Glycomic Analyses of Glycoproteins in Bile and Serum during Rat

Aug 23, 2010 - Graduate School of Pharmaceutical Sciences, Nagoya, Japan, and .... for studying hepatocarcinogenesis, as well as Wilson's disease.16...
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Glycomic Analyses of Glycoproteins in Bile and Serum during Rat Hepatocarcinogenesis Tsutomu Nakagawa,† Shunsaku Takeishi,‡ Akihiko Kameyama,§ Hirokazu Yagi,| Tomoko Yoshioka,† Kenta Moriwaki,† Tomomi Masuda,† Hitoshi Matsumoto,† Koichi Kato,| Hisashi Narimatsu,§ Naoyuki Taniguchi,⊥ and Eiji Miyoshi*,† Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, Osaka, Japan, Glycomics Research Laboratory Moritex Corporation, Yokohama, Japan, Reseach Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan, Department of Structural Biology and Biomolecular Engineering, Nagoya City University Graduate School of Pharmaceutical Sciences, Nagoya, Japan, and Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Received April 8, 2009

Fucosylated alpha-fetoprotein (AFP) is a specific tumor marker for hepatocellular carcinomas (HCC). However, the mechanisms underlying the increase in fucosylated AFP in serum of patients with HCC are largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of glycoproteins into bile in the liver. This finding might lead to the selective secretion of fucosylated AFP into bile and the selective secretion might be disrupted in hepatocarcinogenesis. In this study, therefore, we analyzed the oligosaccharide structures of glycoproteins in bile and serum of LEC rats, which are a rat model of spontaneous hepatocarcinogenesis. Lectin microarraying showed enhanced binding of 13 lectins to bile, compared with in serum from normal LEC rats, and the binding of these lectins to serum of LEC rats bearing HCC was higher than in normal rats. Structural analyses involving HPLC and mass spectrometry showed that the fucosylation levels of serum glycoproteins were not increased in CH rats but were in HCC rats, although the fucosylation levels of biliary glycoproteins were increased in both CH and HCC rats. These results suggested that the sorting machinery through fucosylation might be disrupted in the liver with HCC. Keywords: fucose • bile • lectin microarray • HCC

Introduction It has long been known that cellular and serum glycosylation profiles change significantly during oncogenesis.1 Hence a search for tumor markers associated with the altered glycosylation patterns associated with the development of cancer has been performed. Fucosylated alpha-fetoprotein (AFP), also referred to as L3 fraction AFP, is a highly specific tumor marker for hepatocellular carcinomas (HCC). Increases in the AFP level in serum are observed in patients with chronic liver diseases such as liver cirrhosis, but fucosylated AFP is scarcely detected in benign liver diseases.2 Alpha1-6 fucosyltransferase (Fut8) is involved in the fucosylation of AFP, and we previously succeeded in the purification and cDNA cloning of Fut8 from porcine brain3 and a human gastric cancer cell line.4 While * To whom correspondence should be addressed. Eiji Miyoshi, M.D. & Ph.D., Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-oka, Suita, Osaka, Japan. Telephone: +81-6-6879-2590. Fax: +81-6-6879-2590. E-mail: [email protected]. † Osaka University Graduate School of Medicine. ‡ Glycomics Research Laboratory Moritex Corporation. § National Institute of Advanced Industrial Science and Technology. | Nagoya City University Graduate School of Pharmaceutical Sciences. ⊥ Osaka University.

