Báo cáo khoa học: A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus docx

The protein that we term Papilio retinol-binding protein Papilio RBP is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peakin

A novel retinol-binding protein in the retina of the swallowtail

Motohiro Wakakuwa1, Kentaro Arikawa1and Koichi Ozaki2

1

Graduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa;2Graduate School of Frontier Biosciences, Osaka University, Toyonaka, Osaka, Japan

Retinoid-binding proteins are indispensable for visual cycles

in both vertebrate and invertebrate retinas These proteins

stabilize and transport hydrophobic retinoids in the

hydro-philic environment of plasma and cytoplasm, and allow

regeneration of visual pigments Here, we identified a novel

retinol-binding protein in the eye of a butterfly, Papilio

xuthus The protein that we term Papilio retinol-binding

protein (Papilio RBP) is a major component of retinal

soluble proteins and exclusively binds 3-hydroxyretinol, and

emits fluorescence peaking at 480 nm under ultraviolet (UV)

illumination The primary structure, deduced from the

nucleotide sequence of the cDNA, shows no similarity to any

other lipophilic ligand-binding proteins The molecular mass

and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplas-mic matrix All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer These results suggest that the Papilio RBP is involved in visual pigment turnover Keywords: retinol-binding protein; rhodopsin; visual pig-ment; visual cycle

Retinalaldehyde (retinal) plays an essential role in animal

vision as the chromophore of visual pigments that are

generically called rhodopsins In the rhodopsin molecule,

retinal is bound to the protein, opsin, in the 11-cis

configuration Light energy first isomerizes the

chromo-phore into its all-trans form that subsequently causes a

conformational change of the opsin into an active form The

activated rhodopsin, usually called metarhodopsin, triggers

the phototransduction cascade, that eventually controls the

flow of ion currents through cation channels in the plasma

membrane of the photoreceptor cell Prolonged illumination

will cause depletion of rhodopsin unless its chromophore is

replenished An important pathway for rhodopsin

replen-ishment in all known photoreceptor cells is the recovery of

all-trans retinal from opsin, its reverse isomerization to the

11-cis form, and subsequent recombination with opsin

Some processes in the pathway do not occur in the

photoreceptive membrane, where rhodopsin molecules

are embedded and function Thus, the retinal has to

be transported, when necessary, in hydrophilic matrices

As retinoids are highly hydrophobic and hardly soluble in water, hydrophilic retinoid-binding proteins are therefore required for stabilizing retinoids in the watery plasma as well as in the cytoplasm, and for transporting retinoids within and/or between cells [1] In addition, recent studies have demonstrated that such protein is not simply a carrier

of retinoid Regulation of retinoid concentration and its delivery to various cells, protection of retinoid from degradation and protection of cells from the potentially toxic properties of free retinoid may also be biologically important functions of retinoid-binding proteins (reviewed

in [2])

The rhodopsin recycling system, the visual cycle, is well characterized in vertebrates (reviewed in [3–5]) Briefly, all-transretinol bound to serum retinol-binding protein (RBP) circulates in the blood and is targeted to the retinal pigment epithelial (RPE) cells There it is possibly transferred to cellular retinol-binding protein (CRBP) and esterified to all-trans-retinyl ester After hydrolysis and isomerization to the 11-cis form, it is transferred to cellular retinal-binding protein (CRALBP) and oxidized to 11-cis retinal Several mechanisms for the isomerization from all-trans to 11-cis isomer have been proposed These include coupling of the hydrolysis of all-trans-retinyl esters to isomerization gener-ating 11-cis-retinol [6], or the presence of an enzyme catalyzing the direct isomerization of all-trans- to 11-cis-retinol through a carbocation intermediate [7] In both cases, the isomerization requires the presence of CRALBP [6,7] Another pathway for isomerization is mediated by RPE retinal G-protein-coupled receptor (RGR) RGR is a vertebrate homolog of squid retinochrome (see below), and catalyzes light-dependent isomerization of all-trans- to 11-cis-retinal [5,8] The 11-cis-retinal formed in the RPE cells is then transported across the interphotoreceptor

Correspondence to K Ozaki, Graduate School of Frontier

Biosciences, Osaka University, 1-1 Machikaneyama, Toyonaka,

Osaka 560-0043, Japan Fax/Tel.: + 81 6 6850 5439,

E-mail: ozaki@bio.sci.osaka-u.ac.jp

Abbreviations: CRALBP, cellular retinal-binding protein; CRBP,

cellular retinol-binding protein; IRBP, interphotoreceptor

retinoid-binding protein; RBP, retinol-binding protein.

Note: The nucleotide sequence reported in this paper has been

deposited in the DDBJ/EMBL/GenBank under the accession number

AB070628.

