RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye

Article abstract of DOI:10.1073/pnas.1706332114

Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common

Full text of DOI:10.1073/pnas.1706332114

RPE65 has an additional function as the lutein to
meso-zeaxanthin isomerase in the vertebrate eye
Rajalekshmy Shyama,b, Aruna Gorusupudia, Kelly Nelsona, Martin P. Horvathc, and Paul S. Bernsteina,b,1
aDepartment of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah, Salt Lake City, UT 84132; bDepartment of Neurobiology and
Anatomy, University of Utah, Salt Lake City, UT 84112; and cDepartment of Biology, University of Utah, Salt Lake City, UT 84112
Edited by John E. Dowling, Harvard University, Cambridge, MA, and approved August 14, 2017 (received for review April 20, 2017)
lutein produce labeled meso-zeaxanthin, while labeled zeaxanthin
feed did not have the same effect (7). Similar results have been
observed in primates as well. When carotenoid-deficient monkeys
were fed lutein, meso-zeaxanthin was present in their retinas, but
no meso-zeaxanthin was detected when these animals were
maintained on feed enriched with zeaxanthin (8). Both of these
studies indicate that lutein undergoes metabolic transformations
to form meso-zeaxanthin in vivo, but the biochemical mechanism
by which this reaction occurs is unknown, although it is efficiently
produced under harsh industrial conditions, such as high tem-
perature and strong base (9). Conversion of dietary zeaxanthin to
meso-zeaxanthin would require inversion of a chiral center at the
3′ position, a reaction rarely encountered in biological systems.
In contrast, conversion of lutein to meso-zeaxanthin proceeds by
the migration of just one double bond from the 4′-5′ position to
the 5′-6′ position, a reaction that should be readily accomplished
by coordinated acid-base catalysis as illustrated in Fig. 1B. Other
mechanisms involving radical chemistry are also plausible.
In an effort to determine the biochemistry behind meso-
zeaxanthin formation, our laboratory undertook studies in de-
veloping chicken embryos. In this isolated system, we determined
that meso-zeaxanthin is produced in a developmentally regulated
manner in the RPE/choroid of chicken embryos from the lutein
and zeaxanthin naturally present in egg yolk (10). In a previous
study, we detected the presence of meso-zeaxanthin in the RPE/
choroid of E17 embryos, with increasing levels as the embryo
neared hatching at E21 (10). Retinal detection of meso-zeaxanthin
occurred only at E19, and all other tissues examined (brain,
liver, serum, and yolk) were devoid of this carotenoid. Since the
eggs were incubated in the dark, we could rule out the role of
light in meso-zeaxanthin production. In the current study, we
present evidence that RPE65, the isomerohydrolase enzyme of
the vertebrate visual cycle responsible for the isomerization of
Carotenoids are plant-derived pigment molecules that vertebrates
cannot synthesize de novo that protect the fovea of the primate
retina from oxidative stress and light damage. meso-Zeaxanthin is
an ocular-specific carotenoid for which there are no common dietary
sources. It is one of the three major carotenoids present at the foveal
center, but the mechanism by which it is produced in the eye is
unknown. An isomerase enzyme is thought to be responsible for
the transformation of lutein to meso-zeaxanthin by a double-bond
shift mechanism, but its identity has been elusive. We previously
found that meso-zeaxanthin is produced in a developmentally reg-
ulated manner in chicken embryonic retinal pigment epithelium
(RPE)/choroid in the absence of light. In the present study, we show
that RPE65, the isomerohydrolase enzyme of the vertebrate visual
cycle that catalyzes the isomerization of all-trans-retinyl esters to
11-cis-retinol, is also the isomerase enzyme responsible for the pro-
duction of meso-zeaxanthin in vertebrates. Its RNA is up-regulated
23-fold at the time of meso-zeaxanthin production during chicken
eye development, and we present evidence that overexpression of
either chicken or human RPE65 in cell culture leads to the production
of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65
function resulted in significant inhibition of meso-zeaxanthin bio-
synthesis during chicken eye development. Structural docking exper-
iments revealed that the epsilon ring of lutein fits into the active site
of RPE65 close to the nonheme iron center. This report describes
a previously unrecognized additional activity of RPE65 in ocular
carotenoid metabolism.
carotenoid isomerase retina lutein zeaxanthin
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he rare carotenoid meso-zeaxanthin is present only in the
T
eyes of higher vertebrates (1, 2). This is a unique phenome-
non in nature, especially since vertebrates normally obtain carot-
enoids through their diet and are incapable of producing these
molecules de novo (2, 3). meso-zeaxanthin is not commonly found
in dietary sources; besides the eyes of vertebrates, this carotenoid is
present in shrimp shells, turtle fat, and fish skin (2, 4, 5). Hundreds
of carotenoids are present in nature, and even though primates
consume more than 50 of them, meso-zeaxanthin is one of only
three carotenoids present in the foveal center of the retina, the
region responsible for sharp, central vision. Degeneration of the
retina and retinal pigment epithelium (RPE) surrounding the fovea
occurs in the disease state known as age-related macular de-
generation (AMD). Carotenoid supplementation has been shown
to be effective in curtailing the progression of this disease, because
these molecules are capable of protecting the fovea from blue light
damage and reactive oxygen species (2, 6). Despite the abundance
of meso-zeaxanthin in the foveal center, its specific function relative
to dietary lutein and zeaxanthin remains unknown.
