Characterization of reduced expression of glycine N-methyltransferase in cancerous hepatic tissues using two newly developed monoclonal antibodies.
Glycine N-methyltransferase (GNMT) is a protein with multiple functions. Recently, two Italian siblings who had hepatomegaly and chronic elevation of serum transaminases were diagnosed to have GNMT deficiency caused by inherited compound heterozygosity of the GNMT gene with missence mutations. To evaluate the expression of GNMT in cell lines and tissues from hepatocellular carcinoma (HCC) patients, we produced two monoclonal antibodies (mAbs) 4-17 and 14-1 using two recombinant GNMT fusion proteins. M13 phage peptide display showed that the reactive epitopes of mAbs 4-17 and 14-1 were amino acid residues 11-15 and 272-276 of human GNMT, respectively. The dissociation constants of the binding between GNMT and mAbs were 1.7 x 10(-8) M for mAb 4-17 and 1.8 x 10(-9) M for mAb 14-1. Both mAbs can identify GNMT present in normal human and mouse liver tissues using Western blotting (WB) and immunohistochemical staining assay (IHC). In addition, WB with both mAbs showed that none of 2 hepatoblastoma and 5 HCC cell lines expressed GNMT. IHC demonstrated that 50% (13/26) of nontumorous liver tissues and 96% (24/25) of HCC tissues did not express GNMT. Therefore, the expression of GNMT was downregulated in human HCC.
Liu
Hsiao-Han
HH
Institute of Microbiology and Immunology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC.
Chen
Kuan-Hsuan
KH
Shih
Yi-Ping
YP
Lui
Wing-Yiu
WY
Wong
Fen-Hwa
FH
Chen
Yi-Ming A
YM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000915:Antibody Affinity; D015153:Blotting, Western; D015536:Down-Regulation / Q000187:drug effects; D018604:Epitope Mapping; D005822:Genetic Vectors; D050938:Glycine N-Methyltransferase; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D008780:Methyltransferases / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C494510:GNMT protein, human; D050938:Glycine N-Methyltransferase; C494511:Gnmt protein, mouse