Glycobiology Advance Access originally published online on September 20, 2007
Glycobiology 2007 17(12):1311-1320; doi:10.1093/glycob/cwm094
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A High Expression of GDP-Fucose Transporter in Hepatocellular Carcinoma is a Key Factor for Increases in Fucosylation
2 Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, 1-7, Yamada-oka, Suita, Osaka 565-0871, Japan
3 Osaka Rosai Hospital, 1179-3, Nagasone-cho, Kita-ku, Sakai, Osaka 591-8025, Japan
4 Departments of Glycotherapeutics Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan
5 Biochemistry Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan
6 Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan
7 Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Center for Advanced Science & Innovation, 2-1, Yamada-oka Suita, Osaka 565-0871, Japan
1 To whom correspondence should be addressed: Tel and Fax: +81-6-6879-2590; e-mail: emiyoshi{at}sahs.med.osaka-u.ac.jp
Received on April 27, 2007; revised on August 9, 2007; accepted on August 31, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Changes in the levels of fucosylation regulate the biological phenotype of cancer cells and a specific fucosylation, such as fucosylated
-fetoprotein (AFP-L3) has been clinically used as a tumor marker for hepatocellular carcinoma (HCC). However, detailed molecular mechanisms that explain the increased fucosylation in HCC remain unknown despite 10 years of study by these researchers. Fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter (GDP-Fuc Tr), and synthetic enzymes of GDP-fucose, such as GDP-mannose 4, 6-dehydratase (GMD), GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase (FX), and GDP-fucose pyrophosphorylase. In this study, the expression of fucosylation-related genes in HCC tissues was studied and it was found that GDP-Fuc Tr is a key factor for increases in fucosylation. A real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed significant increases in GDP-Fuc Tr and FX expression in HCC, and levels of the GMD protein were upregulated by posttranslational modification in HCC tissues. In vitro cell experiments showed that the level of GDP-Fuc Tr was the most significantly correlated with the level of cellular fucosylation and the overexpression of GDP-Fuc Tr dramatically increased fucosylation in Hep3B cells. The importance of GDP-Fuc Tr in the increase of fucosylation was also confirmed with immunohistochemical analyses. These findings suggest that the upregulation of GDP-Fuc Tr plays a pivotal role in increased fucosylation in HCC and represents an attractive target for new treatments and diagnosis for HCC. Key words: AFP-L3 / fucose / GDP-fucose / GDP-fucose transporter / hepatocellular carcinoma
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Oligosaccharides are one of the most important factors in the posttranslational modification of proteins. It is a well-known fact that oligosaccharide structures are changed during malignant transformations (Hakomori et al. 1989
). Therefore, oligosaccharides have been used as bio-markers for various types of tumors and have the potential to become targets for effective treatment. While alpha-fetoprotein (AFP) has been clinically used as a tumor marker for hepatocellular carcinoma (HCC) (Alpert et al. 1968), its levels are also increased in chronic liver diseases, such as chronic hepatitis (CH) and liver cirrhosis (LC). Therefore, it is difficult to make a diagnosis of HCC that is differential from chronic liver diseases based on low or medial elevations of AFP – making it of questionable value as a tumor marker (Chen et al. 1977; Ebara et al. 1986). Under these circumstances, fucosylated AFP (AFP-L3 fraction) is more effective for the specific diagnosis of HCC because it increases in patients with HCC, but not CH and LC (Aoyagi et al. 1985; Taketa et al. 1993). The measurement of fucosylated AFP for the diagnosis of HCC has been clinically applied in Japan since 1996 and was approved by the Food and Drug Administration (FDA) in the United States in 2005.