Significance
Carotenoids are plant-derived pigment molecules that cannot
be synthesized de novo by higher organisms. These physio-
logically relevant compounds function as potent antioxidants
and light screening compounds, and their supplementation has
been shown to ameliorate the progression of such diseases as
age-related macular degeneration. Hundreds of carotenoids
are present in the plant world, but the primate macula contains
only three: lutein, zeaxanthin, and meso-zeaxanthin. The
presence of meso-zeaxanthin in the foveal region of primates
is an unexplained phenomenon, given its lack of dietary sour-
ces. We show that RPE65 is responsible for the conversion of
lutein to meso-zeaxanthin in vertebrates, a unique role for
RPE65 in carotenoid metabolism beyond its well-known reti-
noid isomerohydrolase function in the vertebrate visual cycle.
Lutein, zeaxanthin, and meso-zeaxanthin are the three carot-
enoids present at the foveal center (2). These molecules share the
same molecular formula, C40H56O2 (Fig. 1A). Since lutein and
zeaxanthin are abundant in a normal diet, it has long been hy-
pothesized that an isomerase enzyme may be responsible for the
metabolic transformations of either lutein or zeaxanthin to pro-
duce meso-zeaxanthin (2). In vivo studies from our laboratory
using quail have shown that birds fed with deuterium-labeled
Author contributions: R.S. and P.S.B. designed research; R.S., A.G., and K.N. performed
research; R.S., A.G., M.P.H., and P.S.B. analyzed data; and R.S. and P.S.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. Email: paul.bernstein@hsc.utah.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1706332114/-/DCSupplemental.
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convert vitamin A1 to vitamin A2 (24), showed a decrease in
transcript abundance between E16 and E21. No significant dif-
ferences in expression were observed for retinoid transporters,
such as IRBP, CRALBP, and RBP1. From the RNA sequencing
data, we concluded that among the relevant genes involved in the
visual cycle and carotenoid metabolism, RPE65 is most likely to be
responsible for meso-zeaxanthin production, especially since it is a
relative of two carotenoid metabolic enzymes, BCO1 and BCO2.
We next determined whether the protein expression profile of
RPE65 showed a similar trend as the mRNA levels. No RPE65
protein was detected in E16 chicken RPE/choroid, whereas
strong expression was observed in E21 tissue (Fig. S1).
Overexpression of RPE65 Leads to the Production of meso-Zeaxanthin
in HEK293T Cells. To determine whether RPE65 can catalyze the
conversion of lutein to meso-zeaxanthin, we used the nonocular
cell line HEK293T, which is derived from human embryonic
kidney and does not express RPE65 or LRAT (12, 13). Over-
expression of pCDNA3.1-CRPE65 (chicken RPE65) resulted in
strong expression of RPE65 at 48 h posttransfection that was
sustained for another 4 d, while nontransfected cells had no
Fig. 1. Structures of macular xanthophylls (A) and simple mechanism of
coordinated acid-base catalysis of lutein to meso-zeaxanthin (B). The first
step involves base-catalyzed proton abstraction from the e-ionone ring at
position C6′. The resulting negative charge on the intermediate is expected
to be resonance-stabilized (double-headed arrow). In the final step, BH+ acts
as a source of proton for attachment to the ionone ring at the new position,
C4′. The mechanism shown here illustrates how conversion might occur
in vivo; alternate mechanisms involving radical chemistry (e.g., hydrogen
atom transfer) are also possible.
all-trans-retinyl palmitate to 11-cis-retinol (11–14), is the lutein
to meso-zeaxanthin isomerase.
Results
RPE65 Transcript and Protein Levels Are Significantly Up-Regulated in
E21 Chicken RPE/Choroid. To identify the enzyme responsible for the
production of meso-zeaxanthin in chicken RPE/choroid, we per-
formed RNA sequencing to ascertain which transcripts of likely
candidates were up-regulated. We used total RNA isolated from
E16 RPE/choroid, a stage at which meso-zeaxanthin is not de-
tectable in the RPE/choroid, and compared the expression profile
of mRNA transcripts with those of E21 RPE/choroid, a stage at
which substantial amounts of meso-zeaxanthin are present. Log2
FPKM values of at least three embryos from each stage were
compared, and the relative abundance of gene transcripts nor-
mally involved in either carotenoid metabolism and transport
(GSTP1, STARD3, STARD1, BCO1, BCO2, SCARB1, SCARB2,
and CD36) or retinoid metabolism and transport (RBP1, IRBP,
CRALBP, RPE65, LRAT, CYP27C1, STRA6, and DES1) are
plotted in Fig. 2. Among the genes that we considered, RPE65
was the most highly up-regulated between E16 and E21. Its tran-
script levels were 23-fold higher at E21 compared with at E16.
