1
Introduction
As
succinctly summarized
by Wolf,
1
lack
of vitamin A (all-trans-retinol) was recognized by
ancient Egyptians as causing a visual deficiency involving the retina
and cornea that could be cured by eating liver. One of the symptoms
of vitamin A deficiency is night blindness or nyctalopia (from Greek
νύκτ-, nykt – night; and αλαός,
alaos – blindness), recognized by ancient Greeks, including
Hippocrates, as affecting the retina.
2
In
1913 McCollum showed that “fat-soluble factor A” was
essential for growth of a rat colony (reviewed in ref (3)). The treatment of factor
A-deficiency included liver or liver extracts, but later in 1930 Moore
found that yellow pigment (carotene) was a good substitute for this
therapy.
4
A major breakthrough occurred
in 1931 when the chemical structures for β,β-carotene
and retinol (its all-trans isomer now known as vitamin
A) were determined by Karrer and colleagues.
5
But, it was Wald who discovered that retinol derivatives constitute
the chemical basis of our vision,
6
a contribution
subsequently recognized by a Nobel Prize award in 1967. In 1950–1960,
a variety of vitamin A metabolic transformations, including oxidation/reduction
and esterification, were elucidated by Olson,
7−20
Goodman,
21−32
Chytil and Ong,
33−41
and Norum and Blomhoff.
42−57
The discovery that one set of these metabolites, namely retinoic
acids, plays a key role in the nuclear regulation of a large number
of genes added a notable dimension to our knowledge of gene expression.
This mechanism is also a critical player in the successful healing
of corneal wounds,
58
a second manifestation
of vitamin A-deficiency recognized earlier.
Further progress
in understanding the multiple physiological roles
of retinoids has been made in recent years, due mainly to the successful
application of modern scientific technology. Examples include enzymology
combined with structural biology, in vivo imaging
based on retinoid fluorescence, improvements in analytical methods,
generation and testing of animal models of human diseases with specific
pathogenic genetics, genetic analysis of human conditions related
to changes in vitamin A metabolism, and pharmacological approaches
to combat these diseases.
In this review we focus on the involvement
of retinoids in supporting
vision via light-sensitive rod and cone photoreceptor
cells in the retina. We begin with a brief description of isopentenyl
diphosphate (IPP) biosynthesis, which is essential for carotenoid
(C40 isoprenoid) production. Certain of these colored compounds, such
as lutein, are deposited in our retina’s macula, appearing
as a “yellow” spot. Other carotenoids containing at
least one unmodified β-ionone ring (represented by β,β-carotene
and cryptoxanthin) serve as precursors of all-trans-retinal. Many different compounds
can be generated from this monocyclic
diterpenoid, which contains a β-ionone ring and polyene chain
with a C15 aldehyde group. Among the numerous enzymatic activities
that contribute to retinoid metabolism, polyene trans/cis isomerization is a particularly
fascinating
reaction that occurs in specialized structures of the retina based
on a two cell system comprised of retinal photoreceptor cells and
the retinal pigment epithelium (RPE). A specific enzyme system, called
the retinoid (visual) cycle, has evolved to accomplish retinoid isomerization
that is required for visual function in vertebrates. Individual enzymes
of this pathway harbor secrets about the molecular mechanisms of this
chemical transformation. Malfunctions of these processes or other
pathological reactions often precipitate severe retinal pathologies.
This review attempts to balance contributions that have been published
over the past decades and does not intend to replace the views of
investigators with different perspectives of retinoid chemistry in
the eye.
59−85
2
Chemistry of Isoprenoids
In animals, carotenoids
and retinoids must be acquired through
the diet, as they cannot be synthesized de novo.
These compounds are involved in critical functions of many organs
in addition to their vital involvement in vision.
86
2.1
Mevalonate and Nonmevalonate Pathways
Naturally occurring carotenoids are all synthesized from two basic
5-carbon precursors: isopentenyl diphosphate (IPP) and its isomer
dimethylallyl diphosphate (DMAPP).
87
A
multitude of additional compounds, including steroids, ubiquinones,
and chlorophylls, are also synthesized from these isoprene precursors
and are thus collectively referred to as isoprenoids or terpenoids.
88,89
Until the early 1990s it was believed that isoprenoid precursors
were synthesized exclusively through the mevalonate pathway, a series
of enzymatically catalyzed reactions in which three molecules of acetate,
in the form of acetyl-coenzyme A (acetyl-CoA), are condensed and modified
by reduction, phosphorylation, and decarboxylation to generate IPP
89
(Figure 1). Research
in eubacteria and plants then revealed a second metabolic route for
IPP synthesis referred to as the methylerythritol 4-phosphate (MEP)
or nonmevalonate pathway, which utilizes the triose derivative, d-glyceraldehyde 3-phosphate
along with pyruvate as starting
materials
90−94
(Figure 2). Following condensation of these
two molecules, a methyl isomerization reaction occurs that converts
the initially linear carbon chain into an isopentyl linkage.
95,96
Subsequent reduction, CDP transfer, phosphorylation, and reduction
serve to eliminate hydroxyl functional groups and introduce a diphosphate
group to generate IPP as well as DMAPP in about a five to one ratio.
97
In many cases, a particular organism encodes
the enzymes for only one of these two metabolic pathways in its genome.
90
Metazoans, fungi, and archaea rely exclusively
on the mevalonate pathway for isoprenoid biosynthesis, whereas cyanobacteria
and algae make sole use of the MEP pathway.
98
Depending on the species, eubacteria and protozoa can employ either
of the two pathways in a mutually exclusive fashion.
99
However, most eubacteria utilize the MEP pathway.
99
In higher plants, most cytosolic IPP and DMAPP
are derived from the mevalonate pathway via enzymes
encoded by genomic DNA. By contrast, these isoprene compounds are
synthesized through the MEP pathway in plastids of plants as a consequence
of the evolutionary relationship of plastids to cyanobacteria.
100
However, mixing of cytosolic and plastid isoprenoid
precursors has been reported.
101,102
Most carotenoids consumed
by humans are synthesized from MEP-derived IPP and DMAPP.
Figure 1
Mevalonate
pathway for the synthesis of IPP. Two molecules of acetyl-CoA
are joined together to form acetoacetyl-CoA (i) in a
reaction catalyzed by thiolase with the release of free CoA (CoASH).
A third molecule of acetyl-CoA is added by 3-hydroxy-3-methylglutaryl
(HMG)-CoA synthase to form HMG-CoA (ii). Compound ii is reduced by HMG-CoA reductase
in an NADPH-dependent manner
to form (R)-mevalonate (iii), which
is the rate-limiting step of the pathway. Compound iii is sequentially phosphorylated
by mevalonate kinase and phosphomevalonate
kinase to form (R)-mevalonate-5-diphosphate (iv). Finally, iv is decarboxylated by
mevalonate-5-diphosphate
decarboxylase in an ATP-dependent manner to form IPP (v). Coloring of oxygen atoms
is intended to assist in tracking of
the chemical origin of the carbon skeleton. P, phosphoryl group; OPi, inorganic phosphate.
Figure 2
MEP (nonmevalonate) pathway for the synthesis of IPP and DMAPP.
First, d-glyceraldehyde-3-phosphate (i) is condensed
with pyruvate to form 1-deoxy-d-xylulose-5-phosphate (DOXP,
ii) catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase
(DXS) using a thiamine diphosphate cofactor with the loss of CO2. ii is isomerized
and reduced by DOXP-isomeroreductase
(IspC) in an NADPH-dependent manner to form 2C-methyl-d-erythritol-4-phosphate, which
is then conjugated with CTP
to form 4-diphosphocytidyl-2C-methyl d-erythritol
(iv) in a reaction catalyzed by 2C-methyl-d-erythritol cytidylyltransferase (IspD).
iv is
then phosphorylated to form 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate
(v) by 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE). v is cyclized with
the loss of CMP to form 2C-methyl-d-erythritol-2,4-cyclodiphosphate (vi) by 2C-methyl-d-erythritol-2,4-cyclodiphosphate
synthase
(IspF). vi is then reduced with cleavage of the cyclodiphosphate
moiety to form 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate
(vii) by the iron-sulfur enzyme IspG using ferrodoxin
as a cofactor. Finally, vii is further reduced by a second
iron–sulfur protein IspH, giving a mixture of IPP (viii) and dimethylallyl diphosphate
(DMAPP, ix). The red
colored oxygen atom is intended to assist in tracking of the chemical
origin of the carbon skeleton. P, phosphoryl group; PPi, inorganic pyrophosphate.
IPP and DMAPP differ only in the position of an alkene double
bond
and are thus classified as geometric isomers (Figure 3). Enzymes known as IPP isomerases
(IDIs) catalyze the reversible
isomerization of IPP into DMAPP. For organisms that solely use the
mevalonate pathway for isoprene biosynthesis, IDIs are essential for
production of DMAPP because only IPP is generated through this pathway.
Although both IPP and DMAPP are produced via the
MEP pathway, IDIs are still required to generate the proper IPP/DMAPP
ratio for isoprenoid biosynthesis.
103,104
Two structurally
unrelated enzymes, called IDI type 1 and IDI type 2, can each carry
out the reaction.
105
Type 1 IDIs were the
first to be characterized and rely on an active site Cys residue as
well as divalent cations for their catalytic function.
106
Biochemical and crystallographic analyses of
type I IDI indicate that an active site, metal-binding Glu residue
transfers a proton to the alkene π bond of IPP, generating a
tertiary carbocation or carbocation-like intermediate.
107,108
Abstraction of a proton from C2 of the isoprene skeleton by an active
site Cys residue regenerates the alkene forming DMAPP.
109,110
Isotope labeling studies demonstrated that the proton transposition
occurs in an antarafacial manner, a mechanism consistent with the
placement of catalytic groups in the active site of type I IDIs.
111
Type II IDIs are a more recently discovered
class of enzymes that catalyze the same reaction as type I IDIs but
are structurally and evolutionarily unrelated.
105,106
In contrast to type I IDIs, type II enzymes rely on flavin mononucleotide
(FMNred) and NADPH as well as divalent cations as cofactors
to perform catalysis.
105
The dependence
on these nucleotide cofactors was somewhat surprising given that IPP
to DMAPP isomerization does not involve net redox changes. Free radical
mechanisms involving transient abstraction of a hydrogen atom
112
as well as protonation–deprotonation
(carbocation intermediate) mechanisms
113,114
have been
proposed as a means to effect the double bond shift. The involvement
of carbocation intermediates turns out to be a major theme in the
enzymatic isomerization of isoprenoids.
Figure 3
Reversible isomerization
of IPP (i) into DMAPP (ii). The reaction,
catalyzed by IPP/DMAPP isomerase, is thought
to proceed via a protonated carbocationic intermediate
shown in brackets.
2.2
Carotenoid
Biosynthesis
The biosynthesis
of carotenoids from IPP and DMAPP begins with the condensation of
one molecule of DMAPP with three molecules of IPP catalyzed by the
enzyme geranylgeranyl diphosphate synthase to form geranylgeranyl
diphosphate (GGPP) (Figure 4).
87
In some plant species, geranyl diphosphate is synthesized
first by the enzyme geranyl diphosphate synthase and then elongated
by the addition of two IPP molecules in a GGPP synthase-catalyzed
reaction. These enzymes belong to the prenyl transferase family, which
use divalent cations, such as Mg2+ or Mn2+,
to carry out the condensation of isoprenoid precursors.
115
Mechanistically, the divalent cation polarizes
the diphosphate moiety of DMAPP to facilitate its dissociation with
consequent formation of an electrophilic carbocation intermediate.
116,117
Proper positioning of IPP in the active site facilitates a nucleophilic
attack of the alkene π bond electrons on C1 of the isoprene
carbocation, resulting in formation of a new carbon–carbon
single bond. Deprotonation of the diphosphate-containing unit results
in C1–C2 π bond formation, allowing chain elongation
to continue in the presence of appropriate enzymes. Two molecules
of GGPP are then joined in a head to head fashion to form phytoene
in a reaction catalyzed by the enzyme phytoene synthase, which is
the first committed step in the synthesis of carotenoids.
100
This reaction again features a carbocation
or carbocation-like intermediate that reacts with a second GGPP to
from a cyclopropylcarbonyl diphosphate compound (prephytoene diphosphate).
118−120
This compound breaks down with loss of pyrophosphate and a proton
to produce 15-cis-phytoene.
121
The colorless, C40 tetraterpenoid product is then subjected
to a series of desaturation and isomerization reactions that culminate
in the production of all-trans-lycopene, the immediate
precursor of β,β-carotene. In bacteria, a single enzyme
called carotene desaturase (CrtI) is responsible for conversion of
phytoene into lycopene. In plants, two desaturase enzymes called phytoene
desaturase (PDS) and ξ-carotene desaturase (ZDS)
122
and two isomerases called ξ-carotene
isomerase
123
and carotenoid isomerase (CrtIso)
124
together convert phytoene into lycopene.
87
Interestingly, CrtIso shares significant sequence
homology with the carotene desaturase (CrtI) as well as a mammalian
enzyme known as retinol desaturase (RetSat).
63,125
Lycopene, a compound with a red hue conferred by a set of 11 conjugated
double bonds, is transformed into β,β-carotene by an enzyme
known as lycopene-β-cyclase.
126,127
Although
not a reaction that involves net redox changes, the cyclization performed
by lycopene-β-cyclase is dependent on an NAPDH cofactor.
128
In keeping with the general theme of terpenoid
isomerization, lycopene cyclization reactions also occur through carbocation
intermediates.
129
Figure 4
The carotenoid branch
of isoprenoid biosynthesis. Synthesis of
β,β-carotene begins with the sequential condensation of
a single DMAPP molecule (i) with three IPP molecules
to form C20 geranylgeranyl diphosphate (ii, GGPP) catalyzed by GGPP synthase (CrtE).
Next, two GGPP molecules
are combined in a head-to-head fashion to form C40 15-cis-phytoene (iii, shown in
the all-trans configuration for ease of presentation) in a reaction catalyzed
by phytoene synthase (CrtB), which is the first committed step in
carotenoid biosynthesis. In bacteria, phytoene is converted to all-trans-lycopene
(iv) by a series of desaturation
and isomerization steps catalyzed by CrtI. In plants this conversion
is catalyzed by phytoene desaturase and ζ-carotene desaturase
together with the isomerases ζ-carotene isomerase and CrtIso.
Finally, lycopene is converted to β,β-carotene (v) in two steps by lycopene β-cyclase.
Lycopene is also
a substrate for lycopene ε-cyclase, which catalyzes formation
of δ-carotene (not shown).
2.3
Retinoid Metabolism in Vertebrates
Mammals efficiently utilize both preformed vitamin A in the form
of all-trans-retinyl esters and pro-vitamin A carotenoids
(mainly β,β-carotene) to sustain the body pool of vitamin
A.
