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Biosynthetic Studies of A2E, a Major Fluorophore of Retinal Pigment Epithelial Lipofuscin*

Open AccessPublished:December 26, 2001DOI:https://doi.org/10.1074/jbc.M108981200
      We have examined questions related to the biosynthesis of A2E, a fluorophore that accumulates in retinal pigment epithelial cells with aging and in some retinal disorders. The use of in vitro preparations revealed that detectable levels of A2-PE, the A2E precursor, are formed within photoreceptor outer segments following light-induced release of endogenous all-trans-retinal. Moreover, experiments in vivo demonstrated that the formation of A2-PE in photoreceptor outer segment membrane was augmented by exposing rats to bright light. Whereas the generation of A2E from A2-PE by acid hydrolysis was found to occur very slowly, the detection in outer segments of a phosphodiesterase activity that can convert A2-PE to A2E may indicate that some portion of the A2-PE that forms in the outer segment membrane may undergo hydrolytic cleavage before internalization by the retinal pigment epithelial cell. The identities of additional minor components of retinal pigment epithelium lipofuscin, A2E isomers withcis olefins at positions other than the C13-C14 double bond, are also described.
      The fluorophore A2E, a pyridinium bisretinoid (
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      ,
      • Ren R.F.
      • Sakai N.
      • Nakanishi K.
      ), accumulates in retinal pigment epithelial (RPE)
      RPE
      retinal pigment epithelium
      FAB
      fast atom bombardment ionization
      HPLC
      high performance liquid chromatography
      PLD
      phospholipase D
      ROS
      photoreceptor outer segment(s)
      MS
      mass spectroscopy
      1RPE
      retinal pigment epithelium
      FAB
      fast atom bombardment ionization
      HPLC
      high performance liquid chromatography
      PLD
      phospholipase D
      ROS
      photoreceptor outer segment(s)
      MS
      mass spectroscopy
      cells as a major component (
      • Eldred G.E.
      • Katz M.L.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      ) of the lipofuscin that is characteristic of senescence and some inherited retinal disorders. Recent evidence indicates that A2E has the potential for causing RPE cell death (
      • Sparrow J.R.
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      ,
      • Holz F.G.
      • Schutt F.
      • Kopitz J.
      • Eldred G.E.
      • Kruse F.E.
      • Volcker H.E.
      • Cantz M.
      ,
      • Sparrow J.R.
      • Nakanishi K.
      • Parish C.A.
      ,
      • Schutt F.
      • Davies S.
      • Kopitz J.
      • Holz F.G.
      • Boulton M.E.
      ,
      • Sparrow J.R.
      • Cai B.
      ) and, as such, may contribute to the RPE cell atrophy that is observed in age-related macular degeneration (
      • Bressler S.B.
      • Bressler N.M.
      • Gragoudas E.S.
      ) and retinal degenerative diseases such as Stargardt's disease (
      • Lopez P.F.
      • Maumenee I.H.
      • de la Cruz Z.
      • Green W.R.
      ,
      • Birnbach C.D.
      • Jarvelainen M.
      • Possin D.E.
      • Milam A.H.
      ), Best's macular dystrophy (
      • Weingeist T.A.
      • Kobrin J.L.
      • Watzke R.C.
      ), and cone-rod dystrophy (
      • Rabb M.F.
      • Tso M.O.
      • Fishman G.A.
      ).
      The biosynthesis of A2E (
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      ,
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ) begins in the photoreceptor outer segment (ROS) membrane as a reaction between phosphatidylethanolamine and a single molecule of all-trans-retinal that generates a phosphatidylethanolamine-all-trans-retinal Schiff base conjugate (N-retinylidene-phosphatidylethanolamine). This adduct undergoes a [1,6]-proton tautomerization generating a phosphatidylethanolamine-retinyl enamine, which reacts with a second molecule of all-trans-retinal. After aza-6π-electrocyclization and auto-oxidation, the fluorescent phosphatidyl-pyridinium bisretinoid A2-PE is formed. A2-PE was established as the precursor of A2E by HPLC detection of A2E after enzyme-mediated hydrolysis of A2-PE, whereas the structure of A2-PE was confirmed by collision-induced dissociation mass spectrometric analysis (FAB collision-induced dissociation mass spectrometry/MS). The A2-PE that forms in the outer segments leads to the deposition of A2E in RPE cells because of the latter cell's role in phagocytosing the outer segment membrane that is discarded daily by the photoreceptor cell. The tendency for A2E to undergo photoisomerization was illustrated by the identification of iso-A2E, a Z-isomer, at the C13-C14 double bond of one of the hydrophobic retinal chains. In the HPLC profile of human RPE extracted in the dark, iso-A2E is a pigment that is slightly less polar than A2E. Using synthetic samples,iso-A2E has been shown to exist in photoequilibrium with A2E at a ratio of ∼4:1, A2E:iso-A2E.
