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Department of Biochemistry [K. N., J. G., C-X. G., S. N., T. K., K. H., N. T.], Department of Molecular Biochemistry and Clinical Investigations [K. N., E. M., C-X. G.], Department of Molecular Therapeutics, Division of Molecular Therapy Science [K. N., N. H.], Department of Internal Medicine and Therapeutics [K. N., M. H.], and Department of General Medicine [A. K.], Osaka University, Graduate School of Medicine, Osaka, Japan; Departments of Gastroenterology [K. S., H. Y.], Surgery [K. Y.], and Pathology [K. K.], Osaka Rosai Hospital, Osaka, 565-0871, Japan; Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, 16132 Genova, Italy [M. T.], and Department of Molecular Genetics, Kochi Medical School, Kochi 783-8505, Japan [K. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6 fucosyltransferase (1-6 FucT) and FX mRNA. Transfection of this gene caused an increase in GDP-L-fucose levels as well as the extent of fucosylation on glycoproteins, including -fetoprotein, as judged by reactivity to lectins. Collectively, the results herein suggest that the high level of fucosylation in HCC is dependent on a high expression of FX followed by increases in GDP-L-fucose, as well as an enhancement in 1-6 FucT expression. Thus, an elevation in GDP-L-fucose levels and the up-regulation of FX expression represent potential markers for HCC. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6 fucosylated (referred to as core fucosylated) glycoprotein synthesis. Core-fucosylated glycoproteins are synthesized via the action of 1-6 FucT.3
In previous studies, we reported on the purification and cDNA cloning of 1-6 FucT, which has been implicated as a key enzyme in the core fucosylation of N-glycans from porcine brains (11)
and a human gastric cancer cell line (12)
. 1-6 FucT catalyzes the transfer of fucose from GDP-L-fucose to the reducing end of GlcNAc in complex N-glycans via an 1-6 linkage (Fig. 1
; for a review on 1-6 FucT, see Ref. 13
). In an earlier study, we reported that the overexpression of 1-6 FucT in hepatoma cells suppressed intrahepatic metastasis after splenic injection in athymic mice partly by a decrease in the adhesion of core fucosylated 5ß1 integrin to fibronectin (14)
. It has also been reported that core fucosylation increases the flexibility of a biantennary N-linked oligosaccharide (15)
and has an effect on the stability of glycoproteins (16)
. The serum half-life of biantennary neoglycoproteins is altered by the addition of sialylation and core fucosylation (17)
. The depletion of core fucosylation of IgG1 was reported to be the most critical role in the enhancement of antibody-dependent cellular cytotoxicity (18
, 19)
.
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The up-regulation of the 1-6 FucT gene in hepatoma cell lines, either by transfection (27)
or induction (28)
, caused an increase in both 1-6 FucT activities and the core fucosylation of AFP. In an investigation of 1-6 FucT expression during hepatocarcinogenesis, using a rodent model, it was dramatically enhanced in precancerous nodules as well as in cancer lesions but not in adjacent nontumor tissues (29)
. However, in cases of human liver tissues, 1-6 FucT expression was unexpectedly increased in chronic hepatitis, especially in liver cirrhosis as well as HCC (27
, 30)
. Thus, it is very likely that the levels of core-fucosylated glycoproteins are modified by as-yet-unknown mechanisms as well as by the direct up-regulation of 1-6 FucT gene expression. In cases of human hematopoietic cell lines, the critical regulator of cell surface sialylation was found to be an UDP-GlcNAc 2-epimerase, an enzyme that catalyzes an early rate-limiting step in the biosynthesis of CMP-NeuAc, the donor substrate nucleotide sugar and not the result of the up-regulation of sialyltransferase itself (31)
. Therefore, the issue of whether the level of biosynthesis of GDP-L-fucose, a sugar nucleotide, which is a common donor substrate for all fucosyltransferases, has a similar regulatory influence on the fucosylation in HCC tissues is of great interest.