(Received 12 February 2003, revised 4 April 2003,

accepted 9 April 2003)

matrix to the photoreceptor cells Involvement of

inter-photoreceptor retinoid-binding protein (IRBP) in this

step has been advocated, but is, however, still in dispute

(reviewed in [2]) In photoreceptor cells, retinal binds to

opsin to form rhodopsin All-trans-retinal, liberated from

opsin after light absorption, is reduced into all-trans-retinol

in the photoreceptor cells and then moved back to retinal

pigment epithelial cells

Regeneration of rhodopsin in invertebrates is somewhat

different from that of invertebrates, as studied intensively in

cephalopods and insects Metarhodopsins of these animals

are usually thermostable, i.e the opsin and the

chromo-phore do not immediately separate as they do in vertebrates

Therefore, metarhodopsins can absorb light whose

wave-length is different from the wavewave-length absorbed by

rhodopsins Upon light absorption by metarhodopsin,

all-trans-retinal is reconverted to 11-cis form, and thus,

rhodopsin is regenerated This pathway is called

photo-reconversion or photoregeneration In addition to this

photochemical reaction, there exists another pathway

through which rhodopsin is metabolically regenerated

(visual cycle) In squid, Todarodes pacificus, metarhodopsin,

resulting from photoconversion of rhodopsin, transfers

its all-trans-retinal to squid retinal-binding protein (squid

RALBP) [9] The protein transports the all-trans-retinal

from the outer segment to the inner segment of the

photoreceptor cell [10,11] In the inner segment, all-trans

retinal is transferred to retinochrome Light absorption by

the retinochrome-all-trans-retinal complex causes

photo-isomerization of the all-trans-retinal to the 11-cis form,

which is then transferred to the squid RALBP and

sub-sequently transported back to the outer segment The squid

RALBP provides the attached 11-cis-retinal to

metarho-dopsin and, in return, receives all-trans-retinal: the

rhodop-sin is thus regenerated In this system, squid RALBP

functions as a shuttle carrying 11-cis- and all-trans-retinal

back and forth between the inner and the outer segments

[10,12] A similar recycling system using retinochrome and

RALBP is also found in gastropods [13,14] Recently,

Robles et al suggested the direct interaction of rhodopsin

with retinochrome, based on immunocytochemical

obser-vations [15] However, this finding does not completely rule

out the involvement of RALBP in chromophore transport

in the cephalopod visual cycle

The visual cycle in insect retina has been studied in several

species In the blowfly retina, metarhodopsin is degraded

slowly into opsin and all-trans-3-hydroxyretinal [16] HPLC

analysis of retinoids suggested that the liberated

all-trans-3-hydroxyretinal might be bound to a protein that mediates

photoisomerization of the all-trans-3-hydroxyretinal to the

11-cis form [17,18] A protein having required properties has

been isolated from the honeybee retina [19,20], but not yet

from fly The 11-cis-3-hydroxyretinal is then reduced to

alcohol (11-cis-3-hydroxyretinol) followed by slow

re-oxi-dation to aldehyde (11-cis-3-hydroxyretinal) The aldehyde

would be used as a chromophore to regenerate rhodopsin

Involvement of 11-cis-3-hydroxyretinol in this pathway was

proposed based on the observation that the amount of

11-cis-3-hydroxyretinol was increased considerably by

light-adaptation [17] Also in the butterfly retina, it has been

demonstrated that metarhodopsin is degraded rapidly [21],

and abundant 3-hydroxyretinol is contained in the soluble

fraction [22,23] These findings suggest that a visual cycle similar to that in the fl y also exists in the butterfl y retina

In addition, it was demonstrated in the Japanese yellow swallowtail butterfly, Papilio xuthus, that the isomer com-position of the 3-hydroxyretinol changes between the light-and dark-adaptation, suggesting that the 3-hydroxyretinol is possibly involved in the visual cycle Although these studies strongly suggest that some retinol-binding protein may be involved in the insect visual cycle, no such a protein has been identified

In addition to the above biochemical studies, we recently found that the Papilio compound eye consists of three distinct types of ommatidia, one of which emits strong fluorescence under ultraviolet light [24] The microspectro-fluorometric study suggested that the fluorescence is due to 3-hydroxyretinol that can act as a UV absorbing spectral filter These previous observations suggested strongly that some kind of retinol-binding protein possibly localized in the Papilio retina, and functions in the visual cycle and/or color vision

In this study, we therefore isolated a soluble retinol-binding protein from the Papilio retina, and performed molecular biological and biochemical analyses of the pro-tein As the protein is a novel species of the hydrophobic-ligand-binding protein and solely binds 3-hydroxyretinol

as an intrinsic ligand, we termed this protein the Papilio retinol-binding protein (Papilio RBP) Further analysis suggested that Papilio RBP is involved in the visual cycle rather than the ommatidial fluorescence