      It was clear from the early work of Katz et al. (
      • Katz M.L.
      • Drea C.M.
      • Robison W.G., Jr.
      ,
      • Katz M.L.
      • Norberg M.
      • Stientjes H.J.
      ), which demonstrated that rats fed a diet lacking in retinoid precursors of 11-cis-retinal exhibited significantly reduced levels of RPE lipofuscin despite normal numbers of photoreceptor cells, that the constituents of RPE lipofuscin are predominantly vitamin A aldehyde-conjugate. Because the all-trans-retinal that is released from photoactivated rhodopsin and cone pigment is reduced to all-trans-retinol by an NADPH-dependent retinol dehydrogenase located in the photoreceptor outer segment, it is generally assumed that only all-trans-retinal that has avoided reduction is available to react with phosphatidylethanolamine. Correspondingly, conditions that allow the generation of all-trans-retinal to surpass its reduction by retinol dehydrogenase can lead to accelerated formation of A2-PE and an enhanced accumulation of A2E.
      In vivo, A2-PE has been identified as the orange-colored fluorophore that accumulates in the photoreceptor outer segment debris of Royal College of Surgeon rats (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ). Significant amounts of A2-PE were also detected in outer segments isolated from mice with a knockout mutation in the gene encoding for the photoreceptor-specific ATP-binding cassette RIM transporter (
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ). The latter protein, a member of the superfamily of ATP-binding cassette (ABC) proteins, is causative for Stargardt's disease, recessive cone-rod dystrophy, recessive retinitis pigmentosa, and some cases of atrophic age-related macular degeneration (
      • Allikmets R.
      • Singh N.
      • Sun H.
      • Shroyer N.F.
      • Hutchinson A.
      • Chidambaram A.
      • Gerrard B.
      • Baird L.
      • Stauffer D.
      • Peiffer A.
      • Rattner A.
      • Smallwood P., Li, Y.
      • Anderson K.L.
      • Lewis R.A.
      • Nathans J.
      • Leppert M.
      • Dean M.
      • Lupski J.R.
      ,
      • Martinez-Mir A.
      • Paloma E.
      • Allikmets R.
      • Ayuso C.
      • del Rio T.
      • Dean M.
      • Vilageliu L.
      • Gonzalez-Duarte R.
      • Balcells S.
      ,
      • Shroyer N.F.
      • Lewis R.A.
      • Allikmets R.
      • Singh N.
      • Dean M.
      • Leppert M.
      • Lupski J.R.
      ,
      • Cremers F.P.
      • van de Pol D.J.
      • van Driel M.
      • den Hollander A.I.
      • van Haren F.J.
      • Knoers N.V.
      • Tijmes N.
      • Bergen A.A.
      • Rohrschneider K.
      • Blankenagel A.
      • Pinckers A.J.
      • Deutman A.F.
      • Hoyng C.B.
      ,
      • Allikmets R.
      • Shroyer N.F.
      • Singh N.
      • Seddon J.M.
      • Lewis R.A.
      • Bernstein P.S.
      • Peiffer A.
      • Zabriskie N.A., Li, Y.
      • Hutchinson A.
      • Dean M.
      • Lupski J.R.
      • Leppert M.
      ,
      • Allikmets R.
      ). It is postulated that the photoreceptor-specific ATP-binding cassette transporter makes all-trans-retinal available to all-trans-retinol dehydrogenase on the cytoplasmic side of the disc membrane (
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      ,
      • Sun H.
      • Molday R.S.
      • Nathans J.
      ,
      • Ahn J.
      • Wong J.T.
      • Molday R.S.
      ). As a consequence of the loss of photoreceptor-specific ATP-binding cassette transporter activity and the accumulation of A2-PE in outer segments, the levels of A2E in RPE cells of abcr−/− mice are reported to be 20 times greater than those in normal mice (
      • Weng J.
      • Mata N.L.
      • Azarian S.M.
      • Tzekov R.T.