The biosynthesis of GDP-L-fucose in mammals consists of two pathways, namely salvage and de novo pathways (Fig. 2)
. In the salvage pathway, GDP-L-fucose is synthesized by the action of an L-fucose kinase and GDP-L-fucose pyrophospholylase (32)
from free fucose, which is delivered to the cytosol either extracellularly or intracellularly. In the de novo pathway, GDP-L-fucose is synthesized from GDP-mannose via an oxidation, an epimerization, and a reduction. These steps are catalyzed by two enzymes, GDP-mannose-4,6-dehydratase (33
, 34)
and GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase. In humans, the latter enzyme is designated FX (35)
. It has been reported that FX-knockout mice exhibit virtually a complete deficiency of cellular fucosylation, demonstrating that the de novo pathway is the major route for cellular GDP-L-fucose biosynthesis in vivo (36)
. However, the direct analysis of GDP-L-fucose levels in cancer tissues has not been reported because of the lack of a convenient assay system for this sugar nucleotide. We have recently established a high-throughput assay that is suitable for the measurement of GDP-L-fucose levels in a variety of biological samples (37)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunostaining for Core-Fucosylated Glycoproteins in Human Liver Tissues
Immunostaining was performed using a CAB4 antibody (a generous gift from Dr. Hudson H. Freeze, Burnham Institute, La Jolla, CA), which is a IgG monoclonal antibody raised against core-fucosylated glycoproteins. Detailed analyses of this antibody have been reported previously (38)
. The antibody does not recognize fucose residues in Lewis a, Lewis b, Lewis y, 3-sialyl Lewis a, and 3-sialyl Lewis x. However, Lewis x, Fuc1,3GlcNAcß, Fuc1,4GlcNAcß, and Fuc1,2Galß1,3GlcNAcß are very weakly recognized by this antibody. This weak reactivity for the different fucosylated glycans suggests that not only the fucose residues but the surrounding glycans and linkages are also important for recognition by the CAB4 antibody. Liver tissues, embedded in Tissue-Tek OCT compound (Sakura), were cut in 6-µm thick sections with a cryostat. Sections were air dried and washed in ice cold PBS three times for 5 min each. The following immunostaining procedures were performed according to the manufacturer’s recommended protocols, with minor modifications using the Dako LSAB 2 kit. Briefly, endogenous peroxidase was blocked using hydrogen peroxide included in the kit. The section was washed in ice-cold PBS twice, 10 min each, and the Dako Biotin Blocking System was introduced to minimize the background avidin and biotin. The sections were washed again with ice-cold PBS twice, 10 min each. Liver sections were incubated overnight with 1/100 diluted CAB4 in the Dako Antibody Diluent with Background Reducing Components. The sequential procedures were performed according to the recommended protocols, and 3,3'-diaminobenzidine was used as a chromogen with hematoxylin as the counterstain.
Cell Culture
Human hepatoma cell lines, Hep3B and HepG2 cells, as well as a hepatocyte lineage, Chang liver cells, were obtained from the American Type Culture Collection (Manassas, VA). Human hepatoma cell lines, HLE and HLF, were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University. These cells were cultured in an incubator under 5% CO2 at 37°C. Hep3B were cultured in RPMI 1640 (Sigma, St. Louis, MO). HepG2, Chang liver cell, HLE, and HLF were cultured in high glucose DMEM (Sigma). Both RPMI and DMEM were supplemented with 100 µg/ml kanamycin, 50 units/ml penicillin, and 10% FCS.
Measurement of Cytosolic GDP-L-Fucose Concentration
Preparation of Cell Lysates and Tissue Homogenates.
The supernatants of microsomal fraction from tissues and cells were prepared for measuring GDP-L-fucose as described in our previous report (37)
. These fractions were quantitated for protein concentration using a BCA kit (Pierce, Rockford, IL) and used as sources for GDP-L-fucose.
Preparation of Samples for GDP-L-Fucose Determinations.
In a typical experiment, 40 µg of the above protein fractions were adjusted to a volume of 18 µl with chilled autoclaved water and then boiled at 100°C for 20 s to inactivate endogenous enzymes that use or degrade GDP-L-fucose. The samples were then cooled in an ice bath for 2 min. After the addition of 5.5 µl of ice-cold 200 mM MES-NaOH (pH 7.0), the samples were centrifuged at 15000 rpm for 15 min at 4°C. The collected samples were mixed with 1 µl of 10% Triton X-100, 0.5 µl of acceptor molecule, GnGn-bi-Asn-PABA (PABA,4-(2-pyridylamino) butylamine) (39)
and 5 µl of purified 1-6 FucT. The mixtures were incubated at 37°C for 2 h to allow the contained GDP-L-fucose to attach to the fluorescence-labeled acceptor molecule by the action of 1-6 FucT. The reactions were then terminated by boiling at 100°C for 1 min. The samples were then centrifuged at 15000 rpm for 10 min, and 10 µl of the 35-µl supernatants were subjected to a high-performance liquid chromatography. Finally, the areas of fluorescence intensity of the core fucosylated acceptor substrates were converted to GDP-L-fucose concentrations using a standard curve of 0–10 pmol GDP-L-fucose.
