ISI]