Characterization of two new variants of human catechol O-methyltransferase in vitro

Abstract 

Catechol O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that human soluble COMT (S-COMT) is genetically polymorphic with a wild type and at least one variant in which a valine has been substituted with a methionine at codon 108. This polymorphism has been the subject of intense molecular epidemiological studies because of the important role of COMT in the metabolism of catecholamines and catechol estrogens. Several epidemiological studies have shown that women, homozygous with the Val108Met variant, have an increased risk of developing estrogen-associated cancers. However, some other studies have shown that this COMT polymorphism is not associated with increased risk of developing cancers. These conflicting data suggest that additional COMT genetic variants might contribute to the increased risk of developing cancers. Although two new single nucleotide polymorphisms (SNP) that cause amino acid substitutions Ala22Ser and Ala52Thr have been identified recently, they have not been fully characterized. In the present study, Ala22Ser and Ala52Thr variants of human S-COMT were produced using recombinant DNA techniques, and then COMT properties were measured including enzymatic activity, thermostability, and sensitivity to inhibition mediated by 4-hydroxyequilenin (4-OHEN). The Ala22Ser variant showed lower methylation capacity and higher thermolability. In addition, this variant is sensitive to 4-OHEN mediated irreversible inhibition. Our data indicate that the Ala22Ser polymorphism might also be of functional significance and might play a role in susceptibility to estrogen-associated cancers.

Keywords: COMT, Gene polymorphism, Estrogen replacement therapy, 4-Hydroxyequilenin

Abbreviations: 2-MeOE₂, 2-methoxyestradiol, 4-MeOE₂, 4-methoxyestradiol, 4-MeOEN, 4-methoxyequilenin, 4-OHE₂, 4-hydroxyestradiol, 4-OHEN, 4-hydroxyequilenin, COMT, catechol O-methyltransferase, DMSO, dimethyl sulfoxide, DTT, dithiothreitol, HPLC, high performance liquid chromatography, MB-COMT, membrane bound COMT, PBS, phosphate-buffered saline, PCR, polymerase chain reaction, SAM, S-adenosyl-l-methionine, S-COMT, soluble COMT, SNP, single nucleotide polymorphism