Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

JBC Papers in Press. Published on November 6, 2019 as Manuscript RA119.011169

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.011169

Jianye Zhang^{1, *}, Elliot H. Choi^{1,2, *}, Aleksander Tworak^{1, *}, David Salom^{1, *}, Henri Leinonen¹,

Christopher L. Sander^1,2, Thanh V. Hoang³, James T. Handa⁴, Seth Blackshaw^3,4,

Grazyna Palczewska⁵, Philip D. Kiser⁶, Krzysztof Palczewski^1,#

¹Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine, CA

92697;

²Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106;

³Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of

Medicine, Baltimore, MD 21205;

⁴Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD

21287;

⁵Polgenix, Inc., Department of Medical Devices, 5171 California Ave., Suite 150, Irvine, CA

92617;

⁶Department of Physiology and Biophysics, University of California, Irvine, CA 92697 and the

Research Service, VA Long Beach Healthcare System, Long Beach, CA 90822.

*equal contributions

Running title: Photic generation of 11-cis-retinal in the RPE

^#Correspondence should be addressed: Krzysztof Palczewski, Ph.D.: Gavin Herbert Eye Institute

and the Department of Ophthalmology, University of California, Irvine, CA 92697, USA;

kpalczew@uci.edu; Tel. (949) 824-6527

Keywords: enzymatic photoisomerization, 11-cis-retinal,13-cis-retinal, visual chromophore,

RPE-retinal G protein-coupled receptor (RGR), retinal pigment epithelium-specific 65 kDa protein

(RPE65), visible light, cellular retinaldehyde-binding protein (CRALBP), rhodopsin, cone opsin

ABSTRACT: Photoisomerization of the 11-cis-

retinal chromophore of rod and cone visual

pigments to an all-trans configuration is the

initiating event for vision in vertebrates. The

regeneration of 11-cis-retinal, necessary for

sustained visual function, is an endergonic

process normally conducted by specialized

enzyme systems. However, 11-cis-retinal also

can be formed through reverse

photoisomerization from all-trans-retinal. A non-

visual opsin known as retinal pigment

epithelium (RPE)-retinal G protein–coupled

receptor (RGR) was previously shown to

mediate visual chromophore regeneration in

photic conditions, but conflicting results have

cast doubt on its role as a photoisomerase.

Here, we describe high-level production of 11-

cis-retinal from RPE membranes stimulated by

illumination at a narrow band of wavelengths.

This activity was associated with RGR and

enhanced by cellular retinaldehyde-binding

protein (CRALBP), which binds the 11-cis-

retinal produced by RGR and prevents its re-

isomerization to all-trans-retinal. The activity

was recapitulated with cells heterologously

expressing RGR and with purified recombinant

RGR. Using an RGR variant, K255A, we

confirmed that a Schiff base linkage at Lys-255

is critical for substrate binding and

isomerization. Single-cell RNA-Seq analysis of

1

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

the retina and RPE tissue confirmed that RGR

is expressed in human and bovine RPE and

Müller glia, whereas mouse RGR is expressed

in RPE, but not in Müller glia. These results

provide key insights into the mechanisms of

physiological retinoid photoisomerization and

suggest a novel mechanism by which RGR, in

concert with CRALBP, regenerates the visual

chromophore in the RPE under sustained light

conditions.

exposure can result in high isomerization rates

that may exceed the regeneration capacity of the

visual cycle, particularly for foveal cones (21).

This imbalance may be countered by the

photochemical production of 11-cis-retinal under

sustained light conditions. For example, it is

known that exposure of all-trans-retinal-

containing solutions to light results in the

formation of a sizable fraction of 11-cis-retinal

even though this isomer makes up a minority of

the thermal equilibrium mixture (4). In vivo, it has

been observed that blue light also can cause the

regeneration of rhodopsin through direct

photoreversal processes (9) or by

INTRODUCTION

photoisomerization of all-trans-retinal-

Our visual system is based on the

phosphatidylethanolamine adducts (22,23).

However, due to the relatively low quantum

yields of these photoisomerization reactions and

photoisomerization of visual pigments in rod and

cone photoreceptors (1-3). The forward

bleaching reaction (cis-trans retinal

the gradual loss of hydrolyzed all-trans-retinal

(through reduction and trapping in the RPE),

these processes would serve only to slow the

depletion of visual pigments rather than provide

a means to sustain their levels in the face of high

illumination. They also lack specificity and could

generate non-physiological toxic isomers such

as 13-cis-retinoids, which are not normally

observed in the eyes of healthy animals. Further,

in humans and other primates, the formation of

cis-retinoids by blue light-dependent

isomerization) must be countered by trans-cis re-

isomerization for continuous regeneration of the

visual pigments. One source of regenerated 11-

cis-retinal is a metabolic pathway referred to as

the retinoid (visual) cycle (4) (Fig. 1) with a

critical step that generates 11-cis-retinol from all-

trans-retinyl esters via the action of a retinoid

isomerase called RPE65 (5-7). This activity is

consistent with an earlier proposal that light-

independent isomerization of all-trans- to 11-cis-

retinoids in the eye occurs at the alcohol

oxidation state (8). It is also in agreement with

genetic evidence of RPE65 playing a key role in

11-cis-retinal biosynthesis (9). An alternate light-

independent pathway has also been described

(10,11), but the physiological relevance of its

proposed enzyme activities has not been

established (12). The overall rate of visual cycle

activity can be estimated by electroretinography

through dark adaptation measurements (13), in

which visual sensitivity is monitored over time

under dark conditions following light exposure

(14-16) or by direct measurement of visual

pigment levels (17-19). The dark adaptation

process can be modulated by visual

mechanisms is suppressed by the filtering of

blue light by xanthophyll macular pigments

before it can reach the photoreceptor and RPE

cell layers (24).

Mollusks possess a well-characterized

system for photic regeneration of 11-cis-retinal

that consists of an opsin protein known as

retinochrome and a retinoid-binding protein,

which are phylogenetically related to RPE-retinal

G protein-coupled receptor (RGR)-opsin and to

cellular retinaldehyde-binding protein (CRALBP)

in vertebrates, respectively (25). RGR was

originally reported to mediate a photic visual

cycle in vertebrates (26), and activity was

observed in Rpe65^-/-, but not Rpe65^-/-Rgr^-/-mice

under light conditions (27). However, low 11-cis-

retinal synthetic activity cast doubt on its

physiological relevance (26,28). The slightly

higher 11-cis-retinal production at a shorter

wavelength from the main Rgr absorbance

maximum suggested non-enzymatic

chromophore delivery to the photoreceptor outer

segments or by the release of spent

chromophore from opsins, which can be

regulated by rhodopsin kinase (20).

Although the eye has mechanisms to limit

retinal illuminance and visual pigment activation,

for example by pupillary constriction, bright light

2

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

photochemical isomerization rather than RGR–

dependent activity. Other studies have also

indicated that RGR plays a more indirect role in

11-cis-retinal synthesis by mobilizing retinyl

esters and enhancing RPE65 activity (29,30),

possibly through G protein signaling. Further,

Fig. 1b). The system covered most of the

absorption spectra of cone and rod opsins (36-

38) (Fig. 2a, b and Supplementary Fig. 1b).

For most experiments, the light power was kept

below 250 µW. At such levels, temperature

changes to the reaction solution were not

RGR inactivity or deficiency has been associated

with recessive rod-cone dystrophy in humans,

thus signifying its potential importance (31),

although a cis-acting mutation in a nearby gene

called CDHR1 likely contributes significantly to

the phenotypes observed in these patients (32).

Recent work on Müller cell Rgr in mice using

electrophysiological approaches has indicated

that this protein, possibly working in concert with

a retinol dehydrogenase enzyme (although see

(33,34)), makes a sizable contribution to cone

visual pigment regeneration under sustained

light conditions (35). However, robust Rgr-

dependent 11-cis-retinal production has never

been demonstrated at the biochemical level, and

the function of RGR present in the RPE remains

incompletely understood. Considering the

importance of cone pigment regeneration in

human visual physiology and pathophysiology, a

more complete description of alternative visual

cycle pathways is highly desirable. The goal of

this study was to characterize a robust

evident. For experiments requiring variable

temperature or higher light power, a

temperature-controlled cuvette holder was

utilized (see Methods section and

Supplementary Fig. 1c). Exogenous all-trans-

retinal was added to the RPE microsomes at a

molar excess of at least 10 times with respect to

RPE65, the most abundant protein in the RPE

(39), and recombinant human CRALBP was

used to prevent any further non-photoenzymatic

isomerization events (40). The photo-products

were extracted with hexanes and analyzed by

normal phase HPLC (Supplementary Fig. 2).

The light-induced 11-cis-retinal production in the

RPE was profiled according to three variables: a)

wavelength, b) illumination intensity, and c)

length of light exposure (Supplementary Fig. 3).