130
Unlike retinyl esters, which are
hydrolyzed to retinol in the small intestine followed by rapid absorption
by enterocytes, uptake of carotenoids is mediated and regulated by
scavenger receptor class-B type-I (SR-BI).
131,132
Upon absorption by enterocytes, β,β-carotene undergoes
oxidative cleavage catalyzed by β,β-carotene 15,15′-monooxygenase
(BCMO1).
133,134
This symmetric split of the
carotenoid produces two molecules of all-trans-retinal
that subsequently enter vitamin A metabolic pathways (Figure 5).
Figure 5
Retinoid metabolism in vertebrates. Dietary all-trans-β,β-carotene (i), obtained primarily
from
plants, is oxidatively cleaved in a symmetric manner by β-carotene
monooxygenase I (BCMO I), yielding two molecules of all-trans-retinal (ii). Retinal
can reversibly combine with an
amino group to form a retinyl imine (Schiff base) (iv). Retinal is also subject to
oxidation and reduction to form retinoic
acid (iii) and retinol (vitamin A) v, respectively,
the latter in a physiologically reversible manner. Retinoic acid can
be converted into several conjugated and/or oxidized derivatives,
some of which exert biological effects. Retinol also can be converted
into several derivatives including retro-retinoids, saturated retinols,
and phosphate conjugates. Retinol is also reversibly esterified to
produce retinyl esters (vi), the main storage form of
vitamin A in the body.
The diverse functions of retinoids are carried out by a few
physiologically
active metabolites of all-trans-retinol that are
produced by enzymatic modification of the functional groups of this
vitamin and geometric isomerization of its polyene chain. Oxidation
of all-trans-retinal catalyzed by retinaldehyde dehydrogenases
(RALDHs) leads to formation of all-trans-retinoic
acid, a ligand for nuclear retinoic acid receptors (RARs) that, coupled
with retinoid X receptors (RXRs), bind to retinoic acid response elements
on the promoter region of target genes and regulate their transcription.
135,136
Because all-trans-retinoic acid formation is irreversible,
an excess of this active retinoid can be cleared only by its further
conversion to more polar metabolites through oxidation by cytochrome
P450 (CYP26) and/or glucuronidation by UDP-glucuronosyl transferases
(UGTs).
137,138
An alternative metabolic pathway of all-trans-retinal leads to formation of 11-cis-retinal,
a visual chromophore that couples to rod and cone opsins
to form photosensitive pigments.
139−141
The thermodynamically
unfavorable isomerization of the 11–12 double bond does not
occur at the aldehyde level in vivo.
142
Instead, all-trans-retinal
is first reduced to all-trans-retinol by short-chain
dehydrogenase/reductase (SDR) or alcohol dehydrogenase enzymes.
136,143,144
Subsequent esterification of all-trans-retinol, mainly by lecithin/retinol acyltransferase
(LRAT), provides both a major storage form of retinoids in the body
and a direct substrate for RPE65-dependent enzymatic isomerization
of the retinoid polyene chain.
145−148
Multiple additional endogenous retinoid
metabolites are derived from all-trans-retinol. These
include all-trans-13,14-dihydroretinol produced by
saturation of the double bond by RetSat. Other examples are retro-retinoids,
such as anhydroretinol and 14-hydroxy-4,14-retro-retinol. Though the
molecular identities of enzymes involved in the production of retro-retinoids
in vertebrates are currently unknown, these metabolites have been
shown to regulate lymphocyte proliferation.
149−151
Transfer of a phospho group onto all-trans-retinol
results in formation of retinyl monophosphate that has been detected
in the liver of rodents.
152,153
Glycophospholipids
consisting of a retinol moiety linked by a phosphodiester bond to
mannose or galactose were postulated to function in the transfer of
sugar units onto proteins to form some glycoproteins.
154
3
Isomerization of Retinoids
Retinoids are reactive compounds that readily isomerize. Here we
take a close look at their important chemical transformations.
3.1
Geometric Isomers of Retinoids
Progress
in understanding vitamin A metabolism would not be possible without
development of adequate analytical methods that allow separation,
detection, and quantification of retinoids. Such methods have gradually
advanced since the discovery of vitamin A about 100 years ago. A complication
is that retinoids exist in several geometrical configurations with
differently modified functional groups (Figure 6A). Lipophilic compounds soluble in
organic solvents, including retinol
and its esters, were initially separated by thin-layer chromatography
on alumina- and silica-based stationary phases.
155,156
However, introduction of modern high performance liquid chromatography
(HPLC) techniques in the early 1970s together with standardized commercially
available compact stationary phase columns enabled precise, reproducible
analyses of vitamin A metabolites and, most importantly, determination
of their isomeric composition.
157−160
Today retinoids can be separated under numerous
chromatographic conditions (summarized in refs (160 and 161)) optimized for
normal and reverse-phase columns. Selection of the most appropriate
methodology depends on the chemical properties of the particular retinoid
as well as the source of the biological sample. The eye exhibits an
especially complex retinoid composition. The highest resolution method
for separating retinyl esters, retinal, and retinol as well as their
isomers present in the eye is normal phase HPLC (Figure 6B).
161
The strength of an analyte’s
interaction with the silica stationary phase depends not only on its
functional groups but also on steric factors and the structure of
the molecule. This feature is especially important for separation
of molecules that are chemically similar but physically different,
e.g. geometric isomers. Moreover, this methodology provides unique
flexibility in tuning chromatographic conditions by adjusting the
polarity of the mobile phase, routinely composed of hexane and ethyl
acetate. Highly hydrophobic hexane simplifies the tissue homogenate
extraction procedure and greatly reduces sample complexity without
sacrificing overall analytical performance.
162
Figure 6
HPLC-based
separation and detection of retinoids. (A) The main
classes of retinoid isomers commonly found in experimental samples
that can be distinguished by analytical methods. (B) Elution profiles
of retinol, retinal, and retinal oxime isomers from a normal phase
HPLC column with an isocratic flow of 10% ethyl acetate/hexane. Primary
analytical methods of retinoid identification and quantification include
UV/vis spectroscopy and mass spectrometry. (C) UV/vis absorbance spectra
of selected retinoids reveal characteristic differences in absorbance
maxima and overall shape of the spectra that are used to classify
the chemical and geometric form of retinoids. (D) Electrospray ionization
of retinol and retinyl esters triggers water or carboxylate dissociation,
resulting in the predominant parent ion of m/z = 269 [M – 17]+, whereas retinal exhibits
the expected molecular ion of m/z = 285 [M + H]+. Characteristic MS/MS fragmentation
patterns
of the parent ions are shown in the bottom panels. The typical m/z = 161 fragment
of retinal in MS/MS
spectra is indicative of ionone ring loss from the parent ion.
Retinoid detection, identification,
and quantification are simplified
by their characteristic spectral properties (Figure 6C and D). The conjugated polyene
chain contributes to relatively
strong absorption at ultraviolet (UV) and visible (Vis) wavelengths.
Thus, absorbance maxima as well as the overall shape of the spectra
provide valuable information about the number of conjugated double
bonds and allow identification of the isomeric states of the compound.
UV/Vis detection offers a limit of retinoid quantification at a low
picomolar range through most photodiode array detectors and provides
excellent linearity over a wide range of concentrations (2–1500
pmol) (Figure 6C).
163
A complementary analytical method allowing precise molecular identification
and quantification of retinoids is mass spectrometry (MS). The greatest
advantage of MS coupled to HPLC is sensitivity in the low femtomolar
range. Moreover, modern tandem mass spectrometry offers definitive
molecular identification based on the induced fragmentation pattern
of the precursor ion (Figure 6D).
3.2
Retro- and anhydro-retinols
In the
1920s two groups
164,165
made the discovery that vitamin
A-containing solutions, in the presence of Brønsted and Lewis
acids, undergo a color change from pale yellow (λmax ∼ 325 nm) to brilliant blue (λmax
∼
600 nm). The blue color is semistable with an effective lifetime of
up to ∼3 min when chloroform is used as a solvent.
166
This color change, when initiated using antimony
trichloride as the Lewis acid, is known as the Carr–Price reaction
and was a primary means of retinol detection and quantification prior
to the advent of chromatographic methods.
167
Investigation into the mechanism of the reaction underlying the
color change revealed that the hydroxyl moiety of retinol readily
combines with acids (e.g., protons or antimony), converting it into
a good leaving group.
166
Hydroxyl dissociation
generates a short-lived retinylic cation that can undergo proton elimination
to form anhydroretinol (Figure 7). Anhydroretinol
can then undergo further chemical reactions to generate relatively
long-lived, blue-color species.
168
In addition
to anhydroretinol, direct protonation of the 13,14 retinol double
bond followed by proton elimination at C4 leads to formation retro-retinol
(Figure 7). Generation of the retinylic cation
can also be accomplished by flash photolysis of retinyl acetate via heterolytic carbon–oxygen
bond dissociation
169
or by reactions of protons, released by radiolytic
pulses, with retinol or retinyl acetate.
170
The special location of the retinol hydroxyl group with respect
to the conjugated polyene chain makes it a much better leaving group
than is typical for regular aliphatic alcohols. Upon dissociation,
the resulting retinylic cation is stabilized by extensive delocalization
along the polyene chain with a half-life on the nanosecond time scale.
169,171
Hydroxyl dissociation can also be facilitated by its conjugation
with an electron-withdrawing group, for example a sulfo moiety, as
occurs catalytically via the enzyme retinol dehydratase.
This member of the sulfotransferase family is responsible for the
catalytic conversion of retinol into anhydroretinol.
172
In this reaction, a sulfo moiety is first transferred from
3′-phosphoadenosyl-5′-phosphosulfate (PAPS) onto the
retinol hydroxyl group to produce retinyl sulfate. Sulfate then readily
dissociates, generating a resonance-stabilized carbocation that is
quenched by proton loss from carbon 4 of the ionone ring to yield
anhydroretinol
172
(Figure 8).
Figure 7
Conversion of all-trans-retinol (i) into anhydroretinol (ii) and retro-retinol (iii)
in the presence of acid via carbocationic
intermediates.
Figure 8
Production of anhydroretinol
from all-trans-retinol
catalyzed by retinol dehydratase. First, a sulfo group is transferred
to all-trans-retinol (i) to form retinyl
sulfate (ii). Loss of sulfate then generates a carbocationic
intermediate (iii) that, in the confines of the enzyme
active site, preferentially rearranges with loss of a proton to form
anhydroretinol (iv).
3.3
Chemical/photochemical isomerization of retinoids
Spectroscopic properties of retinoids can be directly related to
their conjugated double bond system. Mobile π electrons of polyenes
are delocalized over the entire molecule, resulting in resonance stabilization
of the compound.
173
Thus, polyene single
bonds display some double-bond characteristics contributing to their
preferred planar conformation. The most stable conformation for retinoids
is all-trans.
174,175
Alternative geometrical
isomers introduce some steric hindrance that increases the conformational
energy compared to the all-trans isomer.
176
Although some retinal isomers introduce only
mild steric clashes, e.g. between 12H and 15H in 13-cis or between 8H and 11H in the
9-cis conformation,
other isomers such as 11-cis and 7-cis constitute examples of severely hindered isoprenoids
in which the
planar conformation of the polyene chain cannot be sustained.
174
To eliminate the strain, a twist is introduced
that further contributes to the increased conformational energy of
the retinoid.
177
Cis/trans isomerization of retinals does not occur efficiently in the dark
at room temperature. However, an equilibrium between retinoid isomers
can be introduced chemically or photochemically (Figure 9). The radical-mediated,
iodine-catalyzed isomerization of
polyenes is a three-step process that involves binding of an iodine
atom to a carbon, an internal rotation, and then detachment of the
iodine.
178,179
Because iodine atoms are easily formed thermally
from I2, retinoid isomerization can be studied independently
of light. Starting from all-trans-retinal, the composition
of retinal isomers at equilibrium was consistent among multiple reports
that included all-trans, 13-cis,
9-cis, 11-cis, and 9,13-di-cis isomers in ratios of about 0.62, 0.23, 0.11, 0.001,
and 0.04, respectively.
177,180,181
The same results were obtained when all-trans-retinol
or its palmitoyl ester was used as the starting material.
181
Interestingly, kinetic studies revealed that
the 9,13-di-cis isomer was a favored intermediate
in the isomerization of 9-cis-retinal, whereas 13-cis-retinal was directly converted
into the all-trans conformation without the contribution of any other isomeric intermediates.
181
Consequently, different isomers lead to a diverse
retinoid composition in the equilibrium state. Nevertheless, 11-cis-retinal was never
preferably formed upon I2 or acid-catalyzed isomerization. In contrast, early work
by Wald
indicated that photolysis of dilute ethanolic solutions of all-trans-retinal resulted
in the formation of about 50% cis-retinals, with an exceptionally high contribution
of
11-cis-retinal representing 25% of the mixture.
182−184
Since then, quantum yields for trans/cis and cis/trans photoisomerization upon light
excitation of retinal
have been studied under a variety of conditions demonstrating both
isomer and solvent dependence. The quantum yields for all-trans-retinal were considerably
lower in polar (0.1–0.2) than in
nonpolar (0.4–0.7) solvents.
185−189
Figure 9
Generation of retinoid isomers from all-trans-retinal.
The all-trans form of retinoids is the lowest in
free energy and thus predominates at equilibrium. Formation of retinoid
isomers can be facilitated chemically by treatment with I2, sulfhydryls, and trifluoroacetic
acid, or by exposure to light.
The composition of retinal isomers found at equilibrium is reported
after Rando et al.
181
and Deval et al.
192
The influence of solvents on the efficiency and composition
of
isomers at photoequilibrium has been attributed to solvent-dependent
shifting of the nπ* and ππ* excited states.
190−192
For example, in hexane light illumination led to almost exclusive
formation of the 13-cis isomer whereas increased
solvent polarity contributed to efficient production of 11-cis, 9-cis, and 7-cis-retinals
in 0.19, 0.06, and 0.005 ratios, respectively.
192,193
Many studies were dedicated to elucidate the mechanism(s) of photoisomerization,
particularly the potential role of singlet and triplet excitation
states of retinals that could provide a mechanistic explanation for
early stages of rhodopsin-mediated visual excitation.
186,190,194−196
Although the directly formed singlet excited state had a major role
in the photoisomerization of either all-trans or
13-cis-retinal, in the case of 11-cis-retinal and 7-cis-retinal, isomerization occurred
also after an intersystem crossing to an excited triplet state, so
the triplet state was responsible for up to 50% of the observed isomerization
with the other half of isomerized retinoid arising from the singlet
state.
