Evolution of mammalian Opn5 as a specialized UV-absorbing Title pigment by a single mutation.

Yamashita, Takahiro; Ono, Katsuhiko; Ohuchi, Hideyo; Yumoto, Akane; Gotoh, Hitoshi; Tomonari, Sayuri; Sakai, Author(s) Kazumi; Fujita, Hirofumi; Imamoto, Yasushi; Noji, Sumihare; Nakamura, Katsuki; Shichida, Yoshinori

Citation The Journal of biological chemistry (2014), 289(7): 3991-4000

Issue Date 2014-02-14

URL http://hdl.handle.net/2433/203075

This research was originally published in [ The Journal of Biological Chemistry, 289, 3991-4000. doi: Right 10.1074/jbc.M113.514075 © the American Society for Biochemistry and Molecular

Type Journal Article

Textversion author

Kyoto University Molecular property and expression pattern of mammalian Opn5

Evolution of mammalian Opn5 as a specialized UV-absorbing pigment by a single amino acid mutation*

Takahiro Yamashita1,6, Katsuhiko Ono2,6, Hideyo Ohuchi3,6, Akane Yumoto1, Hitoshi Gotoh2, Sayuri Tomonari4, Kazumi Sakai1, Hirofumi Fujita3, Yasushi Imamoto1, Sumihare Noji4, Katsuki Nakamura5, and Yoshinori Shichida1

1Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, 2Department of Biology, Kyoto Prefectural University of Medicine, Kyoto 603-8334, Japan, 3Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan, 4Department of Life Systems, Institute of Technology and Science, University of Tokushima Graduate School, Tokushima 770-8506, Japan, 5Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan.

*Running title: Molecular property and expression pattern of mammalian Opn5

Correspondence should be addressed to Y.S. ([email protected])

6These authors contributed equally to this work.

Keywords: ; Photoreceptors; ; G ; Molecular evolution; non- visual photoreception

Background: Opn5 is considered to regulate non-visual photoreception in the and brain of . Results: Mouse and UV-sensitive Opn5 along with retinoid isomerase are localized in the preoptic . Conclusion: Mammalian Opn5 can function as a high-sensitivity photosensor in the deep brain with the assistance of 11-cis- supplying system. Significance: Mammals including may detect short-wavelength within the brain via Opn5.

ABSTRACT Opn5 is one of the recently identified groups that is responsible for non-visual photoreception in animals. We previously showed that a homolog of mammalian Opn5 (Opn5m) is a Gi- coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse and are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle, is expressed near the preoptic hypothalamus of the mouse and common marmoset brain, but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short-wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal- supplying system.

Opsins are the universal photosensitive coupled receptors (GPCRs) having a seven proteins responsible for visual and non-visual transmembrane α-helical structure. They photoreception in animals, and are G - contain -derivative retinal as their

1/23 Molecular property and expression pattern of mammalian Opn5 light-absorbing , and the retinal libitum. Postnatal and adult mice were binds to a conserved residue located at purchased from Shimizu Laboratory the center of helix VII through a Schiff base Center (ICR and C57BL/6 strains, Shizuoka, linkage. A variety of opsins have been Japan). Animals were anesthetized and identified so far and are classified into at least euthanized at Zeitgeber time 6-10. The seven groups according to their sequence and brains of common marmosets were obtained similarity (1-2). Opn5 is the most recently through the Cooperation Research Program of identified opsin in the human and mouse Primate Research Institute, Kyoto University. genomes (3) and is categorized in an The use of animals in these experiments was in independent group on the basis of its low accordance with the guidelines established by sequence to the other opsin groups. the Ministry of Education, Culture, Sports, Opn5 have been identified in various Science, and Technology, Japan, and University from fishes to and are of Tokushima and Kyoto Prefectural University classified into several subgroups (4-5). Most of Medicine (chick; mouse, Mus musculus mammals have only one Opn5 (Opn5m), domesticus) or Kyoto University (common whereas non-mammalian vertebrates have marmoset, Callithrix jacchus). The protocol additional Opn5 genes. This is probably was approved by the Committee on the Ethics of because of the nocturnal period in the early Animal Experiments of the University of , during which mammals Tokushima (Permit Number: 08089, 11091, lost several non-visual opsins as well as color 11120), Kyoto Prefectural University of opsin genes (6). Medicine (M22-197) or Kyoto University In our previous reports, we succeeded in the (2011-142, 2012-B-35). All surgery was purification of recombinant Opn5 proteins performed under deep anesthesia (pentobarbital, obtained by the expression of chicken Opn5 100 mg/kg body weight), and all efforts were genes and reported that these proteins are UV- made to minimize suffering. sensitive bistable pigments that activate Gi-type of (4,7). We also speculated from the Preparation of Opn5 proteins - The cDNAs of complete conservation of the amino acid human (GenBank accession number; residues surrounding the chromophore between AY377391), mouse (AY318865), Xenopus chicken and mammalian Opn5m proteins that tropicalis (XM_002935990), and zebrafish mammalian Opn5m proteins should be UV- (Danio rerio) (AY493740) Opn5m were tagged sensitive pigments, which was recently by the epitope sequence of the anti-bovine confirmed in mouse and human Opn5m proteins rhodopsin monoclonal antibody Rho1D4 (8). In the present study, we compared the (ETSQVAPA) at the C-terminus and were molecular properties of Opn5m proteins from inserted into the mammalian expression vector zebrafish (fish), Xenopus tropicalis (amphibian), pCAGGS (11). Site-directed mutations were chicken (bird), mouse (mammal) and human introduced by the QuikChange kit (Agilent (primate), paying particular attention to the Technologies) according to the manufacturer’s differences between mammals and non- method. The plasmid DNA was transfected mammalian vertebrates. In addition, we also into HEK293 cells using the calcium phosphate analyzed the expression patterns of mouse and method. After 1 day of incubation, 11-cis- or primate (common marmoset) Opn5m and all-trans-retinal was added to the medium (final RPE65, an enzyme essential for generating 11- retinal concentration, 5 μM) (7). After cis-retinal in the visual cycle (9-10), to compare additional incubation for 1 day in the dark, the with those of chicken Opn5m and RPE65. cells were collected. The pigments were extracted with 1% digitonin (for human and EXPERIMENTAL PROCEDURES mouse Opn5m proteins) or 1% n-dodecyl-β-D- Animals and ethics statement - Fertilized maltoside (DM) (for Xenopus and zebrafish chicken eggs (Gallus gallus domesticus) were Opn5m proteins) in buffer A (50 mM HEPES purchased from a commercial farm (Goto- [pH 6.5] and 140 mM NaCl) and were purified furanjyo, Inc., Gifu, Japan; using Rho1D4-conjugated agarose. The http://www.gotonohiyoko.co.jp/) and incubated purified pigments were eluted with 1% digitonin at 37.5 °C in a humidified incubator until (for human and mouse Opn5m proteins) or hatching. Post-hatch chicks were housed under 0.02% DM (for Xenopus and zebrafish Opn5m a 12:12 light-dark cycle with food and water ad proteins) in buffer A containing the synthetic

