Biochemistry 2003, 42, 5809-5818
5809
All-trans-retinyl Esters Are the Substrates for Isomerization in the Vertebrate Visual Cycle† Deviprasad R. Gollapalli and Robert R. Rando* Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115 ReceiVed January 17, 2003; ReVised Manuscript ReceiVed March 27, 2003
ABSTRACT: The identification of the critical enzyme(s) that carries out the trans to cis isomerization producing 11-cis-retinol during the operation of the visual cycle remains elusive. Confusion exists in the literature as to the exact nature of the isomerization substrate. At issue is whether it is an all-trans-retinyl ester or all-trans-retinol (vitamin A). As both putative substrates interconvert rapidly in retinal pigment epithelial membranes, the choice of substrate can be ambiguous. The two enzymes that effect interconversion of all-trans-retinol and all-trans-retinyl esters are lecithin retinol acyl transferase (LRAT) and retinyl ester hydrolase (REH). The retinyl ester or all-trans-retinol pools are radioactively labeled separately in the presence of inhibitors of LRAT and REH, effectively preventing their interconversion. Pulse-chase experiments unambiguously demonstrate that all-trans-retinyl esters, and not all-trans-retinol, are the precursors of 11-cis-retinol. When the all-trans-retinyl ester pool is radioactively labeled, the resulting 11-cis-retinol is labeled with the same specific activity as the precursor ester. The converse is true with vitamin A. These data unambiguously establish all-trans-retinyl esters as the precursors of 11-cis-retinol.
Dark adaptation in vertebrates depends on the enzymatic processing of all-trans-retinol (vitamin A) into an 11-cisretinoid (1, 2). The visual pigment rhodopsin contains an 11-cis-retinal protonated Schiff base, and the absorption of light by rhodopsin causes a cis to trans photoisomerization, initiating the phototransduction cascade (2-4). Hydrolysis of the resultant all-trans-retinal Schiff base and dehydrogenase-mediated reduction of the all-trans-retinal product results in the formation of all-trans-retinol in the retina (5, 6). The all-trans-retinol is transported to the retinal pigment epithelium (RPE)1 where it is processed into 11-cis-retinal and then returned to the photoreceptors (7, 8) (Scheme 1). The nature of the enzymatic machinery in the RPE responsible for the overall transformation of all-trans-retinol into 11-cis-retinal is only partially understood (7, 9). Central to investigations on this problem is the nature of the substrate and product of the isomerization pathway. Since the all-transand 11-cis-retinoids can exist as aldehydes, alcohols, or esters, the question is not a trivial one inasmuch as there are nine possible pathways. Double labeling experiments using 15-14C,15-3H-all-trans-retinol to probe this issue demonstrated that isomerization occurred at the alcohol oxidation state, eliminating a retinal isomerase from consideration (10). † The work described here from the authors’ laboratory was supported by the U.S. Public Health Service N.I.H. Grant EY-04096. * To whom correspondence should be addressed. Phone: (617) 4321794. Fax: (617) 432-0471. E-mail:
[email protected]. 1 Abbreviations: BSA, bovine serum albumin; CRALBP, cellular retinaldehyde binding protein; DMSO, dimethyl sulfoxide; DPPC, L-dipalmitoylphosphatidylcholine; EA, ebelactone; LRAT, lecithin retinol acyl transferase; REH, retinyl ester hydrolase; RBA: all-transretinyl bromoacetate; RPE, retinal pigment epithelium; a.u., arbitrary units.
