![]() Received 14 March 2003 | Accepted 30 April 2003 | Published 16 May 2003 |
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A novel Xenopus SWS2, P434 visual pigment: Structure, cellular location, and spectral analyses
Alix G. Darden,1,2 Bill X.
Wu,1 Sergey L. Znoiko,1 E. Starr
Hazard, III,1 Masahiro Kono,1
Rosalie K. Crouch,1 Jian-Xing
Ma1
1Storm Eye Institute, Department of Ophthalmology, Medical University of South Carolina, Charleston, SC; 2Department of Biology, The Citadel, Charleston, SC
Correspondence to: Alix G. Darden, Ph.D., Department of Biology, The Citadel, 171 Moultrie Street, Charleston, SC, 29409; Phone: (843) 953-7873; FAX: (843) 953-7264; email: dardena@citadel.edu
Abstract
Purpose: The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis.
Methods: The cDNA for the Xenopus "green rod" pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcription polymerase chain reaction and the 5' end cloned by rapid amplification of the cDNA ends. The cellular localization of the Xenopus opsin was determined by immunolabeling of flat-mounted retinas using a specific antibody against this opsin. Spectral properties of the expressed protein were determined by absorption spectroscopy using recombinant pigment.
Results: A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids, forming 7 hydrophobic helices. Sequence analysis indicates that it belongs firmly to the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, tiger salamander, and newt SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2 opsin-specific polyclonal antibody demonstrated that the SWS2 pigment is expressed in green rods. After expression in COS cells, reconstitution with 11-cis retinal, and purification, the SWS2 pigment exhibits an absolute absorption maximum of 434 nm Thus, the name "SWS2, P434" was assigned for this opsin. The pigment decays rapidly in hydroxylamine in the dark, unlike the red rod pigment, rhodopsin.
Conclusions: A novel green rod opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis, which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties although it was identified only in the Xenopus green rod cells.
Introduction
Amphibian photoreceptor cells are used extensively as models for vision research, especially in electrophysiological studies. Some of the first observations of visual pigments were made using frog retinas [1,2]. The first two-dimensional rhodopsin crystal was obtained from the disk membranes of frog retinal rod outer-segments, and a number of early physiological studies were done with the frogs as well [3].
Unlike mammalian retina, many amphibians have two types of rod cells: red rods, which absorb at long wavelengths (around 500 nm), and green rods, or blue-sensitive rods, which absorb at shorter wavelengths (<450 nm). These two rod types are different in size, density, and absorbance spectra [4-7]. The two types of rod cells were named red rod and green rod (or blue-sensitive rod) because of their characteristic colors when a flat mount of retina was viewed through a microscope in white light [8,9]. The red rods are more prevalent in the retina while the green rods only constitute 1-15% of the total photoreceptor cells in amphibians [4,8,10]. In frogs, the red and green rods have similar morphologies but slightly different sizes. The red rods are 5-7 x 35-50 μm whereas the green rods are 4 x 30-40 μm lying on their side [7]. In addition, the red and green rods have distinct absorbance spectra [4,5,7,11]. Nevertheless, the similar morphology and photon sensitivity at the maximally absorbed wavelength of the red and green rods would suggest that the green rods are more like rods than cones [12]. The biological function of the green rod is currently unknown. It was speculated that the two types of rods act in conjunction to give limited hue discrimination at dim-light levels [13,14]. They may also play the role of blue cones in trichromatic color vision in an environment in which light at the blue end of the visual spectrum is attenuated. The latter environment might arise, for example, in turbid water, in which short-wavelength light would scatter more than longer-wavelength light, and would thus not penetrate as deeply [12].
Although these two types of rod cells have been known for many years, the molecular basis underlying the distinct absorbance peaks is only now being elucidated. The genes for the two rod pigments, as well as the three cone pigments, have recently been isolated and sequenced in many amphibians including the tiger salamander, newt, clawed frog, and bull frog [15-24]. In the African tree-frog, Xenopus laevis, the genes encoding the red rod, red cone, and violet cone pigments have been cloned and sequenced [22-24]. However, the gene for the green rod pigment in Xenopus laevis has yet to be cloned and sequenced. As more photopigment genes have been isolated, cloned, and characterized it has become clear that the historically used color-name nomenclature of photopigments is inappropriate and confusing [25]. The commonly used nomenclature to date reflects absorbance spectral properties and amino acid sequence of the pigments [26]. Thus, the green rod pigment discussed above is considered an SWS2 opsin.
