Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: Comparison with the known marmoset CYP2D19

Hiroyuki Hichiya, Shino Kuramoto, Shigeo Yamamoto, Sumio Shinoda, Nobumitsu Hanioka, Shizuo Narimatsu, Kazuo Asaoka, Atsuro Miyata, Shinichi Iwata, Masahiro Nomoto, Tetsuo Satoh, Kimio Kiryu, Nobuhiko Ueda, Shinsaku Naito, Geoffrey T. Tucker, S. Wynne Ellis

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1″-hydroxylase activities, similar to CYP2D6. BF 1″-hydroxylation by CYP2D30 exhibited product- enantioselectivity of (1″R-OH-BF ≪ 1″S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1″R-OH-BF ≥ 1″S-OH-BF). BF 1″-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1″-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.

Original languageEnglish
Pages (from-to)165-175
Number of pages11
JournalBiochemical Pharmacology
Volume68
Issue number1
DOIs
Publication statusPublished - Jul 1 2004

Fingerprint

Callithrix
Cloning
Liver
Cytochrome P-450 Enzyme System
Organism Cloning
Cytochrome P-450 CYP2D6
Hydroxylation
Complementary DNA
Enzymes
Mixed Function Oxygenases
Debrisoquin
Messenger RNA
Quinidine
Enantioselectivity
Polymerase chain reaction
Transcription
bufuralol
Rats
Liver Microsomes
Nucleotides

Keywords

  • 1″-hydroxybufuralol
  • 1″-OH-BF
  • 4-hydroxydebrisoquine
  • 4-OH-DB
  • BF
  • bufuralol
  • CYP
  • cytochrome P450
  • DB
  • debrisoquine
  • G-6-P
  • glucose-6-phosphate
  • Kagoshima University
  • KAU
  • Kyoto University
  • KYU
  • reverse transcriptase-polymerase chain reaction
  • RT-PCR

ASJC Scopus subject areas

  • Pharmacology

Cite this

Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30 : Comparison with the known marmoset CYP2D19. / Hichiya, Hiroyuki; Kuramoto, Shino; Yamamoto, Shigeo; Shinoda, Sumio; Hanioka, Nobumitsu; Narimatsu, Shizuo; Asaoka, Kazuo; Miyata, Atsuro; Iwata, Shinichi; Nomoto, Masahiro; Satoh, Tetsuo; Kiryu, Kimio; Ueda, Nobuhiko; Naito, Shinsaku; Tucker, Geoffrey T.; Ellis, S. Wynne.

In: Biochemical Pharmacology, Vol. 68, No. 1, 01.07.2004, p. 165-175.

Research output: Contribution to journalArticle

Hichiya, H, Kuramoto, S, Yamamoto, S, Shinoda, S, Hanioka, N, Narimatsu, S, Asaoka, K, Miyata, A, Iwata, S, Nomoto, M, Satoh, T, Kiryu, K, Ueda, N, Naito, S, Tucker, GT & Ellis, SW 2004, 'Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: Comparison with the known marmoset CYP2D19', Biochemical Pharmacology, vol. 68, no. 1, pp. 165-175. https://doi.org/10.1016/j.bcp.2004.03.018
Hichiya, Hiroyuki ; Kuramoto, Shino ; Yamamoto, Shigeo ; Shinoda, Sumio ; Hanioka, Nobumitsu ; Narimatsu, Shizuo ; Asaoka, Kazuo ; Miyata, Atsuro ; Iwata, Shinichi ; Nomoto, Masahiro ; Satoh, Tetsuo ; Kiryu, Kimio ; Ueda, Nobuhiko ; Naito, Shinsaku ; Tucker, Geoffrey T. ; Ellis, S. Wynne. / Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30 : Comparison with the known marmoset CYP2D19. In: Biochemical Pharmacology. 2004 ; Vol. 68, No. 1. pp. 165-175.
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T1 - Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30

T2 - Comparison with the known marmoset CYP2D19

AU - Hichiya, Hiroyuki

AU - Kuramoto, Shino

AU - Yamamoto, Shigeo

AU - Shinoda, Sumio

AU - Hanioka, Nobumitsu

AU - Narimatsu, Shizuo

AU - Asaoka, Kazuo

AU - Miyata, Atsuro

AU - Iwata, Shinichi

AU - Nomoto, Masahiro

AU - Satoh, Tetsuo

AU - Kiryu, Kimio

AU - Ueda, Nobuhiko

AU - Naito, Shinsaku

AU - Tucker, Geoffrey T.

AU - Ellis, S. Wynne

PY - 2004/7/1

Y1 - 2004/7/1

N2 - Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1″-hydroxylase activities, similar to CYP2D6. BF 1″-hydroxylation by CYP2D30 exhibited product- enantioselectivity of (1″R-OH-BF ≪ 1″S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1″R-OH-BF ≥ 1″S-OH-BF). BF 1″-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1″-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.

AB - Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1″-hydroxylase activities, similar to CYP2D6. BF 1″-hydroxylation by CYP2D30 exhibited product- enantioselectivity of (1″R-OH-BF ≪ 1″S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1″R-OH-BF ≥ 1″S-OH-BF). BF 1″-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1″-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.

KW - 1″-hydroxybufuralol

KW - 1″-OH-BF

KW - 4-hydroxydebrisoquine

KW - 4-OH-DB

KW - BF

KW - bufuralol

KW - CYP

KW - cytochrome P450

KW - DB

KW - debrisoquine

KW - G-6-P

KW - glucose-6-phosphate

KW - Kagoshima University

KW - KAU

KW - Kyoto University

KW - KYU

KW - reverse transcriptase-polymerase chain reaction

KW - RT-PCR

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