Highly Efficient Retinal Metabolism in Cones

Highly efficient retinal metabolism in cones

Sadaharu Miyazono*, Yoshie Shimauchi-Matsukawa*, Shuji Tachibanaki*†, and Satoru Kawamura*†‡

*Graduate School of Frontier Biosciences and †Department of Biology, Graduate School of Science, Osaka University, Suita, Osaka 565-0871, Japan

Edited by Carter Cornwall, Boston University School of Medicine, Boston, MA, and accepted by the Editorial Board August 26, 2008 (received for review July 8, 2008) After bleaching of visual pigment in vertebrate photoreceptors, has been suggested: Mu¨ller cells can isomerize all-trans retinol to all-trans retinal is reduced to all-trans retinol by retinol dehydro- 11-cis retinol (11). When this 11-cis retinol is transported to genases (RDHs). We investigated this reaction in puriﬁed carp rods cones, it can be oxidized to 11-cis retinal with a hypothesized and cones, and we found that the reducing activity toward all- enzyme, 11-cis retinol dehydrogenase, that was assumed to be trans retinal in the outer segment (OS) of cones is >30 times higher present in cones and requires NADPϩ as a cofactor (12). than that of rods. The high activity of RDHs was attributed to high In the present study, by using purified carp (Cyprinus carpio) content of RDH8 in cones. In the inner segment (IS) in both rods and rods and cones (13), we investigated the metabolism of all-trans cones, RDH8L2 and RDH13 were found to be the major enzymes retinal and 11-cis retinal within the photoreceptor cells. We among RDH family proteins. We further found a previously unde- found that the reduction of all-trans retinal and the production scribed and effective pathway to convert 11-cis retinol to 11-cis of 11-cis retinal are both much more efficient in cones. In retinal in cones: this oxidative conversion did not require NADP؉ addition, it was found that 11-cis retinal is formed from 11-cis and instead was coupled with reduction of all-trans retinal to retinol by a previously undescribed retinal-retinol redox all-trans retinol. The activity was >50 times effective than the (ALOL)-coupling reaction. -oxidizing activity of RDHs that require NADP؉. These highly effec tive reactions of removal of all-trans retinal by RDH8 and produc- Results tion of 11-cis retinal by the coupling reaction are probably the Highly Efficient Reduction of all-trans Retinal in Cones. All-trans underlying mechanisms that ensure effective visual pigment re- retinal formed in the bleached visual pigment is reduced to generation in cones that function under much brighter light con- all-trans retinol. Fig. 1A shows the time courses of the reduction ditions than rods. of the exogenously applied all-trans retinal to all-trans retinol measured in the lysate of purified carp rods and cones. We 11-cis retinal ͉ 11-cis retinol ͉ all-trans retinal ͉ retinol dehydrogenase ͉ routinely quantified the amount of rods and cones by measuring visual cycle the amount of visual pigment in a sample. Therefore, the activity of the reduction of all-trans retinal, defined as the RDH activity ight detection in vertebrates is mediated by rods and cones. in this study, is expressed in units per visual pigment present. LBoth rods and cones consist of two parts, the outer segment This expression is useful in the comparison of the RDH activities (OS) and the inner segment (IS). The OS contains the machinery per unit volume of the OS between rods and cones, because the responsible for conversion of a light signal to an electrical signal. density of the visual pigment in the OS is similar between rods The light-absorbing molecule, the visual pigment, is present in and cones (14). However, most of our purified rods and cones the OS and is composed of a chromophore, 11-cis retinal, and a retained both the OS and the IS [see Materials and Methods and protein moiety, opsin. Light isomerizes 11-cis retinal to all-trans supporting information (SI) Fig. S1 A and E], and in addition, retinal, which induces a conformational change in opsin to lead both the OS and the IS showed the RDH activity (see below). to activation of an enzymatic cascade to evoke a light response For this reason, we show (Fig. 1) the activity in units per cell (rod (1, 2). or cone) by assuming that, on an average, a rod contains 1.6 ϫ All-trans retinal, the product formed after light absorption, 108, and a cone contains 7.8 ϫ 107 visual pigment molecules (13). then