[From proteomics to functional glycomics: importance of functional analysis of sugar chains].
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Animals↗
Biomedical subjects
Eiji Miyoshi
Publications and source records attributed to Eiji Miyoshi.
Expression and cellular localization of p8 protein in thyroid neoplasms.
OBJECTIVES: p8 protein has mitogenic activity and is linked to the development of pancreatic carcinoma. However, little is known about the expression and physiological significance of this protein in other human carcinomas. METHODS: In this study, we immunohistochemically investigated p8 expression in thyroid neoplasms as well as in the normal thyroid gland. RESULTS: p8 was expressed in normal follicular cells, but no normal thyroid was regarded as overexpressing p8. On the other hand, 44.3% of papillary carcinoma overexpressed p8 and the incidence was directly linked to the tumor size (p=0.0340) and lymph node metastasis (p=0.0145). In follicular tumors, the incidence of p8 overexpression did not depend on histological type. In anaplastic (undifferentiated) carcinoma, p8 was overexpressed only in 5.0%, which was significantly lower than in papillary (p=0.0006) and follicular carcinomas (p=0.0049). In normal follicules and follicular tumors, p8 was localized mainly in the nucleus except for two adenomas. On the other hand, p8 localization was more cytoplasmic in papillary carcinoma larger than 1.0 cm (p=0.0186) and with a poorly differentiated lesion (p=0.0313). CONCLUSIONS: These results suggest that the overexpression and cytoplasmic localization of p8 protein may reflect disease progression of papillary carcinoma, whereas this protein plays little part in thyroid carcinoma after anaplastic transformation.
Adenocarcinoma, Follicular↗
N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan.
Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.
Brain↗
14-3-3 sigma possibly plays a constitutive role in papillary carcinoma, but not in follicular tumor of the thyroid.
14-3-3 sigma is a negative regulator of the cell cycle and contributes to G2 arrest. Thus far, the lack of its expression due to hypermethylation of the CpG islands has been reported in some carcinomas. In this study, we investigated the expression of 14-3-3 sigma in thyroid neoplasms by means of immunohistochemistry as well as Western blot analysis. Normal follicules did not express 14-3-3 sigma. In 82 papillary carcinomas, all the cases expressed 14-3-3 sigma and its expression was not reduced but even enhanced in the advanced stage and in poorly differentiated types. Furthermore, 21 of the 23 anaplastic carcinomas expressed 14-3-3 sigma and its expression level tended to be higher than in papillary carcinoma. On the other hand, none of the 34 follicular carcinomas or 29 follicular adenomas expressed 14-3-3 sigma. These results suggest that 14-3-3 sigma plays a constitutive role in papillary carcinoma rather than acting as a cell cycle regulator, whereas it is not required for the occurrence and development of follicular tumor.
14-3-3 Proteins↗
Expression of alpha1,6-fucosyltransferase (FUT8) in papillary carcinoma of the thyroid: its linkage to biological aggressiveness and anaplastic transformation.
Previous studies have demonstrated that terminal fucosylation is associated with the biological aggressiveness of carcinomas, but the significance of core fucosylation (alpha1,6-fucosylation) through alpha1,6-fucosyltransferase (FUT8) has not been studied in depth. Herein, we investigated the expression of alpha1,6-fucosyltransferase (FUT8) in 133 cases of thyroid carcinomas using an immunohistochemical approach. The expression of FUT8 was quite low in normal follicules. A high expression of FUT8 was observed in 33.3% of papillary carcinoma and the incidence was directly linked to tumor size and lymph node metastasis. In contrast, this phenomenon was less frequently observed in follicular carcinoma and anaplastic (undifferentiated) carcinoma. These results suggest that FUT8 expression may be a key factor in the progression of thyroid papillary carcinomas, but not follicular carcinomas, and decreases in FUT8 expression might be linked to anaplastic transformation.
Carcinoma↗
Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells.
A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.
Animals↗
Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin.
beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.
Amino Acid Sequence↗
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.
