Immunohistochemical localization of Papilio RBP in the eye of butterflies
1 Graduate School of Integrated Science, Yokohama City University, Yokohama,
Kanagawa 236-0027, Japan
2 Graduate School of Frontier Bioscience, Osaka University, Toyonaka, Osaka
560-0043, Japan
* Author for correspondence (e-mail: arikawa{at}yokohama-cu.ac.jp)
Accepted 2 February 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: butterfly, Papilio xuthus, Papilionoidea, retinoid binding protein, immunohistochemistry, 3-hydroxyretinol, pigment cell, tracheal cell, visual cycle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Visual pigments will be depleted by prolonged illumination unless they are
regenerated and/or newly synthesized. A key process of the visual pigment
regeneration, the visual cycle, is to generate 11-cis retinal from
all-trans retinal, retinol and other retinoids. As a result of
extensive studies of this process in recent decades, a number of retinoid
binding proteins have been identified in both invertebrates and vertebrates
(Hara and Hara, 1991;
McBee et al., 2001
;
Ozaki et al., 1987
;
Saari, 1999
;
Stavenga et al., 1991
). We
recently identified a retinol binding protein, Papilio RBP, whose
ligand is 3-hydroxyretinol, in the compound eye of the Japanese yellow
swallowtail butterfly Papilio xuthus
(Wakakuwa et al., 2003
). The
Papilio RBP is a novel protein, for it has little homology with any
other retinoid binding proteins so far reported. We found that the isomer
composition of the ligand of Papilio RBP changes between light- and
dark-adaptation: the content of 11-cis isoform increases in eyes in
the light. In addition, illumination significantly increases the amount of
11-cis isoform, especially in the distal part of the retinal layer
(Wakakuwa et al., 2003
).
This observation fully agrees with the results described in a previous
report, where retinoid composition was measured under various conditions of
adaptation (Shimazaki and Eguchi,
1995). We thus concluded that the protein is involved in the
regeneration of Papilio visual pigment whose chromophore is
11-cis 3-hydroxyretinal (Wakakuwa
et al., 2003
).
Where in the eye does the Papilio RBP function? Is this novel binding protein strictly specific to Papilio, or shared by other species? To answer these questions, we raised a specific antiserum against Papilio RBP. We then carried out light and electron microscopic immunohistochemistry in the Papilio retina to localize the protein in the retinal tissue. We also performed combined native PAGE and immunoblot analyses on several other insect species to evaluate the distribution of Papilio RBP-like protein among insects.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the comparative study, we used four papilionid species, Papilio machaon, Papilio protenor, Papilio helenus and Graphium sarpedon, a nymphalid Vanessa indica, a pierid Pieris rapae, and a hesperid Parnara guttata, captured in the field around the campus of Yokohama City University. We also investigated a dragonfly Orthetrum albistylum speciosum (Odonata, Libellulidae), a cicada Graptopsaltria nigrofuscata (Hemiptera, Cicadidae), a locust Oedaleus infemalis (Orthoptera, Acrididae) and a honeybee Apis mellifera (Hymenoptera, Apidae). These insects were captured in the field around the campus of Yokohama City University, except for Apis mellifera, which was taken from a hive culture.
Gel electrophoresis
For native PAGE, Laemmli's buffer system was used
(Laemmli, 1970) but without SDS
and 2-mercaptoethanol in the gel, running and sample buffer solutions. Unless
otherwise stated, whole compound eyes were used for extraction. The compound
eyes were homogenized in 63 mmol l1 Tris-Cl buffer (pH 6.8),
and the homogenate was centrifuged at 15 000 g for 30 min at
4°C. The supernatant was loaded onto a 10% polyacrylamide gel, and soluble
proteins were electrophoretically separated. After electrophoresis, the gel
was illuminated with UV light, which visualizes the RBP as a single
fluorescing band. The RBPs were recovered from cut pieces of the gel
containing the fluorescing band (purified RBP). When necessary, the gel was
stained with Coomassie Brilliant Blue (CBB). Regular SDS-PAGE was also carried
out using a 12% polyacrylamide gel
(Laemmli, 1970
). The gel was
then stained with CBB.