GSTP1 and STARD3 are zeaxanthin- and lutein-binding proteins,
respectively (15, 16), and their transcript levels did not show any
significant increase between E16 and E21. BCO1 and BCO2 are
carotenoid oxygenases (17–20), and both of their genes were
slightly up-regulated at E21. STRA6 is a retinol transport protein
(21), and DES1 is a vitamin A isomerase expressed in the Müller
cells of the retina (22). While STRA6 mRNA was up-regulated at
E21, its magnitude of increase was lower than that observed for
RPE65. DES1 mRNA levels were not changed during develop-
ment. SCARB1, SCARB2, and CD36 are carotenoid transport
proteins in the eye (23). While SCARB1 showed increased levels
between E16 and E21, it is an unlikely candidate to catalyze the
production of meso-zeaxanthin. LRAT, the acyl transferase en-
zyme required for visual pigment regeneration, was moderately
up-regulated at E21 (12–14). CYP27C1, a protein known to
Fig. 2. Comparison of gene expression profiles in E16 and E21 chicken RPE/
choroid. The FPKM value of each gene is compared with its expression across
all samples to obtain the average expression. The ratio of gene expression in
each sample is compared with the average, and the values are plotted on a
log base2 scale. Positive values indicate above-average expression; negative
values, below-average expression.
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Fig. 3. CRPE65 overexpression followed by lutein
treatment gives rise to meso-zeaxanthin in HEK293T
cells. Nontransfected HEK293T cells do not express
RPE65, but transient transfection results in expres-
sion of this gene for several days (A). Treatment of
CRPE65-transfected cells with 4 μM lutein resulted in
meso-zeaxanthin production (C), whereas the cells
overexpressing control plasmid were devoid of
meso-zeaxanthin (B). The ratio of meso-zeaxanthin
to lutein shows an increase in the latter in a time-
dependent manner (D). Treatment with 4 μM zeax-
anthin in control plasmid-overexpressing cells (E) or
CRPE65-overexpressing cells did not result in meso-
zeaxanthin production (F). n = 3. Error bars repre-
sent SEM. **P < 0.005; ***P < 0.0005.
RPE65 (Fig. 3A). We supplied HPLC-purified lutein with no de-
tectable meso-zeaxanthin and <0.5% zeaxanthin (Fig. 4B) to
HEK293T cells with no endogenous carotenoids (Fig. 4C).
Treatment with 4 μM lutein for up to 4 d resulted in the pro-
experimental and control cells that increased in a time-dependent
manner (Fig. 3 B and C). This is likely because our lutein stock,
even though devoid of meso-zeaxanthin, contains ∼0.5% zeax-
anthin. To rule out zeaxanthin as the precursor to meso-zeaxanthin
duction of progressively higher levels of meso-zeaxanthin in in CRPE65-overexpressing cells, we treated these cells with 4 μM
RPE65-overexpressing cells, while control cells transfected with isomerically pure zeaxanthin with no detectable lutein. Neither the
pCDNA3.1-GFP did not show the presence of meso-zeaxanthin control nor the experimental cells showed any detectable levels of
(Figs. 3 B and C and 4 D and E). The identity of biosynthesized meso-zeaxanthin or lutein (Fig. 3 E and F).
meso-zeaxanthin was confirmed by comparing its retention time
with that of authentic carotenoid standards (Fig. 4A) and by the
Previous studies have shown that CRPE65 is a better iso-
merohydrolase than its human counterpart (25). We conducted
observation of its characteristic tripeak spectrum with the highest our next set of experiments to determine whether there were any
peak at 450 nm using in-line photodiode array detection (Fig. 4F differences between human RPE65 (HRPE65) and CRPE65 in
and Fig. S2). We observed small amounts of zeaxanthin in our meso-zeaxanthin isomerization (Fig. S3). We observed that
Fig. 4. Chromatograms showing the presence of
carotenoids in HEK293T cells. (A) Retention times of
authentic carotenoid standard mixture. (B) The HPLC-
purified lutein used in our incubations did not contain
any meso-zeaxanthin. (C) HEK293T cells were free of
carotenoids. (D and E) Overexpression of pCDNA3.1-
GFP followed by treatment with 4 μM lutein for 4 d did
not result in meso-zeaxanthin production (D), whereas
cells overexpressing CRPE65 when treated with 4 μM
lutein for 2 d gave rise to detectable levels of meso-
zeaxanthin (E). (F) Characteristic tripeak spectrum
obtained for meso-zeaxanthin from authentic stan-
dard (black) and CRPE65-overexpressing cells (blue)
(Fig. S2). (Insets) Zoom-in views (20×). L, lutein; mZ,
meso-zeaxanthin; O, oxo-lutein; Z, zeaxanthin.
