Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain

Abstract

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.

Figure 1

Effect of the Val/Met SNP on S-COMT and MB-COMT protein immunoreactivity. A, Comparison of protein immunoreactivity (analyzed by Western blotting with the use of a specific anti-human COMT antibody) for S-COMT and MB-COMT proteins in postmortem DLPFC tissues with genotypes Val/Val, Val/Met, and Met/Met. The asterisk (*) indicates difference from Val/Val at a significance level of P<.05. B, A representative Western blotting image with MB-COMT, S-COMT, and internal control (Actin) bands.

Figure 2

Significantly higher activity of COMT-Val than of COMT-Met in human DLPFC tissue and in lymphocytes at 37°C. A, High correlation of the relative COMT activity with protein concentration (r=0.999) in a linear relationship. B, COMT activity in protein samples from human postmortem DLPFC tissues with genotypes Val/Val, Val/Met, and Met/Met. C, COMT activity in protein samples from lymphoblast cultures from subjects with genotypes Val/Val and Met/Met. N = sample size. Two asterisks (**) indicate difference from Val/Val at a significance level of P<.01.

Figure 3

Effect of the Val/Met genotype on COMT activity in human postmortem DLPFC tissue from two different populations (African Americans and whites). The effects of the Val/Met genotype in African Americans (A) and whites (B) were similar. N = sample size. One asterisk (*) indicates difference from Val/Val at a significance level of P<.05; two asterisks (**) indicate difference from Val/Val at a significance level of P<.001.

Figure 4

Comparison of the ratio of the enzyme activity between the heat-treated and cold-treated MB-COMT-Met and MB-COMT-Val. MB-COMT-Val is more stable than MB-COMT-Met at 37°C. The ratios (± SE) of COMT activity in the synthesized MB-COMT-Val and MB-COMT-Met samples exposed to a temperature of 37°C for 0–120 min (H) or kept on ice at 4°C (C), measured in triplicate. One asterisk (*) indicates difference from COMT-Met at a significance level of P<.05; two asterisks (**) indicate difference from MB-COMT-Met at a significance level of P<.01.

Figure 5

Comparison of the human and mouse cDNA sequences and COMT activity from three assays. The activity of mouse COMT-Leu is higher than that of human COMT-Val and COMT-Met. S-COMT proteins from human COMT-Val and COMT-Met, as well as mouse COMT-Leu, were synthesized using an in vitro transcription-coupled transcription system with ³⁵S-methionine as a radioactive substrate. A, Representative autoradiogram of the synthesized proteins with radioactive ³⁵S-methionine residue and the amino acid sequences of the three COMT proteins. B, Averaged COMT activity from three independent assays, normalized with the radioactivity of the ³⁵S-labeled COMT proteins. The activity of mouse COMT-Leu is significantly higher than that of human COMT-Val or COMT-Met. Two asterisks (**) indicate difference from mouse COMT-Leu at a significance level of P<.0001.

Figure 6

Comparison of the activity of the proteins in mouse COMT-Leu, COMT-Met, and COMT-Val. Mutations from Leu to Val and Met in mouse COMT resulted in a dramatic reduction in COMT activity. The Leu codon in mouse COMT cDNA was mutated into Val or Met by site-directed mutagenesis, and the wild-type and mutant mouse COMT proteins were synthesized using an in vitro protein synthesis system. A, Representative autoradiogram of the wild-type and mutant mouse S-COMT proteins labeled with ³⁵S-methionine and partial protein sequences. B, Averaged COMT activity from three independent assays, normalized with COMT protein levels. COMT activity differs significantly between the wild-type and mutant proteins. Two asterisks (**) indicate difference from wild-type COMT-Leu at a significance level of P<.0001.

Figure 7

Effect of the P2 promoter SNP on COMT activity in human DLPFC (A) and lymphoblast cell cultures (B) of different genotypes at the promoter SNP site. Two asterisks (**) indicate difference from genotype 1/1 at a significance level of P<.01. Similar effects were seen in lymphocytes of subjects with the homozygous Met/Met genotype. C, Abundant MB-COMT protein, detected by Western blotting with the use of a specific anti-human COMT antibody, in lymphocytes (L) and brain (B) protein samples.

Figure 8

Effect of the subject's sex on COMT activity in human DLPFC. Females had significantly lower COMT activity in the samples as a whole (A) as well as in each of the Val/Met genotypes (B). The asterisk (*) indicates difference from males at a significance level of P<.05.