With a fixed amount of RPE microsomes and the

substrate under near-UV visible light (385-455

nm, overlapping with the absorbance of blue

cone opsin, Fig. 2b), the maximum level of 11-

cis-retinal was achieved in less than 20 min, and

corresponded to approximately 30% of the

original, exogenous all-trans-retinal

photoisomerase activity that we discovered in

the microsomal fraction from RPE cells.

(Supplementary Fig. 3a-e). Increasing the near-

UV light power did not increase 11-cis-retinal

generation (Supplementary Fig. 3a-e). In

contrast, medium wavelength light (490–565 nm,

overlapping with the absorbance of human green

and red cone opsin and rhodopsin (36-38), Fig.

2b) converted all-trans-retinal into the 11-cis

configuration almost quantitatively, and almost

completely depleted the available exogenous

substrate (Supplementary Fig. 3g-j). The

reaction was efficient with well-tuned light power

as low as 25 µW and illumination time of 60-120

min (Supplementary Fig. 3g-j). Further shift in

the illumination wavelength towards the infrared

region resulted in a dramatic drop in

RESULTS

Bovine RPE microsomes contain a high-level

photoisomerase activity responsive to green light

- all-trans-Retinal contained in isolated bovine

RPE microsomes is not isomerized to its cis

products if kept in the dark. However, when RPE

microsomes were exposed to white light, we

observed the formation of 11-cis-retinal, in

amounts that fluctuated from experiment to

experiment. To further explore this phenomenon

and minimize the variability of 11-cis-retinal

production, we designed and built a precise

illumination system capable of delivering narrow

bandwidth light to the bovine RPE microsomal

samples (Supplementary Fig. 1a). The

photoisomerization efficiency. Generation of 11-

cis-retinal became marginal when the 625 nm

peak wavelength was used (Supplementary

Fig. 3l). Production of 11-cis-retinal was not

observed in darkness (Supplementary Fig. 2a,

illumination was generated by LEDs with peak

wavelengths ranging from 385 to 625 nm and

bandwidths of about 50 nm or less, except for

two diodes at long wavelength (Supplementary

3

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

b), whereas trace amounts of cis-retinals

2c). The maximum amount of 11-cis-retinal

observed in the samples even before illumination

(Supplementary Fig. 2c, d) were most likely

derived from the incomplete depletion of

retinoids in RPE microsomes pre-treated with

intense UV light at 302 nm. In 395 nm light the

amount of 9-cis-, 11-cis- and 13-cis-retinal

plateaued at 20 min, suggesting that

photoisomerization had achieved equilibrium

(Supplementary Fig. 2a). In contrast, the

accumulation of 11-cis-retinal under 530 nm light

continued over the 60 min course of the

experiment, accompanied by the gradual

decrease of both 13-cis-retinal and all-trans-

retinal (Supplementary Fig. 2b). After 60 min of

illumination, the amount of 11-cis-retinal reached

2,825 ± 189 pmol, more than 87% of the amount

of the original all-trans-retinal, whereas the

levels of the other retinoids were minimal

(Supplementary Fig. 2j). We found that under

optimized conditions, the photic regeneration

system can produce 11-cis-retinal at a rate of 1

nmol/min/mg of crude protein, which is about ten

times greater than the rate of 11-cis-retinol

formation by the RPE in the dark (41), indicating

a substantial capacity of this system to support

physiological visual pigment regeneration.

generated upon green light illumination of native

RPE suggested the presence of a wavelength-

specific all-trans-retinal to 11-cis-retinal

photoisomerase, activated optimally in the 505-

530 nm range, prompting us to further explore

this hypothesis.

Dependence of the photoisomerase activity

on the retinaldehyde isomeric state - In

subsequent RPE microsomal experiments, if not

indicated otherwise, photo-reactions were

carried out using 530 nm light at 100 µW for 15

min. Under such conditions increased amounts

of bovine RPE proportionally accelerated the

generation of 11-cis-retinal (Fig. 3a). Similarly,

higher concentrations of all-trans-retinal

increased the production of 11-cis-retinal, but the

effect saturated above 15 µM (Fig. 3b). The

RPE microsomes also converted both pure 13-

cis-retinal, and to a lesser extent 9-cis-retinal, to

the 11-cis conformer (Fig. 3c). The efficiency of

11-cis-retinal production with 13-cis-retinal as

substrate (964 ± 88 pmol) was comparable to

that of all-trans-retinal (1,363 ± 125 pmol) (Fig.

3c). These data are consistent with the thermal

isomerization of 13-cis-retinal during

photoisomerization, which exists in

Green light-stimulated photoisomerase

activity in bovine RPE cells is mediated by

protein rather than lipid adducts - A previous

report showed that non-enzyme associated

photoisomerization of all-trans-retinal to cis-

retinals was boosted by amines through Schiff

base formation, independent of the RPE (42).

We therefore compared the production of cis-

retinals in native and denatured RPE samples

illuminated by 12 different wavelengths. Under

constant light power output (50 µW) and a set

illumination time (20 min), the highest rate of 11-

cis-retinal production by native RPE microsomes

was observed at a wavelength of 505 nm,

corresponding to the UV-vis spectrum of

rhodopsin (Fig. 2c). However, upon an increase

in the energy of the light source to 100 µW the

production of 11-cis-retinal was higher at a

wavelength of 530 nm (Supplementary Fig. 3h,

i). In contrast, generation of 11-cis-retinal in

denatured RPE, as well as 9-cis-retinal in non-

denatured RPE, was consistently low and

correlated with the energy of the photons (Fig.

thermodynamic equilibrium ~1:9 with all-trans-

retinal (4). Interestingly, a more complex

conversion of 9-cis- to 11-cis-retinal was also

observed. Double isomerization is highly

unlikely, so the conversion probably requires all-

trans-retinal as an intermediate. The relatively

low 11-cis-retinal yield in the presence of 9-cis-

retinal therefore could be due to a lower amount

of all-trans-retinal generated thermally.

Impact of CRALBP on the RPE

photoisomerase activity - The fact that CRALBP

has been shown to serve as a retinoid

isomerase facilitating the conversion of 9-cis-

retinal to 9,13-di-cis-retinal (43) prompted us to

quantify its contribution to the generation of 11-

cis-retinal in our experimental protocol. The 11-

cis-retinal production was marginal in the

presence of denatured RPE and non-denatured

CRALBP, even with an excess of the latter (Fig.

3d). CRALBP added to non-denatured RPE

improved the yield of 11-cis-retinal to ~1,400

pmol, but the effect was saturated when the

4

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

molar ratio of CRALBP exceeded that of all-

Supplementary Fig. 1c). The

trans-retinal. Yet, when CRALBP was boiled and

mixed with non-denatured RPE, the generation

of 11-cis-retinal in light fell to ~800 pmol (Fig.

3d). These observations suggested that

CRABLP facilitates the photoisomerization in

RPE and protects the resulting 11-cis-retinal

from re-isomerization to all-trans-retinal.

photoisomerization of all-trans-retinal by RPE

microsomes was only moderately temperature-

dependent in the range of 10 ºC to 40 ºC (Fig.

3f). The enzymatic activity at 10 °C remained

surprisingly high, and was equal to 54% of the

value observed at 37 °C. The observed pH- and

temperature-dependence pattern provided more

support for the existence of a physiological

photoisomerase of the receptor type rather than

a classic enzyme. Many enzymes are acutely

pH-sensitive due to the protonation of key

residues in their active sites, and there is

Photoisomerase activity in the RPE is not

dependent on retinol dehydrogenase enzymes -

Recent data has suggested that RGR in Müller

glia works in concert with retinol dehydrogenase

10 to achieve photic production of 11-cis-retinol

(35). To test if RDH enzymes are involved in the

photoisomerase activity of RGR in the RPE we

carried out photoisomerase assays in the

presence of dinucleotide cofactors with RPE65

activity pharmacologically eliminated

typically a two-fold increase in enzymatic activity

with every 10 ºC increase of temperature up to

the point of protein denaturation (44).

Light-dependent 11-cis-retinal production is

independent of RPE65 and DES1 - In the

classical visual cycle, RPE65 isomerizes all-

trans-retinyl esters to 11-cis-retinol without the

assistance of light (Fig. 4a) (45). The

(Supplementary Fig. 4). NAD⁺/NADP⁺

(oxidation) did not markedly affect the

photoisomerase activity in RPE microsomes

when compared with the dinucleotide-free

controls. As expected, addition of NADH

subsequent oxidation of 11-cis-retinol to the

opsin chromophore requires retinal

promoted 11-cis-retinol formation via the action

of 11-cis-dehydrogenases (e.g. RDH5), but the

total amount of 11-cis-retinoid was somewhat

reduced compared to the nucleotide-free control.