The levels of fucosylated glycoproteins in various cancers and inflammatory processes have been a subject of intense study. The level of fucosyltransferases and intracellular GDP-L-fucose, a sugar nucleotide and a common donor substrate for all fucosyltransferases, may regulate the level of fucosylated glycoproteins. This study reports on the determination of GDP-L-fucose levels in human hepatocellular carcinoma (HCC) and surrounding tissues, using a recently established high-throughput assay system. Levels of GDP-L-fucose in HCC tissues were significantly increased compared with adjacent nontumor tissues or normal livers. The mean +/- SD for GDP-L-fucose level was 3.6 +/- 0.2 micro mol/mg in control liver, 4.6 +/- 0.9 micro mol/mg in adjacent noninvolved liver tissues (chronic hepatitis, 4.4 +/- 0.7 micro mol/mg; liver cirrhosis, 4.8 +/- 0.9 micro mol/mg), and 7.1 +/- 2.5 micro mol/mg in HCC tissues. The level of GDP-L-fucose in HCC decreased in proportion with tumor size (r = -0.675, P = 0.0002). When expression of the series of genes responsible for GDP-L-fucose synthesis was investigated, the gene expression of FX was found to be increased in 70% (7 of 10) of the HCC tissues examined compared with that in their surrounding tissues. The levels of GDP-L-fucose were positively correlated with the expression of FX mRNA (r = 0.599, P = 0.0074). The levels of FX gene expression in some human hepatoma and hepatocyte cell lines were determined. FX mRNA production was strongly increased in HepG2 and Chang liver, moderately increased in Hep3B and HLF, and, in HLE, was similar to that of a normal human liver tissue. To investigate the effect of GDP-L-fucose on core fucosylation, FX cDNA was transfected into Hep3B cells, which express a relatively low level of GDP-L-fucose:N-acetyl-beta-D-glucosaminide alpha1-6 fucosyltransferase (alpha1-6 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 alpha-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 alpha1-6 FucT expression. Thus, an elevation in GDP-L-fucose levels and the up-regulation of FX expression represent potential markers for HCC.
Carbohydrate Sequence↗
14-3-3 sigma possibly plays a constitutive role in papillary carcinoma, but not in follicular tumor of the thyroid.
Jun activation domain-binding protein 1 (jab1) is known as a coactivator of the AP1 transcription factor. Recent studies have demonstrated that jab1 contributes to carcinoma progression by degrading p27/Kip1 protein. In this study, we immunohistochemically investigated jab1 expression in thyroid neoplasms. jab1 overexpression tended to be frequently observed in follicular carcinoma compared to adenoma. Larger-sized papillary carcinoma overexpressed jab1 in higher incidence than microcarcinomas (1.0 cm or less in maximum diameter). In anaplastic carcinoma, jab1 was more frequently overexpressed than in papillary and follicular carcinomas. An inverse relationship was detected between jab1 overexpression and p27 expression in papillary and anaplastic carcinomas, but not in follicular tumor. These results suggest that jab1 plays a role in the progression of thyroid carcinomas, especially those of aggressive phenotypes, and it may be responsible for p27 degradation in anaplastic and papillary carcinomas.
14-3-3 Proteins↗
Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma.
BACKGROUND & AIMS: Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment. However, the differential diagnosis between HCC and benign hepatic lesions is sometimes difficult and new biochemical markers for HCC are required. It has been reported that glypican-3 (GPC3) messenger RNA (mRNA) is significantly increased in most HCCs compared with benign liver lesions or normal liver. The goal of this study is to determine whether GPC3 is also overexpressed at the protein level and whether GPC3 is detectable in the serum of patients with HCC. METHODS: GPC3 was assessed in liver tissue sections by immunohistochemistry and in serum by enzyme-linked immunosorbent assay. Serum alpha-fetoprotein (AFP) level was also measured in the same patients. RESULTS: Immunohistochemical studies showed that GPC3 is expressed in 72% of HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver and benign liver diseases. Consistent with this, GPC3 was undetectable in the serum of healthy donors and patients with hepatitis, but its levels were significantly increased in 18 of 34 patients (53%) with HCC. In addition, only 1 of 20 patients with hepatitis plus liver cirrhosis displayed elevated levels of serum GPC3. Interestingly, in most cases, there was no correlation between GPC3 and AFP values. Thus, at least 1 of the 2 markers was elevated in 82% of the patients with HCC. CONCLUSIONS: GPC3 is specifically overexpressed in most HCCs and is elevated in the serum of a large proportion of patients with HCC. The simultaneous determination of GPC3 and AFP may significantly increase the sensitivity for diagnosis of HCC.
Animals↗
Y-box binding protein expression in thyroid neoplasms: its linkage with anaplastic transformation.
Recent studies have demonstrated that Y-box binding protein (YB-1) regulates the transcription of genes linked to carcinoma progression. In this study, we investigated the expression of this protein in thyroid neoplasms to elucidate its significance. The expression of YB-1 was immunohistochemically investigated using the monoclonal antibody for various thyroid neoplasms. Normal follicles did not overexpress YB-1, and only moderate overexpression of YB-1 was observed in some follicular tumors and papillary carcinoma, especially those of a larger size. In contrast, 92.9% of anaplastic carcinoma strongly overexpressed YB-1. YB-1 immunoreactivity was seen in both cytoplasms and cell nuclei, but the former was more predominant. These findings suggest that YB-1 plays a role in regulating the transcription as well as translation of genes contributing to the anaplastic transformation of thyroid carcinoma.
