VIROLOGY AND GLYCOBIOLOGY DIVISION

10.VIROLOGY AND GLYCOBIOLOGY DIVISION

Research Works

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

　A family of glycoconjugates, the gangliosides, have important functions in various biological phenomena such as cell growth and differentiation, embryogenesis and carcinogenesis. In vertebrates, the ganglio- series gangliosides are synthesized from a common precursor, ganglioside GM3. We have for the first time cloned a cDNA for human Ganglioside GM3 synthetic sialyltransferase (SAT- 1), and also subsequently for murine SAT- 1. Three different transcripts (L- ,B1- and B2- type) have been detected in mice, whereas only a single transcript was detectable in human organs. The L- type transcript was specifically expressed in murine liver, while the B1- type was generally detected in various organs, and B2- type expression was rarely detected.
　The genomic structures of human and murine SAT- 1 are being determined, and programs for the production of gene- knockout mice and of human gene- knockout somatic cells are underway, and the cDNA cloning of amino CTH synthase (one of the GlcNAc transferases)has also been started. Antisense oligo- nucleotide therapy for human melanoma was initiated using a GD3 synthase cDNA, since GD3- dominant melanoma was reported to have a worse prognosis. We are producing SAT- 1 soluble forms as MBP- /GST- fusion proteins which will be used in the development of automatic carbohydrate- chain synthesizers. We have also devised a new toroidal coil counter- current chromatography to isolate less polar alkali- labile glycolipids and proteins with higher sensitivity. ^(165-¹⁶⁸⁾
Docking Proteins Involved in Malignant Transformation

　Cas was first identified as a substrate of Src family tyrosine kinases, which is strongly phosphorylated during cellular transformation. It is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas- negative fibroblasts were recently established from knockout mice. These fibroblasts cannot be transformed by activated Src(527F Src), have no actin stress fiber formation, and show abnormal cell migration.⁽¹⁶⁹⁾ These phenotypes are rescued by expressing wild- type Cas protein. Cas has a 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. To understand the biological function of these domains, 9 different deletion mutants lacking each domain have been constructed and introduced into Cas- negative fibroblasts with/without 527F Src. Stable transfectants are being established and analyzed by the soft agar, immuno- staining, and other biological assays to check whether the phenotypes of Cas- negative fibroblasts can be rescued. Preliminary results indicate the importance of the central YDxP motifs and the Src- binding domain in transformation. These series of Cas mutants were tagged with GFP, and domains responsible for the change in the subcellular localization during cell migration or transformation are being analyzed. Co- existing glycolipids and sphingolipids have also been analyzed by IP- TLC analysis.
　ShcB and ShcC, neuro- specific members of the Shc family which transduce signals from receptor tyrosine kinases to Grb2/ Ras /MAP kinase pathways, have recently been found. They are tyrosine phosphorylated by the stimulation of growth factors such as neurotrophins and epidermal growth factor. Neuroblastoma, glioblastoma and melanoma cell lines have been screened for tyrosine phosphorylation by IP- western analyses, and marked tyrosine phosphorylation of ShcC was found in some neuroblastoma cell lines including NB39- nu, Nagai, and YT- nu cells. To elucidate the signaling pathway activated by tyrosine phosphorylation of ShcC, ShcC- associated molecules are being identified by IP- Western assay in these cell lines, and the effect of dominant- negative mutants are also being analyzed.
　To identify new docking proteins in malignancy, a series of GST- SH2 fusion proteins have been expressed in bacterial cells, purified, and coupled to CNBr sepharose columns. Using these affinity columns, the spectra of binding proteins are currently being analyzed in a number of cancer cell lines. Unique proteins will be subjected to peptide sequencing by mass- spectrometry (Q- TOF).
Hepatitis C Virus (HCV): Its Replication in vitro and Inhibition of Infection with Lactoferrin

　We developed a culture system using two human cell lines, MT- 2 and PH5CH for analysis of the infectivity of sera from HCV- positive blood donors. Using retroviral vector systems, PH5CH subclones which constitutively express HCV nonstructural regions could be established from the parental PH5CH cell line to allow efficient HCV replication. ^(170,¹⁷¹⁾ In these sublines, the positive strand of HCV- RNA could be detected by tagged RT- PCR after the negative strand of the synthesized consensus HCV- RNA clones was transfected.
　Lactoferrin (LF)was found to effectively prevent HCV infection in human hepatocytes. Subsequently, LF was demonstrated to be a candidate anti- HCV reagent that was well- tolerated and effective in the treatment of patients with chronic hepatitis. ⁽¹⁷²⁾