This research group previously succeeded in the purification and cDNA cloning of 1-6 fucosyltransferase (1-6 FucT), which catalyzes the transfer of a fucose residue to the reducing end GlcNAc in complex N-glycans via an 1-6 linkage (Uozumi et al. 1996; Yanagidani et al. 1997). 1-6 FucT is responsible for the fucosylation of AFP. The expression level of 1-6 FucT mRNA was found to be increased in hepatic tumor lesions, but not in adjacent lesions in a hepatocarcinogenic model rat, the Long-Evans with cinnamon-like coat color rat (LEC rat) (Noda et al. 1998). However, in human liver, 1-6 FucT was increased in both HCC tissues and the adjacent tissues, which exhibited CH or LC (Noda et al. 1998). These results indicate that the fucosylation of AFP in HCC is regulated not only by the direct upregulation of 1-6 FucT, but also by other mechanisms.
Guanosine 5'-diphosphate (GDP)-fucose is a donor substrate common to all fucosyltransferases, including 1-6 FucT, and its level is increased in HCC (Noda et al. 2003). GDP-fucose is synthesized in the cytosol via two pathways, namely the salvage pathway and the de novo pathway (Figure 1). The salvage pathway synthesizes GDP-fucose from free L-fucose derived from extracellular or lysosomal sources via two steps: catalyzation by L-fucokinase (Park et al. 1998) and GDP-fucose pyrophosphorylase (Pastuszak et al. 1998). The de novo pathway transforms GDP-mannose into GDP-fucose via three steps: catalyzation by GDP-mannose 4, 6-dehydratase (GMD) (Ohyama et al. 1998; Sullivan et al. 1998) and GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase, also known as FX (Tonetti et al. 1996). The salvage pathway contributes to only about 10% of the cellular pool of GDP-fucose. Thus, the de novo pathway mainly produces cellular GDP-fucose. This research group previously established a unique method for the determination of GDP-fucose levels in cytosolic fractions using isocratic reverse phase high performance liquid chromatography (HPLC) (Noda et al. 2002). Using this method and Northern blot analysis, we found that both the levels of GDP-fucose and the expression of FX mRNA were significantly increased in HCC tissue compared with adjacent tissue or the tissue of normal livers (Noda et al. 2003). However, the level of cellular fucosylation did not always correlate with these parameters.
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While fucosylation catalyzed by fucosyltransferases takes place in the Golgi apparatus, GDP-fucose is synthesized in the cytosol. Therefore, GDP-fucose must be transported by a GDP-fucose transporter (GDP-Fuc Tr), a transmembrane protein in the Golgi, to serve as a substrate of fucosyltransferases. Kumamoto et al. reported that the expression level of UDP-galactose transporter mRNA was significantly increased in colon cancer tissues compared with nonmalignant mucosal tissues, resulting in a possible enhancement of metastatic capacity (Kumamoto et al. 2001
). This study showed that the UDP-galactose transporter could regulate the influx of UDP-galactose from the cytosol into the Golgi lumen, leading to the modulation of oligosaccharide structures via reactions of galactosyltransferases. These facts suggest that a transporter as well as synthetic enzymes of GDP-fucose are likely to play pivotal roles in the regulation of fucosylation.
Zipin et al. proposed that GDP-fucose controls the metastatic capacity of colorectal cancer and could become a target for metastatic prevention (Zipin et al. 2004). In this study, the expression levels of genes responsible for the transport and synthesis of GDP-fucose in HCC tissues were investigated, and the main contributers to the fucosylation of HCC were determined by in vitro experiments. Furthermore, these results were confirmed with an immunohistochemical study using 59 cases of HCC tissue. GDP-Fuc Tr was found to play the most critical role in the fucosylation of HCC among many factors, including fucosyltransferases and GDP-fucose synthetic enzymes.