4888 Journal of Proteome Research 2010, 9, 4888–4896 Published on Web 08/23/2010

overexpression of Fut8 in Hep3B cells increased the rate of fucosylation of AFP, high expression of Fut8 was also observed in noncancerous liver cirrhotic tissues as well as HCC ones.5 Therefore, other factors must be linked to the specific incidence of fucosylated AFP in HCC. GDP-fucose is a donor substrate for Fut8. When we determined the GDP-fucose levels in liver tissues, using an assay system for the GDP-fucose levels in cells/ tissues, the levels in HCC tissues were found to be significantly higher than those in liver cirrhosis and normal liver.6,7 The increases in GDP-fucose in HCC were due to enhancement of FX (human homologue of GDP-4-keto-6-deoxymannose-3,5epimerase-4-reductase) expression, which contributed to the synthesis of GDP-fucose8 and knock down of FX in HepG2 cells suppressed the rate of fucosylation of AFP (manuscript in preparation, Nakagawa, Miyoshi et al.). Therefore, both Fut8 and FX must regulate the production of fucosylated AFP in HCC. However, the increases in Fut8 and FX in HCC tissues were twice or three times compared to in the surrounding tissues, and thus another factor could be involved in terms of an increase in fucosylated AFP in serum of patients with HCC. Hepatocytes, major epithelial cells in the liver, produce a variety of serum glycoproteins and nonglycosylated proteins including albumin. In the hepatocyte system, there are two 10.1021/pr100414r

 2010 American Chemical Society

Changes in Fucosylation during Hepatocarcinogenesis secretion pathways. One pathway is to the apical surface of hepatocytes, which is followed by secretion into bile ducts. The other is to the basolateral surface, which is followed by secretion into blood vessels. Interestingly, the same serum proteins including albumin are detected in bile.9 Previously, however, few cellular or protein factors were found to regulate these secretion pathways.10 Recently, we reported that many glycoproteins in human and mouse bile were strongly fucosylated, compared to those in serum, and suggested that fucosylation is a possible signal for the secretion of glycoproteins into bile in the liver.11 This finding might lead to the selective secretion of fucosylated glycoproteins, including fucosylated AFP, into bile. However, it remains unknown how this sorting machinery for hepatic glycoproteins via fucosylation is disrupted during hepatocarcinogenesis. Increases in fucosylation in serum of patients with HCC are not limited to AFP. Recent glycan analysis of serum demonstrated that increases in the levels of fucosylation of many serum glycoproteins, such as fetuin A, haptoglobin, hemopexin and GP73, occur with the development of HCC.12-14 These results might support our hypothesis that fucosylated glycoproteins secreted into bile normally are secreted into serum on disruption of selective secretion of glycoproteins through fucosylation in the liver with HCC. The Long-Evans Cinnamon color (LEC) rat is a mutant strain that was established from a closed colony of the Long-Evans strain. Although the exact onset of disease varies, depending on the report, LEC rats spontaneously develop acute hepatitis at 12 to 24 weeks after birth, followed by chronic hepatitis (CH). At 40 weeks of age, they begin to develop nodules in the liver that progress to HCC after 1 year.15 These changes are the result of autosomal recessive inheritance, and are associated with the copper-transporting ATPase gene, which is a homologue of the Wilson’s-disease gene. Therefore, the LEC rat is an ideal model for studying hepatocarcinogenesis, as well as Wilson’s disease.16 In this study, we compared the oligosaccharide structures on biliary glycoproteins with those on serum glycoproteins in LEC rats during hepatocarcinogenesis. Lectin microarraying revealed enhanced binding to biliary glycoproteins of several kinds of lectins including AOL that recognize fucosylated structures, compared with serum glycoproteins. Increases in binding to these lectins of serum proteins were observed in rats with HCC, compared with in normal rats. Structural analyses involving HPLC and mass spectrometry showed that the fucosylation levels of glycoproteins in serum of rats with HCC were higher than those in CH and normal rats. Increases in fucosylation of biliary glycoproteins were observed in CH rats, although there were no significant changes in their serum levels, compared with in normal rats. These results suggested that the sorting machinery through fucosylation might be disrupted in the liver with HCC.

