Isomerization of all-trans-retinol to cis-retinols in bovine retinal pigment epithelial cells: dependence on the specificity of retinoid-binding proteins

Abstract

In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that ultimately results in phototransduction and visual sensation. The production of 11-cis-retinal in adjacent retinal pigment epithelial (RPE) cells is a fundamental process that allows regeneration of the vertebrate visual system. Here, we present evidence that all-trans-retinol is unstable in the presence of H(+) and rearranges to anhydroretinol through a carbocation intermediate, which can be trapped by alcohols to form retro-retinyl ethers. This ability of all-trans-retinol to form a carbocation could be relevant for isomerization. The calculated activation energy of isomerization of all-trans-retinyl carbocation to the 11-cis-isomer was only approximately 18 kcal/mol, as compared to approximately 36 kcal/mol for all-trans-retinol. This activation energy is similar to approximately 17 kcal/mol obtained experimentally for the isomerization reaction in RPE microsomes. Mass spectrometric (MS) analysis of isotopically labeled retinoids showed that isomerization proceeds via alkyl cleavage mechanism, but the product of isomerization depended on the specificity of the retinoid-binding protein(s) as evidenced by the production of 13-cis-retinol in the presence of cellular retinoid-binding protein (CRBP). To test the influence of an electron-withdrawing group on the polyene chain, which would inhibit carbocation formation, 11-fluoro-all-trans-retinol was used in the isomerization assay and was shown to be inactive. Together, these results strengthen the idea that the isomerization reaction is driven by mass action and may occur via carbocation intermediate.

Figure 1

Retinoid cycle in the mammalian retina (see text for details).

Figure 2

Schemes for synthesis of [15-²H,¹⁸O]-all-trans-retinol and [15-²H,¹⁸O]-retinyl palmitate and stability of these compounds to H⁺ and base. (A) [15-²H,¹⁸O]-all-trans-Retinol was synthesized by, first, exchanging ¹⁶O of all-trans-retinal with H₂¹⁸O (3:1 CH₃-CN/H₂¹⁸O) in the presence of para-toluenesulfonic acid. [¹⁸O]-all-trans-Retinal was reduced with NaBD₄. [15-²H,¹⁸O]-all-trans-Retinyl palmitate was synthesized from [15-²H,¹⁸O]-all-trans-retinol and palmitoyl chloride. The products were purified by normal phase HPLC as described in Materials and Methods. (B) [15-²H,¹⁸O]-all-trans-Retinol or [15-²H,¹⁸O]-retinyl palmitate (in 90% CH₃CN) was subjected to 0.1 M HCl or 0.1 M HCl plus 10% EtOH for 30 min at 37 °C or by 0.1 M NaOH for 4 h at 37 °C.

Figure 3

Influence of acid and base on all-trans-retinol and all-trans-retinyl palmitate. (A) Traces a, b and c, respectively, show HPLC chromatograms of untreated [15-²H,¹⁸O]-all-trans-retinol, retinol (in 90% CH₃CN) treated with 0.1 M HCl for 30 min at 37 °C, and retinol treated with 0.1 M HCl and 10% EtOH for 30 min at 37 °C. The peak 1 shows the characteristic UV–Vis (left panel) and MS (right panel) spectra of all-trans-retinol. Equal amounts of [15-²H,¹⁶O]-all-trans-retinol and [15-²H,¹⁸O]-all-trans-retinol were present. The peak 2 shows the product of H⁺ degradation of [15-²H,¹⁸O]-all-trans-retinol with characteristic UV–Vis and MS spectra of [15-²H]-anhydroretinol. Peak 3 shows characteristic UV–Vis and MS spectra of [15-²H]-all-trans-retro-retinyl ethyl ether formed during H⁺ elimination in the presence of EtOH. (B) Traces d, e, and f, respectively, show HPLC chromatograms of untreated [15-²H,¹⁸O]-all-trans-retinyl palmitate, the ester treated with 0.1 M HCl for 30 min at 37 °C in 90% CH₃CN, and the ester treated with 0.1 M NaOH for 4 h at 37 °C. Peak 4 shows UV–Vis spectrum (left panel) and MS fragmentation patterns (right panel) consistent with those of [15-²H,¹⁸O]-all-trans-retinyl palmitate. Peak 2 was analogous to peak 2 in panel A, and it has been identified as [15-²H]-anhydroretinol. Peak 5 displays UV–Vis and MS spectra of [15-²H,¹⁸O]-all-trans-retro-retinyl palmitate. Finally, peak 1 represents the product of hydrolysis of [15-²H,¹⁸O]-all-trans-retinyl palmitate by NaOH to [15-²H,¹⁸O]-all-trans-retinol.