188,193,197,198
Because the triplet state of chromophore bound to
rhodopsin has never been spectroscopically observed, it is not clear
whether these observations can be translated into a biologically relevant
model. Nevertheless, these early studies on retinoid isomerization
clearly showed that the mixture of isomers obtained by any of the
available methods did not recapitulate the equilibrium observed in
living tissue. Thus, they indirectly facilitated a shift in research
focus toward the potential role of enzymes and specific binding proteins
in maintaining the composition of retinoid isomers in vivo.
3.4
Dihydroretinol
As mentioned above,
retinoid isomers can be directed toward equilibrium by free radicals via addition–elimination
processes which implicate
transient disintegration of the double bond prior to an internal rotation.
178,181
By analogy, reduction of a selected double bond that allows free
rotation followed by its desaturation could represent an alternative
route to polyene isomerization (Figure 10).
Although the conjugated double bond system of retinoids is resistant
to chemical reduction, this scenario can be effectively completed in vivo.
199
Cyanobacterial and
plant CrtIso catalyze the isomerization of (7Z,9Z,9′Z,7′Z)-tetra-cis-lycopene (prolycopene)
to all-trans-lycopene.
200−203
This enzyme-mediated isomerization employing redox cofactors occurs
through a reversible saturation–desaturation reaction of the cis-double bonds.
204
Animal homologues
of the plant enzyme are inactive toward prolycopene.
125,205
Instead, the mouse CrtIso-related enzyme accepts all-trans-retinol as a substrate
in carrying out the saturation of the retinoid
13–14 double bond.
125,206
However, in contrast
to plant CrtIso, this reaction is not accompanied by isomerization
of the retinoid, yielding (R)-all-trans-13,14-dihydroretinol
as a final product (Figure 11).
206
Consequently, this animal enzyme has been named
retinol saturase or RetSat.
125
The enzymatic
activity of RetSat does not contribute to visual chromophore regeneration
in the eye but rather influences processes involving peroxisome
proliferator-activated receptor γ (PPARγ) activity,
which regulates lipid accumulation in mice.
125,207,208
Figure 10
Retinoid isomerization
by sequential saturation–desaturation
chemistry. This strategy is used, for example, by plant lycopene isomerase
(CrtIso).
Figure 11
Conversion of all-trans-retinol (i) into all-trans-(R)-13,14-dihydroretinol
(ii) by RetSat, an enzyme evolutionarily related to CrtIso.
4
Photoreceptor
Cells and Pigmented Helper Cells
To place retinoid chemical
transformations in response to light
in a proper context, we must review the evolution of the visual system.
Ancient photoreceptors composed of light-sensing cells (proto-eyes)
mediated phototaxis with the assistance of pigmented cells, precursors
of the retinal pigment epithelium (RPE) in the retina of the eye.
209,210
The proto-eye could likely detect directionality of light and could
also be considered a precursor of a primitive circadian clock. As
living organisms evolved over time, the cell numbers, complexity,
and functional capability of the light-sensing organ increased. For
example, whereas Drosophila melanogaster eye has
10 different cells, that number has grown to 400 in humans, with a
parallel development of retinal circuitry.
211−215
Darwin wrote “Reason tells me, that if numerous gradations
from a simple and imperfect eye to one complex and perfect can be
shown to exist, each grade being useful to its possessor, as is certainly
the case; if further, the eye ever varies and the variations be inherited,
as is likewise certainly the case and if such variations should be
useful to any animal under changing conditions of life, then the difficulty
of believing that a perfect and complex eye could be formed by natural
selection, though insuperable by our imagination, should not be considered
as subversive of the theory”.
216
A large number of light sensitive molecules are needed to
increase
the probability of photon capture. These light-sensitive molecules
must also couple to a receptor protein capable of initiating the required
biochemical chain of events. Such 30–60 kDa proteins were given
the name “opsins”, and in bacteria they function as
cation and anion pumps,
217−223
whereas in higher organisms they are coupled to G proteins and therefore
are called G protein-coupled receptors (GPCRs). In every case, the
chromophores are retinals bound to opsin via a Schiff
base (retinylidene) group with a transmembrane domain (TMD) Lys residue.
60,61,79,84,224−228
Because opsins are membrane proteins, they
are densely packed into specific regions of the cell to prevent their
random diffusion.
229
For example, in bacteria
they form 2-dimensional crystalline membrane patches, whereas invertebrate
opsins occupy cellular membrane protrusions called microvilli of rhabdomeric
photoreceptor cells (Figure 12). A different
solution is employed by vertebrate photoreceptors which are modified
ciliary elongated protrusions called outer segments (OS).
230−232
Rhabdomeric and ciliary photoreceptors also employ fundamentally
different mechanisms for regeneration of their visual pigments following
light stimulation.
233
Light-activated rhabdomeric
opsins can be restored to their ground state by absorption of a second,
lower energy photon in a process known as photoreversal. Light activated
ciliary opsins, on the other hand, do not undergo photoreversal but
instead rely on biochemical regeneration of the visual chromophore via the retinoid
(visual) cycle. Notably, there are exceptions
to these typical routes of visual chromophore regeneration, as has
been described, for example, in Drosophila.
234
Rods and cones are the two morphologically
distinct types of ciliated photoreceptor cells found in human retina
(Figure 12). Cones are further divided based
on the opsin they express, such as short-wavelength responsive cells
(S cones that express blue opsin with an absorption peak at 420–440
nm), medium-wavelength responsive cells (M cones that express so-called
“green” opsin with an absorption peak at 530–540
nm), and long-wavelength responsive cells (L cones that express “red”
opsin with a maximum absorbance at 560–580 nm).
66,84,235−241
Figure 12
Comparison of photoreceptor structure between invertebrates represented
by Drosophila and vertebrates represented by man.
Invertebrates utilize a rhabdomeric photoreceptor cell whereas vertebrate
photoreceptors are modified ciliary cells. Notably, a few invertebrates,
such as Amphioxus, employ ciliary photoreceptors.
4.1
Structure of the Mammalian Retina
The vertebrate
eye evolved to utilize key optical principles along
with the physicochemical properties of retinoids. First, light is
focused by passing through two proteinaceous biological lenses at
the front of the eye, namely the cornea and lens.
242
Then the focused wave of photons is absorbed by photoreceptors
of the retina (Figure 13), a highly layered
tissue, where rod and cone photoreceptors constitute more than 75%
of the cells. Detailed phylogenetic analyses across species demonstrated
that the last common ancestor of all jawed vertebrates evolved ∼400
million years ago with a key role for transcriptional master regulator
paired box gene 6 (PAX6) in controlling development of eyes and other
sensory organs.
243−245
Vision is initiated by absorption of light
by photoreceptors, with rod cells acting as photon counters because
under dim illumination they can respond to a single photon.
246,247
The extreme photosensitivity of the 11-cis-retinylidene
chromophore is modulated by the chemical environment of the opsin
chromophore binding pocket, yielding different visual pigments that
respond maximally to light at different wavelengths, causing the so-called
“opsin shift” that allows us to discern differences
in color.
235,248
Forced twisting of the polyene
backbone away from its normally planar configuration induced by the
binding pocket as well as the electrostatic environment surrounding
the chromophore are major factors that influence its absorbance maximum.
249
The key role of binding pocket electrostatics
in spectral tuning of the retinylidene chromophore has been vividly
demonstrated in a rationally engineered cellular retinol-binding protein
(CRBP).
250
Figure 13
Structure of the mammalian
retina. The retina consists of several
layers of neuronal cells. The innermost photoreceptor layer is embedded
in an epithelial monolayer, known as the RPE. Electron micrographs
show stacks of membranous disks within ROS (left) and COS (right),
which contain visual pigments and the associated phototransduction
machinery. The leftmost and rightmost electron micrographs display
the bacillary structure of the outer segments and their interaction
with the RPE, respectively. A portion of this figure is reproduced
with permission from ref (235). Copyright 2009 Elsevier.
There are similarities and differences between rods and cones.
Rod cells are more light-sensitive than cones but saturate at relatively
low levels of light. At the light level at which rods reach saturation,
cones generate measurable responses but have an extremely high photon
saturation threshold.
242
The number and
types of cones that collect photons in ambient light differ dramatically,
from less than 1% in rats to more than 95% in the ground squirrel.
251,252
Both rod and cone cells feature the same structural design. The
most distal portions, called rod/cone outer segments (ROS/COS), are
in close contact with the RPE cell layer. These ciliary structures
connect with the soma via an inner segment endowed
with a high density of mitochondria that supply energy to the highly
metabolically active photoreceptor. The soma in turn connects with
synaptic termini via inner fibers (Figure 13). The specialized photoreceptor cilia
contain
visual pigments responsible for absorption of light and house all
phototransduction proteins needed for amplification and quenching
of the light signal. Here there is yet another difference between
rods and cones. ROS are made up of a stack of individualized disks
surrounded by the ROS plasma membrane, whereas COS differ by having
a series of invaginations continuously connected with the COS plasma
membrane.
The structure of ROS is better known than that of
COS because more
advanced methods were developed for its isolation,
253,254
including mouse ROS, which can be altered genetically.
255−258
ROS structure has recently been reviewed.
66
In short, a mouse ROS with a length of ∼24 μm and a
diameter of ∼1.2 μm
259
contains
about 600–800 membranous stacked disks which increase the density
of rhodopsin available for photon absorption. ROS extend up to the
apical part of the RPE and are tightly enveloped by microvilli of
RPE cells that increase the contact area of these two cell types to
facilitate transfer of substances, including retinoids.
4.2
Structure of the RPE
Without RPE
cells, our vision would not be sustainable. The RPE is a monolayer
of highly polarized, quasi-hexagonal, epithelial cells. The apical
membrane of RPE cells lies adjacent to the photoreceptor OS, whereas
the RPE cell basal surface faces Bruch’s membrane.
260−262
Contact with ROS is maintained by a highly elaborate network of
microvilli visible under the electron microscope (Figure 14) which in some species
changes shape and elongates
when the eye is exposed to light. About 20–40 photoreceptor
cells project toward a single RPE cell. In humans, this ratio depends
on the location of these cells in the eye because the gradient of
rod/cone cells and also slight differences in the dimension of photoreceptors
dictates their packing density. In the periphery, the rod to RPE cell
ratio is 29 whereas, in the fovea, the cones to RPE cells ratio is
22.
263
Functions of the RPE cell layer
are diverse,
59,264
but from the perspective of
this review, two roles are the most critical.
Figure 14
Structure of the RPE.
Electron micrographs show the apical processes
that extend out from the cell body and interdigitate with photoreceptor
outer segments (A, lower resolution; B, higher resolution). In part
C, a cross section though an RPE cell shows its cuboidal morphology
and numerous melanin granules. Panel D depicts interactions between
an RPE cell and photoreceptor outer segments at high resolution. Transmission
electron micrographs from a C57BL/6J mouse retina were taken at postnatal
days 60–66. The RPE cell intimately interacts with the photoreceptor
outer segment via apical microvilli, thereby supporting
photoreceptor cell function.
The first function of the RPE is to facilitate photoreceptor
cell
renewal. To provide optimal signal amplification, membranes of ROS
and COS contain high levels of unsaturated lipids.
265
These lipids are prone to oxidation in the presence of
light, (photo)reactive retinal, and high oxygen tension,
266,267
which are physiological conditions encountered in the retina. Photochemical
reactions induced by light in transparent retinal tissue require a
delicate balance between protein, lipid, and metabolite renewal and
damaged component disposal, which when disturbed can lead to rapid
and massive retinal degeneration. Impressively, postmitotic rod and
cone photoreceptor cells undergo a daily regeneration process wherein
∼10% of their OS volume is shed, subsequently phagocytosed
by adjacent RPE cells
268
and replaced with
newly formed outer segments. Thus, RPE cells dispose of but also accumulate
an immense amount of oxidized cellular debris. Indeed it was estimated
that each RPE cell phagocytoses hundreds of thousands of OS disks
over a human lifetime.
269
Several potentially
toxic byproducts are condensation compounds derived from all-trans-retinal.
69,270
Dysfunction of such processes
as phagocytosis, lysosomal degradation, and removal of waste products
by the RPE can lead to severe retinopathies, including age-related
macular degeneration (AMD).
270−274
As a second major function, the RPE expresses key metabolic
enzymes
required for production of the visual chromophore, 11-cis-retinal, and thus comprises
an integral part of the retinoid cycle.
59,63−65,68,69,71−78,80,81,83,85,261,275−281
In the first step of this metabolic pathway, LRAT, through its ability
to catalyze the formation of retinyl esters that are readily sequestered
by aggregation into lipid droplets called retinosomes, changes the
mass action ratio to favor retinoid uptake from both photoreceptors
and the choroidal circulation.
278,282,283
In addition to LRAT, another critical enzyme called RPE65 (retinoid
isomerase) catalyzes the conversion of all-trans-retinyl
esters into 11-cis-retinol. This cis-retinol is oxidized and sent back to ROS/COS
to re-form photoactive
visual pigments. Diffusion likely suffices for this process because
the chromophore forms, especially in rods, a highly stable covalent
complex with opsin, thereby driving the transfer of retinoids to photoreceptors.
5
Transformation of Retinoids within the Eye
The photosensitive active retinoid, 11-cis-retinal,
is produced in the RPE and delivered to the photoreceptors.
59,64,68,69,71,83,278,279,284−287
It has been postulated that additional but less understood transport
processes take place between Müller cells and cone photoreceptors.
85,285,288
Because retinols and retinyl
esters (but not retinals) are intrinsically fluorescent, their transformation
can be followed by fluorescence induced by two-photon excitation.
278,282,283,289−293
Further development of this method guarantees improved understanding
of retinoid flow at the subcellular level.
5.1
Canonical
Retinoid (Visual) Cycle
The initial discovery of a light-sensitive
pigment in the retina
is generally attributed to a German physiologist by the name of Böll,
who, in ca. 1876, observed the “purple red color of the bacillary
(i.e. rod) layer of the retina” while dissecting
the retina of a frog kept in the dark just prior to the procedure.
1,294
The red color of frog rod cells had actually been noted some 25
years earlier by Müller, who incidentally first described retinal
Müller cells,
295
but the color was
attributed to hemoglobin and not further explored.
1
Böll made the key observation that the red hue of
the retina was fleeting, gradually transforming to a yellow color
and then fading over the course of several seconds, leaving the tissue
colorless. Böll insightfully inferred that this light-induced
bleaching of the retina must be reversed when the animal was maintained
in the dark.
A second German physiologist by the name of Kühne
greatly expanded on Böll’s research by showing that
a photobleached retina regained its red color when placed in contact
with the RPE and stored in the dark.
296,297
This critical
experiment demonstrated that at least two different tissues were required
for the bleach–regeneration cycle proposed by Böll.
Kühne used bile salts, which he had on hand from his experiments
on digestion, to solubilize the red pigment and then used the preparation
to demonstrate a correspondence between its absorption spectrum and
the spectral sensitivity of the retina to light, firmly establishing
the red substance as the visual pigment of rod cells. Kühne
named the pigment “sehpurpur” or “visual purple”
and later referred to it as “rhodopsin”.