Antiserum production
To produce antigen by expression, we first carried out overexpression of
Papilio RBP. We prepared a pair of oligo nucleotide primer
(ROLBP-forward, 5'-GTGAAGACATATGTCTTCACGAATATATCC-3';
ROLBP-reverse, 5'-GAACTCGAGTTCAACTTTTGCCCCAAATATTTTG-3') based on
the full-length cDNA sequence of Papilio RBP
(Wakakuwa et al., 2003). Using
these primers, the entire coding region of Papilio RBP (714 bp) was
amplified. We subcloned the polymerase chain reaction (PCR) product into the
pET-21 expression vector; the vector was designed to enable recombinant
expression of the Papilio RBP as a fusion protein to which a 6
x His-tag was added at the C-terminal end for one-step purification by
nickel chelate affinity chromatography.
Female Wistar rats were immunized intradermally with 0.1 mg of purified recombinant Papilio RBP in 200 µl of phosphate-buffered saline emulsified 1:1 with Freund's complete adjuvant (Difco Laboratories, Detroit, USA). The rats were boosted every 2 weeks in a similar manner using Papilio RBP in incomplete Freund's adjuvant (Difco Laboratories): the rats were injected antigen six times in total. Immune serum was obtained 7 days after the final boost. Monospecificity of the antiserum was confirmed by immunoblot analysis (see below).
Immunoblot
Water-soluble extracts of retinal homogenates or the RBP purified from the
native PAGE gel were separated by 12% SDS-PAGE. The protein samples were then
blotted onto a polyvinylidene difluoride (PVDF) membrane. Nonspecific binding
sites were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered
saline (PBS), followed by overnight incubation with the antiserum. After
washing with PBS, the PVDF membrane was incubated with alkaline
phosphatase-conjugated secondary antibody. After washing with PBS, the PVDF
membrane was incubated in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue
tetrazolium (BCIP/NBT) in alkaline phosphatase buffer (100 mmol
l1 Tris-HCl, pH 9.5, 100 mmol l1 NaCl and
5 mmol l1 MgCl2), until adequate stain intensity
was obtained.
Immunohistochemistry
For light microscopic immunohistochemistry, isolated compound eyes were
fixed in 2% paraformaldehyde and 0.2% picric acid in 0.1 mol
l1 phosphate buffer, pH 7.4 (PB) for 30 min at room
temperature. After a brief wash with 0.1 mol l1 PB, the eyes
were then dehydrated in a graded ethanol series, infiltrated with xylene and
embedded in paraffin. Thin sections (8 µm) mounted on slides were incubated
in 0.3% H2O2 in water for 5 min to quench endogenous
peroxidase activity (Larsson,
1988). Non-specific binding sites for antibodies were blocked by
treating the sections with 10% normal goat serum in PBS for 30 min, and then
the sections were incubated in the anti-Papilio RBP in 1% BSA in PBS
overnight at 4°C. The sections were subsequently reacted with biotinylated
secondary antibody for 30 min, and further incubated with Vectastain ABC
Reagent (Vector Laboratories, Burlingame, USA). After washing with PBS, the
sections were incubated in peroxidase substrate solution (0.2%
3,3'-diaminobenzidine in 50 mmol l1 Tris-HCl, pH 7.4)
until adequate stain intensity was obtained.
For electron microscopic immunohistochemistry, isolated eyes were prefixed by immersing in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol l1 cacodylate buffer, pH 7.3 (CB) for 30 min on ice. After a 10 min wash with 0.1 mol l1 CB, the eyes were postfixed in 2% OsO4 in 0.1 mol l1 CB for 30 min on ice. After dehydration with graded methanol series, the eyes were embedded in LR White resin. Ultrathin sections, mounted on nickel grids, were treated with 4% BSA in PBSG (0.25% fish gelatin in PBS) for 20 min to block non-specific antibody binding sites, and then incubated with anti-Papilio RBP in PBSG overnight at 4°C. After washing with PBSG, the sections were reacted with secondary antibody-conjugated 15 nm colloidal gold particles for 1 h. After washing with PBSG, the sections were stained with 4% uranyl acetate in distilled water, and observed in a transmission electron microscope (JEM 1200EX; Tokyo, Japan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Localization of Papilio RBP in the eye of Papilio xuthus
We studied the possible localization of Papilio RBP via
the distribution of anti-Papilio RBP immunoreactivity in the compound
eye (Fig. 2). An ommatidium of
a Papilio eye consists of a dioptric apparatus, the corneal facet
lens and crystalline cone, and a retinula, containing nine photoreceptor cells
(Fig. 2A). The photoreceptor
cells together construct a photoreceptive rhabdom, which is a long, slender
cylinder in the center of the ommatidium. Neighboring ommatidia are optically
separated by the primary and secondary pigment cells, both containing
dark-brown pigment granules. The primary pigment cells wrap the crystalline
cones, whereas the secondary pigment cells are located between the ommatidia
along the entire length of the retinal layer. We found that
anti-Papilio RBP labeled these pigment cells but not the
photoreceptor cells (Fig.
