Functional Analysis of Genetic Variation in Catechol-O-Methyltransferase (COMT): Effects on mRNA, Protein, and Enzyme Activity in Postmortem Human Brain

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Functional Analysis of Genetic Variation in Catechol-O-Methyltransferase (COMT): Effects on mRNA, Protein, and Enzyme Activity in Postmortem Human Brain Jingshan Chen, Barbara K. Lipska, Nader Halim, Quang D. Ma, Mitsuyuki Matsumoto, Samer Melhem, Bhaskar S. Kolachana, Thomas M. Hyde, Mary M. Herman, Jose Apud, Michael F. Egan, Joel E. Kleinman, Daniel R. Weinberger The American Journal of Human Genetics Volume 75, Issue 5, Pages 807-821 (November 2004) DOI: 10.1086/425589 Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2004 75, 807-821DOI: (10.1086/425589) Copyright © 2004 The American Society of Human Genetics Terms and Conditions