Experimental Section Reagents. Biotinated E4-PHA (Phaseolus vulgaris) and betagalactosidase (Jack bean) were purchased from Seikagaku Corp. (Tokyo, Japan). Biotinated AOL (Aspergillus oryzae) was from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Alpha-fucosidase (bovine kidney) was from Sigma (St. Louis, MO), and beta1-3 galactosidase (Xanthomonas manihotis) from Merck (Darmstadt, Germany). Animals. According to the clinical stage, LEC rats were divided into three groups: 11 weeks (normal stage), 26 weeks

research articles (CH stage), and 56-64 weeks (HCC stage). After the rats had been anesthetized with sodium pentbarbital (5 mg/100 g body weight), bile and serum were collected. Bile specimens were collected by bile duct cannulation. Briefly, the abdomen was opened and the common bile duct was cannulated with a 25gauge needle connected to polyethylene tubing. The protein concentrations of bile specimens were measured with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard after delipidation according to the method of Wessel and Flugge.17 There were no significant differences in the protein concentrations in bile and serum between the three stages. All procedures for animal experiments were performed in accordance with the Osaka University Medical School guidelines. Lectin Microarraying. Lectin microarraying was performed by means of the method reported by Kuno et al.18 with a slight modification. Shortly, bile and serum were equilibrated against IgG Binding Buffer (Pierce) on NAP-5 columns (GE Healthcare UK Ltd., Buckinghamshire, England), and immunoglobulins were removed with a Protein A/G column (Pierce). The obtained proteins (1 µg) were labeled with 100 µg of Cy3succimidyl ester at room temperature for 1 h in the dark. Excess reagent was removed by gel filtration chromatography on a Zeba Desalt Spin Column (Pierce). The resultant Cy3-labeled glycoprotein solution (80 µL, 125 ng/mL) was applied to a lectin microarray. After incubation at 20 °C for 15 h, the glass slide was scanned with an evanescent-field fluorescence scanner, GlycoStation (Moritex, Co., Tokyo, Japan). All of the data were analyzed with Array Pro analyzer version 4.5 (Media Cybernetics, Inc., Bethesda, MD). The net intensity value for each spot was calculated by subtracting the background value. The signal intensity value for each lectin was expressed as the average of the net intensity values for three spots. WGA signals were used to normalize the signal intensity of each lectin, because binding to WGA lectin was relatively stable and almost the same in serum and bile samples from normal, CH, and HCC rats. Representative sugar binding specificities of the lectins examined in this study are shown in the Supplementary Table, Supporting Information. Structural Analysis of Oligosaccharides Derived from Biliary and Serum Glycoproteins. Structural analyses of oligosaccharides on glycoproteins in bile and serum were performed by means of the modified method described in the previous paper.11 Briefly, bile and serum were equilibrated against IgG Binding Buffer on PD-10 columns (GE Healthcare U.K. Ltd.), and immunoglobulins were removed with a Protein A/G column (Pierce). The solutions were subjected to trichloroacetic acid precipitation and the precipitated fractions were washed with ethanol twice. Oligosaccharides were released from the precipitated proteins by hydrazinolysis (100 °C for 10 h) followed by N-acetylation.19 The oligosaccharides were applied to a PALSTATION (Takara Bio Inc., Shiga, Japan) for pyridylamination.20 Excess reagents were removed by chromatography on a Sephadex G-15 column (GE Healthcare U.K. Ltd.). Sialic acids of the purified pyridylamino (PA)-oligosaccharides were removed by neuraminidase treatment (Arthrobacter ureafaciens., Nacalai Tesque, Kyoto, Japan) in 0.2 M acetate buffer, pH 7.4, at 37 °C overnight. The asialo PAoligosaccharides were separated by reverse phase HPLC on a Shim-pack CLC-ODS column (Shimadzu Corp., Kyoto, Japan) and subsequent normal phase HPLC on a TSK-gel Amide-80 column (Tosoh Corp., Tokyo, Japan). Elution and detection of PA-oligosaccharides were performed as described by Tomiya Journal of Proteome Research • Vol. 9, No. 10, 2010 4889

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Figure 1. Differential glycan profiling of bile and serum from normal rats. Rat bile and serum, with immunoglobulins removed, were labeled with Cy3-SE and then applied to a lectin microarray. The fluorescence intensity of each lectin was normalized as to the intensity of WGA. The fluorescence intensities of lectins with biliary and serum proteins are indicated by black and white columns, respectively. Each column represents the mean plus SD for three different rats. *, significantly different (p < 0.05); **, significantly different (p < 0.01); ***, significantly different (p < 0.001).

