Investigating the Role of the Microsomal Epoxide Hydrolase Membrane Topology and Its Implication for Drug Metabolism Pathways

  • Thomas Friedberg
  • Bettina Löllmann
  • Roger Becker
  • Romy Holler
  • Michael Arand
  • Franz Oesch
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 387)

Summary

The microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of reactive epoxides which are formed by the action of cytochromes P450 from xenobiotics. In addition the mEH has been found to mediate the transport of bile acids. For the mEH it has been shown that it is cotranslationally inserted into the endoplasmic reticulum. Here we demonstrate that the amino-terminal twenty amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can be also supplied by a cytochrome P450 (CYP2B1) anchor signal sequence.

In addition we present data showing that the membrane anchor signal sequence of the mEH is dispensable for the catalytic activity of this protein. Our results indicate that it might be feasable to invert the topology of the mEH in the membrane of the endoplasmic reticulum without affecting the catalytic activity of this protein. With this strategy it will be possible to investigate whether the membrane topology of xenobiotic metabolizing enzymes is important for their role in chemical carcinogenesis.

Keywords

Epoxide Hydrolase Membrane Anchor Membrane Topology Cell Free Translation Microsomal Epoxide Hydrolase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

ER

endoplasmic reticulum

mEH

microsomal epoxide hydrolase

δmEH

truncated epoxide hydrolase

CYP

EH

cytochrome P450

mEH fusionprotein

PAGE

polyacrylamide gel electrophoresis

PMSF

phenylmehylsulfonylfluoride

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References

  1. 1.
    Davies, R.L., Crespi, C.L., Rudo, K., Turner, T.R. and Langenbach, R. (1989) Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens. Careinogenesis, 10, 885–891.CrossRefGoogle Scholar
  2. 2.
    Gesch, F. (1973) Mammalian epoxide hydrases. Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica, 3, 305–340.CrossRefGoogle Scholar
  3. 3.
    Grover, P.L. (1986) Pathways involved in the metabolism and activation of polycyclic aromatic hydrocarbons. Xenobiotica, 16, 915–931.PubMedCrossRefGoogle Scholar
  4. 4.
    Wood, A.W., Levin W., Lu A.Y.H., Yagi H., Hernandez O., Jerina D.M. and Conney A.H. (1976) Metabolism of benzo (a) pyrene and benzo (a) pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes. J. Biol. Chem., 251, 4882–4890.PubMedGoogle Scholar
  5. 5.
    Bar-Nun, S., Kreibich G., Adesnik M., Alterman L., Negishi M. and Sabatini D.D. (1980) Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc. Natl. Acad. Sci. USA., 77, 965–969.PubMedCrossRefGoogle Scholar
  6. 6.
    De Lemos-Chiarandini, C, Frey A.B., Sabatini D.D. and Kreibich G. (1987) Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site specific antibodies. J. Cell. Biol., 104, 209–219.PubMedCrossRefGoogle Scholar
  7. 7.
    Szczesna-Skorupa, E., Browne N., Mead D. and Kemper B. (1988) Positive charges at the NH2 terminus convert the membrane anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc. Natl. Acad. Sci. USA., 85, 738–742.PubMedCrossRefGoogle Scholar
  8. 8.
    Okada, Y., Frey A.B., Guenthner T.M., Oesch F., Sabatini D.D. and Kreibich G. (1982) Studies on the biosynthesis of microsomal membrane proteins. Eur. J. Biochem., 122, 393–402.PubMedCrossRefGoogle Scholar
  9. 9.
    Gonzales, F.J. and Kasper C.B. (1980) Biochem. Biophys. Res.Commun., 93, 1254–1258.CrossRefGoogle Scholar
  10. 10.
    Craft, J.A., Baird S., Lamont M. and Burchell B. (1990) Membrane topology of epoxide hydrolase. Biochem. Biophys. Acta, 1046, 32–39.PubMedCrossRefGoogle Scholar
  11. 11.
    Waechter, F., Bentley P., German M., Oesch F. and Stäubli W. (1982) Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment. Biochem. J., 202, 677–686.PubMedGoogle Scholar
  12. 12.
    Alves, C, Vondippe P., Amoui M. and Levy D. (1993) Bile acid transport into hepatocyte smooth endoplasmic reticulum vesicles is mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two distinct topological orientations. J Biol Chem, 268, 20148–20155.PubMedGoogle Scholar
  13. 13.
    Porter, T.D., Beck T.W. and Kasper C.B. (1986) Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase. Arch. Biochem. Biophys., 248, 121–129.PubMedCrossRefGoogle Scholar
  14. 14.
    Yabusaki, Y., Murakami H., Sakaki T., Shibata M. and Ohkawa H. (1988) Genetically engineered modification of P450 monooxygenases: Functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme. DNA, 7, 701–711.PubMedCrossRefGoogle Scholar
  15. 15.
    Sagara, Y., Barnes H.J. and Waterman M.R. (1993) Expression in Escherichia coli of functional P450c17 lacking its hydrophobic amino-terminal signal anchor. Arch. Biochem. Biophys., 304, 272–278.PubMedCrossRefGoogle Scholar
  16. 16.
    Larson, J.R., Coon M.J. and Porter T.D. (1993) Alcohol inducible cytochrome P450IIEl lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane bound when expressed in E. coli. J. Biol. Chem., 266, 7321–7324.Google Scholar
  17. 17.
    Friedberg, T., Lollmann B., Becker R., Holler R. and Oesch F. (1994) The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein. Biochem J, 303, 967–972.PubMedGoogle Scholar
  18. 18.
    Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  19. 19.
    Glatt, H.R., Gemperlein I., Setiabudi F., Platt K.L. and Oesch F. (1990) Expression of xenobiotic metabolising enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis, 5, 241–249.PubMedCrossRefGoogle Scholar
  20. 20.
    Clark, B.J. and Waterman M.R. (1991) Heterologous expression of mammalian P450 in COS cells. In Waterman, M.R. and Johnson, E.F. (eds), Methods in Enzymology. Vol. 206. Academic Press, San Diego, pp. 100–108.Google Scholar
  21. 21.
    Clark, B.J. and Waterman M.R. (1991) The hydrophobic amino-terminal sequence of bovine 17a hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells. J. Biol. Chem., 266, 5898–5904.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Thomas Friedberg
    • 1
  • Bettina Löllmann
    • 1
  • Roger Becker
    • 1
  • Romy Holler
    • 1
  • Michael Arand
    • 1
  • Franz Oesch
    • 1
  1. 1.Institute of ToxicologyUniversity of MainzMainzGermany

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