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Results |
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Levels of fucosylation-related genes among normal livers, HCC tissues, and adjacent tissues
The expression of GDP-Fuc Tr, FX, GMD, GDP-fucose pyrophosphorylase and
1-6 FucT mRNA was investigated by real-time RT-PCR analysis using 13 human HCC tissues and two normal liver tissues (Figure 2). The expression of GDP-Fuc Tr and FX were significantly increased in HCC compared with chronic liver disease (CH and LC) or normal liver (HCC versus chronic liver disease, P = 0.035 and 0.019, HCC versus normal liver, P = 0.022 and <0.01, respectively, by one-way ANOVA followed by the protected least significant differences (PLSD) test). In contrast, no significant differences were observed in the expression of GMD, GDP-fucose pyrophosphorylase, and 1-6 FucT among HCC, chronic liver disease, and normal liver. In individual cases, GDP-Fuc Tr and FX were also significantly increased in HCC compared with the adjacent tissue (P = 0.036 and <0.01, respectively, by the paired t test) (Supplementary data, Figure 1). None of the other genes showed significant differences. In all the genes investigated, no significant difference was observed between CH and LC, suggesting that the upregulation of fucosylation-related genes is not associated with the progression of chronic liver diseases. The expression of fucosylation-related genes, except for GDP-fucose pyrophosphorylase, in chronic liver diseases was increased compared with normal livers, but the increases were not significant due to the small number of normal liver samples (3 patients). A study using a larger number of samples would allow us to detect statistically significant differences between chronic liver disease and normal liver. These results indicate that GDP-Fuc Tr is increased in HCC compared with chronic liver disease, and might be involved in the increased fucosylation in HCC.
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Western blot analysis of GMD
GMD is one of the most important rate limiting enzymes in the de novo pathway of GDP-fucose synthesis. While the results of real-time RT-PCR using human HCC tissues did not show a significant increase in the level of GMD mRNA, we investigated the expression level of the GMD protein by western blot analysis. As shown in Figure 3A, the expression level of the GMD protein was markedly increased in HCC tissues. Moreover, the expression level of GMD protein in HCC was increased compared with chronic liver disease and normal liver, as analyzed densitometerically (HCC versus chronic liver disease, P < 0.01, HCC versus normal liver, P = 0.014, by one-way ANOVA followed by the PLSD test) (Figure 3B). The protein and mRNA levels of GMD were not correlated, particularly in cases of a low expression of its mRNA, suggesting that unknown posttranslational regulations are responsible for the increase of GMD protein in HCC (Figure 3C). These results indicate that the increased level of GDP-fucose in HCC is due to the upregulation of the expression levels of FX and GMD, followed by activation of the de novo pathway.
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Fucosylation level of hepatoma cells in terms of GDP-fucose transporter, FX, and GMD
To determine which factors are important for the increased fucosylation among GDP-Fuc Tr, FX, and GMD, we compared the expression levels of these molecules with the fucosylation levels in five hepatoma cells (Hep3B, HepG2, HLE, HLF, and Huh7). We first determined the expression levels of GDP-Fuc Tr, FX, and GMD in hepatoma cells by either real-time RT-PCR analysis or western blot analysis (Figure 4A and B). The expression level of the GDP-Fuc Tr was higher in HepG2 and Huh7 cells, but lower in Hep3B, HLE, and HLF cells. The expression levels of FX and GMD were higher in Hep3B, HepG2, and Huh7 cells, but lower in HLE and HLF cells. We subsequently performed a lectin blot analysis with Aleuria aurantia lectin (AAL) to investigate the fucosylation level in each of the hepatoma cells. The AAL lectin recognizes fucosylated oligosaccharides. In HepG2 and Huh7 cells, numerous proteins were strongly fucosylated, but in Hep3B, HLE, and HLF cells, the fucosylation was weak (Figure 4C). These results suggest that the upregulation of GDP-Fuc Tr is much more important for increases in fucosylation.