2/23 Molecular property and expression pattern of mammalian Opn5 peptide that corresponds to the C-terminus of with subsequent yellow light (>500 nm) for 1 bovine rhodopsin. The expression levels of min. After irradiation, the GDP/GTPγS human and Xenopus Opn5m proteins were very exchange reaction was initiated by the addition low, and thus to obtain sufficient amounts of of [35S]GTPγS solution to the mixture of the recombinant proteins we truncated 37 and 21 pigment and G protein. After incubation for amino acid residues from the C-terminus of the indicated time in the dark, an aliquot (20 μl) human and Xenopus Opn5m proteins, was removed from the sample into 200 μl of respectively. stop solution (20 mM Tris/Cl (pH 7.4), 100 mM NaCl, 25 mM MgCl2, 1 μM GTPγS and 2 μM Spectrophotometry and HPLC analysis - GDP), and it was immediately filtered through a Absorption spectra were recorded at 10 °C (for nitrocellulose membrane to trap [35S]GTPγS human and mouse Opn5m proteins) or 0 °C (for bound to G proteins. The amount of bound Xenopus and zebrafish Opn5m proteins) with a [35S]GTPγS was quantitated by assaying the Shimadzu UV-2400 spectrophotometer. The membrane with a liquid scintillation counter sample was irradiated with UV light through a (Tri-Carb 2910 TR; PerkinElmer). UV-D35 glass filter (Asahi Technoglass), with yellow light through a Y-52 cutoff filter Antibodies - Specific antibodies were raised to (Toshiba) or with orange light through an O-57 the N-terminus of mouse Opn5m and the C- cutoff filter (Toshiba) from a 1 kW halogen terminus of human Opn5m using guinea pigs. lamp (Master HILUX-HR; Rikagaku). The Each polyclonal antibody was commercially absorption spectra of visible and UV light- produced against a 15-amino-acid synthetic absorbing forms of Opn5m proteins were peptide conjugated to Keyhole Limpet calculated using the methods previously Hemocyanin by Protein Purify Ltd. (Gunma, described (7). Briefly, the spectral region at Japan), according to their standard procedures wavelengths longer than the maximum of the (mouse Opn5m: MALNHTALPQDERLPC; main peak of the spectrum difference between human Opn5m: CVRKSSAVLEIHEEWE). the visible and UV light-absorbing forms of the We confirmed that the sequence of this portion pigment was best-fitted with a template of human Opn5m (the C-terminus) is identical spectrum previously described (12-13). The to that of common marmoset Opn5m deposited best-fitting spectrum was considered to be the in the public database. We also confirmed that visible light-absorbing form of the pigment. this isoform of human Opn5m cDNA was The absorption spectrum of the UV light- isolated from human retina cDNA (Marathon- absorbing form was then calculated by adding Ready cDNA, Clontech) with 3’RACE. These the visible light-absorbing form to the difference antibodies were affinity purified prior to use by spectrum. The chromophore configurations of Protein Purify Ltd. The primary antibodies the sample were analyzed by HPLC (LC-10AT used in this study included: guinea pig anti- VP; Shimadzu) equipped with a silica column chick Opn5m antibody (7), and rabbit anti- (150 x 6 mm, A-012-3; YMC) according to the RPE65 antibody (a gift from Dr. Rosalie K. previous report (14). Crouch, Medical University of South Carolina, USA) (15). We performed western blotting G protein activation assay - A radionucleotide analysis using the antibodies and confirmed that filter-binding assay, which measures the anti-mouse and human/monkey Opn5m GDP/GTPγS exchange by G protein, was antibodies recognized the recombinant proteins performed as described previously (4,7). All of full-length mouse and human Opn5m procedures were carried out at 0 °C. The assay expressed in HEK293 cells, respectively (Fig. 1). mixture consisted of 50 mM HEPES (pH 7.0), The secondary antibody used for 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, immunostaining in this study was Alexa Fluor 0.01% DM, 1 μM [35S]GTPγS and 2 μM GDP. 488 anti-guinea pig IgG (A-11073, Life Mouse Opn5m pigments after reconstitution Technologies Corp.). with 11-cis-retinal were purified in 0.02 % DM in buffer A for the assay. Purified mouse Fixation and sectioning - The animals were Opn5m pigments (final concentration: 300 nM) perfusion-fixed with 4 % paraformaldehyde in were mixed with G protein solution (final phosphate-buffered saline (PBS) under deep concentration: 600 nM) and were kept in the pentobarbital anesthesia, and the and brain dark or irradiated with UV light for 1 min or