Materials and methods

Animals

We used both sexes of the Japanese yellow swallowtail butterfly, Papilio xuthus Linnaeus The butterflies were reared on fresh citrus leaves at 25C under a light regime of 8-h light : 16-h dark The pupae were stored at 4C for

at least 3 months and then allowed to emerge at 25C When necessary, the butterflies were dark-adapted for 48 h

in complete darkness, or light-adapted for 12 h by posi-tioning the animals 5-cm from a 15 W white fluorescent lamp For light-adaptation, butterflies were immobilized by clipping their wings and fixed in appropriate positions Column chromatography

PapilioRBP was purified from a water-soluble fraction of the retina by two-step column chromatography All of the following procedures were conducted under dim red light Retinas of the dark-adapted butterflies were detached from the corneal cuticle of the compound eyes and homogenized

in 63 mMTris/HCl buffer (pH 6.8) The homogenate was centrifuged at 15 000 g for 15 min at 4C yielding a clear supernatant containing only soluble proteins The proteins

in the extract were first separated by anion-exchange chromatography using the SMART System (Amersham Pharmacia Biotech) equipped with a Mono Q column that was equilibrated with 20 mMbis/Tris/HCl buffer (pH 6.5)

at room temperature The proteins were eluted with a linear gradient from 0–0.4M NaCl in the same buffer The fractions that emit bluish fluorescence under UV-irradiation

were collected, concentrated by ultrafiltration, and subjected

to further purification using size-exclusion chromatography

The chromatography was performed using the SMART

System equipped with a Superdex 75 column The column

was equilibrated with 150 mM bis/Tris/HCl (pH 6.5)

con-taining 0.15MNaCl, and proteins were eluted with the same

buffer at room temperature The absorbance of the eluent

was monitored at 280 nm and 330 nm

Gel electrophoresis

Besides the column chromatography, native PAGE was

also used for purification of Papilio RBP as follows The

compound eyes were homogenized in 63 mM Tris/HCl

buffer (pH 6.8), and the homogenate was centrifuged at

15 000 g for 30 min at 4C The supernatant was put on a

5% polyacrylamide concentrating gel (125 mM Tris/HCl,

pH 6.8), and proteins in the supernatant were separated in

a 10% polyacrylamide gel (375 mM Tris/HCl, pH 8.8)

under electrophoresis using Tris/glycine (25/192 mM)

run-ning buffer After electrophoresis, the gel was illuminated

with UV light that visualizes a single band of Papilio RBP

by strong whitish fluorescence A piece of gel containing

the fluorescing band was then cut out, and Papilio RBP

was eluted electrophoretically out of the gel Alternatively,

the gel was placed in a whole gel elutor (Bio-Rad)

immediately after electrophoresis, and fluorescing fractions

were retrieved electrophoretically Regular SDS/PAGE

was also performed according to Laemmli (1970) by the

use of 12% polyacrylamide gel [25] The gel was then

stained with Coomassie Brilliant Blue to visualize the

proteins

Protein digestion and sequencing

PapilioRBP was purified from 100 compound eyes as

described above The purified protein was digested with

10 pmol of lysyl-endopeptidase in 83 mM Tris/HCl buffer

(pH 9.2) for 5 h at 37C The reaction was stopped by

adding trifluoroacetic acid to the reaction mixture at a final

concentration of 0.04% Peptides were separated and

isolated by reverse-phase HPLC (SMART System) using

a lRPC C2/C18column equilibrated with 0.1%

trifluoro-acetic acid Peptides were eluted with a 0–80% linear

gradient of acetonitrile containing 0.1% trifluoroacetic acid

Elution was monitored at 215 nm and peaks were collected

separately Amino acid sequences of isolated peptides were

determined using a protein sequencer (Model G1005A,

Hewlett Packard) For nucleotide sequencing of

Papi-lioRBP cDNA, Poly(A)+RNA was prepared from 40

compound eyes using a QuickPrep mRNA Purification Kit

(Amersham Pharmacia Biotech), and used for synthesis of

cDNA with oligo(dT) primer We prepared three pairs of

oligo nucleotide primers (ROLBP1-forward, 5¢-AARGAR

GAYGTNTGG-3¢; ROLBP1-reverse, 5¢-CCANACRTC

YTCYTT-3¢; ROLBP2-forward, 5¢-AARGCNGGNAT

HYT-3¢; ROLBP2-reverse, 5¢-ARDATNCCNGCYTT-3¢;

