title: Increased expression and phosphorylation of liver glutamine synthetase in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.
abstract: Hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and chronic infection with hepatitis C virus (HCV) is thought to be one of the main causes in Japan. To identify diagnostic or therapeutic biomarkers for HCC associated with HCV (HCV-HCC), we tried to elucidate the factors related to the products from cancerous tissues of HCV-infected patients. From proteomic differential display analysis of liver tissue samples from HCV-HCC cancerous tissues and corresponding non-cancerous tissues from patients, three protein spots of the same molecular mass (42 kDa), whose expression increased in well-differentiated cancerous tissues, were detected. Although their pI were different, they were identified as glutamine synthetase (GS) by PMF with MALDI-TOF MS and by Western blotting using anti-GS specific mAb. Immunohistochemical analysis showed that tumor tissue consists of two parts, GS-positive cell and GS-negative cell regions, suggesting that GS-producing cells grew in the tumor tissue as a nodule in nodules. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da, corresponding a peptide of SASIRIPR, and gained a signal of 1059.5 Da, which was submitted to PSD analysis. PSD analysis showed the neutral loss by elimination of two phosphate groups, supposed to be on serine residues of the 899.5-Da peptide, from serine 320 to arginine 327 in GS. PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation sites of proteins showing molecular heterogeneity.
authors.affiliation: Department of Biochemistry and Biomolecular Recognition, Yamaguchi University School of Medicine, Yamaguchi, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kuramitsu
authors.forename: Toshio
authors.initials: T
authors.lastname: Harada
authors.forename: Motonari
authors.initials: M
authors.lastname: Takashima
authors.forename: Yuuichirou
authors.initials: Y
authors.lastname: Yokoyama
authors.forename: Isao
authors.initials: I
authors.lastname: Hidaka
authors.forename: Norio
authors.initials: N
authors.lastname: Iizuka
authors.forename: Tosifusa
authors.initials: T
authors.lastname: Toda
authors.forename: Masanori
authors.initials: M
authors.lastname: Fujimoto
authors.forename: Xiulian
authors.initials: X
authors.lastname: Zhang
authors.forename: Isao
authors.initials: I
authors.lastname: Sakaida
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Okita
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oka
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000209:etiology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000378:metabolism; D019698:Hepatitis C, Chronic / Q000150:complications / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005974:Glutamate-Ammonia Ligase