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Effect of the addition of exogenous L-fucose on hepatoma cell line, Hep3B
To demonstrate the direct involvement of GDP-fucose in the regulation of fucosylation in hepatoma cells, various concentrations of L-fucose were added to the condition medium of Hep3B cells. L-Fucose was incorporated into the cells and converted to GDP-fucose via the salvage pathway (Smith et al. 2002
; Luhn et al. 2004). After culturing for 72 h in the presence of L-fucose, cytosolic GDP-fucose was increased in proportion to the concentration of L-fucose in the condition medium and was increased by about 16-fold in the presence of 5000 µM L-fucose (Figure 5A). In the case of the presence of 100 µM L-fucose, no elevation in cytosolic GDP-fucose was detected. We subsequently analyzed the change in fucosylation level by means of an AAL lectin blot. As shown in Figure 5B, cellular fucosylation was not increased at all. When we cultured the cells in the presence of 5000 µM L-fucose for 4 weeks, we observed no increase in fucosylation (data not shown). Accordingly, even when cytosolic GDP-fucose was extremely increased, cellular fucosylation was not changed, suggesting that GDP-fucose levels are not a rate-limiting factor for the total cellular fucosylation in Hep3B cells.
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The effect of overexpression of GDP-fucose transporter on fucosylation level
To determine whether or not GDP-Fuc Tr is directly involved in regulating fucosylation in HCC, we investigated the effects of the GDP-Fuc Tr gene transfection on the level of fucosylation in Hep3B cells. Two transfectants of GDP-Fuc Tr were established through selection by G418 treatment (clone 1 and 2). Western blot analysis using anti-V5 antibody, which recognized a tag sequence in GDP-Fuc Tr expression vector, showed a high expression of GDP-Fuc Tr in the transfectants (Figure 6A). To investigate changes in the fucosylation levels, we performed a lectin blot analysis using AAL lectin. As shown in Figure 6B, fucosylation levels were dramatically increased in the transfectants. Clone 2, which expressed higher level of GDP-Fuc Tr than clone 1, showed a much higher increase in fucosylation. To deny secondary effects of overexpression of GDP-Fuc Tr on other fucosylation-related genes, expressions of FX and GMD were investigated by real-time RT-PCR analysis. As expected, expression of FX and GMD mRNA was not changed among transfectants of GDP-Fuc Tr, parent and mock (Supplementary data, Figure 2). These results show that GDP-Fuc Tr is able to efficiently upregulate the influx of GDP-fucose from the cytosol to the Golgi lumen followed by increases in the cellular fucosylation. Rather than cytosolic GDP-fucose, it is GDP-Fuc Tr that is mainly involved in the regulation of cellular fucosylation in Hep3B cells.
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Correlation between the expression of GDP-fucose transporter and the cellular fucosylation in HCC tissues
The expression level of GDP-Fuc Tr and how it relates to cellular fucosylation in vivo were investigated using tissue microarray (Figure 7A, B, and C). This microarray includes 59 HCCs of various stages. The HCC serial sections that showed positive stainings for AAL, GDP-Fuc Tr, and
1-6 FucT are shown in Figure 7D, E, and F. In contrast, the sections that showed positive stainings for AAL and GDP-Fuc Tr, but negative for 1-6 FucT are shown in Figure 7 G, H and I, indicating that the expression of GDP-Fuc Tr resulted in the increases of fucosylation even below the detectable level of 1-6 FucT. The positive stainings of GDP-Fuc Tr and 1-6 FucT were observed as a Golgi-localized pattern. Of the 59 cases, 38 cases showed AAL (+) and GDP-Fuc Tr (+), and 8 cases showed AAL (–) and GDP-Fuc Tr (–), indicating that the expression of GDP-Fuc Tr was highly correlated with the cellular fucosylation in vivo (Table IA). There was no correlation between 1-6 FucT and AAL stainings (Table IB). These results indicate that GDP-Fuc Tr directly regulates the cellular fucosylation in vivo and is the most important factor for the increases of fucosylation in HCC.
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Discussion |
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Alterations in the expression of fucosylated oligosaccharides are observed in a variety of biological and pathological conditions. Above all, fucosylated oligosaccharides have clearly been related to malignant tumors in numerous studies conducted over the past several decades (Tatsumura et al. 1977
). Therefore, an advanced understanding of the underlying mechanism of fucosylation would provide new strategies for the treatment and diagnosis for malignant tumors.
