Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form.
A cDNA clone for human catechol-O-methyltransferase (hCOMT; S-adenosyl-L-methionine:catechol O-methyltransferase; EC 2.1.1.6) was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (COMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The endogenous COMT activity, which was approximately 9.98 units per mg of protein in the untransfected cells, increased to 206 units per mg of protein upon transfection with a plasmid containing the COMT cDNA. The COMT activity of recombinant protein was inhibited competitively (IC50 = 700 nM) by the selective COMT inhibitor Ro 40-7592. An anti-COMT monoclonal antibody recognized, on immunoblots, a major polypeptide with apparent molecular mass of 29 kDa, in reasonable agreement with the predicted molecular mass. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A)+ RNA.
Bertocci
B
B
Pharma Research Central Nervous System, F. Hoffman-La Roche Ltd, Basel, Switzerland.
Miggiano
V
V
Da Prada
M
M
Dembic
Z
Z
Lahm
H W
HW
Malherbe
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002394:Catechol O-Methyltransferase / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D015723:Gene Library; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014162:Transfection
D004273:DNA, Neoplasm; D007527:Isoenzymes; D002394:Catechol O-Methyltransferase