et al.21 The structures of PA-oligosaccharides were determined from the elution positions of individual peaks on the basis of the GALAXY database.22 The structures of PA-oligosaccharides so far not registered in the GALAXY database were trimmed by exoglycosidase treatment to make them identical to known ones. Mass Spectrometry. MS spectra were acquired in the positive ion mode with a MALDI-TOF mass spectrometer (Reflex IV; Bruker Daltonik, Bremen, Germany). Ions were generated with a pulsed 337 nm nitrogen laser and were accelerated to 20 kV. All of the spectra were obtained in the reflectron mode with delayed extraction of 200 ns. MS/MS spectra were acquired with a MALDI-QIT-TOF mass spectrometer (AXIMA-QIT; Shimadzu Corp., Kyoto, Japan). Argon was used as the collision gas. All MS/MS spectra were obtained for Na+ adduct ions. Sometimes K+ adduct ions abundantly appeared in MS spectra. In such cases, the analyte solutions were desalted by NuTip 10 (C18/ carbon mix. Glygene Corp., Columbia, MD), and then doped with small amount of a 10 mM NaCl solution. The collisional energy was adjusted to reduce the intensity of the precursor ion to less than 15% of the area of a base peak.23 For sample preparation, 0.5 µL of an approximately 10 µM analyte solution was deposited on the target plate and allowed to dry. Then, 0.5 µL of a 2,5-dihydroxybenzoic acid (2,5-DHB; Wako Pure Chemical Industries Ltd.) solution (10 mg/mL in 30% ethanol) was used to cover the analyte on the target plate, followed by drying. The deposited analyte/matrix mixtures were recrystallized by the addition of 0.2 µL of 100% ethanol for AXIMAQIT. Lectin Blot Analysis. Lectin blot analyses were performed as described previously.24 Briefly, bile and serum were equilibrated against IgG Binding Buffer (Pierce) on NAP-5 columns (GE Healthcare U.K. Ltd.), and immunoglobulins were removed with a Protein A/G column (Pierce). The obtained proteins (3 µg) were subjected to 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis, 4890

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the gels were blotted onto nitrocellulose membranes. The membranes were incubated with 3% BSA in Tris-buffered saline (20 mM Tris, 0.5 M NaCl, pH 7.5, TBS) overnight, and then incubated with 1.0 µg/mL of biotinated lectins (AOL, E4-PHA) in TBST (TBS containing 0.05% Tween 20) for 1 h. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated avidin (VECTASTAIN ABC kit. Vector Laboratories, Burlingame, CA) for 1 h, and then washed with TBST. Staining was performed with ECL Western blot detection reagents (GE Healthcare U.K. Ltd.).

Results Differential Glycan Profiling of Rat Biliary and Serum Glycoproteins Using a Lectin Microarray. To determine the glycan profiles of glycoproteins in rat bile and serum, Cy3labeled proteins derived from bile and serum of normal rats were subjected to lectin microarray analyses. As shown in Figure 1, the intensities of 13 lectins (LCA, AOL, AAL, L4-PHA, E4-PHA, GSL-II, ConA, BPL, EEL, ABA, Calsepa, PTL-I and GSL-I-B4) were higher for biliary glycoproteins than for serum glycoproteins. However, the signal intensities of L4-PHA, GSLII, EEL, BPL, PTL-I, and GSL-I-B4 were out of dynamic range (NI; 1,000-50,000). Enhanced intensities of lectins that recognize fucose residues, that is, LCA, AOL and AAL, were observed in bile, compared with in serum (Figure 1). These results were in accordance with those obtained for humans and mice on lectin blot analyses in our previous study.11 Next, to investigate changes in the oligosaccharide structures of serum glycoproteins during hepatocarcinogenesis, Cy3labeled proteins derived from serum of normal and HCC rats were subjected to lectin microarray analyses (Supplementary Figure, Supporting Information). The binding intensities of biliary proteins-binding lectins such as LCA, AOL, AAL, E4-PHA, ConA, ABA, and Calsepa to sera from normal and HCC rats were shown in Figure 2. The intensities of all the lectins except

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Figure 2. Focused differential glycan profiling of serum from normal and HCC rats. Serum of normal and HCC rats, with immunoglobulins removed, was labeled with Cy3-SE and then applied to a lectin microarray. The fluorescence intensities of the seven focused lectins were normalized as to the intensity of WGA. The fluorescence intensities of the lectins with serum proteins from normal and HCC rats are indicated by white and black columns, respectively. Each column represents the mean plus SD for three different rats. *, significantly different (p < 0.05) from normal rats; **, significantly different (p < 0.01) from normal rats.