297
Despite the rapid progress made by Böll
and Kühne
on the mechanism of light perception, the field was essentially dormant
for the next 50 years. In the 1930s the task of identifying the molecular
components of the visual system was taken up by Wald and his colleagues.
Among his many achievements, Wald discovered that the chromophore
imparting a red color to rhodopsin was a vitamin A-derived compound
called 11-cis-retinal. Moreover, he found photons
striking this chromophore could induce a change in its configuration
from an 11-cis, through a series of photointermediates,
to an all-trans state, and that this photochemical
reaction, which initiates the series of retinal color changes observed
by Böll and Kühne, represents the first step in the
sensing of light by the eye.
6
He also showed
that, following photoactivation, rhodopsin decomposes into its protein
and retinal components. Wald was the first to lay out a general scheme
of chemical reactions, termed the visual or retinoid cycle, which
underlies visual perception and regeneration.
6
Dowling, a student of Wald, through detailed measurements of retinoid
flow between photoreceptors and RPE during light exposure and dark
adaption, established that the retinal liberated from photooactivated
rhodopsin is rapidly taken up by the RPE and esterified. During dark
adaption, the flow of retinoids proceeds in a reverse manner from
the RPE to photoreceptors for rhodopsin regeneration to occur. These
findings firmly established the role of the RPE in visual chromophore
regeneration.
298
This visual cycle scheme
was refined over the years, and the enzymes responsible for catalyzing
the individual reactions were identified. Our current understanding
of the retinoid (visual) cycle is summarized in Figure 15.
Figure 15
Retinoid (visual) cycle. Enzymes (red) and binding proteins (blue)
involved in 11-cis-retinal regeneration are found
in both photoreceptor and RPE cells. Metabolic transformations occurring
in the RPE take place in the smooth ER, where key enzymes of the visual
cycle are located. PC, phosphotidylcholine.
5.2
Cone Visual Cycle
In addition to
the canonical visual cycle, several lines of evidence indicate that
cone photoreceptors might have access to a special source of visual
chromophore not available to rods
85
(Figure 16). For example, after equal levels of bleaching,
cones dark-adapt faster than rods by a factor of ∼10.
299
In contrast to rods, cones operate in bright
light without saturating, implying that the rate of visual chromophore
delivery to these two cell types must be substantially different.
300,301
Additionally, studies have found that the canonical visual cycle
is too slow to provide enough visual chromophore to maintain cone
light responsiveness under bright light conditions.
302
The distribution of retinoids in cone-dominant retinas
is substantially different from that of rod-dominant species, with
an abundance of 11-cis-retinyl esters found in the
retina as opposed to the stores of all-trans-retinyl
esters in the RPE.
303,304
Three enzymatic activities,
namely isomerase, 11-cis- and all-trans-retinyl ester synthase, and retinol dehydrogenase/reductase,
are
associated with membrane fractions from chicken neural retina.
302
Cultured primary Müller cells from chickens
were found to convert all-trans-retinol added to
the media into 11-cis-retinol and 11-cis-retinyl esters, suggesting that this cell
type could be a site of
visual chromophore production in vivo.
305
Moreover, the 11-cis-retinol-binding
protein, cellular retinaldehyde-binding protein (CRALBP), is known
to be expressed in Müller cells, providing additional support
for the above hypothesis.
306,307
Although significant
progress has been made in elucidating this alternative pathway, most
of the responsible enzymes have not yet been molecularly characterized.
A candidate protein responsible for the alternative retinoid isomerization
(all-trans-retinol to 11-cis-retinol)
activity found in chicken retinas was identified as dihydroceramide
desaturase-1 (DES-1), a member of the integral membrane hydroxylase/desaturase
enzyme family that contains an eight-His-coordinated di-iron active
site.
308,309
This enzyme is a sphingolipid Δ4-desaturase
that converts dihydroceramide into ceramide, with the latter being
a more active signaling molecule.
310,311
Interestingly,
DES-1 produces mainly 9-cis- and 13-cis-retinoids rather than 11-cis-retinoids from
all-trans-retinol, which is unexpected given the abundance
of 11-cis-retinoids in the chicken retina.
308
Further research is required to determine the
physiological relevance of DES-1 to the synthesis of a visual chromophore.
Figure 16
Putative
cone-specific retinoid (visual) cycle. This metabolic
pathway is postulated to involve enzymes located in cone photoreceptor
and Müller glial cells. The proposed direct isomerization of all-trans-retinol into
11-cis-retinol is
a key difference between this pathway and the canonical retinoid cycle.
5.3
Retinoid-Containing
Structures: Retinosomes
and Retinal Condensation Products Found in Healthy and Diseased Eyes
Imaged by Two-Photon Microscopy (TPM)
Once released from
ROS, all-trans-retinal is reduced to all-trans-retinol which then diffuses into the
RPE. There, this alcohol is
esterified by fatty acid in a reaction catalyzed by LRAT. As highly
hydrophobic substances, in part because of the physicochemical properties
of their long fatty acid esters, these retinoid ester products then
coalesce into lipid droplets termed retinosomes, which are found in
vertebrate RPE.
291
These esters can be
minor or major components of lipid droplets that along with phospholipids
total about 160 molecular species including other fatty acid esters,
triglycerides, cholesterol, cholesterol esters, and various proteins.
312
Retinosomes are reminiscent of lipid droplets
in other tissues that store other hydrophobic substances. The highly
efficient enzymatic activity of LRAT traps retinol delivered from
the photoreceptors or from the circulation, whereas retinosomes are
absent in the eyes of Lrat
–/–
mice deficient in retinyl ester synthesis. The lipid core
content of retinosomes appears to be homogeneous.
312
Retinosomes recruit proteins such as caveolin-1, perilipins
PLIN1-3, sterol carrier protein-2, structural proteins, chaperone
proteins, and redox enzymes.
290,312
We postulated that
retinosomes in RPE cells perform functions similar to those of lipid
droplets in other types of cells, suggesting that they are rather
dynamic tissue-specific organelles that change their composition in
response to fatty acid, cholesterol, and all-trans-retinol availability. Because retinoids
are naturally fluorescent,
the study of retinosomes could provide important insights into the
formation and metabolism of lipid droplets in general, especially
because these structures are accessible for real time TPM imaging in vivo.
282,289,291,313
Capitalizing on the intrinsic
fluorescence of all-trans-retinyl esters, noninvasive
TPM revealed that retinosomes are elongated structures approximately
8 μm long and 1 μm wide with their long axis oriented
perpendicularly to the RPE basal surface (Figure 17).
291
Retinyl esters in retinosomes
accumulate in Rpe65
–/–
mice lacking retinoid enzymatic isomerization. Retinosomes
are located close to the RPE plasma membrane and are essential components
for 11-cis-retinal production.
282,312,313
Figure 17
Retinoid-containing
structures found in healthy and diseased retinas
imaged by two-photon microscopy. The top panel shows a schematic of
the photoreceptor outer segment–RPE interaction with retinoid-containing
retinosomes (red ovals) and retinoid conjugate-containing particles
(orange circles) shown with their approximate dimensions (bottom left).
In healthy eyes (WT), numerous peripherally located retinosomes (punctuate
green spots) can be visualized. The number and size of these vesicles
are elevated in Rpe65
–/–
mice, owing to excessive accumulation of retinyl esters (bottom
center). In Abca4
–/–
Rdh8
–/–
mice with delayed all-trans-retinal clearance,
retinoids are diffusely present throughout the cell, presumably in
the form of all-trans-retinal-conjugates (bottom right). Scale bars represent 20 μm.
The RPE also accumulates other retinoids, including
retinal condensation
products.
314,315
These metabolites also can be
detected by TPM, and since they are characterized by unique spectral
properties, the intracellular accumulation and distribution of these
compounds can be monitored independently from other retinoids (Figure 17).
313
Resulting from
inadequate reduction/clearance of all-trans-retinal,
they are often detected as small 1 μm deposits scattered throughout
RPE cells. These condensation products accumulate most prominently
in mice lacking the ABCA4 transporter and retinol dehydrogenase 8
(RDH8) (Figure 17).
313
They also increase with age because they are delivered from photoreceptors
(at least the first steps of condensation reaction occur in ROS) by
daily phagocytosis.
270,314
Some investigators consider
these retinoid condensation products as major contributors to retinal
dysfunction in Stargardt and age-related macular degeneration (AMD)
diseases.
270,316,317
Others propose that these biomarkers are merely indicative of inadequate
clearance of all-trans-retinal, which at elevated
levels may contribute to photoreceptor/RPE degeneration.
318−320
6
Proteins and Enzymes of the Retinoid Cycle
Here we describe the molecular properties of proteins involved
in retinoid transformation in the retina including the light receptor
rhodopsin.
6.1
Rhodopsin
At the center stage of
phototransduction is rhodopsin, the most extensively studied GPCR.
Rhodopsin is the main component of disk and plasma membranes of ROS,
accounting for 90 and 75% of their protein content. In disk membranes,
the density of rhodopsin translates into about 50% of the entire volume
or surface area.
61,66,79,225,226,235,321−324
Thus, it is not surprising that the expression level of rhodopsin
dictates the size of the ROS.
259,325−327
Although rhodopsin is not uniformly distributed throughout disks,
84,328
its local high density within each disk allows efficient absorption
of light. Corresponding visual pigments in cone cells of frog retina
are so dense that they form crystalline structures.
329
Physiologically, the minimal building block of rhodopsin
is a dimer in which only one monomer is activated under normal lighting
conditions
330−335
(reviewed in refs (336−338)).
The fundamental photochemical reaction of our visual system
is isomerization of 11-cis-retinylidene to all-trans-retinylidene (Figure 18). The
Schiff base between 11-cis-retinal and Lys296
of opsin is protonated to allow a spectral shift to longer wavelengths
(at least in rhodopsin, green and blue pigments). It is remarkable
that a ligand, retinal, which is just slightly larger than tryptophan,
when photoisomerized causes a reliable change in the conformation
of rhodopsin to its activated form that couples with G protein. This
activation is accomplished through geometric cis/trans isomerization of the chromophore,
182
deprotonation
of the Schiff linkage,
339
and reorganization
of water molecules within the TMD of this receptor.
333,340−342
Figure 18
Structure and photoactivation of rhodopsin.
(a) Crystal structure
of ground-state bovine rhodopsin. The Schiff base-linked 11-cis-retinal chromophore
is shown in stick representation
(red). (b) Photoactivation and regeneration of rhodopsin. (c) Primary
conformational changes observed between ground-state (red, PDB accession
code 1U19) and
activated, meta II-like (yellow, PDB accession code 3PXO) rhodopsin.
Photoactivation of rhodopsin causes
conformational changes that
provide a binding site for the rod G protein called transducin.
343,344
No high resolution structure of the complex is yet available, but
lower resolution methods have been informative. On the basis of structural
mass spectrometry techniques, we found that the transition of ground
state rhodopsin to its photoactivated state causes a structural relaxation
that then tightens upon transducin binding.
333
Using affinity chromatography, we trapped and purified the photoactivated
rhodopsin–transducin complex. Scanning transmission electron
microscopy demonstrated about a 221 kDa molecular weight for this
complex. A 22 Å structure was calculated from projections of
negatively stained photoactivated rhodopsin–transducin complexes.
The determined molecular envelope accommodated two rhodopsin molecules
together with one transducin heterotrimer, indicating a heteropentameric
structure for the photoactivated rhodopsin–transducin complex.
334
The dimeric structure of rhodopsin in the complex
was confirmed using succinylated concanavalin A as a labeling probe.
345
Recently, we used the retinoid chromophores,
11-cis-retinal, 9-cis-retinal, and all-trans-retinal to monitor each dimeric rhodopsin
monomer
within a stable complex with transducin. We found that each of the
dimeric rhodopsin monomers contributed differently to the pentameric
complex, indicating a functional distinction between rhodopsin monomers
in their oligomeric form.
332
For a more
detailed description of the activation events, recent reviews are
available (refs (66,79, and 346−349)).
Two other important
questions need to be answered about the rhodopsin
cycle. First, how is the chromophore released and how do opsins recombine
with 11-cis-retinal to regenerate rhodopsin and cone
visual pigments? Mechanistically, more information is available for
rhodopsin than for cone pigments. Key residues in rhodopsin’s
active site are Lys296, Glu113, and Glu181 (Figure 19).
60,139,226,227,240,350−355
The 11-cis-retinylidene bond is protonated, and
Glu113 is the counterion of this linkage. A counterion is essential,
as positively charged groups are extremely rare in the TMD of membrane
proteins. Upon illumination of rhodopsin (λmax =
500 nm), the chromophore undergoes geometrical isomerization.
356,357
Next, rapidly formed and decaying intermediates have been detected
before Meta I is observed (λmax = 478 nm). Though
the difference in absorption results from relaxation of the chromophore,
only small changes in the protein moiety take place at this stage
358−360
and it is believed that a switch in the counterion occurs from Glu113
to Glu181.
324,361−364
Further relaxation combined with Schiff base deprotonation results
in a signaling form called Meta II (λmax = 382 nm)
343
which then interacts with G protein-coupled
receptor kinase 1 and arrestin.
60,62,239,365,366
Especially in cold blooded vertebrates, a prominent fraction of
Meta I relaxes into Meta III (λmax = 465 nm), the
difference in the latter’s light absorption derived from an anti to syn thermal isomerization
of the
Schiff base double bond.
367
From both Meta
II and Meta III, a protonated carbinol ammonium ion is formed before all-trans-retinal
is released from opsin. When regeneration
with 11-cis-retinal subsequently occurs, formation
of the Schiff base requires polarization of the carbonyl group of
11-cis-retinal and deprotonation of the Lys296 side
chain amine as well as exclusion of water from the active site (Figure 19). Regeneration
and recombination of 11-cis-retinal with opsin restores the dark state condition
needed for subsequent photon absorption. Although there is convincing
evidence for the roles proposed for Glu113 and Glu181, independent
verification of this switch and the whole mechanism of chromophore
regeneration is still lacking.
Figure 19
Chemical changes in the rhodopsin chromophore
during photoactivation.
The pathway is initiated when 11-cis-retinylidene
(i) absorbs a photon, leading to cis/trans isomerization. Then the Glu113 counterion
of the protonated Schiff
base becomes protonated, leading to the formation of Meta I rhodopsin
(ii). Meta I, in turn, can convert to Meta II rhodopsin
(iii), the active signaling form of rhodopsin, or, rarely,
to Meta III rhodopsin (iv), a non-signaling form of rhodopsin.
Both forms decay through a carbinol ammonium intermediate (v) to form a non-covalent
opsin–all-trans-retinal
complex (vi), which then dissociates to yield free all-trans-retinal and opsin (vii).