ROLBP3-forward, 5¢-AARGTNTGGWSNGA-3¢; ROLB

P3-reverse, 5¢-TCNSWCCANACYTT-3¢) based on the

amino acid sequences determined above (KEDVW, KAG

IL, KVWSE) Using these primers, we amplified the Papilio

retinal cDNA by PCR, and determined the nucleotide

sequences of amplified cDNA products The 3¢- and 5¢-RACE were employed to complete sequencing of the entire coding region of the Papilio RBP cDNA For 3¢-RACE, the primer containing EcoRI–SacI–KpnI sites and poly(T) sequences (ROLBP-RT1, 5¢-GCCGAATT CGAGCTCGGTACCTTTTTTTTTTTTTTTTT-3¢) was prepared to synthesize the first strand cDNA from the Papilioretinal mRNA Based on the nucleotide sequence of the above PCR products, specific forward primers (ROL-BP4-F, 5¢-TTGCTTCCTCACGGCACCAG-3¢;

ROLBP5-F, 5¢-GACTAGTGGTGAACATGTGTATGCCGCAG-3¢) were synthesized and used for PCR with the first strand cDNA (template) and the partial sequence of ROLBP-RT1 (T-RAP, 5¢-GCCGAATTCGAGCTCGGTACC) as a reverse primer To synthesize the first strand cDNA for 5¢-RACE, a specific reverse primer (ROLBP-RT2, 5¢-TCTGCTCAATGATTGATGTC-3¢) was prepared The poly(A) sequence was attached to the 5¢-end of the cDNA, which was then amplified by PCR, using a set of primers, ROLBP-RT1 and ROLBP7-R (5¢-GACTAG TATCGCTTCAGGGTCCTCCGCTG-3¢) The product was again amplified with the second set of primers, T-RAP and ROLBP7-R

Ligand analysis The ligand analysis experiments were carried out under dim red light Ten compound eyes were used for each experi-ment In order to analyze the geometric isomers of retinoids using HPLC, Papilio RBP was isolated by native PAGE from the crude extract of the light-adapted or dark-adapted retinas, and finally dissolved in 200 lL PAGE running buffer Each sample (200 lL) was mixed with 60 lL of 2M hydroxylamine (NH2OH) and 400 lL of cold 90% meth-anol to convert 3-hydroxyretinals, if any, to retinaloximes Retinoids and retinaloximes were extracted with 500 lL dichloromethane and 6 mL n-hexane The extract was then concentrated and separated with a Hitachi model 635 HPLC system equipped with a YMC A-012 column (5-lm silica gel, 6· 150 mm, Yamamura Chemical Laboratory) Elution was carried out with n-hexane containing 25% ethyl acetate and 2% ethanol at a flow rate of 1.2 mLÆmin)1, and the eluent was monitored for absorbance at 340 nm In this elution condition, isomers of 3-hydroxyretinaloximes and 3-hydroxyretinol were eluted between 10 and 35 min, while isomers of retinaloximes and retinol were eluted just after the solvent front without enough resolution In the present study, we did not carry out further analysis of retinaloxims and retinol, as neither retinal nor retinol are contained in the Papilio retina [22] Standard isomers of 3-hydroxyretinal were synthesized by M Ito (Kobe College of Pharmacy, Japan) [22] Isomers of 3-hydroxyretinol were prepared

by reducing the corresponding isomers of 3-hydroxyretinal

in ethanol with a trace amount of sodium borohydride For routine analyses, isomers of 3-hydroxyretinal and 3-hydroxyretinol extracted from Drosophila heads were also used as a standard mixture The molar ratio of retinol isomers was calculated by using their extinction coefficients

at 340 nm in the eluent (all-trans, 39 100; 11-cis, 22 700; 13-cis, 42 500) In order to measure absorption and fluorescence spectra of Papilio RBP, the fluorescing protein was collected from dark-adapted compound eyes using a