10.VIROLOGY AND GLYCOBIOLOGY DIVISION



	Research Works 　Research in the Virology and Glycobiology Division is focused on the molecular mechanism(s)and practical application(s) of genetic and epigenetic changes in the dynamic glycosylation or phosphorylation of membraneous and cytoplasmic regulatory molecules during viral infection or cancer formation. Biological Functions of Glycoconjugates in Cancer Cells 　A family of glycoconjugates, the gangliosides, have important functions in various biological phenomena such as cell growth and differentiation, embryogenesis and carcinogenesis. In vertebrates, the ganglio- series gangliosides are synthesized from a common precursor, ganglioside GM3. We have for the first time cloned a cDNA for human Ganglioside GM3 synthetic sialyltransferase (SAT- 1), and also subsequently for murine SAT- 1. Three different transcripts (L- ,B1- and B2- type) have been detected in mice, whereas only a single transcript was detectable in human organs. The L- type transcript was specifically expressed in murine liver, while the B1- type was generally detected in various organs, and B2- type expression was rarely detected. 　The genomic structures of human and murine SAT- 1 are being determined, and programs for the production of gene- knockout mice and of human gene- knockout somatic cells are underway, and the cDNA cloning of amino CTH synthase (one of the GlcNAc transferases)has also been started. Antisense oligo- nucleotide therapy for human melanoma was initiated using a GD3 synthase cDNA, since GD3- dominant melanoma was reported to have a worse prognosis. We are producing SAT- 1 soluble forms as MBP- /GST- fusion proteins which will be used in the development of automatic carbohydrate- chain synthesizers. We have also devised a new toroidal coil counter- current chromatography to isolate less polar alkali- labile glycolipids and proteins with higher sensitivity. ^(165-¹⁶⁸⁾ Docking Proteins Involved in Malignant Transformation 　Cas was first identified as a substrate of Src family tyrosine kinases, which is strongly phosphorylated during cellular transformation. It is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas- negative fibroblasts were recently established from knockout mice. These fibroblasts cannot be transformed by activated Src(527F Src), have no actin stress fiber formation, and show abnormal cell migration.⁽¹⁶⁹⁾ These phenotypes are rescued by expressing wild- type Cas protein. Cas has a 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. To understand the biological function of these domains, 9 different deletion mutants lacking each domain have been constructed and introduced into Cas- negative fibroblasts with/without 527F Src. Stable transfectants are being established and analyzed by the soft agar, immuno- staining, and other biological assays to check whether the phenotypes of Cas- negative fibroblasts can be rescued. Preliminary results indicate the importance of the central YDxP motifs and the Src- binding domain in transformation. These series of Cas mutants were tagged with GFP, and domains responsible for the change in the subcellular localization during cell migration or transformation are being analyzed. Co- existing glycolipids and sphingolipids have also been analyzed by IP- TLC analysis. 　ShcB and ShcC, neuro- specific members of the Shc family which transduce signals from receptor tyrosine kinases to Grb2/ Ras /MAP kinase pathways, have recently been found. They are tyrosine phosphorylated by the stimulation of growth factors such as neurotrophins and epidermal growth factor. Neuroblastoma, glioblastoma and melanoma cell lines have been screened for tyrosine phosphorylation by IP- western analyses, and marked tyrosine phosphorylation of ShcC was found in some neuroblastoma cell lines including NB39- nu, Nagai, and YT- nu cells. To elucidate the signaling pathway activated by tyrosine phosphorylation of ShcC, ShcC- associated molecules are being identified by IP- Western assay in these cell lines, and the effect of dominant- negative mutants are also being analyzed. 　To identify new docking proteins in malignancy, a series of GST- SH2 fusion proteins have been expressed in bacterial cells, purified, and coupled to CNBr sepharose columns. Using these affinity columns, the spectra of binding proteins are currently being analyzed in a number of cancer cell lines. Unique proteins will be subjected to peptide sequencing by mass- spectrometry (Q- TOF). Hepatitis C Virus (HCV): Its Replication in vitro and Inhibition of Infection with Lactoferrin 　We developed a culture system using two human cell lines, MT- 2 and PH5CH for analysis of the infectivity of sera from HCV- positive blood donors. Using retroviral vector systems, PH5CH subclones which constitutively express HCV nonstructural regions could be established from the parental PH5CH cell line to allow efficient HCV replication. ^(170,¹⁷¹⁾ In these sublines, the positive strand of HCV- RNA could be detected by tagged RT- PCR after the negative strand of the synthesized consensus HCV- RNA clones was transfected. 　Lactoferrin (LF)was found to effectively prevent HCV infection in human hepatocytes. Subsequently, LF was demonstrated to be a candidate anti- HCV reagent that was well- tolerated and effective in the treatment of patients with chronic hepatitis. ⁽¹⁷²⁾