Figure 3. HPLC separation of PA-oligosaccharides in bile and serum from normal rats. Representative elution profiles of PAoligosaccharides in bile [A] and serum [B] from normal rats on an ODS column. The framed chart showed the elution profile of the peak 1 or 4 material on an ODS column after beta1,3-galactosidase digestion. The procedures are described in detail in the Experimental section. The numbers of the peaks and the symbols for monosaccharides correspond to those in Table 1.

Calsepa in HCC rats were higher than those in normal rats. These results showed that a glycosylation pattern of the bile type was observed in serum of HCC rats, but not in normal rats, suggesting that the selective secretion of glycoproteins into bile might be partially disrupted in HCC rats. Structural Analysis of PA-Oligosaccharides Derived from Rat Biliary and Serum Glycoproteins. As shown in Figure 1, some obvious differences in the signal patterns for N-glycans were observed on comparison between biliary and serum glycoproteins, while the signal intensities for O-glycans were low. Therefore, we examined N-glycans on glycoproteins in the following study. To examine the oligosaccharide structures on biliary and serum glycoproteins in more detail, two-dimensional mapping HPLC analyses were performed. Representative

elution profiles of PA-oligosaccharides derived from biliary and serum glycoproteins on reverse phase HPLC are shown in Figure 3. The elution position of each peak was recorded in glucose units and the structures were determined based on the GALAXY database (Table 1). The structural assignments were supported by the results of MS/MS analyses involving a MALDIQIT-TOF MS (Figure 4 and Table 1). The peak 1 and 4 materials were not registered in the GALAXY database. The MS spectral data showed that the peak 1 and 4 materials exhibited the same mass values as the peak 2 and 5 ones, respectively, and there were no differences in the fragment profiles obtained on MS/ MS between peaks 1 and 2 or 4 and 5. However, the fragment patterns including the relative intensities were apparently different (Figure 4). These results indicated that the peak 1 and Journal of Proteome Research • Vol. 9, No. 10, 2010 4891

research articles Table 1. Assignment of the Major PA-Oligosaccharides by Two-Dimensional Mapping HPLC and MALDI-TOF MS Analyses

a Numbers at each peak indicated correspond to those in Figures 3, 4, and 5. b Monosaccharides were denoted by O, galactose; 0, N-acetylglucosamine; b, mannose; 2, fucose. c Elution positions on HPLC columns were expressed as glucose units (GU). The chromatographic conditions are described in the Experimental section. d Ions correspond to [M + K]+.

2 or 4 and 5 materials might be linkage isomers. Peaks 1 and 2 or 4 and 5 moved to the same elution positions on betagalactosidase digestion, suggesting the materials might be linkage isomers as to galactose residues (data not shown). Furthermore, after beta1,3-galactosidase digestion, the peak 1 and 4 materials gave two apparent peaks by the difference in position of the remaining beta1,4-galactose (Figure 3, framed), suggesting that two peak materials in frames were position isomers. Thus, the PA-oligosaccharides derived from rat biliary and serum glycoproteins were mainly separated into ones with eight different structures. Glycomics Analyses of Biliary and Serum Glycoproteins during Hepatocarcinogenesis. As shown in Figure 3, the peak 6 material, which does not contain galactose residues, could be secreted into bile. Therefore, we paid attention to the oligosaccharide structures with fucose and bisecting GlcNAc residues in the following study, that is, the structures of the peak 1 and 2 or 4 and 5 materials, linkage isomers of galactose residues, were considered to be the same in terms of the signal structure for secretion into bile. Changes in the percentage of each oligosaccharide structure during hepatocarcinogenesis are shown in Figure 5. Peak 6 could not be detected at any of the clinical stages in serum while the percentage of the structure increased in bile during hepatocarcinogenesis. This structure could also not be detected in the serum of humans and mice in our previous study.11 These results suggested that the structure of the peak 6 material might be one of the signals for the secretion into bile. If the structure of the peak 6 material is one of the signals for the secretion of glycoproteins into bile, bisecting GlcNAc 4892