In contrast, NADPH reduced total cis retinoid

production likely because of contamination of the

isolated bovine RPE with photoreceptor-derived

RDH8, which stereospecifically reduces the all-

trans-retinal substrate of RGR, converting it to

all-trans-retinol. In summary, dinucleotides did

not enhance photoisomerization activity, but they

did affect the ratio of retinol/retinal as expected.

dehydrogenase 5 (RDH5). Sphingolipid Δ4-

desaturase 1 (DES1) may also contribute to 11-

cis-retinol generation, which can be inhibited by

fenretinide (11). To evaluate their relative roles

in the regeneration of 11-cis-retinoids, both

retinol and retinal were exposed to 530 nm light

at 37 ºC in RPE samples. The HPLC analysis of

the isomerization products showed that the 11-

cis-retinal yield was almost twice that of 11-cis-

retinol when their respective all-trans substrates

were added separately to the RPE (Fig. 4b), and

the generation of 13-cis or 9-cis-retinoids was

negligible. The presence of both retinol and

retinal in the RPE samples did not impair the

photoisomerase activity, whereas the activity of

RPE65 was greatly inhibited by all-trans-retinal

(Fig. 4b). Emixustat, a vision cycle modulator

that is currently in clinical trials for dry age-

related macular degeneration (AMD), non-

covalently binds to the active site of RPE65 and

strongly inhibits 11-cis-retinol production, leading

to undetectable levels of 11-cis-retinol (Fig. 4c).

Here, the visual cycle modulator did not interfere

with the activity of the photoisomerase (Fig. 4b,

c). Due to the strong inhibition of RPE65, adding

all-trans-retinol did not increase the total amount

of 11-cis-retinal, whereas an increase was

Impact of temperature and pH on the RPE

photoisomerase activity - To further explore the

properties and physiological relevance of the

presumed photoisomerase, we tested the pH-

and temperature-dependence of the 11-cis-

retinal production. Maximal photoisomerization

was observed at pH values in the physiological

range, suggesting that the protonation to a Schiff

base derived from retinal binding to the active

site of the enzyme is critical to the production of

11-cis-retinal (Fig. 3e). We also utilized a

variable temperature apparatus to test the effect

of temperature on the enzymatic

photoisomerization (see Methods,

5

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

evident in the absence of emixustat (Fig. 4b, d).

The presence of fenretinide (10 µM) in RPE

samples demonstrated marginal effects on both

and retinal isomerization (47-49).

Hydroxylamine, an aldehyde scavenger through

stable Schiff base formation, competed with the

the production of 11-cis-retinol and 11-cis-retinal,

suggesting that RPE65 dominated the 11-cis-

retinol production and DES1 was irrelevant to

the photoisomerization (Fig. 4d). Des1 is more

likely involved in lipid metabolism and not

retinoid transformations. In summary, enzymatic

photoisomerization is the major pathway of 11-

cis-retinal regeneration under illumination,

independent of the classical visual cycle.

binding of all-trans-retinal to the isomerase (Fig.

5a). The inhibition of isomerase activity required

hydroxylamine at a concentration at least 32

times higher than the substrate, suggesting the

specific binding of retinal to the protein also

involved the non-covalent interaction between

the retinoid backbone and the protein residues in

the active site. The reduction of the retinylidene

Schiff base with cyanoborohydride resulted in

the formation of an irreversible bond with the Lys

residue in the active site, which substantially

inhibited photoisomerization (Fig. 5b). The

substitution of all-trans-retinal with benzaldehyde

abolished the inhibition, suggesting that

benzaldehyde was incapable of fitting into the

active site. The fluorography of RPE microsomal

proteins incubated with radioactive [³H]-all-trans-

retinal revealed that all-trans-retinal specifically

bound to a single protein migrating to band with

an apparent molecular mass of around 27 kDa

(Fig. 5c). In order to identify the all-trans-retinal

binding protein, detergent-solubilized RPE

microsomes were subjected to cation-exchange

chromatography followed by size-exclusion

chromatography (SEC). SDS-PAGE of the SEC

fractions containing isomerase activity again

revealed a 27 kDa band (Fig. 5d) accompanied

by substantial amounts of RPE65, rhodopsin,

and RDH5 among other minor impurities. This

band was further analyzed via mass

Further biochemical properties of the RPE

photoisomerase - Because the photoisomerase

candidate appeared to be a membrane protein,

solubilization was critical for its separation and

identification. The washing of RPE microsomes

with 3 M urea did not affect the activity of the

isomerase and removed RPE constituent

proteins associated with the membrane

(Supplementary Fig. 5a). Incubation with 1%

trypsin for 30 min reduced the enzymatic

photoisomerization (Supplementary Fig. 5b).

The presence of high concentration of salts

(Supplementary Fig. 5c) and transition metal

chelators such as EDTA and EGTA

(Supplementary Fig. 5d) did not alter the

activity of the photoisomerase. In contrast, the

isomerase was sensitive to most detergents

(Supplementary Fig. 5e), except for LMNG.

With 10 mM LMNG, most membrane proteins

were solubilized, and the isomerase retained at

least 75% of its activity, even after being kept on

ice for more than 12 h; however, it was

spectrometry, showing bovine RGR as its major

component (over one third of total peptide-to-

spectrum matches), and absence of any

significant alternative isomerase candidate

(Supplementary Table 1).

deleterious to the function of RPE65

(Supplementary Fig. 5f). The preincubation of

RPE microsomes with phospholipase 2 (PLA2)

altered the membrane composition and inhibited

RPE65 function, but did not impact

photoisomerization (Supplementary Fig. 5g)

(46). The repeated freezing and thawing of the

RPE microsomal suspension in 10 mM LMNG

did not cause a substantial decrease in

photoisomerization activity (Supplementary Fig.

5h).

Origin of the all-trans-retinal substrate of

RGR in bovine RPE – The concentration of all-

trans-retinal in the RPE is known to be very low,

which raises the question of how RGR within the

RPE gains access to its substrate. When RPE

microsomes were treated with an excess of

exogenous all-trans-retinol (30 µM) and

emixustat (10 µM), the esterification of retinol by

LRAT became much less favored (Fig. 6a)

which resulted in a substantial elevation of all-

trans-retinol level, whereas 11-cis-retinol

Identification of the RPE photoisomerase as

RGR - The formation of a Schiff base at the

active site of the putative photoisomerase has

been proposed to be critical for substrate binding

6

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

generation was marginal due to the inhibition of

RPE65. After 30 min of light exposure (530 nm,

200 µW) at 37 ºC, the observed small amount of

11-cis-retinal (108.2 ± 2.3 pmol) came mainly

from the enzymatic photoisomerization of

endogenous retinals in RPE microsomes. The

11-cis-retinal generation by RGR reached 532.1

± 13.1 pmol if NAD⁺(2 mM) was added to the

reaction mixture (Fig. 6b). This boost of 11-cis-

retinal production with NAD⁺addition indicated

that the all-trans-retinol escaping from the LRAT

esterification could be oxidized by retinol

dehydrogenase in the presence of the

absorption spectrum of bRGR bound to all-trans-

retinal showed a peak around 470 nm, as

reported previously (47-49), with little change

observed upon 530 nm light illumination.

However, the 470 nm peak completely

disappeared after 10 min incubation with

hydroxylamine (Fig. 8b). The HPLC analysis

confirmed that all-trans-retinal bound to RGR

was isomerized to 11-cis-retinal upon 530 nm

light illumination, and that the incubation with

hydroxylamine induced the formation of retinal

oximes (Supplementary Fig. 6a). In line with

the previous cell lysate-based assays the

purified bRGR robustly photoisomerized

exogenous all-trans-retinal to 11-cis-retinal upon

530 nm light illumination in the presence of

CRALBP, yet 11-cis-retinal was almost absent in

the negative control sample (Fig. 8c,

dinucleotide cofactor, and the product, all-trans-

retinal, served as the substrate of

photoisomerization.

Heterologously expressed RGR exhibits

spectral and biochemical properties matching

those of the native RPE photoisomerase - To

further confirm that bRGR accounts for the

isomerase activity found in bovine RPE

microsomes, we heterologously expressed it in

HEK293S GnTI^-cells. Along with the wild type

(WT) bRGR, we also expressed a mutant bRGR

that has an alanine residue instead of a Lys

residue at position 255 in the active site. The

K255 residue is conserved from primates to

zebrafish and is known to bind all-trans-retinal

through a covalent Schiff base bond (50).

Immunoblotting of cell lysates showed a

comparable level of bRGR and K255A bRGR

expression (Fig. 7a). Next, we incubated cell

homogenates with all-trans-retinal in 530 nm

light to measure photoisomerization activity.