Shyam et al.
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injected embryos’ RPE/choroid (Fig. 6D). The RPE/choroid lu-
tein and zeaxanthin contents of the ACU-5200–injected embryos
remained comparable to those of control embryos (Fig. 6 C and
D). These results indicate that in an in vivo system, the function
of RPE65 is necessary for the production of meso-zeaxanthin.
Structural Docking Experiments Reveal That the Epsilon Ring of Lutein
Can Fit into the Active Site of RPE65. To determine whether lutein
fits into the substrate tunnel of RPE65, we docked lutein mole-
cules into a homology model for chicken RPE65. Fig. 7 shows a
representative outcome that minimizes steric conflicts and maxi-
mizes hydrogen bonds. The «-ionone ring of lutein rests on a ledge
comprising the iron-coordinating histidine residues, and the
β-ionone ring protrudes from an opening at the surface of the
enzyme. The buried hydroxyl group is positioned in proximity with
two hydrogen-bonding groups (indole amine of Trp-331 and car-
boxylate of Glu-417; Fig. S4). Steric complementarity is evident,
with phenylalanine residues making a close approach on either
side of the «-ionone ring and also stacking with the polyene chain.
The edge of the ionone ring containing atoms C4′, C5′, and C6′,
which are involved in isomerization, was consistently found closer
to the iron center and histidine residues. Docking outcomes with
these atoms pointed away from the iron center were not observed
among well-fitting outcomes, likely because in this orientation, the
curvature observed for lutein molecules does not match the cur-
vature of the substrate tunnel found in RPE65.
Fig. 5. Endogenous RPE65 in chicken RPE primary cells can catalyze the
production of meso-zeaxanthin. (A) RPE primary cells from E21 chicken
embryos retain RPE65 expression. (B) When treated with 2 μM lutein for 2 d,
these cells produce meso-zeaxanthin, and higher levels of meso-zeaxanthin
production are observed when cells are treated with 4 μM lutein. n = 3. Error
bars represent SEM. **P < 0.005; ***P < 0.0005.
HRPE65 transfection resulted in slightly lower meso-zeaxanthin
production compared with CRPE65 at day 2; however, by day
4, both CRPE65- and HRPE65-overexpressing cells contained
similar levels of meso-zeaxanthin (Fig. 3C and Fig. S3C). As with
CRPE65, treatment of HRPE65-overexpressing cells with 4 μM
zeaxanthin resulted in no detectable meso-zeaxanthin, and no
meso-zeaxanthin was present in the control cells (Fig. S3 E and F).
Comparison of the ratio of lutein to meso-zeaxanthin in CRPE65-
and HRPE65-overexpressing cells revealed a conversion rate of
<0.05% over a 4-d period (Fig. 3D and Fig. S3D).
Discussion
RPE65 is an important enzyme in the visual cycle, responsible
for the key all-trans to 11-cis-retinoid isomerization step of the
visual cycle (11–13). Such diseases as Leber congenital amaurosis
and retinitis pigmentosa arise from the loss of function of this
gene (12, 13). Here we show that RPE65 is capable of carrying
out an additional function in which it converts lutein to meso-
zeaxanthin, an eye-specific carotenoid with no common dietary
sources. Since meso-zeaxanthin is accumulated in high concen-
trations at the fovea of the retina, a region crucial for visual
acuity, its hypothesized function is to protect the region from
blue light damage and oxidative stress and to potentially enhance
visual function. In support of this hypothesis, a previous study
E21 Chicken RPE Primary Cells Retain RPE65 Expression and Can Produce
Meso-Zeaxanthin. In these experiments, we explored whether chicken
RPE primary cultures from E21 embryos are capable of producing
meso-zeaxanthin on lutein treatment. Unlike human primary RPE
cells, these cells retain the expression of RPE65 even after five
passages (26) (Fig. 5A). Treatment of 107 cells with 2 μM lutein for
2 d resulted in detectable levels of meso-zeaxanthin (Fig. 5B).
Between days 2 and 4, we observed a progressive increase in meso-
zeaxanthin levels. To determine whether a higher precursor con-
centration will result in increased production of meso-zeaxanthin,
we treated these cells with 4 μM lutein. They produced signifi-
cantly higher amounts of meso-zeaxanthin at days 2, 3, and 4
relative to the cells treated with 2 μM lutein (Fig. 5B).
Pharmacologic Inhibition of RPE65 in the Developing Chicken Embryo
Decreases meso-Zeaxanthin Levels. We next examined whether
pharmacologic inhibition of RPE65 activity could specifically
inhibit meso-zeaxanthin production. Our previous studies have
shown that meso-zeaxanthin is first present at detectable levels in
the chicken RPE/choroid at E17. Therefore, we decided to in-
troduce a competitive inhibitor of RPE65 activity into the yolk
sac of chicken embryos at E17.