Northern Blot Analysis
Total RNAs were prepared from liver tissues and cells according to the method reported by Chomczynski and Sacchi (40)
. Twenty µg of RNAs were subjected to Northern blot analysis. The 1-6 FucT probe was a 909-bp cDNA fragment prepared by digesting the 1-6 FucT cDNA (11)
with HindIII. The probes prepared for FX, GMD, and GDP-L-fucose pyrophosphorylase were 506-, 520-, and 464-bp DNA fragments prepared by PCR using the spleen cDNA library (Clontech) as a template and the following primers based on published sequences [GMD, 5'-GCAGTGAACGGCATTCTCTTC-3' and 5'-CTCAGGCATTGGGGTTTGT-3' (34)
; FX, 5'-CAAGACGACCTACCCGATAGA-3'; 5'-GCTTGCTGTTACTGGCTGTC-3' (35)
; GDP-L-fucose pyrophosphorylase, 5'-GCATTTTCCCAGTTTGTTCTT3'; and 5'-CAATGCTCCACAAATCAATCA-3' (32)
]. These PCR fragments were analyzed by sequencing. The detailed procedures for Northern blot were performed according to our previous study (27)
. The mRNA bands for these enzymes were quantitated by densitometry (Microcomputer Imaging Device, version 2.1- 3.9, Imaging Research Inc.).
Transfection of FX cDNA Gene
The human FX cDNA was prepared by PCR using the spleen cDNA library (Clontech) as a template and the primers based on published sequences (5'-GACATGGGTGAACCCCAGGGA-3' and 5'-GCTTCACTTCCGGGCCTGCTC-3'; Ref. 35
). The obtained DNA fragment was directly inserted into the pTarget Mammalian Expression Vector (Promega, Madison, WI), which is driven by a cytomegalovirus promoter. Then the vector insertion was confirmed by sequencing. For the stable transfection of the FX gene, 1 µg of this vector was transfected to a human hepatoma cell line, Hep3B cells, by a modified polycationic transfection method using the Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s recommendation. Selection was performed by the addition of 600 µg/ml G418 (Sigma).
Lectin Blot Analyses of Core-fucosylated Oligosaccharides
Hep3B cell lysates were prepared for SDS-PAGE according to our previous study (27)
. Duplicate samples of 8 µg were subjected to 10% SDS-PAGE under reducing conditions. One gel was subjected to standard Coomassie Brilliant Blue R-250 staining and the other was transferred to a polyvinylidine difluoride membrane (Millipore) used for AAL lectin blot. AAL lectin interacts preferentially with oligosaccharides that contain a fucosyl residue attached to the innermost N-acetylglucosamine of N-glycans that contain two -mannosyl residues, namely core-fucosylated glycoproteins, although this lectins interacts weakly with 1-2/1-3-fucosylated glycoproteins (41)
. Sequential procedures were done as previously reported (27)
, and final results were visualized using an enhanced chemiluminescence system (Amersham) according to the manufacturer’s protocols.
Immunoelectrophoresis of Core-fucosylated AFP Proportion from Culture Media of Hepatoma Cell Cultures
Core-fucosylated AFP, designated the L3 fraction in conditioned media of hepatoma cells, was measured using an AFP Differentiation Kit L (Wako Pure Chemical Industries Ltd., Osaka, Japan; Ref. 25
). The concentration of total AFP levels from cell culture media were measured by the Immuno Radio Metric Assay (IRMA) method (Otsuka Assay Laboratory, Osaka, Japan). In this study, 2 µl of culture media containing recommended levels of AFP were electrophoresed on an agarose gel containing LCA lectin. The LCA lectin interacts preferentially with N-glycans that contain core fucose (42)
. The following procedures and determination of the results were done according to the manufacturer’s protocols.