Accumulation of 11-cis-retinal was much more

profound in bRGR cell homogenates in the

presence of CRALBP than in its absence,

reaching over a 7-fold excess compared to

K255A bRGR or the non-transfected control

(Fig. 7b, c). Because LMNG has proven to

preserve the photoisomerase activity of bovine

RPE microsomes we utilized this detergent to

solubilize and purify recombinant bRGR. A one-

step immunoaffinity chromatography procedure

performed in the dark and in the presence of all-

trans-retinal achieved significant enrichment of

the target protein and limited non-specific

contaminants (Fig. 8a). The UV-visible light

Supplementary Fig. 6b). The action spectrum

for 11-cis-retinal production by purified bRGR,

exhibited a maximum at ~550 nm, in line with the

action spectrum obtained from native bovine

RPE microsomes, which corroborates the

assignment of RGR as the photoisomerase in

RPE microsomes (Fig. 2c, 8d). Together, these

findings demonstrate that bRGR efficiently

photoisomerizes all-trans-retinal to 11-cis-retinal

upon 530-550 nm light exposure, and that the

Schiff base linkage at K255 is critical for this

process.

Analysis of RGR photoisomerase activity in

other vertebrates - To test the green light-

dependent photoisomerization potential of RGR

in other species we first prepared microsomal

fractions with the same amount of protein from

porcine and chicken RPE. Both were able to

produce 11-cis-retinal upon 530 nm light

illumination, though the efficiency of the latter

was about 25% that of bovine or porcine RPE

(Supplementary Fig. 5i). Microsomal fractions

derived from bovine rod outer segments, mouse

brain and mouse liver were incapable of

generating 11-cis-retinal, confirming that RPE is

the specific tissue contributing the

photoisomerase activity (Supplementary Fig.

5i). To further explore the evolutionary

conservation of this phenomenon we

heterologously expressed and tested chicken

and mouse RGR, in a fashion similar to bRGR.

In concert with the bovine data, cRGR cell

7

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

lysates exhibited robust 11-cis-retinal production

(Supplementary Fig. 7a, b). In contrast,

photoisomerase activity of mRGR was much

lower than that of cRGR even though the

expression of mRGR and cRGR was

the assay demonstrates that hRGR can robustly

photoisomerize all-trans-retinal to 11-cis-retinal.

RNAseq analysis revealed species-specific

differences in RGR and RLBP1 co-expression -

Because our finding suggests that the co-

presence of CRALBP is necessary for efficient

RGR-dependent photoisomerization of all-trans-

retinal, we examined the expression pattern of

both RLBP1 and RGR transcripts in different

retinal cell types and the RPE through a single-

cell sequencing analysis. We performed a

single-cell RNA sequencing of retina and RPE

tissue from mouse, bovine and human, and

analyzed the data with t-SNE (52) to visualize

clusters corresponding to distinct cell types (Fig.

10a, d, g). In human and bovine samples, both

RGR and RLBP1 were simultaneously

expressed in Müller cells and RPE, supporting a

possible interaction between the two proteins

during RGR-dependent photoisomerization. (Fig.

10b, e). In mice, Rlbp1 was expressed in both

Müller cells and RPE (as observed in bovine and

human), whereas Rgr was expressed only in

RPE, but only barely detectable in Müller cells

(Fig. 10h). Furthermore, the single-cell RNA

sequencing allowed us to compare the

expression level of RGR with other genes

involved in the classical visual cycle. We found

that RGR is expressed at a relatively lower level

compared to that of RPE65 in RPE from bovine

and human (Fig. 10c, f). Interestingly, the

expression level of Rgr in RPE from mouse was

significantly higher than that of Rpe65 (Fig. 10i).

These data show that RGR and RLBP1 are co-

expressed in RPE and Müller cells of bovine and

human, but in the mouse eye the co-expression

of Rgr was far higher in RPE, with only minimal

expression detected in Müller cells.

comparable (Supplementary Fig. 7a, b).

Impact of human RGR splicing variation on

photoisomerase activity - The notion that human

RGR (hRGR) contributes to chromophore

regeneration has been controversial, partly due

to its low activity (26). Moreover, since multiple

splice variants of the hRGR gene have been

reported (Fig. 9a), our next goal was to quantify

the relative abundance of these variants in the

human eye and assess their photoisomerization

capacity to produce 11-cis-retinal. Using

available human retina and RPE RNA-seq

datasets (51) (https://doi.org/10.1101/501882)

we confirmed the existence of an alternative

splice acceptor site of hRGR intron 2 leading to

an insertion of 4 additional in-frame codons

(amino acids 79-82, hRGR variant 1) in a small

(<5%) fraction of the total transcripts. Another,

more common variant of hRGR transcripts (10-

20% abundance) originates from the deletion of

exon 6, encompassing 114 nt (amino acids 211-

248, hRGR variant 2) which removes the

transmembrane domain 6 from the encoded

protein. Next, we heterologously expressed the

hRGR together with its two minor splice variants

in HEK293S GnTI^-cells to compare their

photoisomerization activity. Immunoblotting

analysis of cell lysates demonstrated efficient

and comparable expression of all transcripts

(Fig. 9b). The WT hRGR transcript generated

the greatest amount of 11-cis-retinal upon 530

nm light exposure in the presence of CRALBP.

Consistent with the findings from our bovine

studies, the K255A substitution basically

abolished this activity (Fig. 9c). hRGR variant 1,

containing the non-conserved 4-amino acid

insertion, exhibited much lower

photoisomerization activity, while variant 2 was

comparable to the K255A mutant. The level of

11-cis-retinal produced upon 625 nm light

exposure was minimal across all test and control

samples, whereas hRGR still produced

DISCUSSION

The need for efficient chromophore

regeneration in moderate to bright light

conditions appears to exceed that supplied by

the classical visual cycle (53,54). One

explanation for sustained visual function in

vertebrates under these conditions could be the

presence of a complementary photic visual cycle

that responds to increasing light exposure, thus

significant amounts of 11-cis-retinal upon 395

nm light exposure compared to the non-

transfected samples (Fig. 9d). Taken together,

8

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

balancing the bleaching of cone visual pigments

with increased 11-cis-retinal production. We

have demonstrated in this study a pathway

located in the RPE that may serve this

function. We have found that 1) the RPE has the

ability to respond to incident visible light with a

robust photic response to regenerate 11-cis-

retinal, 2) RGR is the enzymatic origin of this

visible light-dependent 11-cis-retinal

regeneration in the RPE, 3) both native and

recombinant RGR show peak activity upon

stimulation with green light (~530 nm) with lower

activity at other wavelengths, and 4) the RGR-

dependent photic response appears to have a

higher capacity for 11-cis-retinal production

compared to the classical visual cycle.

retina > 9-cis-retinal (Fig. 3c). This is opposite to

the order of rod and cone opsin affinity for

retinals. Despite the lower specificity for the 13-

cis-retinal and 9-cis-retinal isomers, enzymatic

photoisomerization in the RPE could play a role

in clearing these compounds that are produced

non-specifically during light exposure. Such a

functional role is consistent with prior studies of

Rgr^-/-mice, which exhibit an accumulation of 9-

and 13-cis-retinoids following exposure to short

light flashes (50).

Prior work indicated that RGR can stimulate

RPE65 activity independent of light (29) and

increase all-trans-retinyl ester hydrolase activity

(30). Further work is needed to reconcile these

findings in the context of our results. Considering

all reports, it is likely that RGR has a

The wavelength selectivity associated with

the photoisomerase activity in native microsomal

membranes and recombinant RGR is similar to

the absorbance spectra of rhodopsin and green

cone opsin (55). This correspondence is

expected to maximize the efficiency of the

system for visual chromophore production when

it is most needed. It is interesting that the

wavelength range (510-550 nm) optimally

stimulating RGR photoisomerase activity only

partially overlaps with the UV-visible absorption

spectrum of purified RGR (Fig. 8b) (47). This

observation is similar to the photoisomerase

activity of retinochrome, which is most efficiently

stimulated with yellow light (> 530 nm) despite

the absorbance maximum of the purified protein

occurring at a shorter wavelength (496 nm)

(56,57). Differences between the absorption and

action spectra likely arise from the multitude of

competing photoisomerization reactions that

occur in the complex mixture of proteins and

membranes. For example, non-specific retinal-

protein Schiff base adducts may exist

multifunctional role in visual physiology.

Although direct signal transduction activity has

not yet been demonstrated for RGR, it is

interesting to note that the protein has conserved

molecular features of G protein-coupled

receptors including the (modified) E/DRY and

NPXXY sequence motifs (58). Additionally, RGR

physically associates with RDH5 and RPE65

(46) in the RPE, suggesting a mechanism by

which light-driven conformational changes in

RGR could be transmitted to these key

components of the classical visual cycle. Despite

the apparent 11-cis-stereospecificity of RPE65 in

vivo, this enzyme can generate substantial

amounts of 13-cis-retinol (59-61). Moreover,

substantial 13-cis-retinoid accumulation occurs

in vivo in the eyes of Rdh5^-/-mice during dark

adaptation (62) or in Rgr^-/-mice after bleaching

(50). These data suggest that RGR, working in

conjunction with RDH5, also may function to

remove the 13-cis-retinol byproduct of the

classical visual cycle in vivo.

predominantly in the trans configuration at

equilibrium during illumination with 440 nm light.