3/23 Molecular property and expression pattern of mammalian Opn5 were quickly dissected, post-fixed for 2 to 3 h synthesized by Medical & Biological (for eyes) or 16 h (for brain) in the same fixative Laboratories, Japan), mouse Rpe65 (AF410461; at 4 °C and then transferred to 20 % sucrose cloned by PCR from mouse eye) and human until they sank. After embedding the samples Rpe65 (BC075035; purchased from IMAGE in optimal cutting temperature compound cDNA Collection) using T7 T3 or Sp6 RNA (Sakura, Japan), the tissues were sectioned with polymerase. Sections were pre-treated with a cryostat (Leica) at 20 μm thickness. Sections methanol for 2 hours and with proteinase K (20 were thaw-mounted onto MAS-coated slides μg/mL) for 40 min at room temperature. After (Matsunami, Japan), dried in air for a few hours, prehybridization, sections were incubated with and stored at -30 °C until use. The anatomy of DIG-labeled RNA anti- or sense probes the chick, mouse and common marmoset brain (15~30 ng/mL in hybridization buffer) at 65 °C was determined according to (16), (17) and (18), overnight. They were washed with x1 SSC respectively, and the nomenclature adopted in buffer containing 50 % formamide three times at this study was based on these atlases and 65 °C, and maleic acid buffer containing 0.1 % previous studies. Tween 20, and subsequently incubated with anti-DIG antibody conjugated to alkaline Immunohistochemistry - Fluorescent phosphatase (1:2000 in PBS containing 0.1 % immunolabeling was performed using standard Triton X-100; Roche) overnight at 4 °C. Color techniques. Briefly, all slides were blocked for was developed with BCIP/NBP. Sections were 30 min at room temperature in PBS Triton X- coverslipped and observed under a microscope. 100 (0.25 %) (PBST) with 5 % serum from the same species as the corresponding secondary Western blotting - Extract from full-length antibodies. Primary antibodies were diluted in human or mouse Opn5m-transfected HEK293 PBST with 5 % serum and secondary antibodies cells was subjected to SDS-PAGE, transferred in PBS. All wash steps were performed 3 times onto a polyvinylidene difluoride membrane, and with PBST for 5 min each. Primary antibodies probed with anti-human or mouse Opn5m (anti-N-terminus of mouse Opn5m [diluted antibody (1:1000) (Fig. 1). Protein extracts 1:2000], anti-C-terminus of human Opn5m from mouse tissues were prepared as follows (8). [diluted 1:2000], and anti-RPE65 [diluted Eight-week old male mice were anesthetized 1:140] were incubated for 5 h at room and perfused with ice-cold PBS. The tissues temperature or for 16 h at 4 °C. Secondary were dissected and solubilized in 1% DM in antibodies were incubated for 1.5 h at room buffer B (50 mM HEPES [pH 6.5], 140 mM temperature and diluted 1:750. The cell nuclei NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4 were stained with 4',6-diamidino-2-phenylindole mg/mL aprotinin, 4 mg/mL leupeptin, 0.1 mM (DAPI) (Vector Laboratories). The slides were phenylmethylsulfonyl fluoride). After mounted with anti-fade mountant, Vectashield centrifugation, the supernatant was collected for (Vector, H-1500). Fluorescent images were SDS-PAGE. Mouse Opn5m or RPE65 in the collected using a Leica TCS-SP5 confocal laser- extracts from mouse neural retina, RPE, scanning microscope, excitation 405, 488, and hypothalamus, other brain area, lung and liver 543 nm with emission wavelengths of 424-489, was detected by anti-mouse Opn5m (1:1500) or 505-539, and 551-618 nm for DAPI, green, and RPE65 antibody (1:1000) (Fig. 7O, P) Cy3, respectively. RESULTS AND DISCUSSION In situ hybridization of mouse brain and retinal Opn5m proteins reconstituted with sections - Frozen sections (20 μm thickness) of 11-cis- or all-trans-retinal mouse (ICR, postnatal day 0, 4 and 10, and 8 We first expressed recombinant proteins of weeks, female; C57BL/6, 8 weeks, male and Opn5m from four vertebrates, zebrafish, female) and adult common marmoset brain and Xenopus tropicalis, mouse and human, in retina were used. In situ hybridization was cultured cells and examined whether or not performed according to standard procedures. these pigments have molecular properties Briefly, digoxigenin (DIG)-labelled RNA similar to those of chicken Opn5m. As already probes were synthesized from full-length cDNA reported, chicken Opn5m is a UV-sensitive of mouse Opn5m (purchased from IMAGE bistable pigment that forms 11-cis- and all- cDNA Collection), common marmoset Opn5m trans-retinal bound states, the latter of which (GenBank accession number; XM_002746623, activates Gi-type of G protein (7). We also

4/23 Molecular property and expression pattern of mammalian Opn5 found that, although the apoprotein of Opn5m The absorption maxima of the UV light- protein is extremely unstable, we could absorbing forms of these Opn5m proteins are all successfully obtain adequate amounts of chicken located at 360 nm (Fig. 2, Insets). In contrast, Opn5m pigment by adding 11-cis- or all-trans- while the absorption maxima of visible light- retinal to the culture medium during the protein absorbing forms of zebrafish, Xenopus and expression. Fig. 2C shows the photoreactions chicken Opn5m proteins are located at 474 nm, of chicken Opn5m that had been purified after those of mouse and human Opn5m proteins are incubation with 11-cis-retinal. The absorption located at 469 nm (Fig. 2, Insets). These spectrum of purified pigment exhibited UV and differences would reflect the differences of the visible absorbances, indicating that the chicken ability to bind all-trans-retinal among these Opn5m protein binds 11-cis-retinal to produce a Opn5m proteins. UV light-absorbing form and also binds all- To confirm that mammalian Opn5m has no trans-retinal, which is formed by thermal ability to directly bind all-trans-retinal, we isomerization of 11-cis-retinal in culture performed similar expression experiments in the medium, to produce a visible light-absorbing presence of all-trans-retinal instead of 11-cis- form (Fig. 2H) (7). UV light irradiation of the retinal in the culture medium (Fig. 2F-J). In pigment caused the conversion of the UV light- zebrafish and Xenopus Opn5m proteins, we absorbing form to the visible light-absorbing observed the production of visible light- form, and subsequent yellow light irradiation absorbing forms that exhibited photoreactions resulted in the complete conversion of the identical with that of chicken Opn5m (Fig. 2H- visible light-absorbing form to the UV light- J). On the other hand, we observed no absorbing form. The light-induced pigments of mouse or human Opn5m under the interconversion between the UV light- and present conditions (Fig. 2F, G). These results visible light-absorbing forms could be observed clearly showed that mammalian Opn5m proteins repeatedly (Fig. 2C). have no ability to directly bind all-trans-retinal. We performed similar experiments using We also confirmed that the visible light- zebrafish, Xenopus, mouse and human Opn5m absorbing form of mammalian Opn5m is the proteins (Fig. 2A, B, D, E). The absorption active state that couples with Gi (Fig. 3). spectra and photoreactions of zebrafish and Purified mouse Opn5m reconstituted with 11- Xenopus Opn5m were quite similar to those of cis-retinal activated Gi in a UV-light dependent chicken Opn5m. That is, the addition of 11-cis- manner, and lost the activity after subsequent retinal to the culture medium caused the yellow light irradiation. These alterations of production of UV light- and visible light- the activity are consistent with the spectral absorbing forms that were interconvertible by changes shown in Fig. 2B, indicating that the light (Fig. 2D, E). In contrast, incubation of visible light-absorbing form is the state mouse and human Opn5m proteins with 11-cis- activating G protein. retinal in the culture medium caused the production of only the UV light-absorbing form. Determination of amino acid residue that In fact, UV light irradiation of mouse and controls the ability of direct binding of all-trans- human pigments caused the production of the retinal visible-light absorbing form, and subsequent Next, we attempted to identify the amino acid yellow light irradiation resulted in the residue(s) whose substitution(s) resulted in loss production of the UV-light absorbing form, of the ability to directly bind all-trans-retinal in whose absorption spectrum overlapped with that mammalian Opn5m proteins. Comparison of of the dark state (Fig. 2A, B). The analysis of the amino acid residues within 4.5 Å from the the retinal configurations of human Opn5m also retinal, which are inferred from the 3D showed that human Opn5m was exclusively structures of bovine and squid (19- reconstituted with 11-cis-retinal and UV light 20), indicates that all the residues are irradiation induced the retinal isomerization completely conserved in mammalian and from 11-cis to all-trans form (Fig. 2K). chicken Opn5m proteins. Thus, we compared Therefore, mouse and human Opn5m showed the residues within 8 Å from the retinal among photoreversibility similar to that of chicken vertebrate Opn5m proteins. There are four Opn5m but had no ability to directly bind all- residues that are conserved in human and mouse trans-retinal. Opn5m proteins but not in non-mammalian Opn5m proteins (Table 1). We introduced each

