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Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49).

Cai H, Jiang J, Yang Q, Chen Q, Deng Y - PLoS ONE (2012)

Bottom Line: All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile.Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein.The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

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In vivo and in vitro expression patterns of CYP2D49.(A) CYP2D49 transcripts in the indicated tissues of the healthy chicken were detected by real-time PCR amplification. The expression of the target gene was calculated relative to that of 18S rRNA according to the 2−ΔCT method. Error bars represent standard deviations obtained by measuring each sample in triplicate. He-Heart; Li-Liver; Sp-Spleen; Lu-Lung; Ki-Kidney; Br-Brain; Sin-Small intestine; Te-Testis; Ov-Ovary. (B) The lysates of the above chicken tissues were separated by 10% SDS-PAGE and then stained by Coomassie brilliant blue R-250 for normalization of sample loadings. Western blotting analysis using anti-CYP2D49 antiserum was exploited to detect the expression of CYP2D49 at the protein level. (C) LMH cells were treated with rifampicin, clotrimazole, β-naphthoflavone, dexamethasone and bufuralol for 24, 48 and 72 h, respectively. Real-time PCR was used to detect the levels of CYP2D49 mRNA. The ratio of CYP2D49 to β-actin in control cells was set to 1 and the values in all treated cells were normalized relative to this value. The experiments were conducted in triplicate and the data are expressed as the mean ± SD.
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pone-0038395-g003: In vivo and in vitro expression patterns of CYP2D49.(A) CYP2D49 transcripts in the indicated tissues of the healthy chicken were detected by real-time PCR amplification. The expression of the target gene was calculated relative to that of 18S rRNA according to the 2−ΔCT method. Error bars represent standard deviations obtained by measuring each sample in triplicate. He-Heart; Li-Liver; Sp-Spleen; Lu-Lung; Ki-Kidney; Br-Brain; Sin-Small intestine; Te-Testis; Ov-Ovary. (B) The lysates of the above chicken tissues were separated by 10% SDS-PAGE and then stained by Coomassie brilliant blue R-250 for normalization of sample loadings. Western blotting analysis using anti-CYP2D49 antiserum was exploited to detect the expression of CYP2D49 at the protein level. (C) LMH cells were treated with rifampicin, clotrimazole, β-naphthoflavone, dexamethasone and bufuralol for 24, 48 and 72 h, respectively. Real-time PCR was used to detect the levels of CYP2D49 mRNA. The ratio of CYP2D49 to β-actin in control cells was set to 1 and the values in all treated cells were normalized relative to this value. The experiments were conducted in triplicate and the data are expressed as the mean ± SD.

Mentions: To determine the distribution of CYP2D49 in chicken tissues, real-time PCR and western blotting analyses were performed on nine tissues. As shown in Fig. 3A, CYP2D49 was ubiquitously distributed at the mRNA level in all normal tissues assayed. It was predominantly expressed in the liver, kidney and small intestine, with lower transcription levels in the brain, lung, heart, spleen, testis and ovary. Using the generated anti-CYP2D49 antiserum, the expression of CYP2D49 at the protein level was further analyzed. As shown in Fig. 3B, CYP2D49 protein exhibited similar expression patterns to its transcription patterns, with protein expression occurring predominantly in the liver, kidney and small intestine, along with weak expression in the testis and ovary. If extending the exposure time, weaker bands could be detected in the brain, lung, heart and spleen (data not shown).


Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49).

Cai H, Jiang J, Yang Q, Chen Q, Deng Y - PLoS ONE (2012)

In vivo and in vitro expression patterns of CYP2D49.(A) CYP2D49 transcripts in the indicated tissues of the healthy chicken were detected by real-time PCR amplification. The expression of the target gene was calculated relative to that of 18S rRNA according to the 2−ΔCT method. Error bars represent standard deviations obtained by measuring each sample in triplicate. He-Heart; Li-Liver; Sp-Spleen; Lu-Lung; Ki-Kidney; Br-Brain; Sin-Small intestine; Te-Testis; Ov-Ovary. (B) The lysates of the above chicken tissues were separated by 10% SDS-PAGE and then stained by Coomassie brilliant blue R-250 for normalization of sample loadings. Western blotting analysis using anti-CYP2D49 antiserum was exploited to detect the expression of CYP2D49 at the protein level. (C) LMH cells were treated with rifampicin, clotrimazole, β-naphthoflavone, dexamethasone and bufuralol for 24, 48 and 72 h, respectively. Real-time PCR was used to detect the levels of CYP2D49 mRNA. The ratio of CYP2D49 to β-actin in control cells was set to 1 and the values in all treated cells were normalized relative to this value. The experiments were conducted in triplicate and the data are expressed as the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366968&req=5

pone-0038395-g003: In vivo and in vitro expression patterns of CYP2D49.(A) CYP2D49 transcripts in the indicated tissues of the healthy chicken were detected by real-time PCR amplification. The expression of the target gene was calculated relative to that of 18S rRNA according to the 2−ΔCT method. Error bars represent standard deviations obtained by measuring each sample in triplicate. He-Heart; Li-Liver; Sp-Spleen; Lu-Lung; Ki-Kidney; Br-Brain; Sin-Small intestine; Te-Testis; Ov-Ovary. (B) The lysates of the above chicken tissues were separated by 10% SDS-PAGE and then stained by Coomassie brilliant blue R-250 for normalization of sample loadings. Western blotting analysis using anti-CYP2D49 antiserum was exploited to detect the expression of CYP2D49 at the protein level. (C) LMH cells were treated with rifampicin, clotrimazole, β-naphthoflavone, dexamethasone and bufuralol for 24, 48 and 72 h, respectively. Real-time PCR was used to detect the levels of CYP2D49 mRNA. The ratio of CYP2D49 to β-actin in control cells was set to 1 and the values in all treated cells were normalized relative to this value. The experiments were conducted in triplicate and the data are expressed as the mean ± SD.
Mentions: To determine the distribution of CYP2D49 in chicken tissues, real-time PCR and western blotting analyses were performed on nine tissues. As shown in Fig. 3A, CYP2D49 was ubiquitously distributed at the mRNA level in all normal tissues assayed. It was predominantly expressed in the liver, kidney and small intestine, with lower transcription levels in the brain, lung, heart, spleen, testis and ovary. Using the generated anti-CYP2D49 antiserum, the expression of CYP2D49 at the protein level was further analyzed. As shown in Fig. 3B, CYP2D49 protein exhibited similar expression patterns to its transcription patterns, with protein expression occurring predominantly in the liver, kidney and small intestine, along with weak expression in the testis and ovary. If extending the exposure time, weaker bands could be detected in the brain, lung, heart and spleen (data not shown).

Bottom Line: All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile.Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein.The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

Show MeSH
Related in: MedlinePlus