Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Oriol Gallego

Olga Belyaeva

Sergio Porté

F Ruiz

*Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain

†Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

^{To whom correspondence should be addressed (email}ude.bau@ilivhsidekn).

Received 2005 Dec 15; Revised 2006 May 11; Accepted 2006 Jun 21.

Abstract

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K_m values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K_m values for retinoids (0.12–1.1 μM), whilst they strongly differ in their k_cat values, which range from 0.35 min^{for AKR1B1 to 302 min}^{for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher}k_cat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.

Keywords: alcohol dehydrogenase, aldo-keto reductase, retinaldehyde, retinoic acid, retinol, short-chain dehydrogenase

Abbreviations: ADH, alcohol dehydrogenase; AKR, aldo-keto reductase; ASMC, aortic smooth muscle cell; CBD, chitin binding domain; CRBP, cellular retinol binding protein; EST, expressed sequence tag; FBS, foetal bovine serum; GST, glutathione S-transferase; HEK, human embryonic kidney cells; holoCRBPI, retinol bound to CRBP type I; LRAT, lecithin–retinol acyltransferase; MDR, medium-chain dehydrogenase/reductase; RoDH or RDH, retinol dehydrogenase; SDR, short-chain dehydrogenase/reductase

Abstract

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Acknowledgments

Supported by the National Institute on Alcohol Abuse and Alcoholism, grant AA12153, and by grants from the Spanish Dirección General de Investigación (BMC2003-09606, BFU2005-02621) and the Generalitat de Catalunya (2005 SGR 00112). We thank Professor Mauro Santos (Departament de Genetica i de Microbiologia, Facultat de Ciencies Universitat Autonoma de Barcelona, Bellaterra. Barcelona, Spain) for his advice in statistical analysis and Emeritus Professor T. Geoffrey Flynn (Department of Biochemistry, Queen's University, Kingston, Ontario, Canada) for support in AKR research.