dissociates from opsin and is reduced to all-trans retinol. Later in the present study, we determine the RDH activity in the This form of retinol is transported to the retinal pigment OS, so that the activity will be expressed in units per visual epithelium (RPE), isomerized, and oxidized to 11-cis retinal with pigment present (after Fig. 2). multistep reactions. The newly synthesized 11-cis retinal is sent The RDH activity in rods was 1.6 Ϯ 0.2 amol of all-trans retinol back to photoreceptors to regenerate visual pigment. This formed per rod per second (amol atROL/rod-sec) (Fig. 1C). A pathway of retinal processing (visual cycle) has been mainly similar value of the RDH activity has been reported in bovine studied in rods or rod-dominant retinas (3, 4). Because cones rod OSs (ROSs) (15). The activity in cones was 12.4 Ϯ 1.4 amol function under much brighter light conditions, the visual cycle atROL/cone-sec (Fig. 1C). It was evident that the reducing for cones could differ from that for rods in a qualitative and/or activity per cell was approximately eight times higher in cones. quantitative way, as is seen in the phototransduction mechanism Our rod preparation contained small amounts of cones (13). in rods and cones (5, 6). Because cones showed much higher RDH activity than rods as The reduction of all-trans retinal to all-trans retinol is attained shown above, a correction for cone contamination in rod prep- by RDHs within a photoreceptor cell. This reduction could be arations was routinely made (see SI Materials and Methods). the rate-limiting step of the visual cycle (7), and the rate of the reduction has been reported to be 10–40 times higher in cones than in rods under in vivo conditions (8). The supply of 11-cis Author contributions: S.M., S.T., and S.K. designed research; S.M. and Y.S.-M. performed PHYSIOLOGY retinal to regenerate visual pigment could be faster in cones. research; S.M. analyzed data; and S.M., S.T., and S.K. wrote the paper. Several lines of evidence suggest that cone pigment is regener- The authors declare no conﬂict of interest. ated in an RPE-independent manner. For example, cone pig- This article is a PNAS Direct Submission. C.C. is a guest editor invited by the Editorial Board. ment, but not rod pigment, can be regenerated in isolated frog Data deposition: The sequences reported in this paper have been deposited in the GenBank retinas separated from the RPE (9). Interestingly, after bleach- database [accession nos. AB439579 (RDH8), AB439580 (RDH8L2), and AB439581 (RDH13)]. ing, photosensitivity recovers with addition of 11-cis retinol in ‡To whom correspondence should be addressed. E-mail: [email protected]. isolated cones but not in rods (10). These results suggest the This article contains supporting information online at www.pnas.org/cgi/content/full/ presence of a cone-specific regeneration pathway. Recently, an 0806593105/DCSupplemental. effective pathway for the supply of 11-cis retinal to opsin in cones © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806593105 PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 16051–16056 Downloaded by guest on September 25, 2021 Fig. 2. RDH activities in the OS and the IS of rods and cones. (A) Examples of SDS/PAGE patterns of ROS-rich, purified rod, and RIS-rich preparation, all Fig. 1. RDH activities in purified carp rods and cones. (A) Time course of containing constant 100 pmol of pigment (arrow). The RIS/ROS ratios in these reduction of all-trans retinal (RDH activity) in the rod and the cone lysate. The samples were 0.62, 1 and 2.9, respectively. (B) RDH activities as a function of activity is expressed as fmol of all-trans retinol formed per cell. The abscissa RIS/ROS ratio. (C) Estimated RDH activities in the ROS and in the RIS. The RDH indicates the time after addition of all-trans retinal. Error bars show standard activities in the ROS and in the RIS were calculated from the result shown in B deviations (n ϭ 4). (B) HPLC elution patterns of retinoids after 5-min incuba- (n ϭ 3). (D) Examples of SDS/PAGE patterns of COS-rich, purified cone, and tion of all-trans retinal (Left) with cone lysate (Upper Right) and rod lysate CIS-rich preparation, all containing constant 5 pmol of pigment (arrow). The (Lower Right). Arrowheads in Upper Right indicate (from left to right) 13-cis CIS/COS ratios in these samples were 0.14, 1 and 3.9, respectively. (E) RDH retinal, all-trans retinal, and