5/23 Molecular property and expression pattern of mammalian Opn5 corresponding mutation (T94S, T168A, D177S proteins, which have alanine residue in this or I204V in the bovine rhodopsin numbering position (Table 1). We speculate that A168T system) into mouse Opn5m and analyzed the mutation occurred after the divergence of alteration of the ability of direct incorporation of eutherian and metatherian mammals. all-trans-retinal. Orange light irradiation of mouse Opn5m mutants incubated with all-trans- Localization of mammalian Opn5m in the retina retinal led to a significant spectral change in and the brain T168A mutant, but not in wild-type or other Mammals have several non-visual opsins, mutants (Fig. 4A). The purified mouse Opn5m including Opn5m. The mRNA transcript of T168A had the peak of its absorption spectrum mammalian Opn4 () is exclusively in the visible region and was photoconverted expressed in the retina (23), whereas the mRNA into a pigment whose absorption maximum was of mammalian Opn3 (encephalopsin) is detected in the UV region (Fig. 4B). This spectral in the brain, but not in the retina (24). On the change is the same as those observed in non- other hand, the mRNA of mammalian Opn5m is mammalian Opn5m proteins reconstituted with present in both the retina and brain (3,8,25), all-trans-retinal. It should be noted that T168A suggesting the multiple physiological roles of mutation of mouse Opn5m resulted in red-shift Opn5m. Therefore it is of interest to of absorption maximum, close to those of non- investigate the detailed expression patterns of mammalian Opn5m proteins. These results mammalian Opn5m. indicated that T168A mutation alters the all- For this, first we performed in situ trans-retinal binding ability as well as the hybridization (ISH) and immunostaining on electrostatic environment of the all-trans-retinal mouse retinal sections. Although our standard chromophore. In addition, we prepared A168T ISH technique could not detect Opn5m mRNA mutant of Xenopus Opn5m reconstituted with in the adult mouse retina, we observed the all-trans-retinal. The purified mutant exhibited expression of Opn5m in the immature retina at no detectable peak of the absorption spectrum in postnatal day 1 (P1) and 11 (P11) (Fig. 5). the visible region and no spectral change after Furthermore, we found Opn5m immunoreactive yellow light irradiation (Fig. 4B). Thus, this cells in the ganglion cell layer (GCL) of the mutant lost the ability to directly bind all-trans- adult retina (Fig. 6A). These signals were not retinal. These data clearly showed that a single seen when immunostaining was performed amino acid residue can modulate the ability of using antigen-adsorbed antibodies (Fig. 6B). direct incorporation of all-trans-retinal as well We also performed immunohistochemical as the electrostatic environment of the all-trans- studies using the common marmoset retina and retinal chromophore. detected signals in the GCL and the inner It has been reported that bovine rhodopsin nuclear cell layer (INL) (Fig. 6C, C’, D). The cannot be reconstituted with all-trans-retinal to expression pattern of Opn5m in the retina was form the active state (21), because in neutral pH similar between mouse and common marmoset, the equilibrium of inactive and active and consistent with those of mouse and rat seen conformations of apoprotein of bovine in the previous studies (8,25). We previously rhodopsin is mostly biased toward the inactive observed immunostaining signals of Opn5m in conformation (22). Bovine rhodopsin has an the GCL and the INL of chick retina (7). Thus, alanine residue at position 168, the same as non- the localization of Opn5m in the retina is mammalian vertebrate Opn5m (Table 1). conserved between mammals and chicken. Therefore, the lack of ability of all-trans-retinal We next investigated the localization of binding in bovine rhodopsin cannot be Opn5m mRNA in the mouse brain. The results explained by the same molecular mechanism. showed that Opn5m was expressed exclusively Thus, the loss of all-trans-retinal binding ability in the preoptic area, specifically, the median in vertebrate visual opsin and mammalian preoptic nucleus and medial preoptic area of the Opn5m is the result of . anterior hypothalamus (Fig. 7A-D). However, Alanine residue at position 168, which is well there was no Opn5m mRNA in the posterior conserved in non-mammalian Opn5m proteins, hypothalamus or hippocampus (Fig. 8), whose is replaced into residue in most counterparts in birds are known to contain mammalian Opn5m proteins. Exceptions are photoreceptive cells (26). Moreover, our platypus (Ornithorhynchus anatinus) and western blotting analysis showed that Opn5m opossum (Monodelphis domestica) Opn5m protein was detected in the hypothalamus as

6/23 Molecular property and expression pattern of mammalian Opn5 well as in the neural retina and other brain areas Opn5m mRNA has also been detected in the with the expected molecular weight (Fig. 7O). human brain by RT-PCR analysis (3). Thus, to analyze the detailed distributions of Opn5m and Comparison of localization of Opn5m and an Rpe65 in the primate brain, we performed ISH 11-cis--generating enzyme, RPE65, in the on the common marmoset brain. We detected mammalian brain the expression of Opn5m mRNA in the anterior We were surprised to find that mouse Opn5m part of the medial preoptic area (Fig. 7I-K), is expressed in the preoptic hypothalamus, a which is in the vicinity of Rpe65-expressing deep brain region, because, in addition to the domains (Fig. 7L-N). These results showed fact that UV light would scarcely penetrate into that Opn5m and RPE65 are expressed in the such a deep brain region, our biochemical primate anterior hypothalamus. analysis showed that mouse Opn5m protein has We also examined the expression pattern of lost the ability to directly bind all-trans-retinal. RPE65 in the chick brain by immunostaining. We previously speculated that chicken Opn5m We found that anti-RPE65 antibody (Fig. 9D, E) in the paraventricular organ (PVO) of the detected a subset of ependymal cells lining the hypothalamus can be regenerated by all-trans- third ventricle (Fig. 9B, C). However, in the retinal binding and work as a photoreceptive PVO region, there were no RPE65- molecule by absorbing visible light even immunoreactive cells in the ependymal layer, without an 11-cis-retinal supply, because visible which abuts the Opn5m-immunoreactive light can more easily be transmitted into the neurons (Fig. 9A). These results support the deep brain than UV light (7). However, mouse notion that, in the chick brain, Opn5m- Opn5m cannot be regenerated by all-trans- expressing cells of the PVO, which are thought retinal, and therefore it appears essential to have to be responsible for the in birds an 11-cis-retinal-supplying system (as in the (28-29), do not require assistance by RPE65 in visual cycle mechanism) in the vicinity. One generating 11-cis-retinal, as chicken Opn5m is a important enzyme constituting the visual cycle bistable pigment that can generate the 11-cis- is retinal pigment epithelium (RPE)-specific 65 retinal binding form from the all-trans-retinal kDa protein, RPE65. RPE65 in the RPE is binding form (7). involved in the conversion of all-trans-retinyl ester to 11-cis-retinol during the visual cycle (9- Photoreception within mammalian retina and 10). We therefore examined the expression brain via Opn5m domain of Rpe65 in the mouse brain. We first In this study, we demonstrated that not only confirmed that a high level of Rpe65 mRNA is rodents but also primates have the UV-sensitive observed in the RPE of the adult mouse retina Opn5 in the retina. Like the expression of (Fig. 5). Then, we performed ISH on mouse melanopsin in a subset of retinal ganglion cells brain sections and showed that Rpe65 is in mammals, including primates (30-31), the expressed in the domains (Fig. 7G, H) near the present findings suggest that retinal Opn5m-expressing cells (Fig. 7E, F). In photoreception in mammals and primates is not addition, by the western blotting analysis we restricted to classical rod and cone detected multiple bands of putative RPE65 photoreceptor cells or intrinsically proteins in the dissected hypothalamus and other photosensitive retinal ganglion cells. More brain areas including a band of the same importantly, we demonstrated here the molecular weight as the band observed in the expression of Opn5m in the preoptic RPE (Fig. 7P). These results clearly showed hypothalamus in the mouse and common that an RPE65-dependent retinal isomerization marmoset deep brain. Mammalian Opn5m system is present near the region where Opn5m proteins exclusively bind 11-cis-retinal due to is expressed. It has been shown that lecithin their loss of the ability to directly bind all-trans- retinol acyltransferase (LRAT), an enzyme that retinal. Direct binding of all-trans-retinal produces the substrate of RPE65 from all-trans- causes the formation of the active state and retinol in the visual cycle, is also expressed in elevates the G protein activation ability without the mouse brain, although its detailed expression light irradiation, which lowers the signal-to- pattern remains unknown (27). Thus LRAT noise ratio in light-dependent signaling. Light may also contribute to Opn5m-RPE65 system in can penetrate the skull in several mammalian the brain. species, including human (26). And mammalian cerebral cortical tissues were