      • Birch D.G.
      • Travis G.H.
      ).
      Determining the steps involved in the biosynthesis of A2E, together with the factors that influence its formation, is fundamental to our understanding of blinding retinal disorders associated with abnormal accumulations of A2E in RPE cells. In the present studies, we have examined several issues related to the biosynthesis of A2E including the mechanisms involved in the hydrolysis of A2-PE and the modulation of A2-PE accumulation by bright light exposure. We also report that previously unidentified components of hydrophobic extracts of human RPE are structurally related to A2E and iso-A2E.

      DISCUSSION

      Whereas we have previously demonstrated the formation of A2-PE in isolated outer segments incubated in the presence of exogenous all-trans-retinal (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ), in the present work we confirm that illumination of whole isolated retina to release endogenous retinal is sufficient to generate detectable levels of A2-PE. In addition, we have shown that the rate of formation of A2-PE in the outer segment membrane can be modulated by light intensity. The most parsimonious explanation for the production of detectable levels of A2-PE after bright light exposure in vivo was an increased availability of all-trans-retinal, with the rate of release of all-trans-retinal from photoactivated rhodopsin presumably exceeding the rate of reduction of all-trans-retinal by retinol dehydrogenase. On the other hand, we cannot rule out the possibility that a light-induced reduction in photoreceptor-specific ATP-binding cassette transporter activity may also have contributed to the accumulation of A2-PE under these conditions (
      • Sun H.
      • Nathans J.
      ). The levels of [14C2]A2-PE formed in the excised whole retinas after illumination may have been accentuated in this preparation by an excess of all-trans-retinal stemming from a depletion of NADPH and consequent failure of all-trans-retinal reduction (
      • Palczewski K.
      • Van Hooser J.P.
      • Garwin G.G.
      • Chen J.
      • Liou G.I.
      • Saari J.C.
      ). Whether an insufficient supply of NADPH, such as that occurring with oxidative stress, contributes to A2-PE deposition in vivo is not known.
      On the basis of their identical molecular weights, similar absorbance spectra, and HPLC retention times, we have also identified additional double bond isomers of A2E as components of RPE lipofuscin. Although Eldred and Katz (
      • Eldred G.E.
      • Katz M.L.
      ) detected a number of fluorescent components in their original separation of RPE lipofuscin, A2E and iso-A2Eare the only lipofuscin fluorophores to be structurally characterized (
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      ,
      • Parish C.A.
      • Hashimoto M.
      • Nakanishi K.
      • Dillon J.
      • Sparrow J.R.
      ,
      • Eldred G.E.
      • Lasky M.R.
      ). Whereas all the double bonds of A2E assume thetrans configuration, iso-A2E contains onecis double bond at position C13-C14 of one of the retinal chains. It is well known that all-trans-retinal also undergoes light-induced isomerization to form various double bond isomers, including 13-cis, 11-cis,9-cis, and 9,–13-di-cis; generally, it is the C13-C14 double bond that is most prone to isomerization (
      • Waddell W.H.
      • Hopkins D.L.
      ,
      • Liu R.
      • Asato A.
      ). Despite the fact that A2E is formed from two molecules of retinal, only one of the retinal-derived chains retains a C13-C14 double bond that can isomerize. The carbon atoms derived from the C13-C14 positions of the second retinal are incorporated into the pyridinium ring system. Thus, a single C13-C14 double bond isomer, iso-A2E, is the most prominent isomer generated during light exposure. Clearly, however, the C11-C12 and C9-C10 double bonds of A2E are available for isomerization and could lead to four additional isomers, each containing a single cis double bond (Fig.9, 1–4). It is also likely that photoisomerization would produce A2E isomers containing twocis double bonds (Fig. 9, 5). It would be expected that the additional cis double bonds would lead to absorbance spectra that are blue-shifted in relation to A2E; indeed, such a shift has been observed here. The range of A2E isomers we have detected could originate in photoreceptor outer segments upon illumination of all-trans-A2-PE. Alternatively, photoisomerization of A2E after phosphate ester hydrolysis of A2-PE would produce the same products. These minor A2E isomers are clearly generated in vivo as evidenced by the isolation of these materials from human RPE harvested under reduced lighting.
      Figure thumbnail gr9
      Figure 9Structures of A2E, iso-A2E, and additional A2E-derived photoisomers are shown.