We previously reported that the expression level of FX was increased in HCC compared with chronic liver disease and normal liver, but that 1-6 FucT was not increased compared with chronic liver disease (Noda et al. 1998; Noda et al. 2003). However, which factors in fucosylation-related genes are responsible for the increases of fucosylation in HCC remains unknown. In the present study, the expression levels of fucosylation-related genes, such as FX and 1-6 FucT, GDP-Fuc Tr, GMD, and GDP-fucose pyrophosphorylase, were comprehensively investigated by real-time RT-PCR analyses. It was found that GDP-Fuc Tr is also significantly increased in HCC compared with chronic liver disease and normal liver. In a recent study by another group, the expression levels of FX, GMD, and GDP-Fuc Tr mRNA were reportedly increased in acute inflammation using a rat kidney allograft for a model system, but GDP-fucose pyrophosphorylase was not increased (Niittymaki et al. 2006). In the present study using human liver tissue, similar changes were observed during chronic inflammation. As shown in Figure 2B, the expressions of FX, GMD, and GDP-Fuc Tr were increased in chronic liver disease compared with normal livers, but the expression of GDP-fucose pyrophosphorylase was not increased. These results suggest that fucosylation-related genes such as GDP-Fuc Tr, FX, and GMD are involved in certain types of inflammation. However, the levels of GDP-Fuc Tr, FX, and GMD were much more increased in HCC tissues, indicating that these fucosylation-related factors are involved in carcinogenesis.
The increased expression of the GMD protein, which is independent of the upregulation of GMD mRNA, suggests the unknown posttranslational regulation of GMD in HCC (Figure 3). Nakayama et al. reported that in Arabidopsis thaliana, AtFX/ GER1 (A. thaliana homolog of human FX) contributed to the maintenance of MUR1 (A. thaliana homolog of human GMD) as the active form by forming a complex, resulting in the stabilization of MUR1 activity (Nakayama et al. 2003). In the case of human HCC, an increased expression of FX might increase the stabilization of GMD, resulting in increases in GDP-fucose levels. Further analyses of interactions between human FX and GMD could shed new light on our understanding of the regulatory mechanisms of fucosylation in HCC.
To know which factors are important for the increases of fucosylation in HCC, expression levels of GDP-Fuc Tr, FX, and GMD were compared with total cellular fucosylation in hepatoma cells. As shown in Figure 4, both HepG2 and Huh7 cells with high expression levels of GDP-Fuc Tr, FX, and GMD showed high fucosylation levels. In contrast, Hep3B cells showed a low fucosylation level in spite of the relatively high expression levels of FX and GMD because of the low expression level of GDP-Fuc Tr. Even when the expression levels of FX and GMD were high, cellular fucosylation did not increase appreciably, indicating that GDP-Fuc Tr could be a rate-limiting factor for fucosylation.
The extreme elevation of cytosolic GDP-fucose by the addition of a large excess of L-fucose in Hep3B cells resulted in no elevation in cellular fucosylation. On the other hand, the elevation of GDP-Fuc Tr could directly lead to a drastic elevation in fucosylation, which would mean, therefore, that GDP-Fuc Tr could effectively upregulate cellular fucosylation in Hep3B cells. These results indicate that cytosolic GDP-fucose has been already in abundance and available for the transport by GDP-Fuc Tr even before the addition of exogenous L-fucose. In a previous study, the overexpression of FX in Hep3B cells resulted in a slight elevation in cellular fucosylation (Noda et al. 2003). Overexpression of FX leads to the increase in cytosolic GDP-fucose via de novo pathway. However, even if cytosolic GDP-fucose is increased, cellular fucosylation does not change without enhancement of GDP-Fuc Tr expression (Figure 5). Therefore, the increase of fucosylation by overexpression of FX in our previous study might be due to an indirect effect of increases in FX expression, which is independent of GDP-fucose synthesis. Functional interaction among FX, GMD, and GDP-Fuc Tr should be investigated in further studies.