7/23 Molecular property and expression pattern of mammalian Opn5 reported to respond to low-intensity light to Amphibian Biology, Hiroshima University, for regulate release (32). In birds, the generous gift of Xenopus tropicalis through the measurement of the light transmittance into the National Bio-Resource Project of MEXT, the deep brain revealed that the hypothalamus Japan. This work was supported by the can receive light enough to trigger opsin- Cooperation Research Program of Primate mediated signal transduction (33) because of the Research Institute, Kyoto University. This high quantum yield of opsins (34). Thus, work was also supported in part by Grants-in- although there is no direct evidence to show that Aid for Scientific Research (to T.Y., K.O., H.O., mammals actually utilize Opn5m to sense short- S.N., K.N. and Y.S.) and the Global Center of wavelength light, by the acquisition of exclusive Excellence Program “Formation of a Strategic 11-cis-retinal binding ability, Opn5m could Base for Biodiversity and Evolutionary function as a high-sensitivity photosensor within Research: from Genome to Ecosystem” of the the mammalian brain. It is known that the Ministry of Education, Culture, Sports, Science sexually dimorphic nucleus is located in a and Technology (MEXT), Japan. cluster of cells of the preoptic area in the hypothalamus of the brain, and that it is related REFERENCES to sexual behavior in animals and its volume is 1. Koyanagi, M., and Terakita, A. (2008) Gq- usually larger in males than in females (35). coupled rhodopsin subfamily composed of Although we found no obvious differences in invertebrate visual pigment and melanopsin. the amount of Opn5m-expressing cells between Photochem Photobiol 84, 1024-1030 males and females (Fig. 10), Opn5m in this 2. Shichida, Y., and Matsuyama, T. (2009) region might have a role in some sexual Evolution of opsins and phototransduction. behaviors. In addition, the preoptic area of the Philos Trans R Soc Lond B Biol Sci 364, hypothalamus is an important region to control 2881-2895 the body temperature (36). Thus, Opn5m 3. Tarttelin, E. E., Bellingham, J., Hankins, M. might contribute to light-dependent W., Foster, R. G., and Lucas, R. J. (2003) thermoregulation (37). In birds, Opn5m in the Neuropsin (Opn5): a novel opsin identified in deep brain can regulate photoperiodic response mammalian neural tissue. FEBS Lett 554, (28). However, in mammals, visual opsins and 410-416 Opn4 in the retina exclusively contribute to the 4. Ohuchi, H., Yamashita, T., Tomonari, S., control of release from the pineal Fujita-Yanagibayashi, S., Sakai, K., Noji, S., gland (38-39), and thus Opn5m in the deep brain and Shichida, Y. (2012) A non-mammalian is not involved in photoperiodism (40). type opsin 5 functions dually in the Compared with chicken Opn5m, mammalian photoreceptive and non-photoreceptive Opn5m has changed its molecular property (i.e., organs of birds. PLoS One 7, e31534 has lost direct binding to all-trans-retinal) and 5. Tomonari, S., Migita, K., Takagi, A., Noji, S., its expression pattern within the brain, although and Ohuchi, H. (2008) Expression patterns of it is difficult to precisely compare the areas of the opsin 5-related genes in the developing the brain between mammals and birds. The chicken retina. Dev Dyn 237, 1910-1922 change of the molecular property and the 6. Kuraku, S., and Kuratani, S. (2011) Genome- expression pattern of Opn5m may be related wide detection of gene extinction in early with the difference of the physiological role of mammalian evolution. Genome Biol Evol 3, Opn5m between mammals and birds. 1449-1462 Therefore, it is intriguing to speculate that 7. Yamashita, T., Ohuchi, H., Tomonari, S., Opn5m might have obtained a new Ikeda, K., Sakai, K., and Shichida, Y. (2010) physiological role in the early evolution of Opn5 is a UV-sensitive bistable pigment that mammals, which will be the subject of our couples with Gi subtype of G protein. Proc future research. Natl Acad Sci U S A 107, 22084-22089 8. Kojima, D., Mori, S., Torii, M., Wada, A., ACKNOWLEDGMENTS Morishita, R., and Fukada, Y. (2011) UV- We are grateful to Dr. Elizabeth Nakajima and sensitive OPN5 in Dr. Take Matsuyama for critical reading of the humans and mice. PLoS One 6, e26388 manuscript. We thank Professor Rosalie K. 9. Cai, X., Conley, S. M., and Naash, M. I. Crouch for the generous gift of the antibody (2009) RPE65: role in the visual cycle, against RPE65. We also thank Institute for