all-trans retinol. Formation of 13-cis retinal is due activities as a function of CIS/COS ratio. (F) RDH activities in the COS and in the to nonspecific thermal isomerization from all-trans retinal (see Fig. 4B). (C) CIS were estimated from the result shown in E (n ϭ 4). RDH activities in the rod and the cone lysate. The activity was calculated from the results shown in A at 5 min (n ϭ 4). (D and E) Activities of reduction and oxidation of all-trans and 11-cis retinoids in the rod lysate (D) and cone lysate As shown in Fig. 2B, in the rod preparations, the activity (E). The activity is shown as a mean Ϯ SD, determined in four independent increased significantly as the content of the IS increased, which experiments for the all-trans retinoids and three independent experiments for indicated that the RDH activity is present in the IS of a rod. the 11-cis retinoids. From these measurements, we calculated the RDH activities in the ROS and in the rod IS (RIS) to be 0.64 Ϯ 0.11 amol The reduction of all-trans retinal is catalyzed by RDHs (15, atROL/ROS-sec and 0.80 Ϯ 0.11 amol atROL/RIS-sec, respec- 16). To characterize the RDH reactions in rods and cones, we tively (Fig. 2C). In rods, the estimated activities in the OS and examined the substrate specificity of the oxidation and the in the IS were nearly equal. In the ROS, the activity was Ϫ reduction of retinoids in the presence of NADPH or NADPϩ, equivalent to 2.4 Ϯ 0.3 ϫ 10 3 molecules atROL/pigment-sec. the essential cofactors for reduction and oxidation of retinoids, Similarly, we measured the RDH activities in cone OS (COS)- respectively (15, 17). All-trans retinal was found to be the best rich and cone IS (CIS)-rich preparations (Fig. 2E). The notable substrate among the substrates examined for RDHs in both rods point was that, in contrast to the result obtained in rod prepa- and cones, because the reducing rate of all-trans retinal was the rations (Fig. 2B), the activity only slightly increased as the highest. The activity of each reaction expressed in units of content of CIS increased. The result indicated that most of the activity per cell was three to eight times higher in cones than in RDH activity is present in the OS in cones. The estimated rods (compare Fig. 1 D and E). It should be mentioned here that reducing activity was 10.2 Ϯ 1.1 amol atROL/COS-sec in the OS in both rods and cones, the catalytic activity toward the forma- and 2.2 Ϯ 1.1 amol atROL/CIS-sec in the IS (Fig. 2F). In the Ϫ tion of 11-cis retinal from 11-cis retinol is remarkably lower than COS, the activity was equivalent to 7.9 Ϯ 0.9 ϫ 10 2 molecules the activities of other reactions even in the presence of the atROL/pigment-sec. cofactor. From the estimated RDH activities in the ROS and the COS, the all-trans retinal reducing activity per unit volume of the OS Ϫ Ϫ RDH Activities in the OS and the IS of Rods and Cones. Visual pigment was found to be 33 times (7.9 ϫ 10 2/2.4 ϫ 10 3) higher in cones is present in the OS almost exclusively. Thus, all-trans retinal than in rods, which suggested that the reduction of all-trans mainly accumulates within the OS, and its reduction to all-trans retinal is much more effective in cones. retinol is thought to predominantly take place within the OS. To investigate whether the measured RDH activity localizes only in RDH8, RDH8L2, and RDH13 as the Major RDH Subtypes Expressed in the the OS of rods and cones, we measured the RDH activities in Carp Retina. To identify which RDH subtypes are responsible for OS- and IS-rich preparations of rods and cones (see the differ- the higher RDH activity in cones, we screened the RDH ence in the SDS/PAGE patterns in Fig. 2 A and D and Fig. S1). subtypes expressed in the carp retina and the RPE with molec- The ratios of IS/OS in these preparations were estimated by ular cloning, and detected RDH5, RDH8, RDH12, RDH13, comparing the content of a mitochondrial enzyme to that of RDH14, and retSDR1 homologous genes (Fig. S2A). visual pigment (see SI Materials and Methods). In Fig. 2, we To determine the relative abundance of these mRNAs, a plotted the RDH activity as a function of the content of the IS semiquantitative RT-PCR study was performed, and we found in rod (Fig. 2B) and cone (Fig. 2E) preparations, each contain- that RDH8, RDH8L2, and RDH13 were expressed at higher ing constant amount of visual pigment. levels in the carp retina, and that RDH5 and retSDR1 were