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Nakagawa et al. must be attached to most of the glycoproteins secreted into bile. To determine whether or not bisecting GlcNAc is a possible signal for the secretion into bile, bile and serum were subjected to lectin blot analyses with E4-PHA and AOL, which bind to bisecting GlcNAc and fucose residues, respectively. As shown in Figure 6, on E4-PHA blotting there were two major bands and some faint bands for bile while AOL binding to bile was detected over a wide molecular weight range. These results suggested that secretion of glycoproteins into bile might not need bisecting GlcNAc but fucose residues. Comparison of Fucosylated Oligosaccharide Structures on Biliary and Serum Glycoproteins during Hepatocarcinogenesis. As mentioned above, the selective secretion of fucosylatiated glycoproteins into bile was observed in rats as well as humans and mice. The disruption of this selective secretion of fucosylated glycoproteins into bile in the liver with HCC could be one of the mechanisms underlying the production of fucosylated AFP. To determine whether or not the sorting machinery through fucosylation is disrupted in the liver with HCC, we examined the levels of fucosylated oligosaccharides on biliary and serum glycoproteins during hepatocarcinogenesis. As shown in Figure 7, the biliary glycoproteins contained many fucosylated oligosaccharide structures at all of the clinical stages, compared with serum glycoproteins. The fucosylation levels of glycoproteins in serum of HCC rats were significantly higher than those in CH and normal rats. On the other hand, increases in fucosylation of biliary glycoproteins were observed in CH and HCC rats, compared with in normal rats.

Discussion In this study, we compared the oligosaccharide structures on biliary glycoproteins with those on serum glycoproteins in LEC rats during hepatocarcinogenesis. In Figure 1, it can be seen that thirteen lectins including three that recognize fucose residues, i.e., LCA, AOL and AAL, bind more material in bile than in serum. BPL also recognizes fucosylated structures weakly. E4-PHA, ABA and Calsepa are lectins that recognize the structure of the peak 6 material, a bile-specific oligosaccharide structure (Figure 3). Therefore, increases of the signals of these seven lectins would be due to increases in fucosylated oligosaccharides including the peak 6 material. On the other hand, we could not determine why the intensities of the other lectins, that is, L4-PHA, GSL-II ConA, EEL and GSL-I-B4, increased. It is thought that an increase in the intensity of ConA would not be involved in an increase in high-mannose structures because enhanced binding of other lectins that recognize high-mannose, that is, NPA, GNA and HHL, to bile was not observed. Also, we could not detect the beta1-6GlcNAc, tri/ tetra-antennary and alpha1-3Gal structures, which are recognized by L4-PHA, GSL-II, EEL, and GSL-I-B4, on structural analyses in this study. Conversely, nine lectins, that is, SNA, SSA, TJA-I, RCA120, DSA, ACG, TxLC-I, LEL and STL, gave much higher signals with serum glycoproteins than with biliary glycoproteins (Figure 1). SNA, SSA, TJA-I, and ACG are lectins that recognize sialic acid residues and TxLC-I also exhibits weak affinity to sialylated glycans. Therefore, the increases in the signals of these five lectins showed that glycoproetins in serum contain high levels of sialic acid residues compared with those in bile. It has been reported that both hepatic uptake and biliary excretion of galactosylated glycoproteins, that is the asialo-forms of glycoproteins, were affected by the extent of galactosylation.25 This report supported the

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Figure 4. MS/MS spectra of PA-oligosaccharides. The MS/MS spectra of the peak 1, 2, 4, and 5 materials are shown. The [M + Na]+ ion was used as the precursor ion in each spectrum. The m/z values of monoisotopic peaks of major fragments are indicated in the spectra. All the major signals were assigned as depicted in the spectra of the peaks 2 and 5 materials. Y-series ions were accompanied by the corresponding dehydrated ions. The procedures are described in detail in the Experimental section. The numbers of the peaks and the symbols for monosaccharides correspond to those in Table 1.