Such a reaction would counteract the activity of

RGR resulting in a red shift of the action

spectrum.

Peropsin orthologs constitute a sister group

to the RGR/retinochrome photoisomerase

subfamily (63). Based on this close phylogenetic

relationship and the fact that peropsin is

expressed in the RPE (64), we considered the

possibility that peropsin could contribute to the

observed photoisomerase activity. However,

mass spectrometry analysis aiming to identify

the RPE microsome component that bound all-

trans-retinal did not show the presence of

The rate at which 11-cis-retinal was

regenerated via photoisomerization in the RPE

was dependent on the isomeric state of the

retinal substrate with the following order of

substrate selectivity: all-trans-retina > 13-cis-

9

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

peropsin (Supplementary Table 1). Additionally,

RGR is a sink for all-trans-retinal, as we

demonstrate in this work, and can be isolated

with bound retinal even after extensive washing

during column chromatography. All-trans-retinol

is esterified by LRAT, and the esters can be

hydrolyzed (70). The conversion of all-trans-

retinol to all-trans-retinal is governed by the

reducing power of the cell and the reaction is

reversible. From a thermodynamic point of view,

there will always be some amount of all-trans-

retinal in RPE cells. To support this view, the

experiments described here (Fig. 6)

demonstrate that 1) RPE microsomes generate

all-trans-retinal from retinyl esters or retinol that

is present in the RPE at high concentration in the

presence of NAD+ and that 2) in the light, all-

trans-retinal is selectively converted to 11-cis-

retinal (Fig. 6). A second source of all-trans-

retinal is derived directly from photoreceptors

under strong light conditions. Note that retinal

transport is not known to be protein mediated.

11-cis-retinal diffuses from the RPE to

a recent study on peropsin function indicated

that peropsin plays a role in vitamin A transit

between the retina and RPE, rather than acting

as a visual chromophore-producing

photoisomerase (65).

CRALBP moderately stimulated photic 11-cis-

retinal production from bovine microsomes (Fig.

3d). However, our experiments using

heterologously expressed RGR or purified bovine

RGR clearly demonstrated the importance of

CRALBP in facilitating RGR-dependent 11-cis-

retinal formation. It is possible that residual

CRALBP present in the RPE microsome samples

could have partially masked the CRALBP-

dependence of the reaction in the native system.

Several lines of evidence have demonstrated that

cephalopod retinal-binding protein is required to

effectively bind 11-cis-retinal produced from

retinochrome and act as a shuttle in the

rhodopsin-retinochrome pathway (66,67). Our

data indicate a similar complementary

relationship between RGR and CRALBP in

vertebrates. Moreover, the co-localization of RGR

and CRALBP in the same cell types of human

and bovine eyes supports their potential

photoreceptors despite the high concentration of

CRALBP in the RPE (with nanomolar affinity for

retinal). This occurs because opsin is a sink for

11-cis-retinal. Likewise, RGR must act as a sink

for all-trans-retinal.

interaction during the regeneration of

chromophore under sustained photic conditions.

Rod opsin was also present in the

photoisomerase-active fractions, due to rod

membrane contamination of the RPE microsomal

preparation, and we considered the possibly that

it could also act as an 11-cis-retinal sink

The mechanism we describe depends on the

availability of all-trans-retinal in RPE to serve as

a substrate for RGR. Prior studies have shown

that all-trans-retinal accumulates in the retina

during periods of light exposure when RGR

could act as a photoisomerase (18,71). This

retinal can likely transfer between the

photoreceptor outer segments and the RPE. For

example, Rando and Bangerter demonstrated

that the intermembranous rates of retinoid (all-

trans-retinol(al) and 11-cis-retinol) transfer from

vesicle to vesicle and vesicle to erythrocyte were

exceedingly rapid, whereas all-trans-retinyl

palmitate did not undergo transfer at an

appreciable rate (72). Fex and Johannesson

showed that retinol, similar to unesterified

cholesterol and long-chain fatty acids but unlike

phospholipids, can transfer rapidly and

spontaneously between liposomal membranes

(73,74). Noy and colleagues suggested that

retinoid transfer between different cell types in

vivo may depend on the geometry of cellular

surfaces (75), which are highly elaborated in the

stimulating 11-cis-retinal production through RGR

by mass action. However, the excess of CRALBP

over rod opsin suggests the latter would play only

a minor role in binding 11-cis-retinal. Moreover, it

is known from prior studies that detergent-purified

rod opsin binds 9- or 11-cis-retinal poorly (68,69).

Finally, photoisomerization reactions carried out

in the presence and absence of bleached ROS

membranes yielded similar production rates and

quantities of 11-cis-retinal. Therefore, the

presence of native CRALBP in the purified

photoisomerase-active samples is the most likely

explanation for the weaker CRALBP-dependence

of the reactions carried out using material from

native RPE microsomes.

10

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

RPE apical membrane. These results suggest

that retinoids may not require active transport or

binding protein-mediated transfer between

membranous compartments of ROS and the

RPE. However, this work does not exclude the

possibility that a yet unidentified active transport

mechanism exists in the mammalian retina.

nocturnal animals. This fundamental difference

in retinal physiology is possibly why it has been

difficult to observe contributions of RGR to light-

dependent visual pigment regeneration in mice

(29,30,50).

Our results indicate that the RGR

isomerization pathway works in a

This study also highlights the importance of

RGR sequence variations in determining

complementary fashion to the classical visual

cycle to regenerate bleached visual pigments via

retinoid transport between photoreceptor outer

segments and the RPE (Fig. 11). In the case of

rods, which desensitize in relatively dim light, it is

likely that the classical visual cycle is of greater

relevance to their function. However, it was

recently shown that rods can maintain some

function under photopic conditions, and that the

photoisomerase activity. The alternative splicing

of human RGR generates at least three different

transcript isoforms, which were identified in

postmortem donor retinas (48,49,76). The

dominant isoform 1, referred to as WT hRGR, is

highly conserved, whereas variant 1 has a 4

amino acid long insertion that is not observed in

other species (Fig. 9a) (49). The second splicing

variant lacks the nucleotide sequence encoding

transmembrane domain 6, which likely renders

the translated protein non-functional (76). Here,

we confirmed that WT hRGR is the most

abundant isoform in both human RPE and Müller

glia, and exhibits the highest photoisomerase

activity, comparable to recombinant bovine and

chicken RGRs.

RGR system may be involved in providing the

chromophore under these conditions (81).

Previous studies have shown that the classical

visual cycle plays a significant role in maintaining

responses from cones, but that cones can

continue to function in circumstances of

profound RPE65 inhibition (54,82). It has been

speculated that 11-cis-retinal bound to CRALBP

in Müller cells could act as a chromophore

reserve to sustain cone function (54). The

presence of RGR in Müller cells of diurnal

animals suggests that the complementary visual

cycle may be at work in the Müller cells as well,

which in turn may explain the source of 11-cis-

retinoids bound to CRALBP in these cells. The

results described here indicate that the RGR

system in the RPE adds another, potentially

more significant source of 11-cis-retinal that

could allow for long-term maintenance of cone

function in the face of sustained bright light

exposure.

The photoisomerase activity of recombinant

mouse RGR was much lower than that of the

human, chicken and bovine orthologs, despite

high overall sequence conservation (81%

identity with human RGR), robust heterologous

expression, and identical assay conditions.

Single cell RNA-seq analysis of mouse retina

and RPE showed a different pattern of RGR

expression compared to the other species, with

mouse RGR transcripts occurring solely in the

RPE cells. These findings are consistent with

prior immunohistochemistry data showing

negligible RGR expression in mouse retinal

tissue harvested at postnatal day 16 (77).

Conversely, RGR expression in human and

bovine Müller glia was readily detectable with

immunostaining (78,79). Considering that the

RGR knock-out mouse exhibits a relatively mild

visual phenotype (50,80), it is possible that the

contribution of the RGR photoisomerase activity

to the visual cycle differs considerably between

the nocturnal mouse and diurnal animals such

as human, cow and chicken, although this

hypothesis will need to be verified in additional

EXPERIMENTAL PROCEDURES

Animals

All experiments were approved by the

Institutional Animal Care and Use Committees at

the University of California, Irvine and were

conducted in accordance with the Association for

Research in Vision and Ophthalmology

Statement for the Use of Animals in Ophthalmic

and Visual Research.