We used the pharmacologic inhibitor ACU-5200-HCl (ACU-
5200) to knock down RPE65 function. ACU-5200 is an analog of
emixustat, a highly specific RPE65 inhibitor that is currently
undergoing clinical trials as a treatment for Stargardt disease (27–
29) (Fig. 6A). ACU-5200 has been shown to inhibit production of
11-cis-retinoids in animals at very low oral doses (ED50
=
0.27 mg/kg in mice; proprietary data provided by Acucela Inc.).
We injected various doses of ACU-5200 into the yolk sac of the
developing embryo at E17 and then again at E19. The ACU-
5200–injected embryos developed normally and had no obvious
phenotypic abnormalities (Fig. 6B). Injection of two 2-mg doses
(2 × 2 mg) of ACU-5200 resulted in significant down-regulation
of RPE/choroid meso-zeaxanthin levels (Fig. 6C). Doubling this
dose resulted in complete absence of meso-zeaxanthin in the
Fig. 6. ACU-5200 injection inhibits meso-zeaxanthin production in the RPE/
choroid of developing chicken embryos. (A) ACU-5200 is an analog of
emixustat. (B) The development of ACU-5200–injected embryos was compa-
rable to that of control embryos. (C and D) No significant differences in lutein
or zeaxanthin levels were observed in ACU-5200–injected embryos compared
with corresponding control embryos. meso-zeaxanthin levels were either sig-
nificantly down-regulated or completely absent following ACU-5200 injection.
n ≥ 5. Error bars represent SEM. *P < 0.05; ***P < 0.0005.
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RPE65 in a manner that could facilitate the double-bond shift
reaction required to convert lutein to meso-zeaxanthin by a
mechanism involving acid-base catalysis (Fig. 1B) or some other
mechanism. We also found that an RPE65 inhibitor, ACU-5200,
was able to specifically inhibit formation of meso-zeaxanthin
during chicken eye development without affecting lutein or
zeaxanthin uptake into the RPE/choroid. Its close analog,
emixustat, is currently in clinical trials as a visual cycle inhibitor
for various eye diseases (27–29). Our finding of RPE65’s addi-
tional role in macular carotenoid metabolism suggests that it may
be of interest to examine whether this compound detectably al-
ters macular pigment levels or distributions in the participants in
these clinical trials.
Mutations in human RPE65 are quite rare and typically cause
severe visual function deficits, and we suspect that individuals
with deleterious mutations in RPE65 may also have abnormali-
ties in their macular pigment levels and distributions. In-
terestingly, SNPs in human RPE65, along with other carotenoid-
associated genes, such as GSTP1, BCO1, and SCARB1, were
identified as determinants of macular optical density in women
participating in the CAREDS study (33).
The notion that RPE65 is the meso-zeaxanthin isomerase is
appealing, since its carotenoid oxygenase family members BCO1
and BCO2 are known carotenoid cleavage enzymes. In fact,
RPE65’s alternate name is BCO3. These three proteins share
significant sequence homology, and each plays a crucial role in
vertebrate retinoid and carotenoid physiology. BCO1 cleaves
β-carotene at the central 15, 15′ site to produce two molecules of
retinal (18). This newly formed all-trans-retinal undergoes re-
duction and conversion into retinyl esters that are substrates for
RPE65-mediated production of 11-cis-retinol; alternatively, retinal
can be oxidized to retinoic acid, which is used for cell signaling and
gene regulation. BCO2 cleaves a variety of xanthophyll carotenoid
substrates at the 9′, 10′ double bond and is involved in the ho-
meostasis of non-provitamin A carotenoids (17).
In other species, a single enzyme can perform the functions of
BCO1, BCO2, and RPE65. Arthropods encode a single carot-
enoid cleavage enzyme, NinaB, that performs the functions of all
three BCO family members (34). Carotenoid cleavage enzymes
in lower organisms have a range of substrate specificities. ACO
from cyanobacteria is capable of cleaving carotenoids of various
lengths, ranging from C20 to C27. This enzyme binds to substrates
with either aldehydes or alcohols at their terminal ends distal to
the ionone ring, and it also can accept apocarotenoids with or
without 3-hydroxyl groups on the ionone rings (35–37). There-
fore, it is not unprecedented that RPE65, whose known inter-
actions until now have been only with retinoids, may interact
with structurally similar molecules, such as carotenoids.
Our findings show that meso-zeaxanthin production from lutein
occurs in the RPE, and that it is catalyzed by RPE65. The specific
accumulation of this carotenoid in the fovea may be mediated by
specific transporters as well as binding proteins. IRBP and class B
scavenger receptor proteins are capable of shuttling carotenoids to
the retinal layers from the RPE via the interphotoreceptor space
(38, 39). GSTP1 is a known zeaxanthin-binding protein present in
the primate RPE and foveal regions that binds meso-zeaxanthin
with equally high affinity (15). It is plausible to hypothesize that
newly formed meso-zeaxanthin from the RPE is shuttled into the
subretinal space and then into the retinal layers by means of
transport proteins, and that once in the retina, it may be held in
place in the foveal region by specific binding proteins.
