VIROLOGY AND GLYCOBIOLOGY DIVISION

10.VIROLOGY AND GLYCOBIOLOGY DIVISION

    Research in the Virology and Glycobiology Division is focused on the molecular mechanism(s) of genetic and epigenetic changes in the dynamic glycosylation or phosphorylation of membranous and cytoplasmic regulatory molecules during cancer formation, and their practical application.
Biological Functions of Glycoconjugates in Cancer Cells

    Gangliosides are a family of glycosphingolipids that contain sialic acid. The first ganglioside synthesized, GM3, is produced by sialyltransferase-1 (SAT-1), is the common precursor of almost all gangliosides, and is involved in cell proliferation, differentiation and carcinogenesis.⁽¹³⁸⁾ The cDNA of human GM3 synthase (SAT-1/ST3GalV) has been successfully cloned by an elaborate expression method, and subsequently a murine SAT-1 cDNA was also obtained. Three transcripts for this enzyme were detected in mice, whereas only a single transcript was detected in human organs. The L-type transcript was specifically expressed in murine liver while the B1-type was generally detected in various organs with B2-type marginally expressed in general. When human cDNA was transfected into colon cancer HCT116 cell line, apoptosis was induced within 3 days of culture, with a concurrent significant increase in SAT-1 activity and GM3 expression. A complete genomic DNA clone of the GM3 synthase gene, was isolated, and it was found to span approximately 56 kb in human genome and consist of 7 exons and 6 introns. The GC content in the region of the transcription start site is high (81%), and the human gene promoter contains no canonical TATA and CCAAT boxes. A number of cis-acting elements for transcription are noted in the 5'-flanking region. The promoter activity was much higher in human neuroblastoma SK-N-MC than in colon cancer HCT116 cell line. Three Sp1 binding sites in the GC-rich region seemed to be a critical positive regulatory element in cell- specific expression of human gene. The genomic structure of murine SAT-1 was determined to consist of 7 exons and 6 introns, with the first exon having 3 forms. A gene-knockout mouse production has proceeded with the successful deletion of a single allele in ES cells. To clarify three-dimensional structure of GM3 synthase and utilize it in the development of carbohydrate-chain synthesizers, its soluble form was produced in E. coli, and its active form was successfully expressed in the periplasm as MBP-fusion protein, using pMAL-p2 vector.
Molecular Structures and Functions of Signal Adaptor (Docking) Proteins Involved in Malignant Transformation

    Cas, a substrate of Src family tyrosine kinases, is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk.⁽¹³⁹⁾ Cas-negative fibroblasts were recently established from knockout mice, which have the following phenotypic features: 1) defective in transformation by activated Src(527F Src); 2) lack of actin stress fiber formation; 3) abnormal cell migration. These are rescued by wild-type Cas protein. Cas has an unique structure, with an SH3 domain in the N-terminal region followed by 15 repeats of similar SH2-binding motifs and a Src-binding domain in the C-terminal region. Using 9 different deletion mutants with/without 527F Src, it was found that the central YDxP motifs and the Src-binding domain are important in transformation. Using Cas mutants tagged with GFP, domains responsible for the subcellular localization of Cas during cell migration or transformation are being analyzed. Neurospecific ShcB and ShcC, which transduce signals from receptor tyrosine kinases to Grb2/Ras/MAP kinase pathways, have recently been discovered.⁽¹⁴⁰⁾ They are tyrosine phosphorylated as a result of cellular stimulation by growth factors. By IP-Western analysis, marked tyrosine phosphorylation of ShcC was found in some neuroblastoma cell lines (NB39-nu, Nagai, YT-nu cells) and ShcC-associating molecules and dominant-negative mutants are being analyzed. Using a series of GST-SH2 fusion protein columns, the spectra of binding proteins are being analyzed in cancer cell lines.
Cloning New Hematopoietic Factors for Stem Cell Growth from Stromal Cells

    From stromal OP9 cell line, which can induce ES cells to differentiate into hematopoietic cells, a cDNA library was constructed and inserted into the pMX-SST retroviral vector. By signal sequence trap method, new hematopoietic factors are being searched for under LIF stimulation with murine pro-B cell line Ba/F3 cells as the target.
Viral Carcinogenesis: Hepatitis C Virus (HCV) Replication in vitro

    In human MT-2 and PH5CH cells, the growth of HCV consensus constructs was detectable.^(141-¹⁴³⁾ In PH5CH subclones which constitutively express HCV nonstructural regions to allow efficient HCV replication, the positive strand of HCV-RNA could be detected by the tagged RT-PCR after the negative strand was transfected.