16052 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806593105 Miyazono et al. Downloaded by guest on September 25, 2021 (Table 1. No. of RDH molecules/OS or IS (؋104 RDH8 RDH8L2 RDH13

Rod OS 1.4 Ϯ 0.2 3.9 Ϯ 2.6 0.1 Ϯ 0.2 IS 0.5 Ϯ 0.2 20.7 Ϯ 2.6 2.2 Ϯ 0.2 Cone OS 29.2 Ϯ 2.2 20.0 Ϯ 22.7 4.3 Ϯ 2.5 IS 2.5 Ϯ 2.2 159.2 Ϯ 22.7 16.2 Ϯ 2.5

Values are shown as a mean Ϯ SD (n ϭ 4).

predominantly expressed in the RPE (Fig. S2B). Other genes including those of RDH12 and RDH14 were not detected significantly. Assuming that the relative abundance of an mRNA reflects that of the corresponding protein, we concluded that the three RDH proteins, RDH8, RDH8L2, and RDH13, are expressed at much higher levels than other RDHs in the carp retina. Thus, carp retinal cDNA library was screened, and the full-length cDNA sequences of these three RDH genes were obtained. These sequences were used for the expression of recombinant proteins (see below).

Expression Levels and Distribution of RDH8, RDH8L2, and RDH13 in Rods and Cones. To determine which subtype of RDH is responsible for the RDH activities in the OS and in the IS, we quantified with immunoblots the amount of each subtype in the OS and the IS of rods and cones. For this purpose, we raised a specific antiserum against each subtype (Fig. S3A) and found that all of Fig. 3. RDH8, RDH8L2, and RDH13 activities in the OS and the IS of rods and the three RDH subtypes were present in the membrane fraction cones. (A) Immunoblots of HEK293 cells expressing the recombinant RDHs, rod of rods and cones (Fig. S3B). By using the OS- and IS-rich and cone samples. RDH8, RDH8L2, and RDH13 were probed by immunoblots preparations together with purified rods and cones as used in the with anti-RDH8 (Left), anti-RDH8L2 (Center), and anti-RDH13 (Right) antiserum in HEK293 cells transfected with an empty vector (Control), vectors study shown in Fig. 2, the contents of the RDHs in the OS and expressing RDH8 (Left), RDH8L2 (Center), or RDH13 (Right). Rod and cone the IS of rods and cones were quantified by immunoblot analyses membrane fractions (rod ppt and cone ppt, respectively) were also probed. (B) with known amounts of RDHs as standards (Fig. S3 C and D). Speciﬁc RDH activities of recombinant RDH8, RDH8L2, and RDH13 expressed The summarized result (Table 1) showed that (i) all RDHs in HEK293 cells (n ϭ 3–6). (C and D) RDH activities in the OS and the IS of rods examined were present in both rods and cones and were more (C) and cones (D) calculated from the speciﬁc activity (B) and the distribution than five times abundant in cones; (ii) in both rods and cones, (Table 1) of each RDH (colored bars). Black bars are taken from the result RDH8 localized mainly in the OS; (iii) among RDHs examined, shown in Fig. 2 C and F. RDH8L2 was the most abundant RDH in both rods and cones; and (iv) RDH8L2 and RDH13 localized predominantly in the IS. retinal in the OS and that the activity in the IS is mainly because The localization of RDH8 and RDH13 is similar to that previ- of RDH8L2 (white area) in both rods and cones. ously reported in primates (16, 18). Carp RDH8L2 is one of the RDH8 homologs but localizes in the IS unlike other vertebrate The 11-cis Retinal Production from 11-cis Retinol with a ALOL-Coupling RDH8s. Reaction. It is known that visual pigment is regenerated with addition of 11-cis retinol in isolated cones, but not in rods (10). RDH8 as the Responsible RDH for Highly Efficient all-trans Retinal It suggests that cones have a mechanism to convert 11-cis retinol Reduction in the COS. As shown, all three subtypes of RDHs to 11-cis retinal. It has been reported that 11-cis retinol is (RDH8, RDH8L2, and RDH13) are expressed in rods and converted to 11-cis retinal in the presence of NADPϩ in a cones. The specific activities of these RDHs were measured by cone-dominant retina (12). This result suggests that NADPϩ- using recombinant RDH proteins expressed in HEK293 cells dependent 11-cis retinol dehydrogenase is present in cones. (Fig. 3). The activity of each RDH sample was measured, and its However, as shown in Fig. 1E, at least in our purified carp cones, amount was quantified by immunoblot analysis. The specific production of 11-cis retinal from 11-cis retinol by RDHs was not activities of RDH8, RDH8L2, and RDH13 obtained were 27.8 Ϯ effective. 10.1, 1.9 Ϯ 0.9, and 1.5 Ϯ 0.3 molecules atROL/RDH molecule- Previous investigations have suggested that the oxidation of sec, respectively (Fig. 3B). The specific activity of RDH8 was retinol to retinal is always coupled with reduction of a cofactor, Ͼ10 times higher than those of the others. The RDH activity was NADPϩ. It is possible that, instead of NADPϩ, other chemicals, not detected in control HEK293 cells transfected with the empty for example, all-trans retinal could substitute the role of NADPϩ.