whole gel elutor as described above, dialyzed to remove

acrylamide contamination, concentrated with a Centricon

YM-10 (Millipore), and re-dissolved in 10 mM Tris/HCl

(pH 8.0) buffer Absorption and fluorescence spectra were

measured with a Hitachi model U-3300

spectropho-tometer and a Hitachi model F-4500 spectrofluorometer,

respectively

Localization ofPapilio RBP in light- and dark-adapted

eyes

Light- or dark-adapted Papilio retina was divided into distal

and proximal portions by pulling out the retina from the

corneal cuticle This manipulation allows the eyes to be

separated into the distal portion, which contains distal

one-third of the photoreceptor layer in addition to the cornea

and the crystalline cone, from the rest that we call the

proximal portion After performing native PAGE (see

above) in the distal and proximal portions separately, we

compared the fluorescence intensity between the portions on

the gel The fluorescence intensity was measured directly

with a CCD camera, stored using an ATTO AE6905C

Image Saver, and quantified withNIH IMAGEprogram The

gel was then stained with Coomassie Brilliant Blue, and

the protein content was measured via the absorbance of the

stained bands using a Sharp JX-350 image scanner We

further analyzed the isomer composition of the intrinsic

ligands of Papilio RBP extracted separately from the distal

and proximal portions of the retina Papilio RBP was

extracted from each portion of the dark-adapted or

light-adapted retina, and purified by native PAGE The ligand

was then analyzed by HPLC as described above

Results

Purification ofPapilio RBP

Figure 1A shows the results of native PAGE of crude

extract from Papilio compound eyes We identified a single

band emitting whitish fluorescence under UV illumination

Coomassie Brilliant Blue staining of the gel indicates that

the fluorescing protein is one of the major components of

soluble proteins in the crude extract The surface of the

fluorescing protein carries negative charge in total, because

the protein expresses high mobility in the native gel

We purified the fluorescing protein from the gel by

two-step column chromatography We first separated the

crude extract with an anion-exchange (Mono Q) column

and then with a size-exclusion (Superdex 75) column (Fig 1B) With this purification procedure, we isolated the protein from other soluble proteins, shown as a single band

in a SDS/PAGE gel (Fig 1C) The apparent molecular mass of this protein was 31 kDa on the SDS/PAGE gel, which was close to 34 kDa estimated from the size-exclusion chromatography in the native state (Fig 1B, inset)

Fig 1 Purification of Papilio RBP (A) Native PAGE of the crude

extract of the Papilio retina Fluorescence under UV (left) and

Coo-massie Brilliant Blue (CBB) staining (right) (B) Anion exchange

(Mono Q, top) and size-exclusion (Superdex 75, bottom)

chromato-graphs of Papilio RBP The fluorescent fraction (arrow) in the anion

exchange chromatography was collected, and re-chromatographed

with Superdex 75 column A well-separated fluorescent peak of

Papi-lio RBP (arrow), whose molecular mass is estimated to be

approxi-mately 34 kDa (open circle in inset) was isolated During

chromatography, eluents were continuously monitored via light

absorption at 280 nm (solid lines) and 330 nm (dotted lines) (C) SDS/

PAGE analysis of the crude extract and the purified Papilio RBP.

This suggests that the protein exists in a monomeric state

in vivo

Biochemistry ofPapilio RBP

To determine the native ligand of the fluorescing protein, we

extracted retinoids from the purified fluorescing protein

collected from dark-adapted Papilio eyes, and analyzed the

composition of retinoids with HPLC (Fig 2) It appeared

that the protein exclusively binds 3-hydroxyretinol We

therefore call the protein Papilio retinol-binding protein, or

Papilio RBP HPLC analysis demonstrated that protein

prepared from dark-adapted animals contained the all-trans

isomer as the major ligand, but significant amounts of 11-cis

and 13-cis isomers were also detected

The UV-induced fluorescence of the Papilio RBP

disap-peared after the intense UV-irradiation, probably because

the ligand was degraded To investigate whether

Papi-lioRBP has the ability to bind exogenous retinol, we

supplied, after irradiating the soluble fraction of the Papilio

retina with UV light, all-trans- or 13-cis-retinol to the

fraction, and analyzed the fluorescence of the proteins with

native PAGE As shown in Fig 3, both all-trans- and

13-cis-retinols restored the fluorescence of Papilio RBP This result

indicated that the protein could bind the exogenously added

retinols in vitro, irrespective of their isomeric form

We next performed spectrophotometry and

spectro-fluorometry of the Papilio RBP Besides the principal peak

at 280 nm, corresponding to the absorption of the

apopro-tein, the absorbance spectrum (Fig 4A) of the Papilio RBP

has a secondary peak at 330 nm, corresponding to the

absorption of 3-hydroxyretinol The rather broad emission

spectrum elicited by 330-nm light (Fig 4B), peaks at

480 nm, and is very similar to that of free 3-hydroxyretinol [24] This indicates that the binding of the apoprotein has little influence on the fluorescence profile of 3-hydroxy-retinol The excitation spectrum (Fig 4B), measured at an emission wavelength of 480 nm, shows two maxima at

332 nm and at 280 nm The principal peak at 332 nm corresponds to the absorbance spectrum of the ligand, 3-hydroxyretinol The distinct secondary peak at 280 nm indicates energy transfer from the apoprotein to the ligand Primary structure ofPapilio RBP