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human retinal disease, and gene therapy. Iuvone, P. M., Hankins, M. W., Tosini, G., Ophthalmic Genet 30, 57-62 and Lucas, R. J. (2006) Evolution of 10. Kiser, P. D., and Palczewski, K. (2010) melanopsin photoreceptors: discovery and Membrane-binding and enzymatic properties characterization of a new melanopsin in of RPE65. Prog Retin Eye Res 29, 428-442 nonmammalian vertebrates. PLoS Biol 4, 11. Niwa, H., Yamamura, K., and Miyazaki, J. e254 (1991) Efficient selection for high-expression 24. Blackshaw, S., and Snyder, S. H. (1999) transfectants with a novel eukaryotic vector. Encephalopsin: a novel mammalian Gene 108, 193-199 extraretinal opsin discretely localized in the 12.Govardovskii, V. I., Fyhrquist, N., Reuter, T., brain. J Neurosci 19, 3681-3690 Kuzmin, D. G., and Donner, K. (2000) In 25.Nieto, P. S., Valdez, D. J., Acosta-Rodriguez, search of the visual pigment template. Vis V. A., and Guido, M. E. (2011) Expression of Neurosci 17, 509-528 novel opsins and intrinsic light responses in 13. Lamb, T. D. (1995) Photoreceptor spectral the mammalian line sensitivities: common shape in the long- RGC-5. Presence of OPN5 in the rat retina. wavelength region. Vision Res 35, 3083-3091 PLoS One 6, e26417 14. Tsutsui, K., Imai, H., and Shichida, Y. 26. Vigh, B., Manzano, M. J., Zadori, A., Frank, (2007) Photoisomerization efficiency in UV- C. L., Lukats, A., Rohlich, P., Szel, A., and absorbing visual pigments: protein-directed David, C. (2002) Nonvisual photoreceptors of isomerization of an unprotonated retinal the deep brain, pineal organs and retina. Schiff base. Biochemistry 46, 6437-6445 Histol Histopathol 17, 555-590 15. Tang, P. H., Wheless, L., and Crouch, R. K. 27. Liu, L., and Gudas, L. J. (2005) Disruption (2011) Regeneration of is of the lecithin:retinol acyltransferase gene enhanced in mouse cone photoreceptors makes mice more susceptible to vitamin A expressing RPE65 protein. J Neurosci 31, deficiency. J Biol Chem 280, 40226-40234 10403-10411 28. Nakane, Y., Ikegami, K., Ono, H., 16. Puelles, L. (2007) The chick brain in Yamamoto, N., Yoshida, S., Hirunagi, K., stereotaxic coordinates : an atlas featuring Ebihara, S., Kubo, Y., and Yoshimura, T. neuromeric subdivisions and mammalian (2010) A mammalian neural tissue opsin homologies, Academic Press (Opsin 5) is a deep brain photoreceptor in 17. Franklin, K. B. J., and Paxinos, G. (2007) birds. Proc Natl Acad Sci U S A 107, 15264- The mouse brain in stereotaxic coordinates, 15268 3rd Edition, Academic Press 29. Oishi, T., Yamao, M., Kondo, C., Haida, Y., 18. Yuasa, S., Nakamura, K., and Kohsaka, S. Masuda, A., and Tamotsu, S. (2001) (2010) Stereotaxic atlas of the marmoset Multiphotoreceptor and multioscillator brain, Igaku Shoin, Tokyo system in avian circadian organization. 19. Murakami, M., and Kouyama, T. (2008) Microsc Res Tech 53, 43-47 Crystal structure of squid rhodopsin. Nature 30. Dacey, D. M., Liao, H. W., Peterson, B. B., 453, 363-367 Robinson, F. R., Smith, V. C., Pokorny, J., 20.Okada, T., Fujiyoshi, Y., Silow, M., Navarro, Yau, K. W., and Gamlin, P. D. (2005) J., Landau, E. M., and Shichida, Y. (2002) Melanopsin-expressing ganglion cells in Functional role of internal water molecules in primate retina signal colour and irradiance rhodopsin revealed by X-ray crystallography. and project to the LGN. Nature 433, 749-754 Proc Natl Acad Sci U S A 99, 5982-5987 31. Provencio, I., Rodriguez, I. R., Jiang, G., 21. Jager, S., Palczewski, K., and Hofmann, K. Hayes, W. P., Moreira, E. F., and Rollag, M. P. (1996) Opsin/all-trans-retinal complex D. (2000) A novel human opsin in the inner activates by different mechanisms retina. J Neurosci 20, 600-605 than photolyzed rhodopsin. Biochemistry 35, 32. Wade, P. D., Taylor, J., and Siekevitz, P. 2901-2908 (1988) Mammalian cerebral cortical tissue 22. Vogel, R., and Siebert, F. (2001) responds to low-intensity visible light. Proc Conformations of the active and inactive Natl Acad Sci U S A 85, 9322-9326 states of opsin. J Biol Chem 276, 38487- 33. Oishi, T., and Ohashi, K. (1993) Effects of 38493 Wavelengths of Light on the Photoperiodic 23. Bellingham, J., Chaurasia, S. S., Melyan, Z., Gonadal Response of Blinded- Liu, C., Cameron, M. A., Tarttelin, E. E.,