How the chromophore migrates into and out of opsin
remains an open
question. Taking into account the hydrophobic nature of retinal, does
it dissociate into the lipid bilayer or the cytoplasm for the reduction
reaction, or could it enter the intradiscal space where condensation
products of retinal start forming? Similar to other GPCRs, which in
addition to orthosteric-binding sites contain other well-defined allosteric
ligand-binding sites, rhodopsin also has two other retinoid-binding
sites within opsin
368,369
in addition to the retinylidene
pocket (site 1). Site II is called an entrance site, and the exit
site (site III) is occupied by retinal after its release from site
I. The crystal structure of opsin,
370
an
opsin structure with another retinoid bound,
371
and mutagenesis studies
372
all suggest
an escape and entrance route for retinal.
6.2
Retinol
Dehydrogenases (RDH’s)
An integral part of the retinoid
cycle is the interconversion of
retinals and retinols.
64,287
Enzymes that catalyze this process
can be classified into three major protein families: cytosolic alcohol
dehydrogenases (ADH’s) that belong to a medium-chain dehydrogenase/reductase
family and selected members of the aldo–keto reductase family
(AKR’s)
373
and microsomal RDH’s
that represent the short-chain dehydrogenase/reductase (SDR) group.
374
However, only RDH’s contribute to vision-related
metabolism of retinoids.
287
The universal
redox carriers for these reactions are the dinucleotide cofactors
NAD(H) and NADP(H). In ADH’s and RDH’s they are bound
by a Rossmann fold, a classic structural element composed of 6 to
7 parallel β-strands flanked by 3–4 α-helices present
in these enzymes (Figure 20).
375
This structural motif contains a Gly-rich sequence (TGXXXGXG)
responsible for its structural integrity and binding of the diphosphate
portion of the nucleotide cofactors. An acidic residue binding to
the 2′ and 3′ hydroxyls of the adenine ribose and located
downstream of the Gly-rich motif confers NAD(H) specificity, whereas
NADP(H) binding is dictated by the presence of a basic residue within
the Gly-rich segment.
375
By contrast, AKR’s
do not contain the canonical Rossmann fold. Preferable NADPH binding
occurs within the characteristic (β/α)8 motif
of this protein family.
376
Despite their
conserved mode of cofactor binding, ADH and RDH families of oxidoreductases
reveal diverse protein domain architectures and mechanisms of catalysis.
ADH’s depend on a catalytic Zn atom bound in the active site,
which electrostatically stabilizes the substrate’s oxygen and
thus increases the acidity of the alcohol proton.
377
In contrast, SDR’s show a Tyr-based catalytic center
with adjacent Ser and Lys residues.
378−381
Here, the deprotonated phenolic
group of Tyr initially forms a hydrogen bond with the alcohol hydroxyl
group and the deprotonated Tyr residue acts as a catalytic base to
extract a proton from the substrate’s hydroxyl group. The hydride
ion, extracted from the substrate, can be directly transferred to
position 4 of the nicotinamide ring. In addition to the interaction
with the nicotinamide ring of the cofactor, the reaction intermediate
is stabilized by the hydroxyl group of an adjacent serine residue
(Figure 20).
382
Because
the phenolic group of the Tyr side chain has a pK
a value around 10, the ε-amino group of Lys is needed
to convert tyrosine to tyrosinate (pK
a 7.6), to facilitate catalysis at neutral pH. Additionally, a Lys
residue forms hydrogen bonds to both the 2′- and the 3′-hydroxyl
groups of the cofactor’s ribose moiety and thus enforces a
proper orientation of the nicotinamide ring to allow a pro-S hydride transfer only.
Recently, an Asn residue was shown
to stabilize the position of this catalytic Lys via a conserved water molecule. Thus,
the final sequence of the catalytic
tetrad of SDR’s is composed of Asn, Ser, Tyr, and Lys residues
(Figure 21).
383
The
catalytic mechanism of AKR’s, in principle, is similar to that
found in SDR’s with an active site Tyr residue and an assisting
Lys residue facilitating the deprotonation of the Tyr hydroxyl group.
384
Figure 20
Structure and catalytic mechanism of RDHs.
(a) Cartoon representation
of a representative SDR family member (type 1 17-β-hydroxysteroid
dehydrogenase, PDB accession code 1A27). (b) Hypothetical structure of an RDH
with bound nucleotide (NAD(H) or NADP(H)) and retinoid (all-trans-retinol or all-trans-retinal)
substrate. The structures
in panels a and b are depicted in the same orientation. (c) Reversible
transfer of hydride from the S4-face of the nucleotide
to all-trans-retinal to produce pro-R-all-trans-retinol.
Figure 21
Sequence alignment of known vertebrate RDH’s of the SDR
family. Glycine residues of the conserved TGXXXGXG, nucleotide-binding
motif are highlighted in blue, whereas residues comprising the catalytic
tetrad are highlighted in orange.
The RDH’s represent a microsomal SDR group with an
overall
sequence similarity of at least 30% (Figure 21) although the catalytic core of these
enzymes reveals a much higher
homology with nearly identical folding. Despite these similarities,
the mode of membrane binding and membrane topology of specific RDH’s
is a matter of controversy. Based on biochemical studies, RDH1 is
anchored in ER membranes with the catalytic domain facing the cytoplasm.
385
The N-terminal residues are essential for the
membrane localization and topology of this enzyme, whereas the C-terminus
was postulated to be involved in stabilization of the protein’s
membrane orientation.
385
In contrast to
this model, RDH12 was reported to be a glycoprotein carrying an endoglycosidase
H-sensitive sugar modification, which suggests a luminal orientation
of this enzyme.
386
Yet other models indicate
that the hydrophobic stretch of the catalytic domain in RDH1 and RDH4
can contribute to membrane binding.
387,388
An interesting
case is RDH8, which localizes to ROS/COS. Efficient transport to the
ROS/COS is mediated by a signaling sequence at the C-terminus of this
enzyme whereas membrane anchoring is achieved by fatty acylation of
conserved Cys residues.
389
Although
all RDH’s identified in vertebrates can utilize
retinol or retinal, some have wide substrate specificities, and retinoids
are not their preferred physiological substrates.
374,390
For example, RDH1, RDH3, RDH4, RDH6, and RDH7 reveal 25–60
times higher affinities for androgens than all-trans-retinol.
391−393
In fact only RDH5, RDH8, RDH10, RDH11, RDH12,
RDH13, RDH14, and retSDR1 were proven to be expressed in the retina
or RPE, and their roles have been studied in the context of the visual
(retinoid) cycle.
68,287
Based on their preferred substrate
geometry and role in the retinoid cycle, this group of enzymes can
be classified into all-trans and 11-cis-retinol dehydrogenases. RDH8 and RDH12 belong
to the first class,
whereas RDH5, RDH11, and RDH10 act on 11-cis-retinoids.
Functions of the two remaining enzymes, RDH13 and RDH14, have yet
to be adequately assigned.
394
Reactions
catalyzed by RDH’s are fully reversible. In a test tube, the
net direction of retinoid interconversion depends on the oxidation
state of the provided cofactor and the ratio between the concentrations
of substrate and product. However, in more complex in vivo systems, the direction
of the enzymatic reaction is determined by
enzyme specificity for binding either NAD(H) or NADP(H). Under physiological
conditions the ratio between NAD/NADH is close to 1000;
287
thus, RDH’s that bind this cofactor
can contribute significantly only to retinol oxidation. In contrast,
the ratio of NADP to its reduced form is about 0.005,
395
such that enzymes utilizing this dinucleotide
reduce retinal to retinol.
The physiological role and significance
of particular RDH’s
in vitamin A homeostasis, retinoic acid signaling, and visual chromophore
regeneration has recently been extensively reviewed
68,287,396
and thus will not be described
here. However, it is worth noting that, in addition to genetic and
biochemical identification of RDH’s, many of these enzymes
were characterized in vivo in the past few years.
397−400
These studies not only provided detailed information about the physiological
function of many RDH’s but also led to the development of animal
models to investigate their roles in human pathological conditions,
including retinal degenerative diseases.
401
6.3
Lecithin/Retinol Acyl Transferase (LRAT)
LRAT is the main enzyme that catalyzes retinyl ester formation
in most tissues,
147,402,403
with the exception being adipocytes that instead exhibit acyl-CoA-dependent
transferase activity of a protein (acyl-CoA:retinol acyltransferase
or ARAT) yet to be identified.
404
Consequently,
LRAT is critical for uptake and storage of retinoids in peripheral
tissues, including RPE cells where it plays a pivotal role in providing
substrate for visual chromophore regeneration via the enzymatic activity of RPE65.
147,148,405
Localized in the endoplasmic reticulum (ER),
LRAT is a 25 kDa bitopic integral membrane protein with a single membrane-spanning
helix localized at the C-terminus
406
and
a potential membrane-interacting N-terminal domain.
407
The C-terminal domain is critical for post-translational
targeting of the enzyme to the endoplasmic reticulum (ER) in a cytosolic
TMD recognition complex-dependent manner.
406,408
On the basis of its amino acid sequence and predicted tertiary structure,
LRAT is classified as a member of the NlpC/P60 thiol peptidase protein
superfamily (Figure 22).
409
Besides LRAT, there are seven genes in the human genome
that encode proteins belonging to the NlpC/P60 family: two neurological
sensory proteins (NSE1-2) and five H-ras-like tumor suppressors (HRASLS1-5).
409,410
The common feature of LRAT and HRASLS proteins is a 6 amino acid
sequence that contains a conserved catalytic Cys residue (NCEHFV).
411
Although the structure of LRAT
has yet to be determined, recently solved structures of two LRAT-like
proteins, human HRASLS2 and HRASLS3, provide important insights into
the molecular organization of this enzyme.
411,412
By analogy to HRASLS proteins, LRAT’s basic structural motif
is largely reminiscent of papain-like proteases and consists of a
four-strand antiparallel β-sheet, three α-helices, and
conserved catalytic residues Cys161, His60, and His72 that define
the active site located at the N-terminus of helix α3 and β-sheets
β2 and β3, respectively (Figure 22).
411
Although the overall folding of
LRAT is similar to that of other NlpC/P60 peptidases, there are significant
topological differences derived from a circular permutation within
the catalytic domain of classical NlpC/P60 proteins.
409,413
Consequently, alternatives to peptidase activity evolved in LRAT-like
proteins. The best characterized example is the acyltransferase activity
of LRAT, which catalyzes the formation of retinyl esters by transferring
an acyl group directly from the sn-1 position of phosphatidylcholine
(PC) onto all-trans-retinol.
145,146,414
Figure 22
Sequence alignment and
structure of LRAT-like acyltransferase enzymes.
A protein sequence alignment of all LRAT-like proteins encoded in
the human genome is displayed on the left, showing conserved His and
Cys residues (orange) that constitute the catalytic triad of this
enzyme family. Hydrophobic C-terminal membrane-anchoring sequences
are colored green. The crystallographic structure of human HRASLS3
is shown on the right with the carbon atoms of residues comprising
the catalytic triad colored orange.
As a consequence of its structural relationship to thiol
peptidases,
LRAT adopts an analogous catalytic strategy whereby the deprotonated
Cys161 serves as a nucleophile attacking the carbonyl carbon of an
ester bond in the lipid substrate, both forcing the carbonyl oxygen
to accept a pair of electrons and transforming the sp
2
-hybridized carbon into an sp
3
-hybridized tetrahedral intermediate (Figure 23).
411,415,416
Collapse
of this intermediate results in transient acylation of the protein
by formation of a thioester bond at the Cys161 sulfhydryl group. Concomitantly,
1-hydroxy-2-acyl-sn-glycero-3-phosphocholine (Lyso-PC)
is liberated as a first product of the reaction. Deprotonation of
the hydroxyl group of retinol then permits decomposition of the thioester
intermediate and transfer of the acyl group from the enzyme onto retinol
to form retinyl esters. Several lines of evidence support this LRAT
model. Initially, a role for Cys161 and His60 in catalysis was derived
from site-directed mutagenesis studies where replacement of either
of these two amino acids abolished retinyl ester formation.
417,418
Recently, the proposed mechanism was proved by trapping the catalytic
intermediate in the absence of an acyl acceptor and directly detecting
the covalent thioester protein modification by mass spectrometry.
415
An identical enzymatic mechanism holds for
other LRAT-like proteins.
411
Importantly,
despite their highly conserved catalytic domains, HRASLS proteins
cannot employ all-trans-retinol as an acyl acceptor
and they lack specificity toward the sn-1 ester cleavage site as well.
411
Instead, they catalyze both lipid hydrolysis
and acyl transfer onto a variety of enzyme-specific substrates, such
as lyso-phospholipids or phosphatidylethanolamine (PE).
411,419−422
These fundamental differences in enzymatic activity do not arise
from changes in the catalytic mechanism but rather are determined
by subtle modifications in the primary sequence and structure of these
proteins. Despite recent advances in understanding the principles
of catalysis, several critical questions remain, including the evolutionary
and structural basis for adaptation of all-trans-retinol
processing by LRAT.
Figure 23
Catalytic mechanism of LRAT. The enzyme utilizes a ping
pong bi
bi catalytic mechanism.
416
In this reaction,
the active site Cys nucleophile, which was crystallographically observed
to exist in two conformations (i), attacks the sn-1 ester
group to form a tetrahedral intermediate (ii) that collapses
into a stable acyl-enzyme intermediate with liberation of Lyso-PC
(iii).
411
The negatively charged
oxygen is stabilized by an oxyanion hole (dotted curve in ii). Next, all-trans-retinol
binds to the active site
and is activated to produce a nucleophilic attack on the acyl-enzyme
thioester bond (iv), resulting in formation of a tetrahedral
intermediate again stabilized by an oxyanion hole (v)
that collapses to release the all-trans-retinyl ester
and regenerate the nucleophilic Cys residue. Catalytic His residues
likely promote catalysis by increasing the nucleophilicity of the
active site Cys and by serving as general proton donors/acceptors.
6.4
Acyl-CoA/Retinol
Acyltransferase
Early studies concerning the esterification
of all-trans-retinol revealed two independent enzymatic
activities that led to
formation of retinyl esters. In addition to lecithin-dependent acyl
transfer facilitated by LRAT described above, profound acyl-CoA-dependent
activity has been reported to exist in a variety of tissues, including
small intestine, liver, adipocytes, skin, testis, and retina.
423−429
In contrast to LRAT, which efficiently utilizes phospholipids as
acyl donors, acyl-CoA/retinol acyltransferase (ARAT) requires a preactivated
acyl moiety coupled to coenzyme A (Figure 24).
Figure 24
Formation of retinyl esters catalyzed by acyl-CoA/retinol acyltransferase.
ARAT has never been purified or
cloned. However, studies of LRAT-deficient mice indicate
that the intestinal absorption
of vitamin A decreased to 50% that of wild type animals challenged
with a physiologic dose of retinol.
