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Molecular Pharmacology

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Research ArticleArticle

CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of ω-3-Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331P Variant

Mariko Nakano, Edward J. Kelly, Constanze Wiek, Helmut Hanenberg and Allan E. Rettie
Molecular Pharmacology October 2012, 82 (4) 679-686; DOI: https://doi.org/10.1124/mol.112.080085
Mariko Nakano
Departments of Medicinal Chemistry (M.N., A.E.R.) and Pharmaceutics (E.J.K.), School of Pharmacy, University of Washington, Seattle, Washington; Department of Otorhinolaryngology, Head and Neck Surgery, Heinrich Heine University, Dusseldorf, Germany (C.W., H.H.); and Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, Indiana (H.H.)
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Edward J. Kelly
Departments of Medicinal Chemistry (M.N., A.E.R.) and Pharmaceutics (E.J.K.), School of Pharmacy, University of Washington, Seattle, Washington; Department of Otorhinolaryngology, Head and Neck Surgery, Heinrich Heine University, Dusseldorf, Germany (C.W., H.H.); and Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, Indiana (H.H.)
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Constanze Wiek
Departments of Medicinal Chemistry (M.N., A.E.R.) and Pharmaceutics (E.J.K.), School of Pharmacy, University of Washington, Seattle, Washington; Department of Otorhinolaryngology, Head and Neck Surgery, Heinrich Heine University, Dusseldorf, Germany (C.W., H.H.); and Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, Indiana (H.H.)
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Helmut Hanenberg
Departments of Medicinal Chemistry (M.N., A.E.R.) and Pharmaceutics (E.J.K.), School of Pharmacy, University of Washington, Seattle, Washington; Department of Otorhinolaryngology, Head and Neck Surgery, Heinrich Heine University, Dusseldorf, Germany (C.W., H.H.); and Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, Indiana (H.H.)
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Allan E. Rettie
Departments of Medicinal Chemistry (M.N., A.E.R.) and Pharmaceutics (E.J.K.), School of Pharmacy, University of Washington, Seattle, Washington; Department of Otorhinolaryngology, Head and Neck Surgery, Heinrich Heine University, Dusseldorf, Germany (C.W., H.H.); and Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine, Indiana University, Indianapolis, Indiana (H.H.)
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Abstract

Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM49054]; and the Drug Metabolism Transport and Pharmacogenetics Research Fund in the School of Pharmacy, University of Washington.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.080085.

  • ABBREVIATIONS:

    BCD
    Bietti's crystalline corneoretinal dystrophy
    P450
    cytochrome P450
    DHA
    docosahexaenoic acid
    EPA
    eicosapentaenoic acid
    PUFA
    polyunsaturated fatty acid
    RPE
    retinal pigmented epithelium
    PCR
    polymerase chain reaction
    GC
    gas chromatography
    PAGE
    polyacrylamide gel electrophoresis
    RT
    reverse transcription
    CI
    chemical ionization
    MS
    mass spectrometry
    FID
    flame ionization detection
    HET0016
    N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine.

  • Received May 29, 2012.
  • Accepted July 6, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (4)
Molecular Pharmacology
Vol. 82, Issue 4
1 Oct 2012
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Research ArticleArticle

Characterization of CYP4V2

Mariko Nakano, Edward J. Kelly, Constanze Wiek, Helmut Hanenberg and Allan E. Rettie
Molecular Pharmacology October 1, 2012, 82 (4) 679-686; DOI: https://doi.org/10.1124/mol.112.080085

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Research ArticleArticle

Characterization of CYP4V2

Mariko Nakano, Edward J. Kelly, Constanze Wiek, Helmut Hanenberg and Allan E. Rettie
Molecular Pharmacology October 1, 2012, 82 (4) 679-686; DOI: https://doi.org/10.1124/mol.112.080085
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