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Pinealectomized Japanese-Quail. Zool Sci 10, protein, as shown for recombinant bovine 757-762 rhodopsin (41). 34. Kim, J. E., Tauber, M. J., and Mathies, R. A. (2001) Wavelength dependent cis-trans Fig. 2. Comparison of the ability to directly isomerization in vision. Biochemistry 40, bind 11-cis- or all-trans-retinal in vertebrate 13774-13778 Opn5m proteins. (A-E) Absorption spectra of 35. Forger, N. G. (2009) Control of cell number human (A), mouse (B), chicken (C), Xenopus in the sexually dimorphic brain and spinal tropicalis (D) and zebrafish (E) Opn5m proteins cord. J Neuroendocrinol 21, 393-399 after incubation with 11-cis-retinal. Spectra 36. Morrison, S. F., Nakamura, K., and Madden, were measured in the dark (curve 1), and after C. J. (2008) Central control of thermogenesis UV light irradiation (curve 2), subsequent in mammals. Exp Physiol 93, 773-797 yellow light (>500 nm) irradiation (curve 3), 37. Cajochen, C., Munch, M., Kobialka, S., UV light re-irradiation (curve 4) or yellow light Krauchi, K., Steiner, R., Oelhafen, P., Orgul, re-irradiation (curve 5). (inset) The calculated S., and Wirz-Justice, A. (2005) High absorption spectra of each Opn5m protein in the sensitivity of human melatonin, alertness, dark (curve 1) and after UV light irradiation thermoregulation, and heart rate to short (curve 2). (F-J) Absorption spectra of human wavelength light. J Clin Endocrinol Metab 90, (F), mouse (G), chicken (H), Xenopus (I) and 1311-1316 zebrafish (J) Opn5m proteins after incubation 38. Hattar, S., Lucas, R. J., Mrosovsky, N., with all-trans-retinal. Spectra were measured Thompson, S., Douglas, R. H., Hankins, M. in the dark (curve 1), and after yellow light W., Lem, J., Biel, M., Hofmann, F., Foster, R. (>500 nm) irradiation (curve 2), subsequent UV G., and Yau, K. W. (2003) Melanopsin and light irradiation (curve 3) or yellow light re- rod-cone photoreceptive systems account for irradiation (curve 4). (K) Retinal configuration all major accessory visual functions in mice. changes by UV light irradiation of human Nature 424, 76-81 Opn5m. (left-hand panel) The retinal 39. Panda, S., Provencio, I., Tu, D. C., Pires, S. configurations were analyzed with HPLC after S., Rollag, M. D., Castrucci, A. M., Pletcher, extraction of the chromophore as retinal oximes M. T., Sato, T. K., Wiltshire, T., Andahazy, (syn and anti forms of 9-cis-, 11-cis-, and all- M., Kay, S. A., Van Gelder, R. N., and trans-retinal oximes). (right-hand panel) Hogenesch, J. B. (2003) Melanopsin is Isomeric compositions of retinal before and required for non-image-forming photic after light irradiation of human Opn5m. responses in blind mice. Science 301, 525- 527 Fig. 3. Time courses of G protein activation 40. Hoffman, R. A., and Reiter, R. J. (1965) ability of mouse Opn5m. Gi activation ability : Influence on Gonads of Male of mouse Opn5m reconstituted with 11-cis- Hamsters. Science 148, 1609-1611 retinal was measured in the dark (circles), after 41. Sung, C. H., Schneider, B. G., Agarwal, N., UV light irradiation (squares), and after Papermaster, D. S., and Nathans, J. (1991) subsequent yellow light (>500 nm) irradiation Functional heterogeneity of mutant (triangles). Data are presented as the means ± rhodopsins responsible for autosomal SEM of three independent experiments. dominant . Proc Natl Acad Sci U S A 88, 8840-8844 Fig. 4. Alteration of the ability to directly bind all-trans-retinal in mouse and Xenopus Opn5m FIGURE LEGENDS mutants. (A) Spectral changes of mouse Fig. 1. Characterization of the anti- Opn5m proteins caused by orange light (>550 mammalian Opn5m antibodies by western nm) irradiation. Extracts containing wild-type, blotting. Signals were detected in Opn5m- T94S, T168A, D177S and I204V of mouse transfected cells but not in mock-transfected Opn5m with 1 % DM after reconstitution with cells. Recombinant mammalian Opn5m all-trans-retinal were irradiated and then the proteins exhibited two bands, probably because difference spectra before and after irradiation of the heterogeneity of the post-translational were calculated. Each panel contains the modification within the cultured cells, such as spectra of wild-type (black curve) and mutant the glycosylation in the N-terminus of the (red curve). (B) Modulation of the ability to directly bind all-trans-retinal by a single

10/23 Molecular property and expression pattern of mammalian Opn5 mutation at position 168. Absorption spectra mRNA near the Opn5m-expression domain in of wild-type and mutant of mouse and Xenopus the mouse brain (approximate lateral level, 0.36 Opn5m reconstituted with all-trans-retinal were mm). The arrows show the localization of measured in the dark (black curve) and after Opn5m mRNA in the medial optic area (E, F), yellow light (>500 nm) irradiation (red curve). and Rpe65 mRNA (G, H) in the . Scale bars: 2 mm in (E, G); 0.2 mm Fig. 5. Expression of Opn5m and Rpe65 in (F, H). Lv, lateral ventricle; 3v, third mRNA in the mouse retina. (A-F) Detection of ventricle; ac, anterior commissure; MPA, medial Opn5m mRNA in the retina of adult (ICR strain, preoptic area; Tu, olfactory tubercle. (I-N) 8 weeks, male), post-natal day 11 (P11) and P1 Detection of Opn5m and Rpe65 mRNA in the mouse. (D-F) Sense probes gave essentially no common marmoset anterior hypothalamus. signals. (G-I) Detection of Rpe65 mRNA in Opn5m mRNA was expressed in the anterior the RPE of adult, P11 and P1 mouse. (I) part of the medial preoptic area (MPO) (I, J) Rpe65 mRNA was undetectable in P1 retina. medially to the diagonal band (DB). Boxed Rpe, retinal pigment epithelium; Onl, outer area in I is magnified in J. Opn5m-positive nuclear layer; Inl, inner nuclear layer; Gcl, cells were observed only in the anterior-most ganglion cell layer; Ob, outer neuroblastic layer; part of the MPO. Rpe65 mRNA was detected Ib, inner neuroblastic layer. Scale bar: 0.2 mm in the paraventricular nucleus (Pa) of the in (A-F); 0.2 mm in (G-I). anterior hypothalamus, slightly caudal to the Opn5m-positive cells (L, M). Boxed area in L Fig. 6. Localization of Opn5m-expressing cells is magnified in M. Signals were not detected in the mammalian retina. Immunostaining of with sense probes (K, N). Scale bars: 1 mm in adult mouse (A, B) and common marmoset (C, (I, L); 200 μm in (J, M); 500 μm in (K, N). (O, C’, D) retina. (B, D) When using the antibody P) Expression of Opn5m and RPE65 proteins in preadsorbed with the antigenic peptide, there the mouse brain analyzed by western blotting. were no signals except weak signals in the GCL Opn5m protein was detected in the neural retina, in (B) and layer in (D). hypothalamus and other brain area, not in the Asterisk and arrow in (C) show Opn5m- RPE, lung or liver (O). REP65 protein was expressing cells enlarged in (C’). Onl, outer detected in the RPE, hypothalamus and other nuclear layer; Inl, inner nuclear layer; Gcl, brain area, not in the lung or liver (P). Ganglion cell layer; Prl, photoreceptor layer. Scale bars: 50 μm in (A, B); 25 μm in (C, C’, D). Fig. 8. Expression of Opn5m mRNA in the mouse posterior hypothalamus and Fig. 7. Expression of Opn5m and Rpe65 in the hippocampus. Coronal sections of mouse brain mammalian brain. (A-D) Detection of Opn5m (ICR strain, female, 12 weeks) at the posterior mRNA in the preoptic area. Coronal sections hypothalamus (A, B) and hippocampus (C, D) of mouse brain (ICR strain, female, 12 weeks) at levels. Approximate Bregma levels are -0.58 the hypothalamus level. Approximate Bregma mm (A, B), and -1.34 mm (C, D). Large levels are 0.50 mm (A-C) and 0.14 mm (D). magnifications of (A, C) are shown in (B, D) as Large magnifications of (A) are shown in (B) as indicated. (A, B) At the posterior indicated. Opn5m mRNA was exclusively hypothalamus, Opn5m mRNA was not detected detected in the median preoptic nucleus and in the paraventricular nucleus along the third medial preoptic area at the anterior ventricle. (C, D) Opn5m mRNA was not hypothalamus as shown in (A, B, D). C, Sense detectable in the hippocampus. vhc, ventral probe did not give rise to any staining. D, a hippocampal commissure; sm, stria medullaris; small number of Opn5m-expressing cells were 3v, third ventricle; f, fornix; fi, fimbria of the detected in the medial preoptic area (arrows) at hippocampus; Lv, lateral ventricle; ic, internal the level of the posterior part of the anterior capsule; cc, corpus callosum; CA3, field CA3 of commissure. Scale bars: 2 mm in (A); 500 μm the hippocampus; MHb, medial habenular in (B, C); 2 mm in (D). cc, corpus callosum; nucleus. Scale bars: 2 mm in (A, C); 500 μm Cpu, caudate putamen (striatum); aca, anterior in (B, D). commissure, anterior part; MnPO, median preoptic nucleus; MPA, medial preoptic area; 3v, Fig. 9. Localization of RPE65-immunoreactive third ventricle; och, optic chiasm; acp, anterior cells in the chick brain. (A) Opn5m-expressing commissure. (E-H) Detection of Rpe65 neurons in the paraventricular organ (PVO).