2B,C). The anti-Papilio RBP strongly labeled the
cytoplasm of the tracheal cells, proximal of the basement membrane, forming
the fenestrated layer between the retina and the lamina, i.e. the first optic
ganglion (Fig. 3D). Non-immune
serum gave no labeling (Fig. 3B
inset).
|
|
The subcellular localization of Papilio RBP was further studied by electron microscopic immunohistochemistry. The results fully confirmed the light microscopical findings. Fig. 3 shows five pairs of electron micrographs, each consisting of one at low magnification and one at high magnification. In the retinal layer the gold particles, which indicate the localization of anti-Papilio RBP, were exclusively found in the pigment cells and not in the photoreceptor cells (Fig. 3AH). Interestingly, the anti-Papilio RBP labeled the nuclei as well as the cytoplasm of the pigment cells at similar density (Fig. 3EH). Electron microscopy revealed that the strongly labeled structures proximal to the basement membrane (Fig. 2D) are the nuclei and the cytoplasm of the tracheal cells (Fig. 3I,J). The photoreceptor axons, which pass through the basement membrane, were not labeled.
We quantified the labeling density in different regions of the eye
(Fig. 4). The labeling of the
pigment cells is significantly higher than the background labeling of the
photoreceptor cells (*, P<0.01, one-way ANOVA, Tukey test). In the
primary and the secondary pigment cells, both nuclei and the cytoplasm were
equally labeled. The strongest labeling was found in the tracheal cells, where
the calculated particle density was even higher in the nucleus than in the
cytoplasm. This is in fact due to the uneven labeling pattern in the
cytoplasm: labeling was rather confined to the region where more
electron-dense materials are concentrated (data not shown). Local labeling
densities in the nucleus and the cytoplasm were comparable (see for example
Fig. 3J). Although the labeling
density is higher in tracheal cells than in all other cells observed
(Fig. 4), this does not
contradict our previous results that the Papilio RBP is mainly
distributed in the distal retinal layer
(Wakakuwa et al., 2003): the
total volume of the pigment cells is overwhelmingly large compared to that of
the tracheal cells.
|
Papilio RBP-like proteins in other insect species
Fig. 5 shows the results of
the native PAGE analysis of crude retinal extracts from seven butterfly
species belonging to the superfamily Papilionoidea. We identified a single
band emitting whitish fluorescence under UV illumination in all species tested
(Fig. 5). The mobility of the
fluorescing proteins in the native gel seems genus dependent. Mobilities in
species of the genus Papilio are similar, whereas those of
Graphium (Papilionidae), Vanessa (Nymphalidae) and
Pieris (Pieridae) are distinctly lower.
|
Fig. 6 shows results of immunoblot analysis of these proteins. We first cut out the gel piece containing the fluorescing band. The proteins were then extracted from the gel pieces and separated by SDS-PAGE. The anti-Papilio RBP revealed a single band in all species. In the Papilionid species the molecular mass of these proteins was about 31 kDa and, in Vanessa and Pieris it was 25 kDa. Although in Vanessa three bands were evident in the SDS-PAGE, the anti-Papilio RBP detected only one of them at 25 kDa.