Figure 4

Theoretical calculations of the transition state energies of carbocation intermediates for all-trans-retinol and 11-fluoro-all-trans-retinol and comparison of the ground and transition state energies for all-trans-retinol, all-trans-retinyl, and 11-fluoro-all-trans-retinyl carbocation. Transition state for trans–cis rotation was modeled by the 90° constraint of the C₁₀–C₁₁–C₁₂–C₁₃ dihedral angle. Energy values shown on the graphs are calculated using the ab initio B3LYP method. The energy of all-trans-isomers was taken as reference zero energy. (A) Isomerization of all-trans-retinol without electron delocalization of the polyene chain requires overcoming an energetic barrier of 36.2 kcal/mol. With carbocation formation, this energy decreases to 17.2 kcal/mol. The activation energy of 11-fluoro-all-trans-retinyl carbocation is only 11.8 kcal/mol. (B) Free energy reaction coordinate profile for isomerization of all-trans-retinol and 11-fluoro-all-trans-retinol to their corresponding 11-cis-isomers through carbocation intermediates. For the carbocation to be formed, the all-trans-isomer is protonated with a hydroxonium ion. Exothermic dehydration of the protonated all-trans-isomer leads to the formation of all-trans-carbocation followed by endothermic trans–cis rotation (for values see above). The energy of endothermic hydration and deprotonation of 11-cis-retinyl and 11-fluoro-11-cis-retinyl carbocations is 1.7 and 0.9 kcal/mol (respectively) less than the energy of exothermic dehydration for formation of the carbocation. For structures refer to Figure 9.

Figure 5

Arrhenius plot of isomerase activity in bovine RPE microsomes. The rate of formation of 11-cis-retinol was measured in duplicate at temperatures ranging from 5 to 53 °C in 6° increments. The log of the rate was plotted against 1/T. A linear least-squares measurement was performed from 5 to 41 °C (47 and 53 °C were excluded because of temperature-dependent inactivation of enzymes of the visual cycle). The measured Arrhenius activation energy was calculated from the slope and was found to be in the 17 kcal/mol range (solid line). The dashed line represents a typical enzymatic reaction when the rate doubles every 10 °C.

Figure 6

Influence of retinoid-binding proteins on the formation of different retinol isomers. Panels A and B show chromatograms of the formation of retinol isomers in the presence of CRALBP and CRBP, respectively. The peaks were identified as follows: 1, retinyl esters; 2, 11-cis-retinol; 3, 13-cis-retinol, and 4, all-trans-retinol. A control experiment without retinoid-binding proteins, panel C, shows the near absence of cis-retinols, while all-trans-retinol was almost completely esterified.

Figure 7

MS analysis of cis-retinols formed from [15-²H,¹⁸O]-all-trans-retinol in the presence of different retinoid-binding proteins in RPE microsomes. Panel a shows the MS fragmentation pattern of standard all-trans-retinol, and panel b shows the fragmentation pattern of the synthesized [15-²H,¹⁸O]-all-trans-retinol. Note the shift from 286 to 289 because the presence of ²H and ¹⁸O and a fragment at 269 due to ²H. Panel c shows a loss of ¹⁸O upon the formation of 11-cis-retinol when CRALBP was used as a retinoid-binding protein. Panel d shows that all-trans-retinol bound to CRBP retains the ¹⁸O-label, yet panels e and f show that 13-cis-retinol and 11-cis-retinol formed in the presence CRBP lost ¹⁸O. Note that the deuterium label is retained in each measurement.

Figure 8

Lack of isomerization of 11-fluoro-all-trans-retinol by RPE microsomes. The isomerization reaction for 11-fluoro-all-trans-retinol (0.5 nmol) was carried out as described in Materials and Methods with (A) and without (B) 25 μM CRALBP. The arrows point to the expected elution time of 11-fluoro-11-cis-retinol. Note that only a small amount of 11-fluoro-11-cis-retinol is formed, most likely nonenzymatically.

Figure 9

Proposed chemical mechanism for the formation of 11-cis-retinol via carbocation intermediate by the isomerization reaction in mammalian RPE microsomes (see text for details).