10.VIROLOGY AND GLYCOBIOLOGY DIVISION



	Research in the Virology and Glycobiology Division is focused on the molecular mechanism(s) of genetic and epigenetic changes in the dynamic glycosylation or phosphorylation of membranous and cytoplasmic regulatory molecules during cancer formation, and their practical application. Biological Functions of Glycoconjugates in Cancer Cells Gangliosides are a family of glycosphingolipids that contain sialic acid. The first ganglioside synthesized, GM3, is produced by sialyltransferase-1 (SAT-1), is the common precursor of almost all gangliosides, and is involved in cell proliferation, differentiation and carcinogenesis.⁽¹³⁸⁾ The cDNA of human GM3 synthase (SAT-1/ST3GalV) has been successfully cloned by an elaborate expression method, and subsequently a murine SAT-1 cDNA was also obtained. Three transcripts for this enzyme were detected in mice, whereas only a single transcript was detected in human organs. The L-type transcript was specifically expressed in murine liver while the B1-type was generally detected in various organs with B2-type marginally expressed in general. When human cDNA was transfected into colon cancer HCT116 cell line, apoptosis was induced within 3 days of culture, with a concurrent significant increase in SAT-1 activity and GM3 expression. A complete genomic DNA clone of the GM3 synthase gene, was isolated, and it was found to span approximately 56 kb in human genome and consist of 7 exons and 6 introns. The GC content in the region of the transcription start site is high (81%), and the human gene promoter contains no canonical TATA and CCAAT boxes. A number of cis-acting elements for transcription are noted in the 5'-flanking region. The promoter activity was much higher in human neuroblastoma SK-N-MC than in colon cancer HCT116 cell line. Three Sp1 binding sites in the GC-rich region seemed to be a critical positive regulatory element in cell- specific expression of human gene. The genomic structure of murine SAT-1 was determined to consist of 7 exons and 6 introns, with the first exon having 3 forms. A gene-knockout mouse production has proceeded with the successful deletion of a single allele in ES cells. To clarify three-dimensional structure of GM3 synthase and utilize it in the development of carbohydrate-chain synthesizers, its soluble form was produced in E. coli, and its active form was successfully expressed in the periplasm as MBP-fusion protein, using pMAL-p2 vector. Molecular Structures and Functions of Signal Adaptor (Docking) Proteins Involved in Malignant Transformation Cas, a substrate of Src family tyrosine kinases, is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk.⁽¹³⁹⁾ Cas-negative fibroblasts were recently established from knockout mice, which have the following phenotypic features: 1) defective in transformation by activated Src(527F Src); 2) lack of actin stress fiber formation; 3) abnormal cell migration. These are rescued by wild-type Cas protein. Cas has an unique structure, with an SH3 domain in the N-terminal region followed by 15 repeats of similar SH2-binding motifs and a Src-binding domain in the C-terminal region. Using 9 different deletion mutants with/without 527F Src, it was found that the central YDxP motifs and the Src-binding domain are important in transformation. Using Cas mutants tagged with GFP, domains responsible for the subcellular localization of Cas during cell migration or transformation are being analyzed. Neurospecific ShcB and ShcC, which transduce signals from receptor tyrosine kinases to Grb2/Ras/MAP kinase pathways, have recently been discovered.⁽¹⁴⁰⁾ They are tyrosine phosphorylated as a result of cellular stimulation by growth factors. By IP-Western analysis, marked tyrosine phosphorylation of ShcC was found in some neuroblastoma cell lines (NB39-nu, Nagai, YT-nu cells) and ShcC-associating molecules and dominant-negative mutants are being analyzed. Using a series of GST-SH2 fusion protein columns, the spectra of binding proteins are being analyzed in cancer cell lines. Cloning New Hematopoietic Factors for Stem Cell Growth from Stromal Cells From stromal OP9 cell line, which can induce ES cells to differentiate into hematopoietic cells, a cDNA library was constructed and inserted into the pMX-SST retroviral vector. By signal sequence trap method, new hematopoietic factors are being searched for under LIF stimulation with murine pro-B cell line Ba/F3 cells as the target. Viral Carcinogenesis: Hepatitis C Virus (HCV) Replication in vitro In human MT-2 and PH5CH cells, the growth of HCV consensus constructs was detectable.^(141-¹⁴³⁾ In PH5CH subclones which constitutively express HCV nonstructural regions to allow efficient HCV replication, the positive strand of HCV-RNA could be detected by the tagged RT-PCR after the negative strand was transfected.