In the present study, we have described a novel function of
RPE65 as the lutein to meso-zeaxanthin isomerase. We have
shown that both chicken and human RPE65 are capable of
converting lutein to meso-zeaxanthin. The reaction rate is slow,
and meso-zeaxanthin isomerization is likely a secondary function
of RPE65. The foveal presence of meso-zeaxanthin, especially
given its lack of common dietary sources, has been a conun-
drum in the field of carotenoid biology. With the identification
of RPE65 as the enzyme responsible for the production of
Fig. 7. Model of RPE65 complexed with lutein. A molecule of lutein (car-
bon, gold; oxygen red) is shown docked into a homology model of chicken
RPE65. In this view, much of the protein structure has been cut away to
reveal the substrate tunnel found in the interior. The palmitate-binding
pocket (p) is above and to the left of the iron center, overlapping with
the «-ionone ring of lutein. The polyene chain extends through the pre-
sumed substrate-binding pocket (s), defined by the structure of the enzyme
in complex with its competitive inhibitor emixustat (29). The β-ionone ring
emerges at an opening found on the surface of the enzyme. A more detailed
view of molecular interactions is provided in Fig. S4. Energy minimization
was not used for these coarse-grid rigid-body docking experiments. The
representative result shown here was selected from >33,000 trials because it
has few steric clashes (n = 9; clash defined as interatomic distance less than
3 Å) and makes two hydrogen bonds. Steric conflicts would likely resolve on
subtle adjustments of torsion angles in the lutein molecule and reposition-
ing of sidechains. For comparison, lutein docked into the tunnel-like cavity
of StARD3 with the same rigid-body protocol experiences 14 clashes (42),
ligands palmitate and emixustat bound to RPE65 have two clashes, and lu-
tein molecules found in structures of chlorophyll-binding proteins frequently
have no clashes (42). This figure was prepared with UCSF Chimera (43, 44).
has shown that meso-zeaxanthin has stronger antioxidant prop-
erties than lutein and zeaxanthin (30), and another study has
shown that oral supplementation with all three macular carot-
enoids can improve contrast sensitivity in normal individuals (31,
32). The process by which meso-zeaxanthin is produced in the
eye has been a mystery. In the present study, we show in both
in vitro and in vivo systems that RPE65 is the enzyme that cat-
alyzes the conversion of lutein to meso-zeaxanthin.
After identifying RPE65 as a prime candidate for the meso-
zeaxanthin isomerase in chicken RPE by RNA sequencing
studies, we conducted overexpression experiments using both
chicken and human RPE65 plasmids in a nonocular cell culture
system. Our studies in HEK293T cells show that RPE65 of both
species is capable of producing meso-zeaxanthin from lutein, but
not from zeaxanthin. HEK293T cells do not endogenously ex-
press LRAT, the acyl transferase enzyme essential to provide all-
trans fatty acid ester retinoid substrates for RPE65 to catalyze
their conversion into 11-cis-retinol (12, 13, 25). By over-
expressing RPE65 in a system free of LRAT and treating these
cells with HPLC-purified lutein, we were able to produce meso-
zeaxanthin independent of LRAT’s catalytic activity. The re-
action is slow in cell culture, with no detectable product observed
until several days after the addition of lutein. This is consistent
with the relatively slow formation of meso-zeaxanthin during
chicken eye development, which also takes several days (10).
Our structural modeling studies show that the epsilon ring of
lutein can coordinate with the active site histidines and iron of
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Protein Isolation and Western Blot Analysis. Cell lysis, protein isolation, and
Western blot analysis were done following standard protocols, as described in
SI Materials and Methods.
meso-zeaxanthin, future studies can further delineate the physio-
logical role of this macula-specific carotenoid.
Materials and Methods
Inhibitor Treatment of Chicken Embryos. E17 and the same chicken embryos at
E19 were injected with appropriate concentrations of ACU-5200 (Acucela Inc.)
diluted in Ringer’s solution containing 1% penicillin-streptomycin (SI Mate-
rials and Methods).
Total RNA Isolation and RNA Sequencing. Total RNA was isolated from E16 and
E21 chicken embryos with the Qiagen RNeasy Kit. Intact poly(A) RNA was
purified from total RNA samples (100–500 ng) with oligo(dT) magnetic beads,
and stranded mRNA sequencing libraries were prepared as described using
the Illumina TruSeq Stranded mRNA Library Preparation Kit. Details are
provided in SI Materials and Methods.
Structural Modeling of Lutein Docking into RPE65. Lutein molecules were docked
into a homology model of RPE65 obtained with Phyre2 (40), by threading the
amino acid sequence of CRPE65 into the structure of bovine RPE65 [Protein
Data Bank (PDB) ID codes 4RSC and 3FSN] (29, 41). Additional details are
provided in SI Materials and Methods.