Statistical Analyses
The results are expressed as the mean ± SD. Differences between the two groups were examined by the unpaired t test and paired t test. If two groups could not be considered to be of equal variance, the t test with Welch’s correction was performed. In the comparison of variables in more than two groups, one-way ANOVA was performed, followed by Fisher’s protected least significant difference (PLSD) test if the former was significant. In addition, nonparametric analyses were performed, in part, by a Krauskal-Wallis analysis. All comparisons of variables in more than two groups were done by one-way ANOVA unless stated as a Krauskal-Wallis analysis. Ps < 0.05 were considered to be significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The hemagioma tissue sections, which were used as a negative control for chronic liver disease (Fig. 3A)
, showed a weak positive staining on putative smooth muscle cells that line the hepatic hemangioma (indicated by arrows) but not in hepatocytes. Among the 4 HCC liver sections determined, strong positive stainings were observed for 2 HCCs (Fig. 3, B and C)
sections, moderate positive staining for 1 HCC, and negative staining for one HCC (data not shown) in comparison with these control sections. In contrast, positive stainings were not observed in adjacent nontumor tissues either with chronic hepatitis (Fig. 3D)
(adjacent nontumor tissue of Fig. 3B
) or liver cirrhosis (Fig. 3E)
(adjacent nontumor tissue of Fig. 3C
).
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using our assay system (37)
. The mean ± SD for GDP-L-fucose was 3.6 ± 0.2 µmol/mg in control liver (n = 5), 4.6 ± 0.9 µmol/mg in adjacent noninvolved liver tissues (n = 27) [CH (n = 13), 4.4 ± 0.7 µmol/mg; LC (n = 14), 4.8 ± 0.9 µmol/mg], and 7.1 ± 2.5 µmol/mg in HCC tissues (n = 23). The levels of GDP-L-fucose in normal livers were significantly lower than those in adjacent nontumor tissue and HCC (P < 0.0001 by a Kruskal-Wallis analysis). The GDP-L-fucose levels in HCC tissues were significantly increased compared with adjacent nontumor tissues or normal livers (HCC versus adjacent tissue, P = 0.0148; HCC versus normal, P = 0.0054). Although the reason for this is presently unknown, the level of GDP-L-fucose in HCC tissues decreased in proportion to the tumor size (n = 23, r = -0.675, P = 0.0002; Fig. 5
).
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. The gene expression of FX was elevated in a HCC-specific manner, whereas the gene expression of GDP-L-fucose pyrophospholylase and GMD were increased in HCC as well as in normal controls. Thus, we focused thereafter on FX, which is responsible for the last two steps of the de novo biosynthesis of GDP-L-fucose. To better understand the frequency and magnitude of the expression levels of FX in contrast with 1-6 FucT in human liver tissues, Northern blot analyses on FX and 1-6 FucT mRNA were performed on 10 HCC patients and 2 control subjects. The up-regulation of FX level was observed in 70% of the HCC tissues (7 of 10; Fig. 7
). Importantly, strong up-regulation of FX was not observed in any of the adjacent nontumor tissues of these patients. In contrast, 1-6 FucT mRNA was elevated in 80% of the HCC tissues (8 of 10) but was also elevated in 70% of the adjacent nontumor tissues (7 of 10) from these patients. The magnitude of FX expression levels, as analyzed densitometerically, was also significantly higher in HCC than in adjacent nontumor tissues (P = 0.0314, by unpaired t test; P = 0.498, by paired t test; Fig. 8A
). On the other hand, the difference in 1-6 FucT expression levels between HCC and adjacent nontumor tissues was not significant (Fig. 8B)
. GDP-L-fucose levels and the expression of FX mRNA using data from 18 liver specimens (2 normal tissues, 10 adjacent nontumor tissues, and 6 HCC tissues) exhibited a positive correlation (r = 0.599, P = 0.0074; Fig. 9
).