Adenocarcinoma, Follicular↗
[Regulation of growth and differentiation by oligosaccharides].
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Animals↗
[Protease modified by sugar chain: beta 1-6 branching and matriptase].
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Animals↗
Involvement of oligosaccharide changes in alpha5beta1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line.
Multiple mechanisms are involved in the resistance of cancer cells to cisplatin, including the expression of multidrug resistance-associated protein (MRP) and enhanced DNA repair. Here, we report findings to show that oligosaccharide changes in alpha5beta1 integrin are associated with cisplatin resistance in a head and neck squamous cell carcinoma cell line, HSC-2. Cisplatin-resistant HSC-2 (HSC-2/CR) cells were established by stepwise treatment with various concentrations of cisplatin. The oligosaccharides containing beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching, detected by leukoagglutinating phytohemagglutinin (L(4)-PHA) lectin blot, were found to be dramatically decreased in alpha5beta1 integrin immunoprecipitated from HSC-2/CR cells. To better understand the mechanisms underlying cisplatin resistance and oligosaccharide alteration, we analyzed the downstream signaling of alpha5beta1 integrin, one of the target glycoproteins of beta1-6GlcNAc transferase [UDP-GlcNAc:alpha-D-mannoside beta1, 6-N-acetylglucosaminyltransferase (GnT-V)]. Cell adhesion to fibronectin and phosphorylation of focal adhesion kinase (FAK), which are associated with alpha5beta1 integrin and involved in a cell survival signaling, were found to be increased in the cisplatin-resistant cells. Enhancement of the inhibition of cell adhesion and FAK phosphorylation also support the above data in GnT-V transfectants of HSC-2 cells. Interestingly, the differences in sensitivity to cisplatin and FAK phosphorylation between cisplatin-sensitive and -resistant cells were completely abolished by treatment with a neutral antibody of alpha5beta1 integrin. These results suggest that modification of oligosaccharides of alpha5beta1 integrin represents one of the possible mechanisms of drug resistance in head and neck cancer cells.
Carcinoma, Squamous Cell↗
An enzymatic method of analysis for GDP-L-fucose in biological samples, involving high-performance liquid chromatography.
To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.
Aminopyridines↗
Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens on colon cancer cells and enhanced liver metastasis.
Cyclooxygenase-2 (COX-2) was recently reported (M. Tsujii and R. N. DuBois, Cell, 83: 493-501, 1995) to affect the metastatic potential of cells. Previous studies (M. Fukuda, Cancer Res., 56: 2237-2244, 1996) indicated that sialyl Lewis antigen expression is correlated with hematogenous metastasis of colon cancer. In the present study, we investigated the interaction between COX-2 activity, expression of sialyl Lewis antigens, in vitro cancer cell adhesion to endothelial cells, and in vivo metastatic potential. Effects of COX-2 activity and prostaglandin E(2) on cell adhesion, expression of sialyl Lewis antigens, and glycosyltransferase genes were determined in Caco-2-m (COX-2 low level), Caco-2-COX-2 (programmed to overexpress COX-2), and HT-29 (COX-2 high level) cells. Metastatic spread of these cells to the liver was also investigated. Caco-2-COX-2 cells had increased SPan-1 levels and increased adherence to endothelial cells via SPan-1 compared with Caco-2-m cells. HT-29 cells expressed sialyl Lewis a and adhered to endothelial cells via sialyl Lewis a. Treatment with a COX-2 inhibitor, celecoxib, decreased SPan-1 and sialyl Lewis a expression and adherence to endothelial cells. beta 3Gal-T5 and ST3Gal III and IV expression was inhibited by celecoxib and was enhanced by prostaglandin E(2) treatment. Caco-2-COX-2 and HT-29 cells metastasized to the liver, whereas Caco-2-m cells did not. Pretreatment with celecoxib reduced the metastatic potential as well as anti-sialyl Lewis antibodies. Our results indicate a direct link between COX-2 and enhanced adhesion of carcinoma cells to endothelial cells, and enhanced liver metastatic potential via accelerated production of sialyl Lewis antigens. COX-2 inhibitors may suppress metastasis.
Animals↗
A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity.
Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that beta1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.
Amino Acid Sequence↗
Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching.
Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that beta1-6 GlcNAc branching, a product of UDP-GlcNAc alpha-mannoside beta1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which beta1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.
Acetylglucosamine↗