402
On
the basis of this evidence, ARAT may contribute to retinyl ester formation
within the intestine and thus facilitate retinoid uptake and packaging
into chylomicrons. A distinct role of acyl-CoA-dependent retinoid
esterification has been proposed for the retina. On the basis of enzymatic
studies, retinas isolated from cone-dominant species such as ground-squirrel
and chicken revealed retinoid isomerase activity that, in contrast
to RPE65, converts all-trans-retinol directly into
11-cis-retinol.
302,430
This enzymatic
reaction is thermodynamically driven by secondary esterification of
newly produced 11-cis-retinol in a palmitoyl-CoA-dependent
manner. Studies involving primary cultures of chicken Müller
cells indicate that inner retinal ARAT enzymatic activity is associated
with this cell type.
85,302,431
Interestingly, the retinal ARAT preferably synthesized 11-cis-retinyl esters
428
distinguishing
it from intestinal all-trans-ARAT and suggesting
the existence of two or more separate enzymes responsible for acyl-CoA-dependent all-trans-retinol
esterification. Intestinal acyl-CoA-diacylglycerol
acyltransferase 1 (DGAT1) has been shown to catalyze formation of
retinyl esters in an acyl-CoA-dependent manner in vitro.
402
432,433
However,
later detailed in vivo studies indicated that DGAT1
deficiency did not cause a decline in retinol esterification but rather
markedly reduced postprandial plasma trigliceride and retinyl ester
excursions by inhibiting chylomicron secretion.
434
6.5
Retinoid Isomerase (RPE65)
Our understanding
of RPE65 biochemistry has changed dramatically since the last Chemical Reviews article
covering the visual cycle.
435
Once thought to function solely as a retinoid-binding
protein, RPE65 has now been conclusively identified as the retinoid
isomerase of the canonical retinoid cycle.
6.5.1
History
and Functional Characterization
RPE65 was identified in the
early 1990s as a conserved, developmentally
regulated, RPE-specific, microsomal membrane protein with an apparent
molecular mass of 65 kDa.
436−438
Rpe65
–/–
mice, generated in the Redmond laboratory,
exhibited early onset blindness and ocular retinoid abnormalities
consisting of a lack of 11-cis-retinoids and overaccumulation
of all-trans-retinyl esters, indicating an important
physiological role for RPE65 in the visual cycle.
439
Furthermore, human RPE65 mutations were
shown to cause Leber Congenital Amaurosis (LCA), a recessive, severe
childhood blinding disease.
440,441
These two important
findings established a key role for RPE65 in retinal physiology.
At about the same time, in the seemingly unrelated field of plant
biology, a carotenoid cleavage enzyme from maize called viviparous
14 (VP14), involved in the production of abscisic acid from 9-cis-violaxanthin,
442
was identified
and shown to possess significant sequence homology to RPE65.
443
Despite the clear relationship of RPE65 to
an established enzyme family, efforts to demonstrate either carotenoid
oxygenase
133
or retinoid isomerase activity
444,445
for purified RPE65 were initially unsuccessful, leading to the conclusion
that RPE65 was not an enzyme but instead a retinoid-binding protein.
Through visual cycle complementation experiments using unbiased RPE
cDNA libraries
405
or candidate gene approaches,
446,447
RPE65 was finally identified as the visual cycle retinoid isomerase
in 2005, about 12 years after the gene was first cloned. This delay
in identifying the catalytic function of RPE65 was primarily due to
its extreme sensitivity to detergents that are required for its solubilization
and purification.
6.5.2
Evolution
RPE65
is evolutionarily
related to carotenoid cleavage oxygenases (CCOs), a group of enzymes
that catalyze the oxidative cleavage of various carotenoids and apocarotenoids
as well as certain other olefin-containing compounds, such as lignostilbenes.
443,447
However, RPE65 is the black sheep of this family, as it is not known
to possess such oxygenase activity.
133
Sequence
identity between RPE65 and other CCO members varies from ∼20
to 40%, but all of these enzymes contain an absolutely conserved set
of four His and three Glu residues that are involved in binding of
a required iron cofactor
448,449
(Figure 25). RPE65 is found only in vertebrates and can usually
be discriminated from other CCOs on the basis of its characteristic
chain length of 533 (±1–2) residues and high (≥70%)
sequence conservation. Additionally, sequence alignments have revealed
several positions where residue type can be predictive of whether
a protein likely has RPE65 activity.
410,450
The steps
in RPE65 evolution from the true CCOs remain unclear, but this process
was undoubtedly critical for the establishment of the vertebrate visual
cycle.
451
Figure 25
Structural alignment of CCO family members.
(A) Iron-binding His
residues are highlighted in orange, and second sphere Glu residues
are highlighted in blue. (B) Structural superposition of CCO members
of known structure (RPE65, orange; ACO, blue; VP14, pink). These enzymes
adopt a 7-bladed β-propeller fold (blades labeled with Roman
numerals) with a helical cap on the top face of the propeller that
houses the active site and membrane-binding domain (curved, dashed
line), which surrounds the active site entrance indicated by a yellow-green
arrow. The iron cofactor located at the center of the propeller is
coordinated by four conserved His residues (green). The two views
in panel B differ by a 90° horizontal rotation.
6.5.3
Structure
The
crystal structure
of RPE65, determined by Kiser in 2009 in the Palczewski laboratory,
has provided a basis for understanding the substantial biochemical
data obtained for this enzyme as well as structural features that
distinguish it from true CCOs
452
(Figure 26). The basic fold is a seven bladed β-propeller
capped on one face by a group of α-helices. This same fold was
also found for a cyanobacterial apocarotenoid oxygenase (ACO) enzyme,
thus confirming that the entire CCO family shares a similar three-dimensional
structure
453
(Figure 25). The propeller is sealed within blade VII in a “Velcro”
manner via interactions between the first and last
strands of the core propeller fold. An iron cofactor is located on
the propeller axis directly coordinated by the four conserved His
residues mentioned above. Retinyl esters gain access to the iron center
through a tunnel that runs along the interface between the helical
cap and the top face of the β-propeller (Figure 27). The tunnel terminates within
the protein interior, suggesting
that the substrate uptake and product egress pathways are the same.
By contrast, the structures of two bona fide CCOs,
ACO, and VP14 show two pathways to the iron center, possibly reflecting
the need for the two products of the cleavage reaction to be released
into different cellular compartments (i.e., membrane vs cytosol).
453,454
In all crystal forms reported to date, RPE65 forms a dimeric assembly
primarily mediated by an extension to the β-propeller structure
455
(Figure 26). The other
structurally characterized CCOs lack this extension and consequentially
are not dimeric (Figure 25).
Figure 26
Crystal structure of
RPE65 obtained in the presence of native microsomal
membranes. Conserved His residues (green sticks) are shown coordinating
the catalytic iron (orange spheres). The dimeric structure of RPE65
has been observed in multiple crystal forms. This results in a parallel
orientation of the membrane-binding surfaces (brown sticks), which
likely promotes membrane attachment. The membrane-binding surface
surrounds the entrance to the active site cavity outlined in magenta
mesh.
Figure 27
RPE65 active site cavity. The cavity
(gray mesh) is predominantly
lined by hydrophobic residues that facilitate retinyl ester uptake
from the membrane. The cavity passes by the catalytic iron and terminates
deep inside the enzyme core. Residues colored green have been shown
through mutagenesis studies to be important in maintaining the 11-cis specificity
of RPE65 isomerase activity.
6.5.4
Expression and Membrane
Binding
RPE65 is expressed almost exclusively in the RPE.
436,437
It localizes to the abundant smooth ER of the RPE where other retinoid
processing enzymes such as LRAT and RDH5 are found.
436,456−458
Independent studies have shown that RPE65
can associate with RDH5, possibly forming a functional complex.
459,460
Our understanding of the interaction of RPE65 with the ER membrane
has evolved considerably.
284
RPE65 was
once thought to be peripherally anchored to the ER by reversible post-translational
palmitoylation.
461
Although palmitoylation
of a particular Cys residue (position 112 in the bovine sequence)
might promote membrane attachment,
452,462
more recent
biochemical studies indicate that RPE65 has substantial integral membrane
protein character even though it has no transmembrane spanning segments.
284,459
Inspection of the RPE65 crystal structure reveals a surface enriched
in residues capable of interacting with the lipophilic core as well
as headgroups of a phospholipid membrane indicating a monotopic mode
of membrane attachment
452,455,463
(Figure 26). This surface surrounds and helps
form the entrance to the active site tunnel, allowing the enzyme to
extract hydrophobic retinoids from the membrane. The structure of
RPE65 determined in the presence of native microsomal phospholipids
455
revealed that these membrane-binding residues
undergo major conformational changes upon phospholipid removal by
detergents, consistent with prior biochemical data showing that RPE65
adopts structurally and functionally different conformations in its
membrane-bound versus detergent-solubilized states.
464
These findings provide a structural explanation for the
well-known inhibitory effects of detergents on RPE65 activity.
465
The membrane-interacting regions are arranged
in parallel (i.e., on the same side) in the RPE65 dimer, allowing
them to reinforce each other in anchoring the protein to the membrane
466
(Figure 26). This hydrophobic
patch is conserved in all CCOs reported to date, indicating that it
is a general structural feature of these enzymes, which allows the
extraction of hydrophobic substrates from lipid membranes (Figure 25).
6.5.5
Active Site and Iron
Cofactor Binding
The active site cavity of RPE65 is lined
primarily by nonpolar
side chains, including several aromatics (Tyr, Phe, and Trp residues),
consistent with the hydrophobic nature of the retinyl ester substrate
(Figure 27). The relatively narrow width of
the tunnel indicates that a retinyl ester would have to enter the
active site in an extended conformation. A complex structure between
RPE65 and an intact retinyl ester has not yet been experimentally
determined, so the orientation of retinyl ester entry remains uncertain.
However, modeling studies
467,468
as well as the location
of a putative fatty acid molecule observed in the active site of several
RPE65 crystal structures
455
(vida
infra) suggest that the retinoid part of the retinyl ester
enters first. Like other CCOs, RPE65 contains a ferrous iron cofactor
within its active site directly coordinated in a distorted octahedral
or trigonal bipyramidal fashion by His residues 180, 241, 313, and
527 (bovine and human sequence numbering) with Fe–Nε bond lengths between 2.1 and
2.2 Å. A second sphere of highly
conserved and functionally important Glu residues 148, 417, and 469
hydrogen bond with the Nδ atoms of His residues 241, 313, and
527, respectively. The open iron coordination sites (i.e., sites not
occupied by protein ligands) contain electron density that has been
attributed to a bound fatty acid molecule coordinating the iron through
its free carboxylate moiety based on crystallographic and extended
X-ray absorption spectroscopy (EXAFS) data.
455
Notably, iron–carboxylate interactions have been observed
in a number of proteins containing the 4-His iron-binding motif, including
photosystem II, 15-lipoxygenase, and the photosynthetic reaction center.
469−471
The functional importance of several RPE65 active site residues
has been probed either directly through site-directed mutagenesis
or indirectly by identifying RPE65 mutations that give rise to type
II (RPE65-associated) LCA. Mutations in the iron-coordinating His
residues,
447
the second sphere Glu residues,
472−474
and even a third sphere of residues that form hydrogen bonding interactions
with the second-sphere Glu residues
468,475
either abolish
or greatly reduce RPE65 activity. In general, any change in active
site amino acid composition is detrimental for activity. Interestingly,
mutations of a few residues in close proximity to but not directly
binding the iron cofactor, namely Phe103, Thr147, Tyr338, and Phe526,
can alter RPE65 product specificity by changing the ratio of 11-cis to 13-cis-retinol
isomers produced
(Figure 27) (vide infra).
467,468
Moreover, even wild-type RPE65 in native membranes produces some
13-cis-retinol.
171,476
7
Mechanisms of Retinoid Isomerization
The heart of the
retinoid cycle is the isomerization reaction.
The reaction catalyzed by RPE65 consists of two steps: a trans/cis alkene bond isomerization
and an ester bond cleavage. Because of
this dual activity, RPE65 is frequently referred to as an isomerohydrolase,
although it is not hydrolytic water that mediates this process.
7.1
Acyl versus O-Alkyl Cleavage
in the Hydrolysis of Esters
The ester cleavage reaction catalyzed
by RPE65 is not an ordinary ester bond hydrolysis whereby attack of
water on the acyl carbon generates a tetrahedral intermediate that
collapses to form a carboxylic acid and an alcohol (Figure 28). Instead, the ester
dissociates by cleavage of
the O-alkyl bond. Owing to this distinction, we prefer
to refer to the process as ester “cleavage” rather than
“hydrolysis.” This unusual reaction was identified by
using various isotopically labeled retinols as substrates for the
isomerization reaction. It was found that a stereochemical inversion
of carbon 15 occurs
477,478
and that the 15-hydroxy oxygen
is lost
171
and replaced by bulk water-derived
oxygen during the isomerization.
452
Because
11-cis-retinol is thermodynamically less stable than all-trans-retinol by ∼4 kcal/mol,
181
it has been argued that ester cleavage, which typically
releases ∼5 kcal/mol of free energy, could be used to drive
the isomerization reaction.
479
The requirement
of CRALBP and other binding proteins for robust 11-cis-retinol production by RPE65
indicates that product release could
be rate limiting.
476
The unusual ester
cleavage reaction instead seems to be more important in overcoming
the ∼36 kcal/mol activation energy barrier of the double bond
isomerization.
171
The retinoid polyene
chain is relatively rigid, owing to favorable continuous π-orbital
overlap. Thus, in order for isomerization of these double bonds to
occur at physiological temperatures, the carbon–carbon bond
order must be temporarily lowered.
Figure 28
Acyl versus O-alkyl
ester cleavage.
7.2
Binuclear
Nucleophilic Substitution Mechanism
for Retinoid Hydrolysis/Isomerization
Taking into account
these biochemical data, three different mechanisms of RPE65-dependent
retinoid isomerization have been proposed. The first, proposed by
Rando and colleagues, involves the attack of an enzyme-associated
nucleophile on C11 of the retinyl ester with simultaneous ester dissociation
and shuffling of the double bonds (Figure 29). The enzyme-linked retinoid intermediate
could undergo low-energy
rotation around the 11–12 single bond to a cis-like conformation. Attack of water
or hydroxide on C15 would lead
to a reshuffling of the double bonds and dissociation of the retinoid–enzyme
covalent intermediate locking the retinoid in an 11-cis configuration. This mechanism,
which can be classified as a dual
SN2′ nucleophilic substitution reaction, predicts
a high degree of 11-cis-retinol product specificity
for RPE65. However, RPE65 does not exhibit such specificity
468
and can readily produce 13-cis or all-trans-retinol, depending on the reaction
conditions and retinoid-binding proteins used for the assay.
