Expression of Functional Membrane‐Bound and Soluble Catechol‐O‐Methyltransferase in Escherichia coli and a Mammalian Cell Line

Publisher Website
Google Scholar
Abstract
Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; Δ membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained. Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37°C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 μM for MB-COMT and 108 μM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of ∼ 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue. Using the polymerase chain reaction (PCR) with primers surrounding the putative membrane anchor region, we have clearly identified a single-size PCR product generated from hCOMT mRNA of various human tissues. Hence, the two forms of the enzyme cannot be the products of an alternative splicing of transcripts. We suggest that S-COMT is generated by proteolytic cleavage between the NH2-terminal membrane anchor and the catalytic domain of the membrane-bound form. Lack of the N-terminal fragments reduces the catalytic activity of the enzyme.