To determine the primary structure of the identified Papilio RBP, we first analyzed the amino acid sequences of lysyl-endopeptidase-digested fragments of purified protein Based

on the sequence results, we designed oligonucleotide primers and carried out RT-PCR to amplify fragments of cDNA encoding the protein, and determined its nucleotide sequence Subsequently, we performed 3¢- and 5¢-RACE protocols, and obtained the complete nucleotide sequence of the full-length cDNA encoding the protein (Fig 5) The cDNA is approximately 1 kb in length, and contains an open reading frame of 708 bases encoding 235 amino acid residues A stop codon (TAA at nucleotides )9 to )7) precedes the ATG at nucleotides 1–3, suggesting that the coding region begins at this ATG A polyadenylation signal, AATAAA, exists 16 bases upstream from the start of the poly(A)+tail

Fig 2 HPLC analysis of the intrinsic ligand of Papilio RBP The

lig-ands were extracted from Papilio RBP purified from the soluble

fraction of the dark-adapted retina Extraction and analysis were

carried out under dim red light as follows Purified protein was first

mixed with 2 M hydroxylamine and cold 90% methanol to convert

aldehydes, if any, to oximes Retinoids and oximes were then extracted

with dichloromethane and n-hexane, and separated by normal phase

HPLC Eluent was monitored for absorbance at 340 nm Each isomer

of 3-hyroxyretinaloxime and 3-hydroxyretinol was identified by its

retention time compared to that of the standard compound Purified

Papilio RBP exclusively binds 3-hydroxyretinol No isomers of

3-hydroxyretinal (detectable as 3-hydroxyretinaloxime, if present) were

detected AT, all-trans 3-hydroxyretinol; 13, 13-cis 3-hydroxyretinol;

11, 11-cis 3-hydroxyretinol.

Fig 3 Binding of exogenous ligands to Papilio RBP Fluorescence of native PAGE (left) and Coomassie Brilliant Blue (CBB) stained gel (right) Soluble proteins from the dark-adapted Papilio retina (lanes 1 and 1¢) were irradiated with intense UV-light to degrade intrinsic ligand (lanes 2 and 2¢) To the UV-irradiated samples, all-trans (lanes 3 and 3¢) or 13-cis (lanes 4 and 4¢) retinol was added, followed by 20-min incubation on ice In this experiment, retinol was used for 3-hydroxyretinol.

Figure 5 shows the amino acid sequence of the

Papi-lioRBP deduced from the cDNA sequence The relative

molecular mass of the protein calculated from the sequence

was 26 412 This value is somewhat smaller than that of

purified Papilio RBP as estimated by SDS/PAGE (31 kDa,

Fig 1) However, this difference is in the range of

experi-mental variation: we have often encountered such an

overestimation of molecular mass with SDS/PAGE when

the proteins are negatively charged (squid RALBP [11];

lipophilic ligand-binding protein in the fly chemosensory

hair [26]) The calculated pI value of the Papilio RBP is 4.92;

the protein is highly acidic This explains the high mobility

of the protein in the native PAGE (Fig 1)

In order to determine the N-terminal sequence of the

PapilioRBP in vivo, we sequenced intact Papilio RBP

without lysyl-endopeptidase digestion We acquired a

sequence, XSRIYPKVWS, although the recovery rate was

extremely low This result indicates that the Papilio RBP

undergoes post-translational modification: the N-terminal

methionine is removed and the second residue, serine,

carries some blocking residue In addition, we could not

identify any N-terminal signal sequence for secretion

Therefore, the Papilio RBP is most likely located in the

cytoplasm

Based on the deduced amino acid sequence, we searched for homologous proteins in databases Two partial sequences of the Papilio RBP, each consisting of less than

60 residues, showed low (<30%) identity to those of the chlorophyll-a/b-binding proteins and N-acetyltransferases

No protein was found that has significant similarity to the full length of Papilio RBP Therefore, we conclude that the PapilioRBP is a member of a novel protein family Papilio RBP in dark- and light-adapted eyes

To address the question of whether Papilio RBP is involved

in the visual cycle, we investigated the isomer composition

of the ligand and the distribution of the protein in dark- and light-adapted eyes In dark-adapted eyes, the molar ratio of all-trans, 11-cis and 13-cis isomer was 48 : 39 : 13 (Fig 6) When light-adapted, the ratio changed to 28 : 60 : 12, i.e the fraction of 11-cis isomer significantly increased, whereas that of all-trans isomer decreased (Fig 6) As the illumin-ation of purified RBP in vitro did not isomerize all-trans ligand to the 11-cis form (data not shown), it is clear that the light-induced change in isomer composition in vivo is not due to the direct isomerization of the all-trans ligand by PapilioRBP Instead, it is possibly interpreted as a result of replacement of all-trans ligand with 11-cis isomer newly produced in the light-adapted eyes