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1-6 FucT were investigated in HLE, HLF, Hep3B, HepG2, and Chang liver cell by Northern blot. The representative result from Northern blot analyses of three independent experiments are presented in Fig. 10
. Endogenous FX mRNA expression was high in HepG2 and Chang liver, moderate in HLF and Hep3B, and at the level in HLE was similar to that of a normal liver tissue. The level of endogenous 1-6 FucT mRNA showed a similar pattern to that of FX mRNA expression, except for Chang liver cells. To address the above issue, Hep3B cell was chosen for a transfection experiment because the endogenous level of 1-6 FucT was the lowest and endogenous FX was relatively low among the hepatoma cell lines examined. HLE was not chosen because it does not secrete AFP. Under phase contrast light microscope examination, FX-transfected Hep3B cells showed similar morphology compared with parent/mock cells (data not shown). Transfection of FX cDNA into the hepatoma cell line, Hep3B, caused an increase in GDP-L-fucose levels 1.73 fold (parent, 4.4 ± 0.76 µmol/mg; mock, 4.8 ± 0.16 µmol/mg; and FX, 8.3 ± 0.64 µmol/mg) compared with mock (parent/mock versus FX, P < 0.01; Fig. 11A
). The representative result of three independent experiments for the level of core-fucosylated AFP by immunoelectrophoresis using the AFP Differentiation Kit L is shown in Fig. 11B
. Although the activities of 1-6 FucT were similar among these cells (data not shown), the dramatic decrease in noncore-fucosylated AFP (designated the L1 fraction) by FX transfectants suggested an increased fucosylation potential by the introduction of the FX gene. The results of densitometric analyses for the core-fucosylated proportion of AFP (namely the L3 fraction) to total AFP are summarized in Fig. 11C
. Regardless of the basal core fucosylation of AFP in Hep3B, the transfection of FX consistently up-regulated the core fucosylation of AFP (parent/mock versus FX, P < 0.05). Finally, the impact of FX transfection on cellular glycoproteins was examined by an AAL lectin blot. As shown in Fig. 12A
, overall protein patterns are similar among the parent/mock and FX transfectants. However, in the case of the FX transfectants, an enhanced intensity of AAL bindings was observed over a wide range of glycoproteins compared with mock or controls, suggesting an increase of core fucosylation in the FX transfectants (Fig. 12B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During intestinal development in the weaning period of rats, fucosyltransferase activities are elevated along with an increase in GDP-L-fucose levels. It has been proposed that the increase in levels of this sugar nucleotide occur through the elevation of GDP-4-dehydro-6-deoxymannose-epimerase-reductase (the human homologue is FX; Ref. 44 ). In terms of cancer biology, cellular GDP-L-fucose levels were related to cell proliferation. Although in Hep3B cells, increased GDP-L-fucose levels by overexpression of FX did not affect the rate of cell growth significantly as judged by cell proliferation assay (data not shown), a high level of GDP-L-fucose has been reported as a characteristic feature of rapidly growing clones of Morris hepatomas (45) . Thus, comprehensive effects of increased GDP-L-fucose levels on proliferation of hepatoma cell lines are still open to discussion. More recently, intimate relationships between FX expression level and the expression of sialyl Lewis a or sialyl Lewis x, well-known selectin ligands that contain fucose residues, have been reported. Namely in head and neck tumors, extracellular signaling by the E48 antibody directed against an E48 receptor, a type of cell membrane glycosylphosphatidyl inositol anchor protein that belongs to the member of the Ly 6 gene family, induced FX (46) , sialyl-, and fucosyl-transferase (47) expression and caused increase in sialyl Lewis a production. In both T- and B-cell lymphocytes, FX expression was shown to increase upon their activation and sialyl Lewis x levels were increased (48) . Conversely, transfection of the antisense FX oligonucleotide to both head/neck tumors and T cells reduced the expressions of sialyl Lewis a (47) and sialyl Lewis x (48) , respectively.
The specific increase in core-fucosylated AFP in patients with HCC has long been considered to be a reflection of the differential up-regulation of 1-6 FucT in HCC. Consistently, immunohistochemical analyses of core-fucosylated glycoproteins from human tissues suggested a predominance of core-fucosylated glycoproteins in HCC. However, although 1-6 FucT catalyzes the core fucosylation of glycoproteins such as AFP, a high expression of 1-6 FucT was observed in HCC as well as their surrounding chronic liver disease tissues (27
, 30)
. This discrepancy to the above notion cannot be explained by the differential expression of a yet unknown 1-6 FucT isozyme because this 1-6 FucT-knockout mouse is deficient of core-fucosylated glycoproteins in all organs,4
denying the existence of an enzyme family analogues to other fucosyltransferases (49)
. Therefore, certain mechanisms for the accumulation of core-fucosylated proteins in HCC could be suggested besides the elevation in 1-6 FucT expression.