Figure 5. Changes in the percentages of oligosaccharide structures during hepatocarcinogenesis. The percentage of each oligosaccharide structure in the total area of the assigned peaks for (A) bile and (B) serum was calculated based on the peak areas of the ODS and Amide-80 elution profiles. The numbers of the peaks and the symbols for monosaccharides correspond to those in Table 1. The oligosaccharides from normal, CH and HCC rats are indicated by white, gray and black columns, respectively. N.D. indicates “not detected”, 6fucosyltransferase. J. Biol. Chem. 1996, 271 (44), 27810–7. (4) Yanagidani, S.; Uozumi, N.; Ihara, Y.; Miyoshi, E.; Yamaguchi, N.; Taniguchi, N. Purification and cDNA cloning of GDP-L-Fuc:Nacetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha16 FucT) from human gastric cancer MKN45 cells. J. Biochem. 1997, 121 (3), 626–32. (5) Noda, K.; Miyoshi, E.; Uozumi, N.; Yanagidani, S.; Ikeda, Y.; Gao, C.; Suzuki, K.; Yoshihara, H.; Yoshikawa, K.; Kawano, K.; Hayashi, N.; Hori, M.; Taniguchi, N. Gene expression of alpha1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of alpha-fetoprotein. Hepatology 1998, 28 (4), 944–52. (6) Noda, K.; Miyoshi, E.; Gu, J.; Gao, C. X.; Nakahara, S.; Kitada, T.; Honke, K.; Suzuki, K.; Yoshihara, H.; Yoshikawa, K.; Kawano, K.; Tonetti, M.; Kasahara, A.; Hori, M.; Hayashi, N.; Taniguchi, N. Relationship between elevated FX expression and increased production of GDP-L-fucose, a common donor substrate for fucosylation in human hepatocellular carcinoma and hepatoma cell lines. Cancer Res. 2003, 63 (19), 6282–9. (7) Noda, K.; Miyoshi, E.; Nakahara, S.; Ihara, H.; Gao, C. X.; Honke, K.; Yanagidani, S.; Sasaki, Y.; Kasahara, A.; Hori, M.; Hayashi, N.; Taniguchi, N. An enzymatic method of analysis for GDP-L-fucose in biological samples, involving high-performance liquid chromatography. Anal. Biochem. 2002, 310 (1), 100–6. (8) Tonetti, M.; Sturla, L.; Bisso, A.; Benatti, U.; De Flora, A. Synthesis of GDP-L-fucose by the human FX protein. J. Biol. Chem. 1996, 271 (44), 27274–9. (9) Kristiansen, T. Z.; Bunkenborg, J.; Gronborg, M.; Molina, H.; Thuluvath, P. J.; Argani, P.; Goggins, M. G.; Maitra, A.; Pandey, A. A proteomic analysis of human bile. Mol. Cell. Proteomics 2004, 3 (7), 715–28. (10) Wang, L.; Boyer, J. L. The maintenance and generation of membrane polarity in hepatocytes. Hepatology 2004, 39 (4), 892– 9. (11) Nakagawa, T.; Uozumi, N.; Nakano, M.; Mizuno-Horikawa, Y.; Okuyama, N.; Taguchi, T.; Gu, J.; Kondo, A.; Taniguchi, N.; Miyoshi, E. Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts. J. Biol. Chem. 2006, 281 (40), 29797– 806. (12) Block, T. M.; Comunale, M. A.; Lowman, M.; Steel, L. F.; Romano, P. R.; Fimmel, C.; Tennant, B. C.; London, W. T.; Evans, A. A.; Blumberg, B. S.; Dwek, R. A.; Mattu, T. S.; Mehta, A. S. Use of

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