|
We also searched for Papilio RBP-like proteins in the eye of insects other than Papilionoidea, namely a skipper Parnara guttata (Lepidoptera, Hesperiidae), a dragonfly Orthetrum albistylum speciosum (Odonata, Libellulidae), a cicada Graptopsaltria nigrofuscata (Hemiptera, Cicadidae) and a grasshopper Oedaleus infemalis (Orthoptera, Acrididae). We identified a fluorescing band in all of these species under UV illumination on native PAGE (Fig. 7). However, none of them cross-reacted with the anti-Papilio RBP on immunoblots. We also checked whether the honeybee Apis mellifera (Hymenoptera, Apidae) has a corresponding protein, but we could not detect any fluorescing band on the native gel and any protein reacting with the anti-Papilio RBP on the immunoblot (data not shown).
|
Localization of Papilio RBP-like protein in the eye of Pieris rapae
Fig. 8 shows the
localization of the anti-Papilio RBP imunoreactivity in the eye of
Pieris rapae. As in Papilio xuthus, specific labeling was
confined to the primary pigment cells, the secondary pigment cells and the
tracheal cells proximal to the basement membrane. No labeling was detected in
the photoreceptor cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The principal function of the pigment cells is to optically isolate the
ommatidia by absorbing off-axis incident light, so to optimize spatial
resolving power. The light-driven isomerase, which may exist in the primary
pigment cells (e.g. Smith and Goldsmith,
1991), therefore, occupies an ideal location for receiving light,
so to serve the visual pigment regeneration cycle. The secondary pigment cells
are elongated in shape, lying between photoreceptor cells along the entire
length of the retinal layer (Fig.
2). Assuming that these cells are also involved in the visual
cycle, a possible function of the Papilio RBP then is to remove
retinoid in the all-trans form from the photoreceptors and/or to
supply retinoid in the 11-cis form back to the photoreceptors, to
replenish visual pigment molecules.
The function of Papilio RBP in the tracheal cells is difficult to understand at present. Of course, the tracheae are not restricted to the eye, but exist throughout the body. We carried out a preliminary immunoblot analysis on the abdominal tracheae and found slight immunoreactivity (data not shown): maybe the Papilio RBP has some function specific to the tracheal system, or it may function in transporting retinoids from and to the haemolymph in turnover and de novo synthesis.
Even more conspicuously, Papilio RBP-like immunoreactivity was found in the nuclei of both the pigment cells and the tracheal cells. To the best of our knowledge, this is the first example of retinoid-binding proteins in cell nuclei.
Comparative aspects
Is Papilio RBP specific to Papilio, or is it shared by
other species? This point is particularly important for elucidating a general
scheme of the function of Papilio RBP in the visual cycle. With this
question in mind, we carried out a comparative biochemical and
immunohistochemical analysis in several other insect species.
In native PAGE, we detected a fluorescing band in all tested butterfly
species. According to Seki et al.
(1987), butterflies use
3-hydroxyretinal as the visual pigment chromophore and most of them also
contain an excess amount of 3-hydroxyretinol. Presumably therefore, the
fluorescing substance in tested butterfly species is 3-hydroxyretinol. The
mobility of the fluorescing proteins in the native gel differs considerably,
indicating that their surface charge, size and/or the three-dimensional
structure rather vary. The proteins with lower mobilities may exist in certain
polymerized forms. In addition to the fluorescence, the proteins were found to
be immunoreactive to the anti-Papilio RBP in all butterflies, except
for the skipper Parnara guttata (Hesperiidae). The molecular mass of
the protein revealed by SDS-PAGE appeared to be similar in seven other
butterflies (2531 kDa; Fig.
6). In Vanessa indica, however, three bands were evident
on SDS-PAGE (Fig. 6), probably
due to overlap of three proteins in the location of the fluorescing protein
(Fig. 5). Immunohistochemical
localization of the Papilio RBP-like protein in the Pieris
eye, using the anti-Papilio RBP, revealed that the distribution
pattern is similar in Pieris and Papilio, indicating a
similar function in both species (Figs
2 and
8).
Our data on the Papilio RBP-like protein in other insects is still
preliminary. For example although we found a fluorescing protein band in the
gel (Fig. 7), we have not yet
identified the fluorescing materials themselves. These insects have actually
been shown to have significant amounts of retinol and/or 3-hydroxyretinol in
the eye (Seki et al., 1987,
1989
). Therefore the proteins
detected here probably have the ability to bind retinols, although they do not
bind to the anti-Papilio RBP.
