A novel retinol-binding protein in the retina of the swallowtail
butterfly,
Papilio xuthus
Motohiro Wakakuwa
1
, Kentaro Arikawa
1
and Koichi Ozaki
2
1
Graduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa;
2
Graduate 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-

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-
trans retinol 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-to11-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

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

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 25 °C under a light regime of
8-h light : 16-h dark. The pupae were stored at 4 °Cfor
atleast3monthsandthenallowedtoemergeat25°C.
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
Papilio RBP was purified from a water-soluble fraction of

NaCl, 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 m
M
Tris/HCl
buffer (pH 6.8), and the homogenate was centrifuged at
15 000 g for 30 min at 4 °C. The supernatant was put on a
5% polyacrylamide concentrating gel (125 m
M
Tris/HCl,
pH 6.8), and proteins in the supernatant were separated in
a 10% polyacrylamide gel (375 m
M
Tris/HCl, pH 8.8)
under electrophoresis using Tris/glycine (25/192 m
M
) 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

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¢)basedonthe
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–KpnIsites
and poly(T) sequences (ROLBP-RT1, 5¢-GCCGAATT
CGAGCTCGGTACCTTTTTTTTTTTTTTTTT-3¢)was
prepared to synthesize the first strand cDNA from the
Papilio retinal 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

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
2438 M. Wakakuwa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
whole gel elutor as described above, dialyzed to remove
acrylamide contamination, concentrated with a Centricon
YM-10 (Millipore), and re-dissolved in 10 m
M
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 of
Papilio
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

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)NativePAGEofthecrude
extractofthePapilio 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.
Ó FEBS 2003 Retinol-binding protein in butterfly eye (Eur. J. Biochem. 270) 2439
This suggests that the protein exists in a monomeric state
in vivo.
Biochemistry of
Papilio

[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 of
Papilio
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¢-and5¢-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 )9to)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 Papi lio RBP. The lig-

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
Papilio RBP 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
Papilio RBP 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

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.
Ó FEBS 2003 Retinol-binding protein in butterfly eye (Eur. J. Biochem. 270) 2441
Discussion
A novel retinol-binding protein,
Papilio
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.
2442 M. Wakakuwa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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

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).
Ó FEBS 2003 Retinol-binding protein in butterfly eye (Eur. J. Biochem. 270) 2443
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

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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