Cell Culture and Transient Transfection. A primary cell culture of E21 chicken RPE
was established, and HEK293T cells were transiently transfected with RPE65 or
GFP for overexpression experiments, as described in SI Materials and Methods.
Carotenoid Treatment. HPLC-purified carotenoid stocks were prepared.
Tween 40 was added to the dried stocks before the addition of medium. Cells
were treated with the carotenoid-containing medium for 0, 1, 2, or 4 d. More
details are provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Acucela Inc. for generously providing the
RPE65 inhibitor ACU-5200, Kemin Health for supplying HPLC-purified lutein,
Dr. Jian-Xing Ma and Dr. Yusuke Takahashi (University of Oklahoma) for
providing RPE65 antibodies and plasmids, and Dr. Wolfgang Baehr (Univer-
sity of Utah) for providing feedback on the manuscript. This work was sup-
ported by National Institutes of Health Grants EY11600 and EY14800 (to
P.S.B.) and Ruth L. Kirschstein Training Grant T32EY024234 (to R.S.), and
an unrestricted departmental grant from Research to Prevent Blindness.
Carotenoid Extraction and HPLC Analysis. Carotenoid extraction and chiral HPLC
analyses were carried out as described previously (10) and in SI Materials
and Methods.
1. Maoka T, Arai A, Shimizu M, Matsuno T (1986) The first isolation of enantiomeric and
meso-zeaxanthin in nature. Comp Biochem Physiol B 83:121–124.
2. Bernstein PS, et al. (2016) Lutein, zeaxanthin, and meso-zeaxanthin: The basic and
clinical science underlying carotenoid-based nutritional interventions against ocular
disease. Prog Retin Eye Res 50:34–66.
3. Li B, Vachali P, Bernstein PS (2010) Human ocular carotenoid-binding proteins.
Photochem Photobiol Sci 9:1418–1425.
4. Katsuyama M, Komori T, Matsuno T (1987) Metabolism of three stereoisomers of
astaxanthin in the fish, rainbow trout and tilapia. Comp Biochem Physiol B 86:1–5.
5. Nolan JM, et al. (2014) Verification of meso-zeaxanthin in fish. J food Process Technol
5:335.
6. Chew EY, et al.; Age-Related Eye Disease Study 2 (AREDS2) Research Group (2014)
Secondary analyses of the effects of lutein/zeaxanthin on age-related macular de-
generation progression: AREDS2 report no. 3. JAMA Ophthalmol 132:142–149.
7. Bhosale P, Serban B, Zhao DY, Bernstein PS (2007) Identification and metabolic
transformations of carotenoids in ocular tissues of the Japanese quail Coturnix ja-
ponica. Biochemistry 46:9050–9057.
8. Johnson EJ, Neuringer M, Russell RM, Schalch W, Snodderly DM (2005) Nutritional
manipulation of primate retinas, III: Effects of lutein or zeaxanthin supplementation
on adipose tissue and retina of xanthophyll-free monkeys. Invest Ophthalmol Vis Sci
46:692–702.
9. Bone RA, Landrum JT, Hime GW, Cains A, Zamor J (1993) Stereochemistry of the
human macular carotenoids. Invest Ophthalmol Vis Sci 34:2033–2040.
10. Gorusupudi A, et al. (2016) Developmentally regulated production of meso-zeax-
anthin in chicken retinal pigment epithelium/choroid and retina. Invest Ophthalmol
Vis Sci 57:1853–1861.
23. Borel P, et al. (2013) CD36 and SR-BI are involved in cellular uptake of provitamin A
carotenoids by Caco-2 and HEK cells, and some of their genetic variants are associated
with plasma concentrations of these micronutrients in humans. J Nutr 143:448–456.
24. Enright JM, et al. (2015) Cyp27c1 red-shifts the spectral sensitivity of photoreceptors
by converting vitamin A1 into A2. Curr Biol 25:3048–3057.
25. Moiseyev G, Takahashi Y, Chen Y, Kim S, Ma J-X (2008) RPE65 from cone-dominant
chicken is a more efficient isomerohydrolase compared with that from rod-dominant
species. J Biol Chem 283:8110–8117.
26. Hamel CP, et al. (1993) Molecular cloning and expression of RPE65, a novel retinal
pigment epithelium-specific microsomal protein that is post-transcriptionally regu-
lated in vitro. J Biol Chem 268:15751–15757.
27. Dugel PU, et al. (2015) Phase II randomized, placebo-controlled, 90-day study of
emixustat hydrochloride in geographic atrophy associated with dry age-related
macular degeneration. Retina 35:1173–1183.
28. Jack LS, Sadiq MA, Do DV, Nguyen QD (2016) Emixustat and lampalizumab: Potential
therapeutic options for geographic atrophy. Dev Ophthalmol 55:302–309.
29. Kiser PD, et al. (2015) Catalytic mechanism of a retinoid isomerase essential for ver-
tebrate vision. Nat Chem Biol 11:409–415.