      It has generally been assumed that the generation of A2E by phosphate hydrolysis of A2-PE occurs within the acidic conditions of RPE lysosomes (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ,
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ,
      • Eldred G.E.
      ). Contrary to expectations, we observed that at pH 5.5, an acidic environment similar to that in lysosomes (pH 5.5), A2E is generated from A2-PE at a slow rate. Only with an overnight incubation in 100 mm HCl is an entire sample of A2-PE converted to A2E (
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ). Although these results do not preclude the possibility that acid hydrolysis contributes to the generation of A2E from A2-PE, the finding that A2E is generated when A2-PE is incubated with phospholipase D (Fig. 3; see also Liu et al., Ref.
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ) indicates that enzyme-mediated mechanisms may play an important role. Moreover, the notion that at least some of the A2-PE that forms in the photoreceptor outer segment can undergo enzyme-mediated cleavage within the photoreceptor outer segment before internalization by RPE cells is supported by our observation of a phosphodiesterase activity in photoreceptor outer segments that is sensitive to inhibitors that act on PLD. The latter finding is consistent with previous reports of a PLD activity in outer segments isolated from bovine eyes (
      • Salvador G.A.
      • Giusto N.M.
      ). Also remarkable in this regard were the observations of Eldred and co-workers (
      • Katz M.L.
      • Eldred G.E.
      • Robison W.G., Jr.
      ,
      • Eldred G.E.
      ) when they analyzed the orange-colored fluorophores present in the outer segment debris that accumulates in Royal College of Surgeons rat eyes. Thus by thin layer chromatography they detected, in addition to the fluorescent fraction later shown to be A2-PE (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ,
      • Katz M.L.
      • Gao C.L.
      • Rice L.M.
      ), a fluorophore with the same chromatographic mobility as the fluorophore they had observed in RPE lipofuscin (fraction VIII) (
      • Eldred G.E.
      • Katz M.L.
      ) and that was later shown to be A2E (
      • Sakai N.
      • Decatur J.
      • Nakanishi K.
      • Eldred G.E.
      ,
      • Eldred G.E.
      • Lasky M.R.
      ). Indeed, they speculated that the fluorophore with lower chromatographic mobility (A2E) was generated, within the outer segment debris, from a fluorophore demonstrating greater mobility (perhaps A2-PE) (
      • Eldred G.E.
      ,
      • Katz M.L.
      • Eldred G.E.
      • Robison W.G., Jr.
      ,
      • Eldred G.E.
      ). Although our observations indicate that a phosphodiesterase activity in the outer segment may contribute to the hydrolysis of A2-PE, we do not doubt that hydrolytic cleavage of A2E can also occur within the RPE cell. The presence of A2-PE in RPE cells of abcr-null mutant mice supports to this assertion (
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ).
      We and others (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ,
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ) have consistently observed that when normal ROS or whole retina is isolated in the dark, as opposed to bleaching conditions (
      • Katz M.L.
      • Gao C.L.
      • Rice L.M.
      ), A2-PE is not detectable. The complete replacement of the photoreceptor outer segment by the processes of disc assembly and shedding approximately every 10 days (
      • Young R.W.
      ) is clearly a factor preventing the accretion of A2-PE in the membrane. Indeed, prevention of the latter accumulation may be one reason for the constant turnover of outer segment membrane. The consequences inherent in the failure to remove effete outer segment membrane at the RPE-photoreceptor interface are evident from the Royal College of Surgeons rat, in which the orange fluorophore A2-PE is amassed in abundance (
      • Liu J.
      • Itagaki Y.
      • Ben-Shabat S.
      • Nakanishi K.
      • Sparrow J.R.
      ,
      • Eldred G.E.
      ). The accumulation of A2-PE within photoreceptor outer segment membrane is not just a generalized feature of degenerating retina, however, because inrd/rd mice, a blind strain that presents with a retinal degeneration caused by homozygous mutations in the gene encoding for the β subunit of rod cGMP-phosphodiesterase (
      • Bowes C.
      • Tiansen L.
      • Danciger M.
      • Baxter L.C.
      • Applebury M.L.
      • Farber D.B.
      ), A2-PE was not detected in retinal extracts by HPLC and FAB-MS.
      Unpublished observation.
      Rather, these observations, together with the report that dark rearing ofabcr−/− mice intervenes in the deposition of A2E (
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      ), corroborate the dependence of A2-PE formation on light-induced release of all-trans-retinal and underscore the association between light exposure and the accumulation of lipofuscin by RPE cells.

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