The significant increase of GDP-L-fucose levels in HCC tissues in comparison with adjacent nontumor tissues represents a possible explanation for the predominance of core fucosylation in hepatoma tissues. Because GDP-L-fucose is synthesized in the cytosol and must be transported to the Golgi to participate in the fucosylation of glycans, GDP-L-fucose transporter levels might alternatively regulate core fucosylation. However, the expression levels of this transporter have been examined only in the normal human tissues and detailed analyses on cancer tissues and cancer cell lines have not been performed. Although the expression of this transporter is reported to be the highest in muscles and liver among various human organs (50) , additional investigations are necessary to elucidate whether the differential expression of this transporter is responsible, in part, for the difference in fucosylation levels between HCC and adjacent nontumor tissues.
Considering our present results on Northern blots of enzymes responsible for GDP-L-fucose biosynthesis, FX appeared to increase in a highly tumor-specific manner. Furthermore, we found a causative link between FX expression and GDP-L-fucose levels. Although the 5'-flanking region of this gene has not been documented and the precise mechanism by which FX gene expression is elevated during hepatocarcinogenesis as well as the above situations (46, 47, 48)
remains unknown, transfection of the FX gene into Hep3B caused an increase in glycoproteins, which bind to AAL lectin (Fig. 12)
rather than LCA lectin (data not shown). The weak binding to 1-2/1-3-fucosylated glycoproteins, besides a strong interaction with core fucosylated glycoprotein, is relatively stronger in AAL compared with LCA (41
, 42)
. This suggests that an increased level of GDP-L-fucose leads to an elevation in both core-fucosylated and 1-2/1-3-fucosylated glycoproteins. More convincingly, significant increases in cell surface sialyl Lewis X [NeuAc2,3Galß1,4(Fuc1,3)GlcNAc] and Lewis X [Galß1,4(Fuc1,3)GlcNAc] were observed by fluorescence-activated cell sorting analysis, using well-characterized monoclonal antibodies, clones KM-93 (51)
and P-12 (52)
, respectively (data not shown). Thus, an increased level of GDP-L-fucose by transfection of FX alone may promote malignant potential of well-established colorectal (4)
and lung (5)
carcinoma cell lines in which malignancy has been defined by degree of these fucosylated oligosaccharide structures.
Collectively, our results suggest that a high expression of FX followed by increases in GDP-L-fucose levels contribute to the elevated core fucosylation in HCC rather than 1-6 FucT. These findings will facilitate our understanding of the complexities of glycoprotein and glycolipids fucosylation in which various fucosyltransferases, acceptors, and GDP-donor synthesis levels are involved. Toward the end, elevation in GDP-L-fucose and FX expression could represent potential markers for HCC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported, in part, by Grants-in-Aid for Scientific Research on Priority Areas 10178104, 15025238 and by the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan and also by Research Fellowship Division of the Japan Society for the Promotion of Science. 
2 To whom requests for reprints should be addressed, at Department of Molecular Biochemistry and Clinical Investigations, Osaka University, Graduate School of Medicine, 1-7 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-2589; Fax: 81-6-6879-2589; E-mail: miyoshi{at}sahs.med.osaka-u.ac.jp
3 The abbreviations used are: 1-6 FucT, GDP-L-fucose:N-acetyl-ß-D-glucosaminide 1-6 fucosyltransferase; GlcNAc, N-acetylglucosamine; GMD, GDP-mannose-4,6-dehydratase; HCV, hepatitis C virus; FX, human homologue of GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase, GDP-L-fucose synthase; AAL, Aleuria aurantica; LCA, Lens culinaris agglutinin; CH, chronic hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma; AFP, -fetoprotein.
Received 3/28/03. Revised 7/10/03. Accepted 7/22/03.