vector. To test this possibility, we measured the conversion of 11-cis PHYSIOLOGY Because we now know both the specific activity of each single retinol to 11-cis retinal in the cone lysate in the presence of RDH molecule (Fig. 3B) and the amount of each RDH in the OS all-trans retinal but in the absence of NADPϩ. and the IS of rods and cones (Table 1), we can simply calculate It has been shown that retinoids are thermally isomerized the total RDH activities in the OS and the IS of rods and cones significantly in the presence of rod membranes (19, 20). We based on these measurements. The calculated results (colored confirmed it in the presence of the cone lysate: 11-cis retinol was bars in Fig. 3 C and D) are consistent with the RDH activities isomerized almost exclusively to all-trans retinol (Fig. 4A), and measured in the OS- and the IS-rich preparations in Fig. 2 C and all-trans retinal was also exclusively isomerized to 13-cis retinal F (redrawn as black bars in Fig. 3 C and D), and showed that (Fig. 4B). In these measurements, 11-cis forms were not de- RDH8 (red area) is responsible for the reduction of all-trans tected. When 11-cis retinol and all-trans retinal were added

Miyazono et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 16053 Downloaded by guest on September 25, 2021 Table 2. Kinetic parameters of the RDH and the ALOL-coupling reaction

Cell type Km, ␮M Vmax*

RDH Rod 6.2 Ϯ 2.2 2.1 Ϯ 0.4 Cone 6.1 Ϯ 2.2 14.3 Ϯ 1.3 ALOL coupling Rod — — Cone 33.0 Ϯ 8.8 3.5 Ϯ 0.3

The parameters were determined for the reduction of all-trans retinal to all-trans retinol, and the values are shown as a mean Ϯ SD (n ϭ 3). *Values are expressed in units of amols all-trans retinal formed per rod or cone per sec.

To examine the physiological relevance of this coupling, we mimicked pseudophysiological conditions to observe pigment regeneration: cone membranes were illuminated in the presence of 11-cis retinol. First, we measured the absorption spectrum of red cone pigment in a carp membrane preparation (Fig. 4H, gray dotted line). Because the endogenous retinal in carp visual pigment is 11-cis 3,4-didehydroretinal (A2 11-cis retinal), the absorption maximum peaks at Ϸ620 nm. As a control, by addition of excess amount of A1 11-cis retinal to the bleached cone membranes, we observed the regenerated visual pigment (␭max ϭϷ570 nm, green line). Then the native cone membranes having A2 11-cis retinal were illuminated, and as a consequence, A2 all-trans retinal should have been formed and released from the bleached pigment. When A1 11-cis retinol of the amount similar to that of the released all-trans retinal was added to these membranes during red light illumination (Ͼ680 nm), visual pigment was regenerated as expected (red line). Although the regeneration Fig. 4. Formation of 11-cis retinal from 11-cis retinol by the ALOL-coupling was Ϸ70% of the control measurement (green line), it was reaction in cones. (A and B) Time courses of thermal isomerization of (A) 11-cis evident that 11-cis retinal was produced most probably by the retinol (11cROL) to all-trans retinol (atROL) (n ϭ 5), and (B) all-trans retinal ALOL coupling with the endogenous all-trans retinal. Pigment (atRAL) to 13-cis retinal (13cRAL) (n ϭ 3) in the cone lysate. (C) Time course of regeneration was not observed without addition of 11-cis retinol retinoids formation in the cone lysate in the presence of both 11-cis retinol and ϭ (black line). Similarly, after bleach, when all-trans retinal was all-trans retinal (n 5). (D) Net effect of the coexistence of 11-cis retinol and removed with NH OH (orange line) or converted to all-trans all-trans retinal. The amounts of thermally isomerized products in A and B 2 were subtracted from those in C.