11

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Spectral illumination

quartz cuvette and irradiated with UV light for 6

min at 4 °C with a ChromatoUVB transilluminator

(model TM-15; UVP, Upland, CA, USA) to

eliminate residual retinoids. After irradiation, 5

mM dithiothreitol was added to the RPE

microsomal mixture.

To provide controlled spectral illumination all

components, including light-emitting diodes

(LEDs) and cuvette holders, were rigidly

mounted on solid aluminum optical breadboards

(Supplementary Fig. 1a). Light from LEDs,

M385F1, M395F3, M405FP1, M420F2, M455F1,

M470F3, M490F3, M505F1, M530F2, M565F3,

M595F2 and M625F2 was guided via a

multimode optical fiber. The fiber was fastened

into a CP02 cage plate (Thorlabs, Newton, NJ,

USA) with the SM1SMA fiber adapter plate

(Thorlabs). To ensure consistent placement

between the cuvette and the light emitting fiber-

end, we used a cage-mounted customized

CVH100 cuvette holder (Thorlabs) and

customized t2Sport cuvette holder with Peltier-

driven temperature control (Quantum Northwest,

Liberty Lake, WA, USA). M395L4, M530L3 and

M625L4 LEDs (Thorlabs) with collimators were

used for photoisomerase activity assays of

recombinant RGR. Light intensity was adjusted

using a DC4100 LED driver (Thorlabs), and light

power was measured with the optical power

meter PM100D equipped with S120C and

S120VC light sensors (Thorlabs).

RPE65 retinoid isomerase activity assay

RPE65 isomerase assays were initiated by

addition of all-trans-retinol in dimethylformamide

(DMF) to a final concentration of 15 µM in BTP

buffer, to a suspension containing 150 μg of

RPE microsomal proteins, 1% bovine serum

albumin (BSA), 2 mM disodium pyrophosphate

and 25 μM human apo-cellular retinaldehyde-

binding protein (CRALBP) (85). The resulting

mixture was incubated at 37 °C for 15 min to 1 h.

The reaction was quenched by adding 400 μL of

methanol (Fisher Chemical, Fair Lawn, NJ,

USA), and the products were extracted with 400

μL of hexanes. Production of 11-cis-retinol was

quantified by normal phase HPLC using a

Zorbax Rx-SIL column (5 µm, 4.6 x 250 mm,

Agilent, Santa Clara, CA, USA) with 10% (v/v)

ethyl acetate in hexanes as the eluent at a flow

rate of 1.4 mL·min⁻¹. Retinoids were detected by

monitoring their absorbance at 325 nm and

quantified based on a standard curve

Reagents

representing the relationship between the

amount of 11-cis-retinol and the area under the

corresponding chromatographic peak.

Solvents and reagents were purchased from

Sigma-Aldrich (St. Louis, MO, USA) unless

otherwise stated. All-trans-retinal was obtained

from Toronto Research Chemicals, Inc.

(Toronto, Canada) or Sigma-Aldrich. 11-cis-

Retinal was obtained by illuminating all-trans-

retinal in acetonitrile and separating the retinal

isomers by normal phase high-performance

liquid chromatography (HPLC) on a silica column

(10 μm, 21.2 mm × 250 mm, Phenomenex,

Torrance, CA, USA) with a mobile phase of 10%

ethyl acetate in hexanes flowing at a rate of 5

mL·min⁻¹(83).

Photoisomerization assay

all-trans-Retinal in DMF was added to a final

concentration of 15 µM in BTP buffer, containing

150 μg of bovine RPE microsomal proteins, 1%

BSA, 2 mM disodium pyrophosphate and 25 μM

CRALBP. The resulting mixture was

subsequently transferred to quartz cuvettes

(Starna Cells, Atascadero, CA, USA, path length

1 mm) and illuminated with LED lights for 5 min

to 2 h. The reaction was quenched by turning off

the light source. Methanol (400 µL) then was

added to the reaction mixture, and retinoid

products were extracted with hexanes (400 µL).

Production of 11-cis-retinal was quantified by

normal phase HPLC with 10% (v/v) ethyl acetate

in hexanes as the eluent at a flow rate of 1.4

mL·min⁻¹. Retinoids were detected by monitoring

their absorbance at 360 nm and quantified

RPE microsomal preparations

Bovine RPE microsomes were isolated from

RPE homogenates by differential centrifugation

as previously described (84). The resulting

microsomal pellet was resuspended in 10 mM

bis-tris propane/HCl, pH 7.4 (BTP buffer), to

achieve a total protein concentration of ~5

mg·mL⁻¹. The mixture then was placed in a

12

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

based on a standard curve representing the

activities of the fractions were evaluated as

relationship between the amount of 11-cis-retinal

and the area under the corresponding

chromatographic peak (46). All isomerization

reactions were performed and analyzed in a

darkroom.

described above.

Mass spectrometry

Fractions collected from size-exclusion

chromatography (SEC) were analyzed by SDS-

polyacrylamide gel electrophoresis (PAGE) and

the gel was stained with Coomassie. The visible

bands in the lane with higher photoisomerase

activity were individually excised, washed and

destained in 50% ethanol/5% acetic acid, and

then dehydrated in acetonitrile. The samples

were then reduced with dithiothreitol and

alkylated with iodoacetamide prior to in-gel

digestion by addition of 50 ng of trypsin in 50

mM ammonium bicarbonate followed by

overnight incubation at room temperature.

Peptides were extracted from the polyacrylamide

gel slices in two aliquots of 30 μL of 50%

acetonitrile/ 5% formic acid. These extracts were

combined and evaporated to <10 μL using a

Speedvac concentrator (Thermo Fisher

Scientific) and then resuspended in 1% acetic

acid to a final volume of ~30 μL for analysis

using a Thermo LTQ-Orbitrap Elite hybrid mass

spectrometer system (Thermo Fisher Scientific).

Peptides were separated on an Acclaim Pepmap

C18 column (15 cm x 75 µm, i.d. 2μm, 100 Å

reversed-phase capillary chromatography

column, Thermo Fisher Scientific) with a 0 to

100% acetonitrile in 0.1% formic acid gradient at

a flow rate of 0.3 μL·min⁻¹, and the eluate was

introduced into the source of the mass

spectrometer on-line. The nano electrospray ion

source was operated at 1.9 kV. The digest was

analyzed using the data-dependent multitask

capability of the instrument acquiring full scan

mass spectra to determine peptide molecular

weights and product ion spectra to determine

amino acid sequence in successive instrument

scans. The data were analyzed using all CID

spectra collected in the experiment to search

against the UniProtKB bovine protein sequence

database using Sequest and Mascot software

(86,87).

Binding of all-trans-retinal to RPE

microsomal protein

RPE microsomes were resuspended in 300

µL of BTP buffer, containing 2 µM [³H]-all-trans-

retinal with or without 12 µM non-radioactive all-

trans-retinal. The resulting mixture was

incubated at 4 ^oC for 1 h, then 7.5 mg of sodium

cyanoborohydride was added. The resulting

samples were loaded onto a Bis-Tris

polyacrylamide gel (4-12%) (Thermo Fisher

Scientific, Carlsbad, CA, USA). After

electrophoresis, the gel was washed with water,

dehydrated with dimethyl sulfoxide (DMSO), and

soaked in a 2,5-diphenyloxazole solution (20% in

DMSO) for 1 h. The gel was subsequently

washed with water, dried under vacuum at 60 ºC

for 1 h and exposed to Kodak BioMax XAR film

(Sigma-Aldrich) at -80 ^oC for 10 h.

Ion-exchange and size-exclusion

chromatography of urea-washed RPE

microsomal proteins

Bovine RPE microsomes were suspended in

6 mL of urea (3 M, pH 8) over ice. The mixture

was centrifuged at 100,000g for 30 min, and the

resulting pellet was gently solubilized with 10

mM lauryl maltose neopentyl glycol (LMNG) in

BTP buffer at 4 ºC. After centrifugation at

100,000g for 30 min, 3.5 mL of the resulting

supernatant were loaded onto a glass column

containing 9.5 mL DEAE-Sepharose (GE

Healthcare, Uppsala, Sweden). The column was

then washed with 20 mL of BTP buffer

containing 0.25 mM LMNG. Bound proteins were

eluted from the column with a 20-mL linear

gradient from 0 to 1 M NaCl. Fractions exhibiting

photoisomerase activity (as described above)

were combined, concentrated to 2.5 mL, and

loaded onto a 125-mL Superdex 200 size-

exclusion column (GE Healthcare), and the

proteins were separated at a flow rate of 0.5

mL·min⁻¹in BTP buffer containing 0.25 mM

LMNG and 100 mM NaCl. The photoisomerase

Protein expression and isomerase activity

assay

Plasmids encoding human RGR (hRGR) and

two splicing variants with C-terminal FLAG-tag

13

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

were purchased from Origene (Rockville, MD,

USA). PcDNA3.1-derived plasmids encoding

chicken RGR (cRGR) and bovine RGR (bRGR)

