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Journal of Lipid Research, Vol. 44, 303-313, February 2003
Copyright © 2003 by Lipid Research, Inc.

Cloning of monkey RALDH1 and characterization of retinoid metabolism in monkey kidney proximal tubule cells

Helene Brodeur^*,, Isabelle Gagnon^*,, Sylvie Mader and Pangala V. Bhat^1,*,

^* Laboratory of Nutrition and Cancer, Universite de Montreal, Montreal, Quebec, Canada
Centre hospitalier de l'Universite de Montreal-Hotel Dieu, Departments of Biochemistry, Universite de Montreal, Montreal, Quebec, Canada
Medicine, Universite de Montreal, Montreal, Quebec, Canada

¹ To whom correspondence should be addressed. e-mail: bhatp{at}medclin.umontreal.ca

All-trans and 9-cis retinoic acids function as ligands for retinoic acid receptors (RARs and RXRs), which are ligand-dependent transcription factors and play important roles in development and cellular differentiation. Several retinal dehydrogenases are likely to contribute to the production of all-trans and 9-cis RAs in vivo, but their respective roles in different tissues are still poorly characterized. We have previously characterized and cloned from kidney tissues the rat retinal dehydrogenase type 1 (RALDH1), which oxidizes all-trans and 9-cis retinal with high efficiency but is inactive with 13-cis retinal. Here we have characterized the retinal-oxidizing activity in monkey JTC12 cells, which are derived from kidney proximal tubules. In vitro assay of cell lysates revealed the presence of a NAD⁺-dependent dehydrogenase that catalyzed the oxidation of all-trans, 9-cis, and 13-cis retinal. Northern blot analysis of JTC12 RNAs and cloning by reverse transcription-polymerase chain reaction demonstrated expression of a monkey homolog of RALDH1. Bacterially expressed JTC12 RALDH1 catalyzed conversion of all three retinal isomers, with a higher catalytic efficiency for 9-cis retinal than for all-trans and 13-cis retinal. Accordingly, live JTC12 produced 9-cis retinoic acid more efficiently than all-trans retinoic acid from their respective retinal precursors.

Only metabolites corresponding to the same steric conformation were formed from 9-cis or all-trans retinal, indicating a lack of detectable isomerizing activity in JTC12 cells.

Abbreviations: ALDH, aldehyde dehydrogenase; GST, glutathione-S-transferase; HPLC, high-pressure liquid chromatography; RA, retinoic acid; RALDH1, 2, and 3, retinal dehydrogenase type 1, 2, 3; RAR, retinoic acid receptor; RXR, retinoid X receptor; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Supplementary key words retinoic acid • retinol • isomers • retinal dehydrogenase type I • retinoid metabolism

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