In order to investigate the distribution of Papilio RBP in the compound eye, we divided the eye into two portions, distal and proximal, by gently pulling the retina off from the corneal cuticle with fine forceps Figure 7A (right) shows a plastic section of the tissue layer containing the cornea, i.e the distal layer This layer contains about one-third of the photoreceptor layer as follows from a comparison with a section of the intact eye (Fig 7A, left) We then prepared crude protein extracts separately from the distal and the proximal portions of the dark-adapted eye, and analyzed them with native PAGE As shown in CBB-stained gel (Fig 7B, left), both portions appeared to contain similar amounts of RBP Also, the fluorescence intensity (Fig 7B, right) is similar in both portions, indicating that the RBP binds the ligand ubiquitously When the eyes were light-adapted, however, the amount of RBP, as well as the fluorescence of the ligand, increased in the distal portion and decreased in the proximal portion This strongly suggests that the RBP together with its ligand migrates distally upon light adaptation

We next analyzed the isomer composition of the native ligands of Papilio RBP extracted separately from the distal and the proximal portions of the retina (Fig 8) As expected from the fluorescence image analysis of the native PAGE (Fig 7B), total amount of 3-hydroxyretinol was increased

in the distal portion, and decreased in the proximal portion,

by light adaptation of the eye Light adaptation also induced the decrease of all-trans isomer both in the distal and proximal portions of the retina In contrast, the increase

of 11-cis ligand was observed in the distal but not in the proximal portion of the retina Together with the results from the native PAGE (Fig 7B), these findings strongly suggest that Papilio RBP exchanges its ligand from all-trans- to 11-cis-3-hydroxyretinol by light adaptation, and migrates from the proximal to the distal region within the retina

Fig 4 Spectrophotometric and spectrofluorometric characteristics of

Papilio RBP Absorbance (A) and fluorescence excitation and emission

(B) spectra of Papilio RBP were measured on the protein purified by

native PAGE The excitation spectrum was measured via emission at

480 nm, and emission spectrum was measured using excitation light

at 330 nm.

A novel retinol-binding protein,Papilio RBP

We identified a novel type of protein that binds retinol,

which we termed the Papilio retinol binding protein (Papilio

RBP) The native ligand of this protein is 3-hydroxyretinol,

whose isomer composition varies between dark- and

light-adaptation The deduced amino acid sequence of

Papi-lioRBP shows no overall similarity to any other proteins so

far described However, part of the sequence has some

similarity to that of chlorophyll-a/b-binding protein, whose

ligand is also hydrophobic [27] Furthermore, the

hydro-phobicity profile of the C-terminal half of Papilio RBP

resembles that of the human CRBP [28] These results

suggest that the binding proteins share some

three-dimen-sional structure that is crucial for binding hydrophobic

ligands Biochemical studies suggest that the butterfly visual

cycle may share a common system with the fly visual cycle (see Introduction) Nevertheless, no isoform of Papilio RBP could be found through the database search of the Drosophila genome We demonstrated previously that a lipophilic ligand-binding protein in the fly chemosensory organs probably had conformational and functional simi-larities to the general odorant-binding protein in the moths, while the similarity between their amino acid sequences was very low [26] This suggests that, in these ligand-binding proteins, amino-acids may be highly variable unless their protein conformation required for ligand binding is disrup-ted Further knowledge on the protein structure of the PapilioRBP would be essential to address above question Possible function of thePapilio RBP

What is the biological function of the Papilio RBP? First, it is important to realize that free retinoids are highly labile and

Fig 5 cDNA and deduced amino acid sequence of Papilio RBP The cDNA (923 bp) encodes an open reading frame for full-length Papilio RBP (708 bp, 235 amino acid resi-dues) The calculated M r and pI values are

26 412 and 4.92, respectively Amino acid sequences revealed by sequencing the peptides with lysyl-endopeptidase digestion are underlined Dotted underline indicates the N-terminal sequence obtained by sequencing the intact Papilio RBP Underline in the 3¢-noncoding region shows a possible polyadenylation signal.