with the C-terminal His8- and 1D4-tags were

produced by Gene Universal (Newark, DE,

USA). The nucleotide sequence for cRGR was

codon optimized for expression in mammalian

cells. The RGR mutants were generated by site-

directed mutagenesis using the Q5 kit (New

England Biolabs, Ipswich, MA, USA) according

to manufacturer’s instructions with primers listed

in Supplementary Table S2 and using the

respective wild-type RGR plasmids as

templates. All RGR variants were expressed in

HEK293S GnTI^-cells (ATCC, Manassas, VA,

USA) cultured in suspension in FreeStyle 293

medium (Thermo Fisher Scientific) with 2% fetal

bovine serum (Atlanta Biologicals, Flowery

Branch, GA, USA) and 1% Pen Strep (Thermo

Fisher Scientific). Cells were transfected using

polyethylenimine Max (Polysciences,

Warrington, PA, USA) at 3:1 PEI to DNA ratio

following previously established protocols (88)

and cultured for 48 h at 37 ^oC, 8 % CO₂in a

shaking incubator. For the retinal isomerization

assay, 8×10⁷HEK293S GnTI^-cells washed in

phosphate-buffered saline (PBS, 10 mM sodium

phosphate, 150 mM sodium chloride, pH 7.4)

were suspended in the 10 mM BTP buffer and

homogenized in a glass tissue grinder (Kimble

Chase, Rockwood, TN, USA). Cell lysates were

complemented with 1 mM sodium

tags (TETSQVAPA), were resuspended in 160

mL of 20 mM HEPES (pH 7.4) plus protease

inhibitor cocktail (Roche Diagnostics GmbH,

Mannheim, Germany), 1 mM MgCl₂and

benzonase nuclease (New England Biolabs),

homogenized with a glass tissue grinder and

centrifuged for 30 min at 50,000g. The

membrane pellet (25 g) was solubilized in 4.5

mM LMNG, 50 mM HEPES, pH 7.4, 0.25 M

NaCl, 1 mM MgCl₂, plus protease inhibitors,

benzonase and 30 μM all-trans-retinal (final

volume 160 mL) and then centrifuged for 50 min

at 50,000g. The supernatant was incubated for 1

h on batch with 6 mL of 1D4 antibody

immobilized in Sepharose beads (CNBr-

activated Sepharose™ 4B, GE Healthcare), then

washed on column with 200 mM HEPES, pH 7.4

with 0.15 M NaCl and 0.2 mM LMNG. Finally,

bRGR was eluted with washing buffer containing

0.8 mg/mL of competing peptide. The purified

protein was then used directly for UV-Vis

spectrometry and for the photoisomerase activity

assay as described above. The purity of the

fractions was assessed by SDS-PAGE on

Coomassie-stained Bis-Tris (4-12%)

polyacrylamide gels (Thermo Fisher Scientific).

Immunoblotting

Total protein fractions were extracted from

HEK293S GnTI^-cells using RIPA lysis buffer

(Millipore, Burlington, MA, USA), separated by

SDS-PAGE on Tris-glycine (4–20%)

polyacrylamide gels (Bio-Rad, Hercules, CA,

USA) and transferred to nitrocellulose

pyrophosphate, 75 µM BSA, 30 µM CRALBP

(85) and 12 µM all-trans-retinal and were

incubated for 30 min in quartz cuvettes in the

dark, or attached to mounted 395/530/625 nm

LEDs matched for 560 µW optical power output.

Retinal isomerization products were extracted

from the 600 µL of reaction mixture by vigorous

shaking with 400 µL of methanol and 400 µL of

hexanes and centrifuged for 5 min at 16,000g.

Then, 100 µL of extracted retinoids were injected

for the HPLC analysis. The retinoids in the

organic phase were quantified as described

above.

membranes (Bio-Rad). Membranes were

blocked in 20 mM Tris, pH 7.5 with 0.5 M NaCl,

0.1% Tween 20, 5% non-fat dry milk (RPI, Mount

Prospect, IL, USA) and proteins were detected

with the following primary antibodies: mouse

anti-FLAG M2 (1:1000, Sigma-Aldrich), mouse

anti-rhodopsin 1D4 (1:1000, produced in-house

(89)), and rabbit anti-GAPDH (1:2000, Abcam,

Cambridge, MA, USA). HRP-conjugated

secondary antibodies were visualized on the

membranes using SuperSignal West Pico Plus

Chemiluminescent Substrate (Thermo Fisher

Scientific). Images were collected with an

Odyssey Fc Imaging System (LI-COR, Lincoln,

NE, USA).

Purification of recombinant bRGR

The pellet from a 2.1-L culture of HEK293S

GnTI^-cells expressing bRGR, which was C-

terminally tagged with His8- and 1D4 antibody

14

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Single-cell RNA sequencing analysis

(Thermo Fisher Scientific) containing 1% fetal

bovine serum (Thermo Fisher Scientific) and 1%

Pen Strep (Thermo Fisher Scientific). The RPE

cells were pelleted through centrifugation for 10

min at 300g. Then, the pellet was triturated with

1 mL of Dulbecco's PBS (Thermo Fisher

Scientific) and filtered by a 50 μm cell strainer to

remove aggregates. The dissociated bovine

retinal cells and RPE cells were washed with 1

mL of Dulbecco's PBS twice and fixed with 100

μL of PBS and 900 μL of methanol. The

A human eye globe from an 86-year-old

Caucasian female who died of a myocardial

infarction and had no known ocular disease

other than cataracts, was obtained from the

Alabama Eye Bank (Birmingham, AL, USA) and

processed within 3.3 h after death. The study

was approved by the Johns Hopkins Institutional

Review Board. The neural retina and

RPE/choroid were dissected from the globe in

ice-cold PBS. First, a circular incision was made

on the sclera, behind the limbus, to remove the

anterior parts, lens and vitreous body. The

neural retina was then peeled from the eyecup,

and retinal cells were dissociated using the

Papain Dissociation System (Worthington

Biochemical, Lakewood, NJ, USA) following the

manufacturer’s instructions. RPE cells were

methanol-fixed bovine samples were rehydrated

and processed by following the protocols

provided from 10x Genomics (10x Genomics,

Pleasanton, CA, USA). Briefly, the methanol-

fixed cells were centrifuged at 3000g for 10 min

at 4 ^oC and washed 3 times in an ice-cold

resuspension buffer containing PBS, 1% BSA

and 0.5 U/μL RNAse inhibitors. The dissociated

dissociated from the eyecup by incubating with 2

mL of 0.05% trypsin-EDTA (Thermo Fisher

Scientific) for 20 min at 37 ⁰C. Dissociated cells

were resuspended in ice-cold PBS, 0.04% BSA

and 0.5 U/μL of RNAse inhibitors. Cells were

then filtered through a 50 μm cell strainer. CD1

mice around 2 months of age were purchased

from Charles River Laboratories (Wilmington,

MA, USA). All experimental procedures were

pre-approved by the Institutional Animal Care

and Use Committee (IACUC) of the Johns

Hopkins University School of Medicine. Mice

were euthanized, and eye globes were removed

and incubated in ice-cold PBS. The neural

retinas were dissected, and cells were

dissociated using the Papain Dissociation

System as mentioned above. In total, 4

biological replicates were used for the mouse

scRNA study. Each replicate contained 4 retinas

from each of 2 male and 2 female mice.

Dissociated cells were resuspended and filtered

as above. Cell count and viability were assessed

by Trypan blue staining. Fresh bovine eyes were

obtained from a local slaughterhouse. The

bovine eyes were hemisected, and the neural

retina was peeled off from the eyecups. The

retinal cells were dissociated using the Papain

Dissociation System as mentioned above. The

RPE layer was detached from the eyecup by

incubating with 2 mL of 0.05% trypsin-EDTA for

20 min at 37 ^oC. The detached RPE cells were

resuspended in 8 mL of DMEM/F12 medium

cells were resuspended in the buffer and filtered

through a 50-μm cell strainer. For human and

mouse samples, freshly dissociated cells

(~10,000) were loaded into a 10x Genomics

Chromium Single Cell system (10x Genomics)

using v2 chemistry following the manufacturer’s

instruction. The bovine samples were processed

using 10x Genomics v3 chemistry. Libraries

were pooled and sequenced on Illumina

NextSeq with ~200 million reads per library.

Sequencing results were processed through the

Cell Ranger 3.0.1 pipeline (10x Genomics) with

default parameters. Seurat v2.3.4 (90) was used

to perform downstream analysis following the

standard pipeline using cells with more than 200

genes and 1000 UMI counts, resulting in 16,659

mouse cells, 14,286 human cells and 34,881

bovine cells. Samples were aggregated, and cell

clusters were annotated based on previous

literature. A t-distributed stochastic neighbor

embedding (t-SNE) dimension reduction was

performed on the top principal components

learned from high variance genes. Gene

expression of each cell cluster was calculated

using the average expression function of Seurat.