Acknowledgments

References

1. Mangelsdorf D., Umesono K., Evans R. M. The retinoid receptors. In: Sporn M. B., Roberts A. B., Goodman D. S., editors. The Retinoids: Biology, Chemistry and Medicine. New York: Raven Press; 1994. pp. 319–350. [PubMed]
2. Vogel S., Gamble M. V., Blaner W. S. Biosynthesis, absorption, metabolism and transport of retinoids. In: Nau H., Blaner W. S., editors. Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoid Action, vol. 139. Berlin: Springer-Verlag; 1999. pp. 31–96. [PubMed]
3. von Lintig J., Wyss AMolecular analysis of vitamin A formation: cloning and characterization of β-carotene 15,15′-dioxygenases. Arch. Biochem. Biophys. 2001;385:47–52.[PubMed][Google Scholar]
4. Napoli J. L. Interactions of retinoid binding proteins and enzymes in retinoid metabolism. Biochim. Biophys. Acta. 1999;1440:139–162.[PubMed]
5. Noy NRetinoid-binding proteins: mediators of retinoid action. Biochem. J. 2000;348:481–495.[Google Scholar]
6. Folli C., Calderone V., Ottonello S., Bolchi A., Zanotti G., Stoppini M., Berni RIdentification, retinoid binding and X-ray analysis of a human retinol-binding protein. Proc. Natl. Acad. Sci. U.S.A. 2001;98:3710–3715.[Google Scholar]
7. Folli C., Calderone V., Ramazzina I., Zanotti G., Berni RLigand binding and structural analysis of a human putative cellular retinol-binding protein. J. Biol. Chem. 2002;277:41970–41977.[PubMed][Google Scholar]
8. Zachman R. D., Olson J. A. A comparison of retinene reductase and alcohol dehydrogenase of rat liver. J. Biol. Chem. 1961;236:2309–2313.[PubMed]
9. Yang Z. N., Davis G. J., Hurley T. D., Stone C. L., Li T. K., Bosron W. F. Catalytic efficiency of human alcohol dehydrogenases for retinol oxidation and retinal reduction. Alcohol Clin. Exp. Res. 1994;18:587–591.[PubMed]
10. Veech R. L., Eggleston L. V., Krebs H. A. The redox state of free nicotinamide–adenine dinucleotide phosphate in the cytoplasm of rat liver. Biochem. J. 1969;115:609–619.
11. Kedishvili N. Y., Gough W. H., Davis W. I., Parsons S., Li T. K., Bosron W. F. Effect of cellular retinol-binding protein on retinol oxidation by human class IV retinol/alcohol dehydrogenase and inhibition by ethanol. Biochem. Biophys. Res. Commun. 1998;249:191–196.[PubMed]
12. Kedishvili N. Y. Multifunctional nature of human retinol dehydrogenases. Curr. Org. Chem. 2002;6:1247–1257.[PubMed]
13. Lapshina E. A., Belyaeva O. V., Chumakova O. V., Kedishvili N. Y. Differential recognition of the free versus bound retinol by human microsomal retinol/sterol dehydrogenases: characterization of the holo-CRBP dehydrogenase activity of RoDH-4. Biochemistry. 2003;42:776–784.[PubMed]
14. Belyaeva O. V., Stetsenko A. V., Nelson P., Kedishvili N. Y. Properties of short-chain dehydrogenase/reductase RalR1: characterization of purified enzyme, its orientation in the microsomal membrane and distribution in human tissues and cell lines. Biochemistry. 2003;42:14838–14845.[PubMed]
15. Crosas B., Cederlund E., Torres D., Jörnvall H., Farrés J., Parés XA vertebrate aldo-keto reductase active with retinoids and ethanol. J. Biol. Chem. 2001;276:19132–19140.[PubMed][Google Scholar]
16. Crosas B., Hyndman D. J., Gallego O., Martras S., Parés X., Flynn T. G., Farrés J. Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism. Biochem. J. 2003;373:973–979.
17. Petrash J. M., Tarle I., Wilson D. K., Quiocho F. A. Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function. Diabetes. 1994;43:955–959.[PubMed]
18. Ruef J., Liu S. Q., Bode C., Tocchi M., Srivastava S., Runge M. S., Bhatnagar A. Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury. Arterioscler. Thromb. Vasc. Biol. 2000;20:1745–1752.[PubMed]
19. Fukumoto S., Yamauchi N., Moriguchi H., Hippo Y., Watanabe A., Shibahara J., Taniguchi H., Ishikawa S., Ito H., Yamamoto S., et al Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers' non-small cell lung carcinomas. Clin. Cancer Res. 2005;11:1776–1785.[PubMed][Google Scholar]
20. Cao D., Fan S. T., Chung S. S. Identification and characterization of a novel human aldose reductase-like gene. J. Biol. Chem. 1998;273:11429–11435.[PubMed]
21. Scuric Z., Stain S. C., Anderson W. F., Hwang J. J. New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma. Hepatology. 1998;27:943–950.[PubMed]
22. Zeindl-Eberhart E., Haraida S., Liebmann S., Jungblut P. R., Lamer S., Mayer D., Jager G., Chung S., Rabes H. M. Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas. Hepatology. 2004;39:540–549.[PubMed]
23. Martras S., Álvarez R., Gallego O., Dominguez M., de Lera A. R., Farrés J., Parés X. Kinetics of human alcohol dehydrogenase with ring-oxidized retinoids: effect of Tween 80. Arch. Biochem. Biophys. 2004;430:210–217.[PubMed]
24. Gough W. H., VanOoteghem S., Sint T., Kedishvili N. Y. cDNA cloning and characterization of a new human microsomal NAD^{-dependent dehydrogenase that oxidizes all-}trans-retinol and 3α-hydroxysteroids. J. Biol. Chem. 1998;273:19778–19785.[PubMed]