To investigate the more direct relationship between the expression of fucosylation-related genes and cellular fucosylation in many HCC tissues, an immunohistochemical study was performed. Positive stainings of AAL and GDP-Fuc Tr were observed in 42/59 cases and 47/59 cases, respectively, and their expression was highly correlated. In contrast, 12/32 cases of 1-6 FucT positive tissues showed negative/low staining for AAL, suggesting that GDP-fucose Tr, but not 1-6 FucT, regulates cellular fucosylation in HCC tissues. When AOL lectin, which recognizes 1-6 linked fucose more specifically (Matsumura et al. 2007), was used for this immunohistochemical study, a positive signal was weak due to the quality of tissue microarray and the data was almost the same with that of AAL (data not shown).
This study demonstrated the importance of the upregulation of GDP-Fuc Tr in cellular fucosylation using human HCC tissues and hepatoma cell lines. Since fucosylation could regulate the malignant potential of cancer cells, we conclude that GDP-Fuc Tr could serve as a new target for the advanced treatment and diagnosis of HCC.
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Materials and methods |
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Patients and liver tissue specimens
Surgical specimens were obtained from 13 HCC patients, aged 63 ± 3.4 years (9 males and 4 females), during surgical operations and were processed as described previously (Noda et al. 1998
). Of these patients, 7, 5, and 1 exhibited CH, LC, and normal liver in the adjacent tissue of HCC, respectively. In addition, 2 patients, aged 58.5, with liver metastasis of colon cancer who were serologically and histologically negative for any chronic liver disease were used as controls (1 male and 1 female). This research was approved by the ethical committee of the hospital. Tumor samples and adjacent liver tissues were obtained and stored in liquid nitrogen until used.
RNA extraction and real-time RT-PCR
RNA was extracted from frozen specimens of 13 patients, two normal livers and five hepatoma cells – Hep3B, HepG2, HLE, HLF, and Huh7 – with the TRIsol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The concentrations of all RNA samples were determined spectrophotometrically and stored at –80°C until used. RNA contained 1x M-MLV buffer, 0.5 mM dNTP mixture, 50 pmol Random 6 mers, 50 U M-MLV RTase and 10 U RNase inhibitor was adjusted to a volume of 10 µL with RNase-free distilled H2O and reverse-transcripted for 15 min at 42°C and 2 min at 95°C (TAKARA BIO, Siga, Japan). Each PCR product was then adjusted to a volume of a 25 µL solution containing 1xSYBR Premix Ex Taq, 0.2 µM forward and reverse primers. Real-time PCR analysis was carried out using the Smart Cycler System (TAKARA BIO). Primers for GDP-Fuc Tr, FX, GMD, GDP-fucose pyrophosphorylase, 1-6 FucT, hypoxanthine guanine phosphoribosyl transferase (HPRT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes used in this study are summarized in Table II. Both HPRT and GAPDH were used as an internal control. The results were normalized as relative values using HPRT for clinical samples and GAPDH for hepatoma cell lines as a reference to compare the mRNA expression.
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Antibody
Anti-GMD and anti-GDP-Fuc Tr polyclonal antibody was developed by immunization of the bovine thyroglobulin-conjugated C-terminated peptide of GMD and GDP-Fuc Tr into a rabbit followed by purification by protein G, respectively (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan). Mouse anti-V5, which is 14 amino acids epitope derived from the P and V proteins of the paramyxovirus, SV5, and anti-
1-6 FucT monoclonal antibody was obtained from Invitrogen and Fujirebio Inc. (Tokyo, Japan), respectively.