possess various biological activities [1] When stored in

tissues, these labile and bioactive molecules need to be

stabilized and inactivated One way to achieve this is to bind

to hydrophilic proteins Apparently, the Papilio RBP

iso-lated from the soluble fraction of the eye is hydrophilic and

contributes to stabilize and inactivate 3-hydroxyretinol, the

native ligand of the protein

We suspect that the primary function of Papilio RBP is

involved in the visual cycle The chromophore of the Papilio

rhodopsins is 11-cis 3-hydroxyretinal [22] The

chromo-phore is converted into the all-trans form upon light

absorption by a rhodopsin molecule This photoconversion

of rhodopsin to metarhodopsin triggers the

phototransduc-tion process, resulting in a change in the photoreceptor

membrane potential (depolarization) To maintain light

sensitivity, photoreceptors have to restore the rhodopsin

content Metarhodopsins of arthropods are usually

thermo-stable; opposite to vertebrate metarhodopsin Therefore,

they have enough time to reabsorb light and change back to

rhodopsin by photoregeneration

However, invertebrate retinas possess additional

proces-ses for rhodopsin recycling that reuproces-ses all-trans retinal

released from metarhodopsin Indeed, it has been

demon-strated that metarhodopsin is bleached more rapidly than

rhodopsin in the fly and butterfly retinas [16,21] In Papilio,

Shimazaki and Eguchi have proposed a process of

rhodop-sin regeneration based on the HPLC analysis of retinoid in

the eye [23,29] According to their hypothesis, the

isomeri-zation process has taken place when the chromophore is

separated from opsin as in vertebrates

All-trans-3-hydroxy-retinol is somehow stored in the distal portion of the eye

The stored 3-hydroxyretinol is oxidized into

all-trans-3-hydroxyretinal that is subsequently isomerized to the 11-cis

form by light, and finally binds to opsin to form rhodopsin

In addition, they suggested that all-trans-3-hydroxyretinol

in the proximal portion of the photoreceptor cell is transported to the distal portion to facilitate biogenesis of rhodopsins Note that the present findings, namely the distal–proximal localization of retinoids (Fig 7) and isomer composition (Fig 8), basically matches the hypothesis

Fig 7 Light-induced relocation of Papilio RBP in the retina (A) Longitudinal sections of the intact (left) and the distal portion (right) of the Papilio eye (B) Native PAGE indicating the distribution of Papi-lio RBP in the distal and proximal portions of the retina Eyes from dark-adapted and light-adapted animals were divided into the distal and proximal portions Soluble proteins were extracted from each portion, and separated by native PAGE The fluorescence of Papi-lio RBP was recorded under UV-illumination (right), and the proteins

in the gel were stained with Coomassie Brilliant Blue (CBB) (left panel) The relative contents of the ligand and apoprotein were esti-mated via the intensity of fluorescence and the density of Coomassie Brilliant Blue, respectively The ligand or apoprotein content in the distal portion (D) was compared with that in the proximal portion (P) Mean ± SEM (n ¼ 4) of the D/P ratio are shown at the bottom of the corresponding electrophoresis records.

Fig 6 Light-induced change in isomer composition of the intrinsic

ligand of Papilio RBP Ligands were extracted from Papilio RBP

purified from dark-adapted or light-adapted retinas, and analyzed by

HPLC The molar ratio of all-trans, 11-cis and 13-cis 3-hydroxyretinol

was then calculated based on the absorbance and the molar extinction

coefficient at 340 nm of each isomer Mean ± SEM of three separate

experiments are presented **P < 0.01 (one-way ANOVA – Tukey test).

proposed by Shimazaki and Eguchi [23,29] The majority of

the Papilio RBP ligand exists in the all-trans form, in

dark-adapted eyes, and is then transformed to the 11-cis form

when eyes are light-adapted Light adaptation causes

relocalization of the Papilio RBP from the proximal to

the distal part of the retina Coincidence of the present data

with the hypothesis strongly suggests that the Papilio RBP

is involved in the visual cycle In order to elucidate how

PapilioRBP functions in the visual cycle, it would be

important to clarify the precise localization of the protein in

the distal and proximal portions of the light- or

dark-adapted retinas Immunohistochemical localization of

Papi-lioRBP would be adequate to address this question

Previously, we reported that the Papilio eye contains the

ommatidia that fluoresce under UV [30] The fluorescing

ommatidia have a concentration of fluorescing material,

most probably 3-hydroxyretinol that acts as a

UV-absorbing spectral filter for the underlying photoreceptors

[24] However, the fluorescence is restricted to the distal

70 lm of the photoreceptor layer, and not detectable in the

proximal portion In addition, the fluorescence is extremely

labile: it disappears in seconds under epi-fluorescence

microscopy [24] These results of fluorescence microscopy

are not explained by the present features of the Papilio

RBP On the other hand, HPLC analysis of retinoids

demonstrated that the amount of all-trans isoform

extrac-ted from the whole eye tissue was greater than that from

the purified Papilio RBP (data not shown) In contrast,

there are no significant differences in the amount of other

isomers between the whole eye tissue and purified samples This result suggests that there may be unknown storage of all-trans-3-hydroxyretinol that is not bound to the Papi-lioRBP and is related to the ommatidial fluorescence Immunohistochemistry would provide further insight on this issue

Acknowledgements

We thank D G Stavenga for critical reading of the manuscript We also thank S Kawamura for useful discussion and kind permission to use analytical instruments This work was supported partly by the Sasagawa Research Grant to M W., and the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science

to K O and K A.

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