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There were no RPE65-immunoreactive cells in Me, median eminence; Os, outer segments of and near the PVO. (B) In the anterior photoreceptors; Onl, outer nuclear layer; Inl, hypothalamus, a subset of ependymal cells were inner nuclear layer. Scale bar: 25 μm. immunoreactive for RPE65. The arrow shows RPE65 protein localized to the apical side of an Fig. 10. Expression of Opn5m mRNA (arrows ependymal cell. (C) More intense expression in A, C) in the mouse anterior hypothalamus of RPE65 protein was observed in the (Bregma 0.50 mm level). (A, B) from male ependymal cells lining the ventral region of the (C57BL6 strain, 8 weeks), (C, D) from female third ventricle (arrowheads). (D) The anti- (C58BL6, 8 weeks). Higher magnification of RPE65 antibody recognized retinal pigment (A, C; arrows) is shown in (B, D), respectively. epithelium (Rpe) in the retina at P10. (E) To The expression pattern appeared similar better visualize nuclei of RPE cells, blue between the two sexes. aca, anterior channel is enhanced. Nuclei are stained with commissure, anterior part; 3v, third ventricle. DAPI. 3v, third ventricle; Arc, arcuate nucleus; Scale bars: 2 mm in (A, C); 0.2 mm in (B, D).

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Table 1. Comparison of the amino acid residues among vertebrate Opn5m proteins

Residue bovine squid zebrafish Xenopus chicken platypus/opossum mouse/human No. rhodopsinb rhodopsin Opn5m Opn5m Opn5m Opn5m Opn5m

94a T M F A S T T 168 A A A A A A T 177 R A N N S N D 204 V I V V V I I a Amino acid residue numbers are designated using the bovine rhodopsin numbering system. b NCBI accession numbers of opsin sequences are as follows: bovine rhodopsin, K00506; squid rhodopsin, X70498; platypus Opn5m, XM_001511941; opossum Opn5m, XM_001369165.

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Figure 1

(kDa) mockmouse Opn5mmockmouse Opn5mmockhuman Opn5mmockhuman Opn5m

50

40

30

N-term of rho1D4C-term of rho1D4 mouse human Opn5m Opn5m

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Figure 2

human Opn5m 0.02 0.03 A 1.0 F 2 1 2 0.5 0.02 Rel. Abs. 0.01 4 0.0 2 400 500 600 Wavelength (nm) 0.01 1 1 5 3 Absorbance 0.00 0.00

300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm) mouse Opn5m 0.03 0.03 1.0 B 1 G 2 2 0.02 0.5 0.02 Rel. Abs. 2 4 0.0 400 500 600 0.01 0.01 1 1 5 Wavelength (nm)

Absorbance 3 0.00 0.00

300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm) chicken Opn5m 0.06 C 1.0 H 1 2 3 0.02 2 4 0.5 0.04 1 Rel. Abs. 1 0.0 0.01 400 500 600 0.02 3 Wavelength (nm) 2

Absorbance 5 4 0.00 0.00

300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm) Xenopus Opn5m 0.06 0.02 D 1.0 1 I 3 1 2 2 0.04 4 0.5 1 Rel. Abs. 0.0 0.01 400 500 600 0.02 Wavelength (nm) 2 3 Absorbance 5 4 0.00 0.00

300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm) zebrafish Opn5m 0.02 0.03 1.0 E 1 J 2 3 1 4 2 0.5 0.02 Rel. Abs. 0.01 0.0 400 500 600 2 1 Wavelength (nm) 0.01 4 Absorbance 5 3 0.00 0.00

300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm)

K syn anti

11 11 0%25% 50% 75% 100% all 9 9 all before irr. 11-cis

UV light irr. all-trans

Rel. Abs. (at 360 nm) 5 10 15 9-cis time (min)

15/23 Molecular property and expression pattern of mammalian Opn5

Figure 3

400 before irr. UV light irr. yellow light irr.

200 S bound (fmol) γ GTP 0 0 4 8 time (min)

16/23 Molecular property and expression pattern of mammalian Opn5

Figure 4

(A) (B)

0.00 0.00 T168 A168 0.03 0.02 mouse WT mouse T168A

Diff. Abs. -0.01 -0.01 0.02 T94S D177S 0.01 400 500 600 400 500 600 0.01

Wavelength (nm) Wavelength (nm) Absorbance Absorbance C 0.00 0.00

300 400 500 600 700 300 400 500 600 700 0.04 0.02 Xenopus A168T Xenopus WT

0.02 0.01

N Absorbance Absorbance 0.00 0.00

0.00 0.00 300 400 500 600 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm)

-0.01 -0.01 Diff. Abs. T168A I204V 400 500 600 400 500 600 Wavelength (nm) Wavelength (nm)

17/23 Molecular property and expression pattern of mammalian Opn5

Figure 5

Opn5m Opn5m Rpe65 antisense sense antisense A D G

Adult

B E H

P11

C F I

P1

18/23 Molecular property and expression pattern of mammalian Opn5

Figure 6

A B

C D

C’

19/23 Molecular property and expression pattern of mammalian Opn5

Figure 7

ABOpn5m antisense Opn5m antisense C Opn5m sense D Opn5m antisense

EFGHOpn5m antisense Opn5m antisense Rpe65 antisense Rpe65 antisense mouse

IJOpn5m antisense Opn5m antisense K Opn5m sense

LMNRpe65 antisense Rpe65 antisense Rpe65 sense marmoset

OP

(kDa) (kDa) liver lung neuralRPE retinahypothalamusbrain liver lung RPE hypothalamusbrain 50 Opn5 RPE65 37 50

20/23 Molecular property and expression pattern of mammalian Opn5

Figure 8

AB

C D

21/23 Molecular property and expression pattern of mammalian Opn5

Figure 9

22/23 Molecular property and expression pattern of mammalian Opn5

Figure 10

23/23