Western blot analysis
The cells were harvested from a 10 cm dish. After precipitation by centrifugation at 2000 rpm for 5 min at 4°C, they were resuspended in TNE buffer (10 mM Tris-HCl [pH 7.8], 1% NP40, 0.15 M NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA]) including a protease inhibitor cocktail (Roche, Basel, Switzerland), and then placed on ice for 30 min to allow solubilization. These samples were then centrifuged at 15,000 rpm for 15 min at 4°C and the supernatants were collected. These cell lysates were quantitated using a Bicinchoninic Acid kit (BCA kit, Pierce, Rockford, IL). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred to a polyvinylidine difluoride (PVDF) membrane (Millipore, Woburn, MA). After blocking with phosphate-buffered saline (PBS) containing 5% skim milk for 1 h at room temperature (RT), the membrane was incubated with rabbit anti-GMD polyclonal antibody or mouse anti-V5, which is 14 amino acids epitope derived from the P and V proteins of the paramyxovirus, SV5, monoclonal antibody for 1 h at RT. After washing the membrane twice with Tris-buffered saline, containing 0.05% Tween 20 (TBST) (pH 7.4), it was incubated with diluted horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling, Beverly, MA) or anti-mouse IgG (Promega, Madison, WI) for 1 h at RT. It was again washed two times and developed with an enhanced chemiluminescence system (ECL kit, GE Healthcare BioSciences, the Chalfont St. Giles, United Kingdom) according to the manufacturer's protocol. Immunoreactivity was quantified by scanning densitometry.
Measurement of cytosolic GDP-fucose concentration
Measurement of the cytosolic GDP-fucose concentration was performed as described previously with minor modifications (Noda et al. 2002). Cells harvested from a 10 cm dish were precipitated by centrifugation at 2000 rpm for 5 min at 4°C and resuspended in a 0.25 M sucrose buffer supplemented with 10 mM Tris-HCl (pH 7.4), 10 mM KCl, 10 mM MgCl2, 5 mM adenosine-5-monophosphate (AMP) (pH 7.4) (Wako, Osaka, Japan) and a protease inhibitor cocktail. The cells were homogenized with a Dounce homogenizer and then centrifuged at 500 x g for 5 min at 4°C to remove cell debris and nuclei. The supernatants were then subjected to ultracentrifugation (Optima TL Ultracentrifuge, TLA-45 rotor, Beckman, Fullerton, CA) at 40,000 rpm for 1 h at 4°C to give the cytosolic fraction. The supernatants were quantitated using a BCA kit.
The above proteins were adjusted to a volume of 20 µL with cold H2O. After the addition of 8 µL of 1 M MES-NaOH (pH 7.0), the samples were boiled at 100°C for 20 s to inactivate endogenous enzymes that use or degrade GDP-fucose. The samples were then centrifuged at 15,000 rpm for 15 min at 4°C. The samples were mixed with 1 µL of 10% Triton X-100, 2 µL of 100 µM fluorescent-labeled acceptor substrate and 4 µL of purified recombinant 1-6 FucT. The mixtures were incubated at 37°C for 2 h to allow the contained GDP-fucose to attach to the acceptor substrate by the action of 1-6 FucT. The reactions were terminated by boiling at 100°C for 3 min. The samples were centrifuged at 15,000 rpm for 15 min. Then, 30 µL of the 35 µL supernatants were subjected to HPLC (Shimadzu, Kyoto, Japan). Finally, the areas of fluorescent intensity of fucosylated acceptor substrates were converted to GDP-fucose concentrations using a standard curve of 0–50 pmol GDP-fucose.
Lectin blot analysis
Duplicate samples were subjected to SDS-PAGE under reducing conditions. One gel was subjected to Coomassie Brilliant Blue R-250 staining and the other was transferred to a PVDF membrane for AAL lectin blot analysis. AAL interacts with fucosylated oligosaccharides (Yamashita et al. 1985). After blocking with PBS containing 3% BSA overnight at 4°C, the membrane was incubated in diluted biotinylated AAL (Seikagaku Corp., Tokyo, Japan) for 40 min at RT. It was then washed three times with TBST and incubated with diluted avidin-peroxidase conjugates (ABC kit, Vector Res. Corp., Burlingame, CA) for 40 min at RT. The membrane was again washed three times with TBST and developed with an ECL system.