(E and F) Initial rates of retinoid formation retinol by endogenous RDHs in the presence of NADPH (blue in the presence of both 11-cis retinol and all-trans retinal in the cone lysate (E) line), pigment regeneration was not observed even with addition and the rod lysate (F). The rates were determined from the amounts of of 11-cis retinol. This regeneration study confirmed that the retinoids formed at 5 min (cones, n ϭ 5; and rods, n ϭ 3). Blue bar, all-trans ALOL coupling is a potential mechanism that may have a crucial retinol thermally isomerized from 11-cis retinol; black bar, 13-cis retinal role in the cone visual cycle. Table 2 summarizes the kinetic thermally isomerized from all-trans retinal; white bar: all-trans retinal formed parameters of the RDH reactions and the ALOL-coupling by the coupling reaction; red bar: 11-cis retinal formed by the coupling reaction in rods and cones. The Km value for all-trans retinal of reaction. (G) Effect of heat treatment on the formation of 11-cis retinal from the ALOL coupling in cones was several times larger than that 11-cis retinol in the presence of all-trans retinal. The amount of formed V retinoid was determined in the cone lysate with heat treatment (65°C for 5 of RDH reaction in cones, and the max was several times smaller min, ϩheat) or without it (Ϫheat) (ϩheat, n ϭ 3; and Ϫheat, n ϭ 5). (H) in the coupling reaction. Difference absorption spectra of red cone pigment regenerated under various conditions (see the text). Discussion Physiological Relevance of the Measured RDH Activity. In Fig. 2 (see Results), we showed that the reducing activity of RDHs toward simultaneously to the cone lysate, 11-cis retinal was additionally all-trans retinal is 2.4 Ϯ 0.3 ϫ 10Ϫ3 molecules atROL/pigment- formed (Fig. 4C, red line). In addition, all-trans retinol was also sec in the ROS and 7.9 Ϯ 0.9 ϫ 10Ϫ2 molecules atROL/pigment- formed in a corresponding amount (compare blue lines in Fig. sec in the COS, or 7.2 Ϯ 0.9 ␮M atROL/sec in the ROS and 4 A and C). The net effect of the presence of both 11-cis retinol 240 Ϯ 30 ␮M atROL/sec in the COS assuming that the visual and all-trans retinal was obtained by subtraction of the control pigment concentration in the OS in both rods and cones is 3 mM values shown in Fig. 4 A and B from the values shown in Fig. 4C, (14). The activity was 33 times higher in the COS than in the which is shown in Fig. 4D. It was evident that 11-cis retinal and ROS. These values show that after 100% bleach of visual all-trans retinol were newly produced. The stoichiometry of the pigment molecules, a half of all-trans retinal is converted to its formation of 11-cis retinal and all-trans retinol was close to 1:1 alcohol at Ϸ5 min in the ROS and 8.5 sec in the COS in the carp (Fig. 4D and also compare white bar and red bar in Fig. 4E). This retina. In living tiger salamander rods and cones, the formation result strongly suggested that, in cones, 11-cis retinol can be of all-trans retinol after bleach was measured (8). The calculated oxidized to 11-cis retinal with accompanied reduction of all-trans times necessary for the half conversion of all-trans retinal to its retinal to all-trans retinol. This ALOL-coupling reaction was not alcohol in their study were 6.4 min in the ROS and 16 sec in the observed in the rod lysate (Fig. 4F). The cone lysate treated with COS. Although these are the values that were obtained in the heat lost this activity (Fig. 4G), which suggested that the ALOL overall reaction including both the decay of the bleaching coupling is an enzymatic reaction. intermediate of visual pigment and reduction of all-trans retinal,