476
Structurally, RPE65 also does not possess a
suitable nucleophile such as a Cys residue in its active site.
452
Figure 29
Binuclear nucleophilic substitution mechanism
of retinoid hydrolysis/isomerization.
The key feature is formation of a covalent enzyme–retinoid
intermediate that allows rotation around the 11–12 bond.
7.3
Unimolecular
Nucleophilic Substitution Mechanism
for Retinoid Isomerization
A second mechanism, postulated
by Palczewski and colleagues, proposes a key role for a carbocation
intermediate in the isomerization reaction (Figure 30). In this reaction involving
an SN1′ nucleophilic
substitution mechanism, ester dissociation via O-alkyl
cleavage removes electrons from the polyene, thereby generating a
retinylic cation with reduced carbon–carbon bond order allowing
bond rotation to take place. Quantum mechanical calculations indicate
that the activation energy of trans/cis isomerization at the 11–12 bond is reduced
to about 18 kcal/mol,
consistent with the experimentally determined energy of activation.
171
The aromatic residues lining the active site
cavity of RPE65 are ideal for stabilizing carbocation intermediates.
480
Following rotation of the 11–12 bond,
likely under steric influence by the enzyme active site, water or
hydroxide attacks C15, quenching the carbocation with the 11–12
double bond in a cis configuration. The interior
cavity of RPE65 indeed has a curved shape that can accommodate cis retinoid isomers
(Figure 27).
Importantly, this mechanism allows for the generation of alternative
isomers such as the experimentally observed 13-cis-retinol.
171,468
As discussed earlier, retinylic
cation formation from retinol or retinyl esters is facilitated by
Brønsted or Lewis acids. The iron-coordinated fatty acid observed via crystallographic
and XAS data suggests that the RPE65
iron cofactor could promote catalysis by coordinating the ester moiety
and facilitating its dissociation.
455
This
mechanism thus bears substantial similarity to the well-characterized
Carr–Price type reactions described earlier, with the exception
that 11-cis-retinol rather than anhydroretinol is
generated in the RPE65 active site.
Figure 30
Unimolecular nucleophilic substitution
mechanism of retinoid isomerization.
The key feature is the generation of a carbocation (retinylic cation)
intermediate with lowered bond order that allows rotation around the
11–12 bond to occur. Dissociation of the ester moiety can be
facilitated by a Brønsted or Lewis acid catalyst (X).
7.4
Radical Cation Mechanism
for Retinoid Isomerization
A third mechanism, recently proposed
by Redmond and colleagues,
posits a role for a retinyl radical cation intermediate in the isomerization
reaction (Figure 31). This proposal is based
on the finding that certain radical spin-trap compounds, for example N-tert-butyl-α-phenylnitrone
(PBN)
can inhibit RPE65-mediated isomerization in an uncompetitive manner.
481,482
In this proposal, removal of a single π electron from the
polyene by an unknown electron acceptor generates a retinyl radical
cation intermediate that can undergo double bond rotation to form cis isomers followed
by quenching of the radical cation
to form an 11-cis-retinyl ester. Ester cleavage then
occurs to produce 11-cis-retinol. Reversal of the
isomerization and ester cleavage steps in relation to the carbocation
mechanism was proposed based on the finding that all-trans-retinyl ester could not
easily be accommodated in the RPE65 active
site whereas 11-cis- and 13-cis-retinyl
esters could.
467
However, more research
is required to develop the mechanistic details of this scheme.
Figure 31
Radical cation
mechanism of retinoid isomerization.
8
Retinoid-Binding Proteins Relevant to Retinoid
Transport to the Eye and the Visual Cycle
Because retinoids
quickly equilibrate between membranes by passage
through the aqueous phase,
483,484
part of their transport
likely takes place by passive diffusion. But this process could be
facilitated by ATP-dependent transporters such as ABCA4
76,485−487
or the channel-like property of STRA6.
488−492
Finally retinoids are readily oxidized and isomerized; thus, a set
of binding proteins evolved to protect them from these undesirable
reactions as well as defend surrounding molecules from retinoid (photo)reactivity.
59,73,171,279,285
STRA6, a key transporter involved
in retinol transfer between the RPE and choroidal circulation. and
its homologue RBPR2 involved in retinol uptake by the liver
493
will be reviewed in detail in another article
in this partial thematic issue of Chemical Reviews. Thus, they will not be discussed
here.
8.1
Retinoid-Binding
Proteins Involved in the
Retinoid Cycle
The hydrophobic nature of retinoids limits
their ability to diffuse in aqueous environments. This property presents
a barrier to retinoid transport from storage sites to sites of utilization
as well as between cellular membranes. Retinoid-binding proteins overcome
this barrier by reversibly binding and sequestering retinoids away
from water in internal cavities, greatly facilitating their diffusion.
A number of retinoid-binding proteins have been characterized.
494
Here, the discussion focuses on those binding
proteins involved in transport of retinols and retinals.
When
required by the body, vitamin A liberated from its hepatic storage
sites in Stellate cells by retinyl ester hydrolysis is complexed with
a 21 kDa retinoid-binding protein belonging to the lipocalin family
called retinol-binding protein 4 (RBP4) to form holo-RBP4.
24
The main structural feature of holo-RBP4, determined
crystallographically in the early 1980s, is an eight-stranded β-barrel
core containing a single retinol-binding site (Figure 32).
495
all-trans-Retinol is oriented in the binding pocket with its hydroxyl group
facing the solvent and the retinyl moiety snuggly bound by a number
of nonpolar residues in a highly complementary fashion, explaining
the high binding specificity toward the all-trans isomer. Holo-RBP4 circulates in
the plasma in complex with a second
protein called transthyretin, which by increasing the molecular weight
of the overall complex prevents holo-RBP4 from being excreted in the
urine.
24,496
Figure 32
Structures of retinoid-binding proteins involved
in the retinoid
cycle. (A) RBP4, (B) Module 2 of X. laevis IRBP,
(C) CRBP I, and (D) CRALBP. Bound retinoids are depicted as sticks
with carbon atoms colored orange.
Inside cells, retinol is transported between membranes in
complex
with a second binding protein called cellular retinol-binding protein
(CRBP). A member of the intracellular lipid-binding protein family,
this compact 15 kDa protein is evolutionarily unrelated to RBP4.
497
Two isoforms of the protein, CRBP I and CRBP
II with ∼50% sequence identity depending on the species, exhibit
differential tissue expression.
494
CRBP
I is expressed in many tissues, including the eye, whereas CRBP II
expression is restricted to the intestine. The structure of CRBP,
similar to that of RBP4, features a β-barrel fold that houses
the retinoid-binding site.
495
However,
the orientation of retinol in the binding pocket is reversed in relation
to that in holo-RBP4 with the β-ionone nearest the mouth of
the pocket and the hydroxyl group hydrogen bonding with a Glu residue
at the base of the cavity. An important role for CRBP in all-trans-retinol trafficking
in the retina has been demonstrated in CRBP I
–/– mice, which show defective
transport of all-trans-retinol from photoreceptors
to ER membranes of the RPE. This is evidenced by accumulation of all-trans-retinol
in the retina and reduced formation of
retinyl esters in the RPE.
498
Two
retinoid-binding proteins are specifically expressed in the
eye.
494
The first of these is an intracellular-binding
protein CRALBP that displays a binding preference for 11-cis-retinoids.
499
CRALBP is expressed predominantly
in two retinal locations, the RPE and Müller glial cells, where
it plays a key role in visual chromophore regeneration by binding
11-cis-retinoids, protecting them from esterification
or from photo- or thermal isomerization and facilitating their intracellular
transport.
476,500,501
11-cis-Retinal and 11-cis-retinol
are the main retinoids associated with CRALBP isolated from bovine
retina.
499,502
It can also effectively bind 9-cis-retinoids as well but not 13-cis-retinoids.
502
The gene encoding CRALBP, RLBP1, was cloned in 1988
503
and was localized
to human chromosome 15.
504
CRALBP is one
of the founding members of the CRAL-TRIO protein family, members of
which share a common three-dimensional architecture.
505−507
The crystallographic structure of the CRALBP–11-cis-retinal complex has been determined,
revealing a curved binding
pocket with high shape complementarity to 9-cis and
11-cis retinoid isomers, consistent with its ligand-binding
preferences.
507
The binding pocket completely
shields the retinoid from solvent. Bound 11-cis-retinal
is found in a 6-s-trans, 11-cis,
twisted 12-s-cis configuration. The near perfect cis configuration of the 11–12 double
bond imposed
by the retinoid-binding pocket is likely important to prevent unwanted
photoisomerization and thermal isomerization. A patch of basic amino
acid residues on the protein’s surface probably mediates the
interaction of the protein with acidic phospholipids, which induce
dissociation of the bound retinoid.
508
Mutations
in the CRALBP gene are associated with several retinal diseases, including
retinitis pigmentosa, Newfoundland rod-cone dystrophy, fundus albipunctatus,
and Bothnia retinal dystrophy.
509
The second eye-specific retinoid-binding protein is a soluble lipoglycoprotein
called interphotoreceptor-binding protein (IRBP). This large, 136
kDa protein is produced by photoreceptors and secreted into the interphotoreceptor
matrix, where it is the most abundant extracellular protein. Unlike
other binding proteins that contain a single retinoid-binding site,
IRBP has at least three high affinity sites. The protein can also
bind several isomeric forms of retinol and retinal but has a preference
for all-trans and 11-cis-retinoids.
510,511
IRBP can also bind a number of nonretinoid, hydrophobic ligands,
but the physiological significance of this capability is not currently
understood.
512
The retinoid-binding preferences
and localization of IRBP indicate that it participates in the retinoid
cycle by transporting retinoids between photoreceptors, RPE, and Müller
cells. Interestingly, IRBP knockout mice do not exhibit acute problems
with vision or dark adaption, and no human disease has been attributed
to mutations in the gene encoding IRBP. The protein consists of four
homologous “modules” generated by gene duplication.
512
Full-length IRBP, as observed by negative stain
electron microscopy, adopts a rod-shaped structure that is flexible
and undergoes major conformational changes in the presence of retinoids.
513
The crystal structure of an isolated module
from X. laevis IRBP has been determined, revealing
a two-domain architecture.
514
Domain A
adopts a ββα-spiral fold whereas domain B forms
an αβα sandwich. Although a retinoid-bound structure
has not yet been obtained, two candidate-binding sites have been identified
through molecular modeling: one in the hinge region connecting the
two domains and a second in domain B. Notably, domain B of the IRBP
module shares structural similarity with the ligand-binding domain
of CRALBP.
83
8.2
Structure
and Function of ATP-Binding Cassette
Transporter Member 4 (ABCA4)
The metabolism of vitamin A
in the eye involves a complex interplay between over twenty different
proteins. The flow of retinoid substrates and products between components
of the cycle depends on specific binding proteins and transporters.
The retinal-specific ATP-binding cassette transporter, ABCA4, plays
a special role in this process. ABCA4, a member of the ABCA transporter
subfamily, is predominantly expressed in the outer segments of photoreceptor
cells where it is located in the rims of rod disk membranes and cone
incisures.
515−517
As indicated by numerous biochemical studies
and the phenotype of Abca4
–/–
mice, the main function of this transporter is to accelerate
the clearance of all-trans-retinal from ROS/COS.
401,518−520
Human ABCA4 is an integral membrane
protein with 2273 residues that form two homologous but nonidentical
parts. Each carries six membrane-spanning helices that constitute
a TMD, a soluble cytoplasmic domain (CD) that hosts a canonical nucleotide-binding
site (NBD) with Walker A and Walker B motifs characteristic of ATP-processing
enzymes, and an exocytoplasmic (intradiscal) domain (ECD) (Figure 33).
76
The overall topological
model for ABCA4 is supported by multiple glycosylation sites identified
in both ECD domains, whereas CD1 hosts multiple phosphorylation sites.
76,521,522
The structure of native bovine
ABCA4 has been determined by negative stain electron microscopy to
a resolution of 18 Å, revealing the overall shape of the molecule.
523
On the basis of the accepted model of ATP-driven
transport across a lipid membrane, binding of a molecule to be transported
increases the affinity of NBDs for ATP. The nucleotide then induces
a conformational change of NBDs that come in close contact to form
a dimer with the two nucleotide molecules positioned at its interface.
This movement is translated onto a TMD that induces the translocation
of the substrate molecule across the lipid membrane. Such conformational
changes have indeed been observed by electron microscopy and hydrogen–deuterium
exchange.
523
Subsequent hydrolysis of ATP
and dissociation of ADP prompt the separation of NBDs, returning the
transporter to its initial state and completing the cycle. In the
absence of substrate, ABC transporters undergo cycles of slow ATP
hydrolysis by individual NBDs, resulting in a basal ATPase activity.
Although numerous lipids stimulate ATP hydrolysis by ABCA4, all-trans-retinal or its
phosphatidylethanolamine (PE) conjugate, N-retinylidene-PE (N-ret-PE) are the preferred
substrates.
524,525
The topology of this protein
in combination with the logic of the visual cycle suggest that ABCA4
acts as an importer, an assumption recently confirmed experimentally.
525
Interestingly, ABCA4 still remains the only
known example of an importer among eukaryotic ABC transporters.
Figure 33
Structure
and function of ABCA4. (A) Two-dimensional topology diagram
of ABCA4. Positions of the Walker A motifs are indicated by blue dashed
lines within the CDs. Glycosylation sites are marked with red stars,
and an intramolecular disulfide bridge is indicated by S–S.
ECD, exocytoplasmic domain; CD, cytoplasmic domain. (B) Electron microscopic
structure of ABCA4 and its dimensions relative to a ROS disk rim.
TMDs, transmembrane domains. (C) Structural differences in ABCA4 in
the absence and presence of ATP. (D) Role of ABCA4 in the visual cycle
and pathology of elevated all-trans-retinal. ABCA4
flips the all-trans-retinal–PE complex, a
product of all-trans-retinal (red line structure)
condensation with PE (black line structure with blue sphere indicating
the headgroup), to the outer leaflet of the disk membrane, allowing
dissociation of the complex and subsequent reduction of all-trans-retinal to all-trans-retinol,
which then reenters
the visual cycle (left). Retinal pathology is observed in mice lacking
ABCA4 and RDH8 activities due to accumulation of all-trans-retinal and its lipid adducts
(right).
The importer activity of ABCA4 has consequences for vitamin
A metabolism
in photoreceptors (Figure 33C). Decay of photoactivated
rhodopsin results in hydrolysis of the opsin-retinylidene Schiff base
bond followed by subsequent release of all-trans-retinal
into the disk membrane.
84
The next step
in the visual cycle is reduction of newly liberated all-trans-retinal to all-trans-retinol
by all-trans-RDHs (mainly RDH8 in mouse retina), which associate with the cytoplasmic
side of the disk bilayer.