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E. Miyoshi, K. Moriwaki, and T. Nakagawa Biological Function of Fucosylation in Cancer Biology J. Biochem., June 1, 2008; 143(6): 725 - 729. [Abstract] [Full Text] [PDF]
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 K. Moriwaki, K. Noda, T. Nakagawa, M. Asahi, H. Yoshihara, N. Taniguchi, N. Hayashi, and E. Miyoshi A High Expression of GDP-Fucose Transporter in Hepatocellular Carcinoma is a Key Factor for Increases in Fucosylation Glycobiology, December 1, 2007; 17(12): 1311 - 1320. [Abstract] [Full Text] [PDF]
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 K. Matsumura, K. Higashida, H. Ishida, Y. Hata, K. Yamamoto, M. Shigeta, Y. Mizuno-Horikawa, X. Wang, E. Miyoshi, J. Gu, et al. Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae: A NOVEL PROBE FOR CORE FUCOSE J. Biol. Chem., May 25, 2007; 282(21): 15700 - 15708. [Abstract] [Full Text] [PDF]
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S. Barrabes, L. Pages-Pons, C. M. Radcliffe, G. Tabares, E. Fort, L. Royle, D. J. Harvey, M. Moenner, R. A. Dwek, P. M. Rudd, et al. Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer Glycobiology, April 1, 2007; 17(4): 388 - 400. [Abstract] [Full Text] [PDF]
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 T. Sasamura, H. O. Ishikawa, N. Sasaki, S. Higashi, M. Kanai, S. Nakao, T. Ayukawa, T. Aigaki, K. Noda, E. Miyoshi, et al. The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila Development, April 1, 2007; 134(7): 1347 - 1356. [Abstract] [Full Text] [PDF]
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Y. Zhao, S. Itoh, X. Wang, T. Isaji, E. Miyoshi, Y. Kariya, K. Miyazaki, N. Kawasaki, N. Taniguchi, and J. Gu Deletion of Core Fucosylation on {alpha}3beta1 Integrin Down-regulates Its Functions J. Biol. Chem., December 15, 2006; 281(50): 38343 - 38350. [Abstract] [Full Text] [PDF]
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B. Ma, J. L. Simala-Grant, and D. E. Taylor Fucosylation in prokaryotes and eukaryotes Glycobiology, December 1, 2006; 16(12): 158R - 184R. [Abstract] [Full Text] [PDF]
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T. Nakagawa, N. Uozumi, M. Nakano, Y. Mizuno-Horikawa, N. Okuyama, T. Taguchi, J. Gu, A. Kondo, N. Taniguchi, and E. Miyoshi Fucosylation of N-Glycans Regulates the Secretion of Hepatic Glycoproteins into Bile Ducts J. Biol. Chem., October 6, 2006; 281(40): 29797 - 29806. [Abstract] [Full Text] [PDF]
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X. Wang, J. Gu, H. Ihara, E. Miyoshi, K. Honke, and N. Taniguchi Core Fucosylation Regulates Epidermal Growth Factor Receptor-mediated Intracellular Signaling J. Biol. Chem., February 3, 2006; 281(5): 2572 - 2577. [Abstract] [Full Text] [PDF]
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 X. Wang, S. Inoue, J. Gu, E. Miyoshi, K. Noda, W. Li, Y. Mizuno-Horikawa, M. Nakano, M. Asahi, M. Takahashi, et al. From The Cover: Dysregulation of TGF-{beta}1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice PNAS, November 1, 2005; 102(44): 15791 - 15796. [Abstract] [Full Text] [PDF]
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L. Sturla, F. Fruscione, K. Noda, E. Miyoshi, N. Taniguchi, P. Contini, and M. Tonetti Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts Glycobiology, October 1, 2005; 15(10): 924 - 934. [Abstract] [Full Text] [PDF]
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 K. F. Aoki, H. Mamitsuka, T. Akutsu, and M. Kanehisa A score matrix to reveal the hidden links in glycans Bioinformatics, April 15, 2005; 21(8): 1457 - 1463. [Abstract] [Full Text] [PDF]
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T. M. Block, M. A. Comunale, M. Lowman, L. F. Steel, P. R. Romano, C. Fimmel, B. C. Tennant, W. T. London, A. A. Evans, B. S. Blumberg, et al. Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans PNAS, January 18, 2005; 102(3): 779 - 784. [Abstract] [Full Text] [PDF]
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 A. Zipin, M. Israeli-Amit, T. Meshel, O. Sagi-Assif, I. Yron, V. Lifshitz, E. Bacharach, N. I. Smorodinsky, A. Many, P. A. Czernilofsky, et al. Tumor-Microenvironment Interactions: The Fucose-Generating FX Enzyme Controls Adhesive Properties of Colorectal Cancer Cells Cancer Res., September 15, 2004; 64(18): 6571 - 6578. [Abstract] [Full Text] [PDF]
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K.-S. Ryu, C. Kim, I. Kim, S. Yoo, B.-S. Choi, and C. Park NMR Application Probes a Novel and Ubiquitous Family of Enzymes That Alter Monosaccharide Configuration J. Biol. Chem., June 11, 2004; 279(24): 25544 - 25548. [Abstract] [Full Text] [PDF]
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