16054 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806593105 Miyazono et al. Downloaded by guest on September 25, 2021 Fig. 5. Scheme of rod and cone visual cycle. (Left) Currently supported visual cycle for rods. After absorption of light, the visual pigment chromophore, 11-cis retinal is isomerized to all-trans reteinal. All- trans retinal is detached from opsin and reduced by RDH8 to all-trans retinol in the presence of a cofactor, NADPH. This retinol is transferred to the RPE where the all-trans form is isomerized to the 11-cis form and is oxidized to 11-cis retinal; 11-cis retinal is transferred back to the OS of a rod to regenerate visual pigment. (Right) Proposed model of visual cycle for cones. A similar pathway of visual cycle is present in cones. However, in cones additional pathway for production of 11-cis retinal is present: 11-cis retinol supplied from Mu¨ller cells is converted to 11-cis retinal by the ALOL- coupling reaction without a cofactor, NADPϩ (see the text for details).

the results of our in vitro measurements of the RDH activity are rate of the conversion of 11-cis retinol to 11-cis retinal was consistent with those measured in living rods and cones. observed by the ALOL coupling in the absence of the cofactor, The RDH activities measured in the present study can be NADPϩ: the rate was 3.5 Ϯ 0.3 amol of 11-cis retinal (equal to expressed in other ways: based on the OS RDH activity, the all-trans retinol) formed/cone-sec (Table 1) and it was 50 (3.5/ production of all-trans retinal by light and its conversion to 0.065) times higher than the rate of production of 11-cis retinal all-trans retinol is approximately balanced at the intensity of light attained by RDHs present in purified cones. Therefore, the bleaching 0.24% of pigment per sec in rods and at that bleaching ALOL coupling is a very effective mechanism to produce 11-cis 7.9% of pigment per sec in cones. These intensities of light seem retinal from its retinol that could be supplied from Mu¨ller cells. to be above the range where rods and cones normally operate. If the activity of the ALOL coupling all resides in the COS, the Therefore, the RDH activity in the OS seems to be high enough maximal rate of coupling is equal to 0.027 Ϯ 0.002 11-cis retinal for both rods and cones to operate under normal conditions. formed/pigment-sec. Assuming that the supply of 11-cis retinal from the RPE is 28 pmol/eye-min (12) and that the total visual Possible Role of Each Subtype of RDH in Photoreceptors. We iden- pigment content in an individual carp eye is Ϸ4 nmol (our tified RDH8, RDH8L2, and RDH13 as major RDHs expressed unpublished result), the supply of 11-cis retinal from the RPE is in photoreceptors (Figs. S2 and S3). RDH8 was considered to be calculated to be 1.1 ϫ 10Ϫ4 molecules/pigment-sec. Then, at the the responsible enzyme to reduce all-trans retinal in the OS (Fig. maximal rate of production of 11-cis retinal by the coupling, the 3 C and D). RDH8L2 and RDH13 localized in the IS, and activity is 240 times [0.027/(1.1 ϫ 10Ϫ4)] higher than the supply accounted for most of the RDH activity in the IS of rods and of 11-cis retinal from the RPE. The rate of the supply of 11-cis cones. The total expression levels of these RDHs were several retinal by the coupling suggests that the supply is sufficient up to times higher in cones (Table 1), which is probably because the IS the light intensity bleaching 2.7% of pigment per sec. volume is correspondingly larger in cones than in rods (our The Km value for all-trans retinal in the ALOL-coupling estimate of the carp CIS/RIS volume ratio is Ϸ13). Judging from reaction is several times higher than that in the RDH reaction lower activity of RDH8L2 and RDH13 toward all-trans retinal (Table 2). However, the fact that the exogenously added 11-cis (Fig. 3B), the physiologically relevant substrate of RDH8L2 and retinol induced pigment formation (Fig. 4H) indicated that the RDH13 may not be retinal. In the mammalian retina, RDH12 is concentration of all-trans retinal was sufficient at least at the site abundant in the IS (18, 21). Interestingly, RDH12 reduces not where the coupling took place. All-trans retinal used in this only all-trans retinal but also medium-chain peroxidic aldehydes coupling was probably that released from the bleached pigment, (22). We assume that carp RDH8L2 has a similar role as because in the dark adapted rods, the level of all-trans retinal is mammalian RDH12 in the IS. RDH13 is present in mitochon- low (21, 25). However, it is possible that significant amount of dria in prostate cancer LNCaP cells (23), but its function is not free all-trans retinal is present in cone membranes and that this clear yet. free retinal was responsible for the coupling. Further studies are required on this point. If only the released all-trans retinal is used Cone-Specific ALOL-Coupling Reaction. It is thought that 11-cis for the coupling, we speculate that the ALOL coupling becomes retinal is supplied from the RPE to the OS of rods and cones. As a major reaction under bright light that produces a large amount discussed by Mata et al. (12), this supply does not seem to fully of all-trans retinal. The ALOL coupling does not require support the amount of 11-cis retinal necessary to regenerate NADPϩ for the oxidation of 11-cis retinol, which suggests that pigment in cones that function under bright light. Instead, in cone pigments can be regenerated even when this cofactor

cones, it has been proposed that 11-cis retinol supplied from produced in an energy-dependent way (26) runs out in the COS. PHYSIOLOGY Mu¨ller cells is converted to 11-cis retinal in cones and that this Fig. 5 summarizes the visual cycle for rods and cones. The mechanism is a cone-specific additional visual cycle pathway to cycle for rods (Left) is adopted from those suggested (3, 4), and regenerate cone pigment effectively (12, 24). It has been pos- that for cones (Right) was drawn based on the scheme similar to tulated that 11-cis retinol dehydrogenase, which converts 11-cis that for rods with addition of our present finding. retinol to its retinal in the presence of NADPϩ, is responsible for As we showed in the present study, the ALOL coupling is this reaction. probably catalyzed by an enzyme, because the activity was lost However, in the present study, as shown in Fig. 1E, this activity by heat treatment (Fig. 4G). This activity is present in the cone was low in purified carp cones: the rate was 0.065 Ϯ 0.020 amol membranes. The entity of this enzyme has not been identified 11-cis retinal/cone-sec. In contrast to this low activity, the higher yet. Our expressed RDH8, RDH8L2, RDH13, or RPE contain-