64,400
The relatively high
hydrophobicity of all-trans-retinal allows its rapid
partition between the inner and outer leaflets of disk membranes,
ensuring its accessibility to RDHs.
526,527
However,
the high chemical reactivity of aldehydes also leads to spontaneous
formation of all-trans-retinal adducts with primary
and secondary amines. In the biological membrane environment, all-trans-retinal reacts
predominantly with PE to form N-ret-PE that, similar to phospholipids, cannot freely
flip
between the inner and outer leaflets of the lipid bilayer. In a test
tube, the formation of N-ret-PE in a mixture of chloroform/methanol
(2:1) occurs with a bimolecular rate constant of about 3.75 ×
10–2 M–1 s–1 while the rate of its hydrolysis is about 7.9 × 10
–6 s–1.
528
This reversible reaction is highly dependent on the presence
of water. The time scale for this process in vivo remains to be elucidated. Upon a
45% bleach of rhodopsin in wild-type
mice, about 24% of released all-trans-retinal was
detected in the form of a conjugate with PE.
529
Thus, the main role of ABCA4 could be aiding the movement of N-ret-PE to the cytoplasmic
side of disk membranes. Because
the Schiff base bond in N-ret-PE is susceptible to
hydrolysis, the resulting all-trans-retinal eventually
can be metabolized to all-trans-retinol. Taking into
account the partition of retinals into the lipid bilayer and a relatively
slow rate of ATP hydrolysis by ABCA4 equivalent to three enzymatic
cycles per second, one can assume that only a fraction of the total all-trans-retinal
is processed by ABCA4.
530
Nevertheless, the activity of ABCA4 turns out to be essential
for lowering all-trans-retinal concentrations below
the threshold that could cause photoreceptor toxicity.
401
8.3
Condensation Reactions
of Retinal
Delayed clearance of all-trans-retinal from the
photoreceptors after light exposure can have dramatic pathophysiological
consequences. In cultured ARPE-19 cells derived from human RPE cells,
exposure to 10 μM all-trans-retinal caused
profound cytotoxicity after less than 1 min of incubation by inducing
a Ca2+-driven apoptotic cell death pathway.
318
Because rhodopsin concentration in ROS reaches
4 mM,
257
bleaching of just 0.5% of the
total amount could potentially generate toxic levels of all-trans-retinal if this
retinoid is not efficiently removed from the retina.
The mechanism of all-trans-retinal toxicity involves
the generation of superoxide radical, singlet oxygen, and peroxides
when irradiated with UVA or blue light.
279
In addition, all-trans-retinal can stimulate increased
levels of reactive oxygen species in a NADPH oxidase-dependent manner.
531,532
Unless quickly dissipated, such reactive oxygen species can cause
oxidative damage to lipids and proteins that compromise photoreceptor
structure and function.
533
Yet another
consequence of elevated all-trans-retinal concentrations
is the accelerated formation and accumulation of bis-retinoid lipofuscin
chromophores within retina and RPE.
314,401,520,534,535
Although formation of the Schiff base adduct of all-trans-retinal with PE is fully
reversible, reaction of N-ret-PE with a second molecule of all-trans-retinal
can initiate a cascade of irreversible nonenzymatic conversions that
lead to the production of fluorescent diretinal compounds, including
pyridinium bisretinoid (A2E) (Figure 34) and
retinaldehyde dimer (RALdi) (Figure 35). The
common precursor in the biosynthesis of these fluorophores is protonated N-ret-PE,
that undergoes spontaneous tautomerization via an H-shift [1,6] generating phosphatidyl
analogs of
enamine.
536,537
The subsequent reaction with
a second molecule of all-trans-retinal can occur
through amine condensation followed by 6π-electrocyclization
to generate a reduced form of A2E, phosphatidyl-dihydropyridine bisretinoid
(A2PE-H2) (Figure 34). Alternatively,
the nucleophilic carbon 20 of the N-ret-PE tautomer
can react with carbon 13 of all-trans-retinal via [1,4] conjugate addition. Consequent
Mannich condensation
then leads to the RALdi precursor (Figure 35).
534
The strong kinetic isotope effect
observed in RALdi synthesis as compared to that found for A2E supports
this reaction mechanism
537,538
Aromatization of the
dihydropyridine in A2PE-H2 eliminates two hydrogens to
yield phosphatidyl-pyridinium bisretinoid (A2PE) whereas the RALdi
precursor spontaneously rearranges to eliminate the amino group of
PE (Figures 34 and 35, respectively). In the final stage of A2E biosynthesis, its
immediate
precursor, A2PE, is either hydrolytically cleaved by enzymatic action
of phosphodiesterase, which can occur in ROS before internalization
by the RPE, or undergoes nonenzymatic acid-catalyzed hydrolysis inside
RPE phagosomes.
314
As a result of incomplete
lysosomal degradation, these byproducts of retinal metabolism accumulate
in the RPE as residual bodies called lipofuscin. These bodies provide
a useful fluorescent marker for lipofuscin quantification in the living
eye, which can serve as long-lasting evidence of excessive past accumulation
of all-trans-retinal.
539−541
Indeed, the primary
fluorescent components of lipofuscin are all-trans-retinal conjugates such as A2E
and RALdi.
542
A2E was shown to be further metabolized by horseradish peroxidase,
543
but there is yet no evidence that this process
occurs in vivo. Lipofuscin granules contain several
chromophores absorbing UV, blue, and green light whereas A2E acts
as an acceptor of energy from their photoexcited states and dissipates
that energy mainly by thermal deactivation and partly by emitting
fluorescence.
544
Considering that lipofuscin
granules contain potent photosensitizers, quenching of excited states
of lipofuscin photosensitizers by A2E may play a protective antioxidant
role. Yet, A2E also exhibits some minor photoreactivity and several
experiments in vitro demonstrated that A2E and its
oxidation products can be cytotoxic to RPE cells and trigger complement
activation and inflammatory signaling.
317
It remains to be established whether or not these deleterious properties
of A2E are physiologically relevant.
Figure 34
Mechanism of A2E formation from all-trans-retinal
and phosphatidylethanolamine (R-NH2).
Figure 35
Mechanism of all-trans-retinal dimer formation
from all-trans-retinal and phosphatidylethanolamine
(R-NH2).
The connection between
delayed all-trans-retinal
clearance, lipofuscin buildup, and retinal degeneration is exemplified
by Stargardt disease, a human condition in which juvenile macular
degeneration is caused by mutations in both alleles of the ABCA4 gene.
487,545
Consequently, retinal samples
collected from these patients revealed elevated levels of N-retinylidene-PE
and overaccumulation of A2E in the RPE.
529
Because the cytotoxicity of retinal conjugates has been widely accepted,
it is believed that the precipitating cause of retinal degeneration
in Stargardt patients is the deterioration of RPE cells responsible
for maintenance of photoreceptors.
546
However,
recent studies of Abca4
–/–
Rdh8
–/–
double knockout mice that closely recapitulate human retinal
pathological conditions indicate that all-trans-retinal
itself may play a decisive role in light-induced photoreceptor degeneration.
318,320,531
9
Aberrations
in the Retinoid Cycle and Human
Retinal Diseases
Studies of mutations in the retinoid cycle
genes can teach us about
the structure–function of key visual proteins and enzymes as
well as the cell biology of rod photoreceptor cells, one of our most
metabolically active neurons. Most of the genes encoding retinoid
cycle enzymes are associated with retinal disease (Figure 36).
64,76,77,285,547−552
For example, point mutations in the rod opsin gene are the most
common cause of autosomal dominant retinitis pigmentosa (RP). The
most frequent mutation and the first identified as causing blindness
is P23H,
553
accounting for 10% of human
cases of autosomal dominant RP. Only a few mutations, including severe
truncation of the opsin gene
554
and the
c.448G > A (p.E150K) mutation,
555,556
are inherited
in an
autosomal recessive manner. RDH12, LRAT, and RPE65, when inactivated
by mutations, cause juvenial forms of blindness called LCA. For RPE65
the severity of disease has been associated with the degree of residual
enzymatic activity.
274,281,557
Mutations in the transporter ABCA4 cause accumulation of condensation
products of all-trans-retinal.
520
Here, lack of a functional transporter causes Stargardt
disease,
545,558,559
a juvenial form of macular degeneration, whereas some other changes
were demonstrated to be associated with AMD.
560,561
Mutations in other genes, such as those encoding RDH5 or IRBP, are
associated with slowly progressive retinal degeneration.
562−565
Figure 36
Retinal diseases caused by defects in visual cycle enzymes. Therapeutic
agents used in the treatment of these conditions are indicated.
This relationship between genetic
mutations and retinal disease
also provides information about the physiological role of these proteins
in the chemistry of the retinoid cycle, as revealed by many animal
model studies. Such generated animal models are also required for
testing possible remedies for these blinding diseases.
566
In some cases the treatment is highly pertinent
because of the significant number of patients involved and the socioeconomic
cost of their associated blindness. In other cases, the small number
of affected individuals decreases the likelihood of commercially developing
a possible treatment. But animal studies are still vital for those
afflicted and also may lead to breakthroughs applicable to more common
retinal diseases.
Natural and genetically altered animal models
have been used to
investigate the effects of retinoid supplementation in treatment.
Thus, blockage in production of 11-cis-retinal was
overcome with oral delivery of another photosensitive but chemically
more stable retinoid, 9-cis-retinal (Figure 36)
567−571
(reviewed in ref (76)). Retinylamine and other primary amines were employed to buffer
the toxic effect of all-trans-retinal either by inhibiting
the visual cycle or by chemically trapping an excess of all-trans-retinal in the form
of a Schiff base when it could not be effectively
cleared from photoreceptor cells by reduction to all-trans-retinol (Figure 36).
318,572,573
The toxicity of all-trans-retinal could be a major contributor to retinal degeneration
in
several human diseases, including Stargardt disease.
318,401,570
Moreover, there are also experimental
therapies that utilize carotenoids, retinoid precursors that comprise
an integral part of human macula.
574
The
therapeutic landscape for inherited and acquired retinal diseases
is rapidly evolving. Just a few years ago, it was difficult to imagine
strategies for treating these diseases with practical pharmacological
approaches. But today, many solid scientific findings provide hope
that these chronic diseases will become manageable.
10
Final Conclusions: The Retinoid Cycle and Whole
Body Retinoid Metabolism
Vitamin A must be adequately distributed
within the body to maintain
the biological function of retinoids in the peripheral tissues and
the production of visual chromophore in the eye. Transport of vitamin
A is facilitated by RBP4, and its cellular receptor, STRA6, functionally
couples with LRAT via CRBP. Recent studies by von
Lintig and co-workers suggest that ocular vitamin A uptake is favored
over other peripheral tissues in vitamin A deficient states.
492
In contrast to other cell types, an overdose
of
all-trans
-retinol does not result
in excessive accumulation of retinyl esters in the RPE.
567,575
Although the pivotal role of STRA6 and LRAT in vitamin A uptake
is widely recognized, the mechanisms that govern “buffering”
of vitamin A within the eye still remain unknown. This also applies
to the potential role of light in the regulation of retinoid metabolism,
especially the rate of the retinoid (visual) cycle. Despite early
work showing that retinal G protein-coupled receptor, expressed in
the RPE, provides light-dependent modulation of all-trans-retinyl ester synthesis
and degradation as well as influences RPE65
activity in mice,
576
the molecular mechanism
of light-induced stimulation of retinoid isomerization remains an
intriguing biological mystery.
A classic unsolved problem is
how retinyl esters traffic from lipid
storage droplets in the retina to the ER and RPE65. It is still unknown
if retinyl palmitate is transported intact or needs to undergo hydrolysis
followed by re-esterification by LRAT in the ER close to RPE65 to
sustain robust retinoid isomerization. Yet another aspect related
to the organization of vitamin A metabolism in the eye is the putative
close interaction of proteins involved in this process. Despite several
reports indicating interaction of particular proteins, the isomerization
complex containing RPE65, RDH5, LRAT, or CRALBP has not been purified
or reconstituted in vitro. Determining the molecular
details of all-trans-retinol processing proteins
at the atomic level through structural biology represents a complementary
approach to biochemical studies. Until recently, progress in this
field has been marked only by NMR or crystal structures of soluble
retinoid-binding proteins such as CRBPs, IRBP, and CRALBP.
507,577,578
The structure of RPE65
452,455
definitely provided an incentive for obtaining structures of other
membrane associated proteins including LRAT and RDHs.
Further
structural studies of RPE65 and other CCOs with the goal
of obtaining high resolution complexes with retinoids and related
compounds will help resolve lingering issues concerning the mechanism
of the retinoid isomerization reaction. Spectroscopic approaches,
which are becoming more feasible with the advent of improved expression
and purification methods, will also be of great utility in unraveling
the fine details of the bioinorganic chemistry of this enzyme family.
Analysis of the isomeric composition of retinoids in rod- and cone-dominant
retinas reveals striking differences.
579
Significant amounts of 11-cis-retinyl esters and
11-cis-retinol in ground squirrel and chicken retinas
not observed in rod-dominant mice, rat, or cow are associated with
alternatives to RPE65 and LRAT enzymatic activities responsible for
formation of these retinoids.
302,305,430
These findings suggest the existence of an unconventional cone-specific
visual chromophore regeneration pathway. Though supported by biochemical
data, this pathway remains only a concept without molecular identification
of its protein components. Thus, efforts to clone or purify enzymes
involved in cone regeneration currently represent an exciting challenge.
Newly generated 11-cis-retinal is needed to reform
light-sensitive visual pigments. Thus, another challenging question
in studies of the rhodopsin (and other visual pigments) cycle is to
determine how the chromophore enters and exits the binding pocket.
In short, recent decades have witnessed an improved understanding
of the retinoid cycle and retinoid metabolism in general. Indeed,
it would be improper to treat the retinoid cycle as a separate entity
because it is so dependent on the metabolism of the whole organism
(Figure 37). The coming years will certainly
bring further molecular characterization of these processes from approaches
involving combinations of human molecular genetics, characterization
of newly generated animal models, advanced imaging techniques, and
structural studies.
Figure 37
Key proteins involved in the transport of retinol from
the liver
to target tissues. Retinol travels in the circulation bound to RBP.
In turn, RBP complexes with a transthyretin (TTR) tetramer, which
prevents filtration of RBP across the glomeruli of the kidney. Holo-RBP
can dissociate from the TTR tetramer and bind to the retinol membrane
transporter, STRA6. all-trans-Retinol is picked up
on the cytoplasmic side of STRA6 by CRBP, which shuttles the retinoid
to the ER of the RPE. There it is esterified by LRAT to form all-trans-retinyl-esters,
which are either used as substrates
for visual chromophore production or stored in lipid bodies known
as retinosomes: atROL, all-trans-retinol; atRE, all-trans-retinyl ester.