Retinyl ester formation in the RPE membrane occurs via the esterification of all-trans-retinol by lecithin retinol acyl transferase (LRAT) (11-14). LRAT rapidly and reversibly transfers an acyl group from the sn-1 position of lecithin to all-trans-retinol, forming an all-trans-retinyl ester (largely palmitate) and lysophospholipids (15). Deciding between retinol and retinyl esters as possible substrates and products is not straightforward because these retinoids interconvert on a more rapid time scale than isomerization (16, 17). To address the issue of substrate and product for the isomerization, a specific inactivator of LRAT, all-trans-retinyl bromoacetate (RBA), was utilized (18). RPE membranes preincubated with RBA are unable to process added all-transretinol into all-trans-retinyl esters and 11-cis-retinoids, while untreated RPE membranes can carry out both conversions (19). Importantly, RBA does not inhibit the processing of all-trans-retinyl esters into 11-cis-retinoids, showing that RBA does not block the formation of an unknown and hypothetical intermediate on the isomerization pathway (19). The straightforward conclusion from these experiments is that RBA blocks isomerization because it blocks the formation of all-trans-retinyl esters (19). Moreover, 11-cis-retinol, but not 11-cis-retinyl esters, were formed when RBA was added subsequent all-trans-retinyl ester formation, demonstrating that 11-cis-retinol is the direct product of isomerization. These, and similar studies (20), indicated that all-trans-retinyl esters are the substrates on the isomerization pathway and that 11-cis-retinol is the product of isomerization. Scheme 1 depicts the overall visual cycle showing the conversion of all-trans-retinyl esters into 11-cis-retinol (21). This unusual isomerohydrolase mechanism is doubtless in place to provide an energy source (ester hydrolysis) to drive the thermody-
10.1021/bi0341004 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/25/2003
5810 Biochemistry, Vol. 42, No. 19, 2003
Gollapalli and Rando
Scheme 1: The Mammalian Visual Cycle
namically uphill (4.1 kcal/mol) trans to cis isomerization (21, 22). Recently, an article was published suggesting that all-transretinol itself is the isomerization substrate rather than an alltrans-retinyl ester (23). In most ways this article happily confirmed already published data. For example, the article (23) confirmed the previous reports (19) that 11-cis-retinol is the direct product of isomerization and that retino(a)l binding proteins stimulate 11-cis-retinol production (16, 17). A later article (24) from the same group also confirmed the previous report that C-O bond cleavage occurs concomitant with isomerization (22). Thus, the results from the earlier (23) and more recent experiments (24) are essentially concordant with previous work (7, 19, 22), save for the issue of the substrate for isomerization. This point though is an important one because ester hydrolysis is considered to be the energy source that drives the endergonic isomerization reaction in one mechanism (22). No identified energy source exists if an all-trans-retinol to 11-cis-retinol conversion were to occur. Previously described 11-cis-retinoid binding protein candidates are not relevant here. For example, the availability of opsin to bind 11-cis-retinal clearly is not essential because isomerization to 11-cis-retinoids proceeds in the RPE in the absence of the retina and photoreceptors (16). The specific 11-cis-retino(a)l binding protein CRALBP (25, 26), found in the RPE, is pre-understood (17) to not be an essential driving force for isomerization because isomerization proceeds in its absence in vitro (17), and CRALBP knockout mice can still synthesize 11-cis-retinoids (29), albeit slowly. The slower in vivo kinetics are understandable because CRALBP, and even nonspecific retinoid binding proteins such as BSA, simply enhance the rate of isomerization in vitro by relieving feed-back inhibition (17). Given these various issues, an important question to address is what is the experimental evidence presented in support of all-transretinol as the isomerization substrate? This view, which is frequently quoted (26-40), is dependent on a single experiment, which is reported in Figure 8 (23). The approach is based on a pulse-chase protocol in which RPE membranes are pretreated with nonradioactive all-trans-retinol to form a pool of nonradioactive all-transretinyl esters (23). A pulse-chase experiment is then performed with 3H-all-trans-retinol as the chase (23). The basis of the experiment lies in temporally correlating the specific activities of the all-trans-retinyl ester/all-trans-retinol pools with the specific activities of the enzymatically formed 11cis-retinol (23). In the experiment as designed, several competing processes occur (Scheme 2). The all-trans-retinyl
Scheme 2: Retinoid Processing in the RPE
esters and all-trans-retinol rapidly interconvert via the combined enzymatic action of LRAT and REH (41). Concomitantly, isomerization occurs via either the all-transretinyl ester or the all-trans-retinol substrates. Superficially, the prediction is that if all-trans-retinyl esters were the substrates for isomerization, then the specific activities of the product 11-cis-retinol would be initially zero and then exponentially increase with time as the specific activity of the all-trans-retinyl ester pool increased because of the LRAT-mediated esterification of 3H-all-trans-retinol (23). Should all-trans-retinol be the substrate, the prediction is that the specific activity of the 11-cis-retinol generated during the course of the experiment would initially be identical to that of the added 3H-all-trans-retinol and then suffer exponential decay as the retinoid pools mix (23). In the actual reported experiment (Figure 8, ref 23), a small rise in the specific activity (