We conclude that the Papilio RBP-like protein is shared by
butterflies belonging to the superfamily Papilionoidea. In addition, native
PAGE indicated that other insects possess their own RBPs. These findings
suggest that insects may share a basic pathway of visual cycle to regenerate
rhodopsin. Nevertheless, the molecular characteristics of RBP could not be
identical between species (or at least beween genuses) (Figs
5 and
7). Such variability in the
protein properties may reflect structural variability in the rhabdom among
species, because the visual cycle involves the removal and incorporation of
visual pigments from and to the rhabdom. In fact many butterflies have
apposition eyes with photoreceptors with small rhabdoms, whereas the eyes of
nocturnal species are of the superposition type containing photoreceptors with
large rhabdoms (Eguchi, 1978).
Different rhabdoms would require a somewhat different mechanism for removal
and incorporation of the visual pigment molecules. At any rate, elucidation of
the function of the Papilio RBP and Papilio RBP-like
proteins in the visual cycle requires further study, including
immunohistochemical localization and the uncovering of other enzymes involved
in the process.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eguchi, E. (1978). Comparative fine structure of lepidopteran compound eyes, especially skippers (Hesperioidea). Zool. Mag. 87,32 -43.
Hara, T. and Hara, R. (1991). Retinal-binding protein: function in a chromophore exchange system in the squid visual cell. In Progress in Retinal Research, vol.10 (ed. N. Osborne and G. J. Chader), pp.179 -206. Oxford-New York: Pergamon Press.[CrossRef]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Larsson, L. (1988). Immunocytochemistry: Theory and Practice. Boca Raton-Florida: CRC Press.
McBee, J. K., Palczewski, K., Baehr, W. and Pepperberg, D. R. (2001). Confronting complexity: the interlink of phototransduction and retinoid metabolism in the vertebrate retina. Prog. Ret. Eye Res. 20,469 -529.[CrossRef][Medline]
Ozaki, K., Terakita, A., Hara, R. and Hara, T. (1987). Isolation and characterization of a retinal-binding protein from the squid retina. Vision Res. 27,1057 -1070.[CrossRef][Medline]
Saari, J. C. (1999). Retinoids in mammalian vision. In Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoids Action, vol. 139 (ed. H. Nau and W. S. Blaner), pp. 563-588. Berlin: Springer-Verlag.
Schwemer, J. (1989). Visual pigments of compound eyesstructure, photochemistry, and regeneration. In Facets of Vision (ed. D. G. Stavenga and R. C. Hardie), pp. 112-133. Berlin-Heidelberg: Springer-Verlag.
Schwemer, J. (1993). Visual pigment renewal and the cycle of the chromohphore in the compound eye of the blowfly. In Sensory Systems of Arthropods (ed. K. Wiese and F. G. Gribakin). Berlin-Heidelberg: Springer-Verlag.
Seki, T., Fujishita, S., Ito, M., Matsuoka, N. and Tsukida, K. (1987). Retinoid composition in the compound eyes of insects. Exp. Biol. 47,95 -103.[Medline]
Seki, T., Fujishita, S. and Obana, S. (1989). Composition and distribution of retinal and 3-hydroxyretinal in the compound eye of the dragonfly. Exp. Biol. 48, 65-75.[Medline]
Shimazaki, Y. and Eguchi, E. (1993). Synthesis of 3-hydroxyretinal in the cytosol of the butterfly compound eye. Vision Res. 33,155 -163.[CrossRef][Medline]
Shimazaki, Y. and Eguchi, E. (1995). Light-dependent metabolic pathway of 3-hydroxyretinoids in the eye of a butterfly, Papilio xuthus. J. Comp. Physiol. A 176,661 -671.
Smith, W. C. and Goldsmith, T. H. (1991). Localization of retinal photoisomerase in the compound eye of the honeybee. Vis. Neurosci. 7,237 -249.[Medline]
Stavenga, D. G., Schwemer, J. and Hellingwerf, K. J. (1991). Visual pigments, bacterial rhodopsins, and related retinoid-binding proteins. In Photoreceptor Evolution and Function, vol. 139 (ed. M. G. Holmes), pp.261 -349. London: Academic Press.
Wakakuwa, M., Arikawa, K. and Ozaki, K. (2003).
A novel retinol-binding protein in the retina of the swallowtail butterfly,
Papilio xuthus. Eur. J. Biochem.
270,2436
-2445.