25. Ruiz A., Winston A., Lim Y. H., Gilbert B. A., Rando R. R., Bok D. Molecular and biochemical characterization of lecithin retinol acyltransferase. J. Biol. Chem. 1999;274:3834–3841.[PubMed]
26. Belyaeva O. V., Korkina O. V., Stetsenko A. V., Kim T., Nelson P. S., Kedishvili N. Y. Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids. Biochemistry. 2005;44:7035–7047.
27. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 1976;72:248–254.[PubMed]
28. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275.[PubMed]
29. Gollapalli D. R., Rando R. R. All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry. 2003;42:5809–5818.[PubMed]
30. Ross R., Kariya B. Morphogenesis of vascular smooth muscle in atherosclerosis and cell culture. In: Stephen G., editor. Handbook of Physiology, Section 2: The Cardiovascular System. Bethesda: American Physiological Society; 1980. pp. 69–91. [PubMed]
31. Bohren K. M., Page J. L., Shankar R., Henry S. P., Gabbay K. H. Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262. J. Biol. Chem. 1991;266:24031–24037.[PubMed]
32. Noy N., Xu Z. J. Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells. Biochemistry. 1990;29:3878–3883.[PubMed]
33. Ong D. E., MacDonald P. N., Gubitosi A. M. Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II. J. Biol. Chem. 1988;263:5789–5796.[PubMed]
34. Yost R. W., Harrison E. H., Ross A. C. Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein. J. Biol. Chem. 1988;263:18693–18701.[PubMed]
35. Li E., Qian S. J., Winter N. S., d'Avignon A., Levin M. S., Gordon J. I. Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli. J. Biol. Chem. 1991;266:3622–3629.[PubMed]
36. Malpeli G., Stoppini M., Zapponi M. C., Folli C., Berni R. Interactions with retinol and retinoids of bovine cellular retinol-binding protein. Eur. J. Biochem. 1995;229:486–493.[PubMed]
37. Wang J., Chai X., Eriksson U., Napoli J. L. Activity of human 11-cis-retinol dehydrogenase (Rdh5) with steroids and retinoids and expression of its mRNA in extra-ocular human tissue. Biochem. J. 1999;338:23–27.
38. Jurukovski V., Markova N. G., Karaman-Jurukovska N., Randolph R. K., Su J., Napoli J. L., Simon M. Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes. Mol. Genet. Metab. 1999;67:62–73.[PubMed]
39. Matt N., Schmidt C. K., Dupe V., Dennefeld C., Nau H., Chambon P., Mark M., Ghyselinck N. B. Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development. Dev. Dyn. 2005;233:167–176.[PubMed]
40. Molotkov A., Ghyselinck N. B., Chambon P., Duester G. Opposing actions of cellular retinol-binding protein and alcohol dehydrogenase control the balance between retinol storage and degradation. Biochem. J. 2004;383:295–302.
41. Harrison E. H., Blaner W. S., Goodman D. S., Ross A. C. Subcellular localization of retinoids, retinoid-binding proteins and acyl-CoA: retinol acyltransferase in rat liver. J. Lipid Res. 1987;28:973–998.[PubMed]
42. Scott W. J., Jr, Walter R., Tzimas G., Sass J. O., Nau H., Collins M. D. Endogenous status of retinoids and their cytosolic binding proteins in limb buds of chick vs mouse embryos. Dev. Biol. 1994;165:397–409.[PubMed]
43. Molotkov A., Deltour L., Foglio M. H., Cuenca A. E., Duester G. Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice. J. Biol. Chem. 2002;277:13804–13811.
44. Haselbeck R. J., Ang H. L., Duester G. Class IV alcohol/retinol dehydrogenase localization in epidermal basal layer: potential site of retinoic acid synthesis during skin development. Dev. Dyn. 1997;208:447–453.[PubMed]
45. Yamamoto H., Simon A., Eriksson U., Harris E., Berson E. L., Dryja T. P. Mutations in the gene encoding 11-cis retinol dehydrogenase cause delayed dark adaptation and fundus albipunctatus. Nat. Genet. 1999;22:188–191.[PubMed]
46. Janecke A. R., Thompson D. A., Utermann G., Becker C., Hübner C. A., Schmid E., McHenry C. L., Nair A. R., Rüschendorf F., Heckenlively J., et al. Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy. Nat. Genet. 2004;36:850–854.[PubMed]
47. Tryggvason K., Romert A., Eriksson UBiosynthesis of 9-cis-retinoic acid in vivo. The roles of different retinol dehydrogenases and a structure–activity analysis of microsomal retinol dehydrogenases. J. Biol. Chem. 2001;276:19253–19258.[PubMed][Google Scholar]
48. Herr F. M., Li E., Weinberg R. B., Cook V. R., Storch J. Differential mechanisms of retinoid transfer from cellular retinol binding proteins types I and II to phospholipid membranes. J. Biol. Chem. 1999;274:9556–9563.[PubMed]
49. Penning T. M. AKR1B10: a new diagnostic marker of non-small cell lung carcinoma in smokers. Clin. Cancer Res. 2005;11:1687–1690.[PubMed]
50. Petrash J. M. All in the family: aldose reductase and closely related aldo-keto reductases. Cell. Mol. Life Sci. 2004;61:737–749.[PubMed]