Miyazono et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 16055 Downloaded by guest on September 25, 2021 ing RDH5 abundantly did not show this activity (data not of primers (Table S1). The nucleotide sequences of the RDH cDNA clones were shown). It could be the case that the enzyme is one that has not determined with a PRISM 3130 Genetic Analyzer (Applied Biosystems). For been characterized previously or that the known RDHs require determination of the full-length nucleotide sequences of RDH8, RDH8L2, and a protein(s) that is expressed specifically in cones. The molecular RDH13, DIG-labeled riboprobes were synthesized to screen the carp retinal cDNA library. mechanisms of this ALOL coupling remain to be identified. For the measurement of the expression levels of RDH mRNAs, total RNA was extracted from the retina and the RPE of three individual carp, and cDNA was Materials and Methods synthesized as described in ref. 30. The primer sequences used are shown in The detailed methods used in this study and materials used are given in SI Table S2. Real-time RT-PCR was performed by using an ABI PRISM 7300 Materials and Methods. (Applied Biosystems).

Preparation of OS- and IS-Rich Rods and Cones. Carp (C. carpio) rods and cones Preparation of Anti-RDH8, RDH8L2, and RDH13 Polyclonal Sera. Partial se- were isolated as described in ref. 13 with slight modification (27). The animal quences of RDH8, RDH8L2, and RDH13 genes corresponding to 196-318, was cared for in accordance with our institutional guidelines. In most of 192-310, and 282-329-aa residues, respectively, were expressed in Escherichia purified rods and cones (both Ͼ90%), the IS was attached to the OS (Fig. S1). coli. Anti-RDH8, -RDH8L2, and -RDH13 antisera were raised against each The purified rods and cones lacked nucleus and terminal regions. By passage purified GST-fused peptide in mice. through a 27-gauge needle, OS (ROS or COS)-rich and IS (RIS or CIS)-rich preparations were obtained (Fig. S1). Expression of Recombinant Proteins. Three kinds of expression systems were The content of the IS relative to that of the OS was estimated by comparing used. For quantification of RDH8L2 and RDH13, recombinants of these RDHs the activity of mitochondrial membrane-bound succinate dehydrogenase were obtained in E. coli. For quantification of RDH8, Sf9 cells were used for (MSD) (28) and the amount of visual pigment as the quantitative markers of expression of recombinant RDH8. For the measurement of specific RDH activ- the IS and the OS, respectively. The MSD activity/pigment ratio in the purified ities of RDH8, RDH8L2, and RDH13, HEK293 cells were used for the expression rod or cone preparation was taken to be 1, because most of purified rods and of these RDHs by using a pEF-BOS-EX vector (31). cones possessed both the OS and the IS (see above). Significant amount of soluble proteins were lost during preparation of OS- and IS-rich preparations Measurement of Visual Pigment Regeneration by 11-cis Retinal Formed from (SI Materials and Methods), and rods were contaminated with cones (see text). 11-cis Retinol in Cones. The lysate of purified cones was incubated under All these contributions were corrected in our estimation in the present study various conditions shown in Results. After incubation, visual pigment was (see SI Materials and Methods). extracted (13), and the absorption spectrum of each sample was measured in the dark. To fully bleach the regenerated red pigment, the sample was Measurements of Reduction or Oxidation of Retinoids. Retinoids prepared by illuminated with Ͼ680-nm light for 2–3 h, and then the absorption spectrum HPLC purification were incubated with the lysed rod or cone preparations in was measured again. The difference of the spectrum before and after the the presence of NADPH for reduction or NADPϩ for oxidation. In the mea- bleach was used to quantify the amount of red pigment regenerated. surement of the ALOL-coupling reaction, oxidation of 11-cis retinol was carried out in the absence of NADPϩ, but in the presence of all-trans retinal. ACKNOWLEDGMENTS. We thank Prof. S. Nagata (Kyoto University) for kindly Retinols were extracted directly, and retinals were extracted as retinal- allowing us to use an expression vector for HEK293 cells and Prof. H. Kanazawa (Osaka University) for help in establishing the RDH expression system. We also oximes (29). The reaction products were identified and quantified by HPLC. thank Dr. T. Seki (Osaka Kyoiku University) for helpful technical advice. This work was supported by the Japan Society for the Promotion of Science Grants Cloning of RDH Genes and Real-Time RT-PCR. The partial coding region of each 20370060 (to S.K.) and 20700350 (to S.T.), by a Human Frontier Science RDH was amplified from the carp retinal cDNA library (30) by PCR with a pair Program grant (S.K.), and by the Naito Foundation (S.T.).

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