Retinoid Metabolism

Figure 3.20. Schematic of the vitamin A cycle showing steps with potential involvement of retinoid binding proteins. Note the absence of binding proteins in the rod outer segment (ROS) . CRALBP, cellular retinaldehyde-binding protein: CRBP, cellular retinol-binding pre tein; IRBP, interstial retinoid-binding protein: Ral, retinaldehyde; Rol, retinol: RE, retinyl ester; RPE, retinal pigment epithelium: and IPM, interphotoreceptor matrix. Kindly provided by John Saari.

Figure 3.20. Schematic of the vitamin A cycle showing steps with potential involvement of retinoid binding proteins. Note the absence of binding proteins in the rod outer segment (ROS) . CRALBP, cellular retinaldehyde-binding protein: CRBP, cellular retinol-binding pre tein; IRBP, interstial retinoid-binding protein: Ral, retinaldehyde; Rol, retinol: RE, retinyl ester; RPE, retinal pigment epithelium: and IPM, interphotoreceptor matrix. Kindly provided by John Saari.

exposure produces an instantaneous isomerization from 11-cis retinal to all-trans retinal, which subsequently degrades thermally through a series of intermediate stages, leading ultimately to the formation of all-trans retinol free of the opsin moiety. In this form, the retinoid is transferred to the RPE, where it is esterified, and eventually isomerized and oxidized to 11-cis retinal for return to the photoreceptors and the reformation of the visual pigment (Fig. 3.20). This series of reactions, from the bleaching of the photopigment to its reformation, is called the "visual cycle."

3.5.1. Retinoid-binding Proteins

Because of their potential toxicity and hydrophobic nature, retinoids are almost never found free outside or inside cells, but are generally stored as retinyl esters or are associated with binding proteins (Saari, 1994). Thus, extracellular retinoids are associated with IRBP or a retinol-binding protein (sRBP), and within retinal cells, retinoids are bound to a group of cellular retinoid-binding proteins: cellular retinaldehyde-binding protein (CRALBP) , cellular retinol-binding protein (CRABP), and cellular retinoic acid binding protein (CRBP) (Saari, 1994). Although direct experimental evidence is lacking, it is generally assumed that the intracellular retinoid-binding proteins are involved in cytoplasmic transport and routing of retinoids, but they may serve also as substrate carriers for enzymatic reactions (Saari and Bredberg, 1982; Ong et al., 1994; Napoli, 1991).

More definitive clues to the functions of retinoid-binding proteins have come from studies that have examined the phenotypes of knockout mice. In the case of CRBP I, the protein appears to be indispensable for efficient retinyl ester synthesis and storage, and its absence results in 6-fold faster turnover of retinol (Ghyselinck et al., 1999). The mice appear normal, which could mean CRBP is not involved in regulating retinoic acid synthesis. The RBP-knockout mice have a markedly impaired ERG during the first few months of life but develop a normal response when fed a vitamin A-suffi-cient diet (Quandro et al., 1999). They are otherwise normal in appearance. Liver retinyl ester stores accumulate but cannot be mobilized. Thus, the animals are dependent on a regular vitamin A intake. The results suggest that an alternate pathway of delivery of retinol to tissues (including RPE) exists but it is inefficient in utero.

In CRALBP-knockout mice, visual pigment synthesis is delayed by about 15-fold (J. Saari, personal communication). HPLC analysis shows that retinyl esters accumulate during the delay, suggesting that the delay occurs at the isomerohydrolase step. Finally, the kinetics of visual pigment renewal

Figure 3.21. CRALBP is localized in Müller cells. Antibodies to CRALBP stain radial fibers of Müller cells in bovine (A) and monkey (B) retinas. P, Pigment epithelium; m, Müller cell; V, vitreous; C, choroid (Bunt-Milam and Saari, 1983). (Copyright 1983 The Rockefeller University Press, reprinted with permission.)

Figure 3.21. CRALBP is localized in Müller cells. Antibodies to CRALBP stain radial fibers of Müller cells in bovine (A) and monkey (B) retinas. P, Pigment epithelium; m, Müller cell; V, vitreous; C, choroid (Bunt-Milam and Saari, 1983). (Copyright 1983 The Rockefeller University Press, reprinted with permission.)

are normal in IRBP-knockout mice (Palczewski et al., 1999; Ripps et al., 2000). However, photoreceptors continue to die even when the animals are raised in the dark.

The availability of highly specific polyclonal and monoclonal antibodies has made it possible to investigate the cellular localization of retin-oid binding proteins (Saari, 1994). Using this approach, Bunt-Milam and Saari (1983) made the surprising observation that CRALBP was localized in Müller cells as well as in the RPE of bovine retina (Fig. 3.21). When isolated from retina, this protein displays a high affinity for 11- cis retinaldehyde as

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