Differential Recognition of the Free versus Bound Retinol by Human Microsomal Retinol/Sterol Dehydrogenases:  Characterization of the Holo-CRBP Dehydrogenase Activity of RoDH-4

Abstract

All-trans-retinol is the precursor for all-trans-retinoic acid, the activating ligand for nuclear transcription factors retinoic acid receptors. In the cytosol of various cells, most retinol exists in a bound form, complexed with cellular retinol binding protein type I (holo-CRBP). Whether retinoic acid is produced from the free or bound form of retinol is not yet clear. Here, we present evidence that holo-CRBP is recognized as substrate by human microsomal short-chain dehydrogenase/reductase (SDR) RoDH-4 with the K(m) value close to the liver concentration of holo-CRBP. The ability to utilize holo-CRBP differentiates RoDH-4 from a related enzyme, RoDH-like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), which is 3-fold more active with free retinol than RoDH-4 but is 15-fold less active toward holo-CRBP. Recognition of the cytosolic holo-CRBP as substrate is consistent with RoDH-4 orientation in the membrane. As established by immunoprecipitation and glycosylation scanning, RoDH-4 faces the cytosolic side of the membrane. Purified RoDH-4, stabilized by reconstitution into proteoliposomes, exhibits the apparent K(m) values for substrates and NAD(+) similar to those of the microsomal enzyme and oxidizes holo-CRBP with the catalytic efficiency (k(cat)/K(m)) of 59 min(-1) mM(-1). Apo-CRBP acts as a strong competitive inhibitor of holo-CRBP oxidation with an apparent K(i) value of 0.2 microM. The results of this study suggest that the human retinol-active SDRs are not functionally equivalent and that, in contrast to RoDH-like 3alpha-HSD, RoDH-4 can access the bound form of retinol for retinoic acid production and is regulated by the apo-/holo-CRBP ratio.

Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

“…This dramatic difference in the activity with free versus bound retinol was similar to that observed for other retinoid active oxidoreductases (18,19,21,28), suggesting that RDH10 also recognizes only the free form of retinol as a substrate.…”

“…In agreement with this observation, silencing of RDH10 expression in HEK293 cells, which contain CRBPI (23), results in a decrease in retinoic acid biosynthesis. This distinguishes RDH10 from other enzymes that also recognize only the free form of retinol but are less able to compete with CRBPI (8,18,19,21,28).…”

“…Similar constants were obtained using three independent preparations of microsomal RDH10. To determine whether RDH10 could utilize retinol bound to CRBPI as a substrate, we saturated CRBPI with all-trans-retinol and purified holoCRBPI from unbound retinol using size-exclusion column chromatography as described previously (28). At 5 M retinol, the reaction rate toward holoCRBPI was significantly lower (0.08 nmol min Ϫ1 mg…”

“…All of these cells contain CRBPI (25)(26)(27). CRBPI was shown to sequester all-trans-retinol from human microsomal retinol dehydrogenases, RoDH4 and RL-HSD (21,28). To determine whether RDH10 can oxidize all-trans-retinol in the presence of CRBPI, the activity of the enzyme was assayed at different molar ratios of apoCRBPI to retinol.…”

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

“…All of the currently known cytosolic ADH and AKR enzymes have been purified and characterized. In contrast, very few microsomal SDRs have been purified to homogeneity (11,17,28), which impedes the comparison of their catalytic properties with those of other retinoid-active SDRs, ADHs, and AKRs.…”