Cells culture and transfection
Human hepatoma cell lines, Hep3B, HepG2, HLE, HLF, and Huh7, provided from the ATCC (American Type Culture Collection, Manassas, VA) were cultured in RPMI1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% FCS.
Human GDP-Fuc Tr cDNA was prepared by PCR using a human spleen cDNA library (TAKARA BIO) as a template and the following forward and reverse primers were designed: 5'- ACCATGAATAGGGCCCCTCTG-3' and 5'-CACCCCCATG- GCGCTCTTCTC-3', respectively. The obtained DNA fragment was inserted into a pcDNA3.1/V5-His TOPO vector (Invitorogen), which is regulated by the cytomegalovirus (CMV) promoter and has the V5 epitope at the C-terminal. Vector insertion was confirmed by sequencing. This construct was transfected to Hep3B cells by the modified polycationic transfection method using the Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Selection was performed by the addition of 700 µg/mL geneticin (G418 disulfate) (nacalai tesque, Kyoto, Japan).
Immunohistochemical analysis
We used the tissue microarray including 59 liver carcinomas (KURABO, Osaka, Japan). In brief, tissue microarray slides were deparaffinized with xylene and ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0). Then, they were pretreated with Peroxidase Blocking Reagent (DAKO, Carpinteria, CA) for 10 min at RT. After washing twice with PBS, they were incubated with TBST containing 5% BSA overnight at 4°C for AAL staining or Protein Block Serum-Free (DAKO) for 30 min at RT for the others. They were incubated with biotinylated AAL (2.0 µg/mL) for 1 h at RT or anti-GDP-Fuc Tr and anti-1-6 FucT antibodies overnight at 4°C. They were then washed three times with PBS and incubated with ABC kit for 30 min at RT for AAL staining or EnVision System Labelled Polymer-HRP Anti-rabbit and mouse (DAKO) for 30 min at RT for GDP-Fuc Tr staining and 1-6 FucT staining, respectively. After washing three times with PBS, positive staining was visualized using diaminobenzidine (DAKO) and counterstaining was performed with hematoxylin. Slides were also stained in the absence of primary antibody to evaluate nonspecific secondary antibody reactions.
Statistical analyses
The results are represented as the mean ± SD. In comparing variables in more than two groups, a one-way ANOVA analysis was performed, followed by a Fisher's PLSD test if the former was significant. Fisher's test was adopted in the immunohistochemical analyses. Results of P < 0.05 were considered to be significant.
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at www.glycob.oxfordjournals.org
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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Japan Science and Technology Agency (JST); 21st Century Center of Excellence program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; New energy and Industrial Technology Development Organization; Health and Labour Sciences Research Grants from Ministry of Health, Labour and Welfare.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Dr. Hideyuki Ihara (Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University) and Dr. Yoko Mizuno-Horikawa (Department of Biochemistry, Osaka University Graduate School of Medicine) for providing a fluorescent-labeled acceptor substrate for the measurement of cytosolic GDP-fucose concentration and technical advice for immunohistochemical analyses, respectively.
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Abbreviations |
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AAL, Aleuria aurantia lectin; AFP, alpha-fetoprotein;
1-6 fucosyltransferase, GDP-L-Fuc : N-acetyl-ß-D-glucosaminide 1-6 fucosyltransferase; CH, chronic hepatitis; CL, liver cirrhosis; FX, GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase; GDP, guanosine 5'-diphosphate; GDP-Fuc Tr, GDP-fucose transporter; GADPH, glyceraldehyde-3-phosphate dehydrogenase; GMD, GDP-mannose 4, 6-dehydratase; GMP, guanosine 5'-monophosphate; HCC, hepatocellular carcinoma; HPLC, high performance liquid chromatography; HPRT, hypoxanthine guanine phosphoribosyl transferase; LEC rat, Long-Evans with cinnamon-like coat color rat; PBS, phosphate-buffered saline; PLSD, protected least significant differences; PVDF, polyvinylidine difluoride; RT-PCR, reverse transcription polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
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