Retinol dehydrogenases in retinoid metabolism : studies on a 9-cis/11-cis-retinol dehydrogenase in adult and embryonic tissues

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Cell and Molecular Biology

Sammanfattning: Retinoids (vitamin A derivatives) are necessary for normal embryonic development and in the adult, for vision, differentiation of various epithelia, reproduction and in the immune system. The physiological effects of retinoids are mediated by binding to the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). In the eye, the active retinoid molecule is 11-cis-retinal, which is the common chromophore of all visual pigments. The present study focused on the molecular mechanisms underlying the generation of active retinoid metabolites in both embryonic and adult tissues. Novel retinol dehydrogenases belonging to the short-chain dehydrogenase/reductase (SDR) family were identified using a RT-PCR screening procedure. RDH4, a 32 kDa membrane bound protein, was identified from embryonic mouse tissues and was further suggested to the mouse homologue of the previously cloned 11-cis-retinol dehydrogenase, abundantly expressed in the retinal pigment epithelium (RPE). RDH4 was proposed to act as a 9-cis-retinol dehydrogenase in embryonic and various non-ocular adult tissues. Thus, a novel pathway for the generation of 9-cis-retinoic acid was proposed in which 9-cis-retinol is generated from all-trans-retinol in a similar way that 11-cisretinol is generated from all-trans-retinol in the eye. Furthermore, RDH4 may have a dual function depending on substrate availability and expression loci; in the eye RDH4 primarily functions as an 11-cis-retinol dehydrogenase whereas in tissues outside of the eye, RDH4 participates in the generation of 9-cis-retinoic acid. Embryonic expression of RDH4 was investigated both using in situ hybridization and immunohistochemical technique. RDH4 was expressed in a cell specific manner throughout the murine development first being detected on embryonic day (E) 8-8.5. Predominat expression was found in the floor plate, in somites and somite derivatives, the developing ear and eye, and in organs of endodermal origin. A co-localization study of RHD4 with three aldehyde dehydrogenases revealed only minor sites of co-localization, suggesting other, yet to be identified, retinal dehydrogenases may exist in the developing mouse embryo. Analysis of the membrane topology revealed that the catalytic domain of the bovine 11-cis-retinol dehydrogenase and mouse RDH4 has a lumenal orientation, which suggest that generation of cis--retinoids is a compartmentalized process. Mutations in the gene that encodes human 11-cis-retinol dehydrogenase (RDH5) have been associated with fundus albipunctatus, an autosomal recessive eye disease. Four mutations, S73F, G238W, R280H and A294P have been further studied with regard to their 9-cis-retinol dehydrogenase activity. Mutants S73F, G238W, and R280H show little or no enzymatic activity using 9-cis-retinol as substrate, in contrast mutant A294P showed enzymatic activity comparable to wild-type protein. Experimental evidence and molecular modeling of RDH5 proposed that functional dimers of RDH5 have to be generated to allow efficient catalytic properties of the enzyme in vivo.

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