Statistical analyses

Data are presented as the means ± standard

deviations or means ± standard errors for the

results of at least three independent

experiments. Significant differences between

15

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

groups were assessed by Student’s t test or one-

for their helpful comments regarding this project.

K. Palczewski is the Leopold Chair of

Ophthalmology at the Gavin Herbert Eye

Institute, Department of Ophthalmology,

University of California, Irvine.

way ANOVA with the Bonferroni post-test for

comparisons of more than two groups. Sigma

plot 11.0 (Systat Software) was used to perform

the statistical analyses. The null hypothesis was

rejected when P values were < 0.05. *P < 0.05;

**P < 0.01; ***P < 0.001; n.s, non-significant.

Conflict of interest: K.P. is Chief Scientific

Officer at Polgenix, Inc. G.P. is an employee of

Polgenix, Inc.

Acknowledgements: We thank Drs. H. Jin

and W. Sun for assistance with 1D4 antibody

production and tissue culture studies. We thank

Dr. S. Gulati and A. Sears for providing rod outer

segments and interphotoreceptor retinoid-

binding protein, respectively. We thank Drs. B.

Willard and L. Li from Cleveland Clinic for

assistance with mass spectrometry. We are also

grateful to members of the Palczewski laboratory

16

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

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FOOTNOTES:

This research was supported in part by grants to K.P. from the National Institutes of Health (NIH)

(EY009339, EY027283, EY025451 and EY019312) and P.D.K. from the U.S. Department of

Veterans Affairs (IK2BX002683). E.H.C was supported by T32GM007250 and T32GM008803. The

authors also acknowledge support from an RPB unrestricted grant to the Department of

Ophthalmology, University of California, Irvine.

The abbreviations used are: BSA, bovine serum albumin; BTP, bis-tris propane; CRALBP, cellular

retinaldehyde-binding protein; DES1, sphingolipid Δ4-desaturase 1; DMF, dimethylformamide;

DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; LED, light-emitting

diode; LMNG, lauryl maltose neopentyl glycol; LRAT, lecithin:retinol acyltransferase; PBS,

phosphate-buffered saline; PLA₂, phospholipase 2; RDH5, retinol dehydrogenase 5; RDH8, retinol

dehydrogenase 8; RGR, RPE-retinal G protein-coupled receptor; RPE, retinal pigment epithelium.

23

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

FIGURES AND FIGURE LEGENDS

Figure 1. Schematic representation of retinoid transport and transformations in vision.

Conversion of all-trans-retinal to 11-cis-retinal occurs through the classical visual cycle (shown by

black arrows) or the pathway described in this work, involving the RPE-retinal G protein-coupled

receptor (RGR) in the RPE under visible light (blue box).

24

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 2. Enzymatic 11-cis-retinal generation is dependent on the wavelength of light. (a)

The wavelength of diodes versus the energy of the photons. The color code indicates the

wavelength of the light. (b) The range of the wavelengths investigated covers the absorbance of

human cone opsins and rhodopsin. (c) Graphs showing the 9-cis- and 11-cis-retinal generation rates

during 20 min light exposure with the given wavelength of light and a fixed power at 50 µW. (●) 11-

cis-retinal generation with native RPE microsomes. The green arrow indicates the optimum

wavelength for the enzymatic reaction. (○) 11-cis-retinal generation with denatured RPE

microsomes. (□) 9-cis-retinal generation with RPE microsomes. Data points are shown as means ±

standard deviations (n = 3).

25

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 3. Optimization of the enzymatic production of 11-cis-retinal in RPE under green

light. Photoreactions were carried out under 530 nm light (100 µW) for 15 min in the presence of

CRALBP. (a) Production of 11-cis-retinal is dependent on the amount of RPE. (b) all-trans-Retinal

serves as substrate for the photoisomerase. (c) all-trans-Retinal is the favored substrate of RGR as

compared to 9-cis-retinal and 13-cis-retinal. The retinal isomer used as substrate in each case, at 15

μM concentration, is marked under each bar. (d) Production of 11-cis-retinal in the presence of

CRALBP (green trace) or denatured CRALBP (black trace) in native RPE microsomes, or

denatured RPE microsomes (gray trace). (e) Photoisomerization is pH sensitive. (f) The influence of

temperature on photoisomerization. Data points are shown as means ± standard deviations (n = 3-

4).

26

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 4. Light-dependent 11-cis-retinal generation is independent of the classical visual

cycle. Retinal/retinol were incubated with RPE microsomes at 37 ºC under 530 nm light (100 μW).

(a) Schematic representation of two routes of isomerization generating 11-cis-retinoids. The 11-cis-

retinal production was investigated with all-trans-retinal (15 μM) only (●) or both all-trans-retinal (15

μM) and all-trans-retinol (15 μM) (■) as the substrate; the 11-cis-retinol production was also studied

with all-trans-retinol (15 μM) only (○) or both all-trans-retinal (15 μM) and all-trans-retinol (15 μM) (□)

as substrates. (b-d) Time course of 11-cis-retinoid generation in RPE microsomes under 530 nm

light (100 μW) at 37 ºC without inhibitors (b), with 10 μM emixustat (c), or with 10 μM fenretinide (d).

Data points are shown as means ± standard deviations (n = 3).

27

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 5. Photoisomerization requires the binding of all-trans-retinal to RGR through a

Schiff base formation. (a) Hydroxylamine competes with the photoisomerase to bind all-trans-

retinal through a Schiff base formation. The X-axis represents the natural logarithm of the

hydroxylamine/all-trans-retinal molar ratio. Data points are shown as means ± standard deviations (n

= 3). (b) 11-cis-retinal generation after RPE membranes had been reduced with sodium

cyanoborohydride, in the presence of all-trans-retinal (Red) or benzaldehyde (Blue). Numbers below

the graph denote the concentration of aldehyde used during the pre-incubation of the RPE samples,

before conducting the 11-cis-retinal generation assay. (c) RPE membranes incubated with [³H]-all-

trans-retinal were analyzed by SDS-PAGE and stained with Coomassie (left) or processed for

fluorography (right). RPE microsomal proteins were labeled with 2 µM [³H]-retinal (lane 1) or 2 µM

[³H]-retinal plus 12 µM non-radioactive retinal (lane 2), then reduced with sodium cyanobrohydride.

Samples in lanes 3 and 4 are 7.5-times dilutions of samples in lanes 1 and 2, respectively. (d) SDS-

polyacrylamide gel of solubilized RPE microsomes after ion-exchange and size-exclusion

chromatography (SEC). Green bars represent the relative photoisomerase activity in each SEC

fraction.

28

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 6. 11-cis-Retinal in RPE also originates from the oxidation of all-trans-retinol and

subsequent photoisomerization. RPE microsomes with exogenous all-trans-retinol (30 µM) was

exposed to 530 nm light (200 µW) for 30 min at 37 ºC. Emixustat (10 µM) was added to inhibit the

isomerization by RPE65. The products were extracted with hexanes and analyzed with normal

phase HPLC. (a, b) The representative chromatographs of products extracted from reactions in the

presence of 0 (a) or 2 mM (b) NAD⁺. Peak 1: retinyl esters; Peak 2: solvent gradient change; peak 3:

13-cis-retinal; peak 4: 11-cis-retinal; peak 5: 9-cis-retinal; peak 6: all-trans-retinal; peak 7: 11-cis-

retinol and 13-cis-retinol; peak 8: all-trans-retinol. The trace amounts of 11-cis-retinoids in (a)

originate from residual retinoid in RPE microsomes.

29

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Figure 7. Bovine RGR expressed in HEK293S GnTI^-cells photoisomerizes all-trans-retinal

to 11-cis-retinal. (a) Immunoblot of bRGR and K255A bRGR expressed in HEK293S GnTI^-cells.

Sample loading was normalized based on the signal for GAPDH. (b) Amounts of 11-cis-retinal

photoisomerized from all-trans-retinal in cell homogenates exposed to 530 nm light. Homogenates of

HEK293S GnTI^-cells transfected with a plasmid encoding bRGR produced a significant amount of

11-cis-retinal. The presence of CRALBP increased the amount of 11-cis-retinal, whereas the

mutation K255A within bRGR nearly abolished its photoisomerase activity. n = 3 for each group.

Data are shown as mean ± S.E.M. (c) Representative chromatograms showing the amount of 11-cis-

retinal and all-trans-retinal from different cell homogenates when exposed to 530 nm light.

30

Article Doi

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

DOI: 10.1074/jbc.RA119.011169

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.RA119.011169

Full text of DOI:10.1074/jbc.RA119.011169

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