30. Li B, Ahmed F, Bernstein PS (2010) Studies on the singlet oxygen scavenging mech-
anism of human macular pigment. Arch Biochem Biophys 504:56–60.
31. Crosby-Nwaobi R, Hykin P, Peto T, Sivaprasad S (2016) An exploratory study evalu-
ating the effects of macular carotenoid supplementation in various retinal diseases.
Clin Ophthalmol 10:835–844.
32. Nolan JM, et al. (2016) Enrichment of macular pigment enhances contrast sensitivity
in subjects free of retinal disease: Central retinal enrichment supplementation trials.
Report 1. Invest Ophthalmol Vis Sci 57:3429–3439.
33. McKay GJ, et al. (2013) Investigation of genetic variation in scavenger receptor class B,
member 1 (SCARB1) and association with serum carotenoids. Ophthalmology 120:
1632–1640.
11. Bernstein PS, Law WC, Rando RR (1987) Isomerization of all-trans-retinoids to 11-cis-
retinoids in vitro. Proc Natl Acad Sci USA 84:1849–1853.
34. Babino D, et al. (2016) The biochemical basis of vitamin A3 production in arthropod
vision. ACS Chem Biol 11:1049–1057.
35. Kloer DP, Schulz GE (2006) Structural and biological aspects of carotenoid cleavage.
Cell Mol Life Sci 63:2291–2303.
12. Moiseyev G, Chen Y, Takahashi Y, Wu BX, Ma J-X (2005) RPE65 is the isomer-
ohydrolase in the retinoid visual cycle. Proc Natl Acad Sci USA 102:12413–12418.
13. Jin M, Li S, Moghrabi WN, Sun H, Travis GH (2005) Rpe65 is the retinoid isomerase in
bovine retinal pigment epithelium. Cell 122:449–459.
36. Kloer DP, Ruch S, Al-Babili S, Beyer P, Schulz GE (2005) The structure of a retinal-
forming carotenoid oxygenase. Science 308:267–269.
37. Sui X, Kiser PD, Lintig Jv, Palczewski K (2013) Structural basis of carotenoid cleavage:
From bacteria to mammals. Arch Biochem Biophys 539:203–213.
14. Redmond TM, et al. (2005) Mutation of key residues of RPE65 abolishes its enzymatic
role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci USA 102:13658–13663.
15. Bhosale P, et al. (2004) Identification and characterization of a Pi isoform of gluta-
thione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the
human eye. J Biol Chem 279:49447–49454.
16. Li B, Vachali P, Frederick JM, Bernstein PS (2011) Identification of StARD3 as a lutein-
binding protein in the macula of the primate retina. Biochemistry 50:2541–2549.
17. Hu K-Q, et al. (2006) The biochemical characterization of ferret carotene-9′,10′-
monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo. J Biol Chem
281:19327–19338.
18. Kiefer C, et al. (2001) Identification and characterization of a mammalian enzyme
catalyzing the asymmetric oxidative cleavage of provitamin A. J Biol Chem 276:
14110–14116.
19. Wyss A, et al. (2001) Expression pattern and localization of β,β-carotene 15,15′-di-
oxygenase in different tissues. Biochem J 354:521–529.
38. Vachali PP, Besch BM, Gonzalez-Fernandez F, Bernstein PS (2013) Carotenoids as
possible interphotoreceptor retinoid-binding protein (IRBP) ligands: A surface plas-
mon resonance (SPR)-based study. Arch Biochem Biophys 539:181–186.
39. Voolstra O, et al. (2006) The Drosophila class B scavenger receptor NinaD-I is a cell
surface receptor mediating carotenoid transport for visual chromophore synthesis.
Biochemistry 45:13429–13437.
40. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web
portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858.
41. Kiser PD, Golczak M, Lodowski DT, Chance MR, Palczewski K (2009) Crystal structure
of native RPE65, the retinoid isomerase of the visual cycle. Proc Natl Acad Sci USA 106:
17325–17330.
42. Horvath MP, et al. (2016) Structure of the lutein-binding domain of human StARD3 at
1.74 Å resolution and model of a complex with lutein. Acta Crystallogr F Struct Biol
Commun 72:609–618.
43. Pettersen EF, et al. (2004) UCSF Chimera: A visualization system for exploratory re-
search and analysis. J Comput Chem 25:1605–1612.
20. Redmond TM, et al. (2001) Identification, expression, and substrate specificity of a
mammalian β-carotene 15,15′-dioxygenase. J Biol Chem 276:6560–6565.
21. Kawaguchi R, et al. (2007) A membrane receptor for retinol binding protein mediates
cellular uptake of vitamin A. Science 315:820–825.
22. Kaylor JJ, et al. (2013) Identification of DES1 as a vitamin A isomerase in Müller glial
cells of the retina. Nat Chem Biol 9:30–36.
44. Huang CC, Meng EC, Morris JH, Pettersen EF, Ferrin TE (2014) Enhancing UCSF Chi-
mera through web services. Nucleic Acids Res 42:W478–W484.
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Shyam et al.