To date, the SWS2 opsin gene has been cloned and sequenced in 26 vertebrate species [25,26]. In the tiger salamander, it has been shown that the identical photopigment, SWS2 P432, is found in both the blue cones and the green rods [27]. This finding is the first example of a single photopigment being shared between two distinctly different cell types: the rods and the cones. In the bullfrog, the SWS2 pigment is expressed only in rod cells, while in the newt, the SWS2 pigment is expressed only in cone cells. In the other 24 vertebrate species studied, the SWS2 pigment is expressed only in cone cells [17,21,27]. It has been hypothesized that the SWS2 pigment in amphibians is expressed only in rods in the anuran order (i.e., bullfrog) and can be expressed in both rods and cones in the caudata order (i.e., newt and tiger salamander) [21]. Because the tiger salamander, newt, bullfrog and African clawed-frog (order anura) are evolutionarily closely related, the cloning and sequencing of the green rod pigment in Xenopus laevis is of great interest.
In this study, we describe the cloning of a cDNA encoding a novel opsin pigment found in Xenopus green rods, belonging to the SWS2 group opsins from the African clawed-frog, Xenopus laevis. Immunohistochemical analysis of the expression of this gene demonstrates that it is expressed in rod cells and not in cones. We have named this pigment "Xenopus SWS2, P434", based on sequence homology and absorbance.
Methods
Animals and tissues
Care, use, and treatment of animals in this study complied strictly with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and with the guidelines for the Care and Use of Laboratory Animals at the Medical University of South Carolina. Tissues and cells used in the experiments were from 7-9 cm long South African clawed-frogs, Xenopus laevis, (Xenopus Express, Homosassa, FL) which were maintained on a 12-h light/12-h dark cycle at 16-20 °C.
Synthesis of Xenopus retinal cDNA
Retinas were dissected from 20 fresh Xenopus eyes. Total RNA was extracted from the retinas using the Trizol reagent (Life Technologies) according to the manufacturer's protocol. Total RNA (2 μg) was reverse transcribed using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Gibco-BRL) using random hexamers as per the Gibco-BRL M-MLV protocol, to produce the cDNA.
Polymerase chain reaction (PCR) amplification and cloning of SWS2, P434 from Xenopus laevis retina cDNA
Nested PCR primers were designed, based on conserved regions in the coding region of green rod pigments from other species and synthesized commercially by Genosys (Fisher Scientific). The PCR reaction was as follows: 2 μL each of the forward and reverse primers (10 μmol/L), 2 μL of cDNA, 2.5 mmol/L dNTP, 2.5 μL 10X PCR buffer, 5 units Taq Polymerase Gold (PE Express), and 3 mmol/L MgCl2, brought to 25 μL volume with water. PCR conditions were 94 °C, 10 min, for one cycle then 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min for 35 cycles, then 72 °C for 5 min. The PCR product was analyzed on a 1.5% agarose gel, isolated and sequenced using an automated DNA sequencer at the Medical University of South Carolina's (MUSC) BioTechnology Resource laboratory.
Rapid amplification of cDNA ends (RACE) was performed to identify the 5' end of the Xenopus SWS2, P434 cDNA from the Xenopus retina using a Smart RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) according to the manufacturer's instruction. The PCR products were cloned into a vector (pCRII; Invitrogen, Carlsbad, CA) and sequenced. All sequences were confirmed by sequencing the complementary strand and were verified in another clone from an independent PCR.
DNA sequence analysis, amino acid prediction and phylogeny
The sequences were compared with existing sequences in GenBank. DNA sequence analysis was conducted using the Wisconsin Genetics Computer Group using a UNIX System software package made available from the Biomolecular Computing Resources (BCR) at the Medical University of South Carolina. The first ATG was used as the translation start codon, and the amino acid sequence was deduced using the GCG PUBLISH program.
Ninety-two invertebrate and vertebrate opsin sequences were selected for sequence analysis. Coding DNA sequences were assembled (Accelrys ASSEMBLE) from Genbank entries and translated to produce protein sequences. Ten-character names were assigned to each sequence to conform to the PHYLIP format convention and to facilitate interpretation of alignments and phylogenetic trees. Table 1 shows the complete list (10 character name, GenBank accession number, species name and common name for the species).
An alignment of the 92 proteins was produced with the CLUSTALW (version 1.7) [28]. The resulting alignment was bootstrap sampled 1000-times to assess the branch groupings. The alignment was submitted to the PHYLIP PROTPARS [29] program to compute a most parsimonious tree. The input order was jumbled three times and the ScalOpsnGq was designated as the root. The same CLUSTALW alignment was bootstrapped 1000 times with SEQBOOT [29]. These 1000 alignments were analyzed by PROTDIST [30], and 1000 trees were produced by the NEIGHBOR program [29]. A majority rule, consensus tree was produced by the PHYLIP CONSENSE program [29].
The Xenopus SWS2 sequence was analyzed for transmembrane regions by comparing its alignment with other opsins and by the Accelrys Transmem and PEPPLOT programs. A consensus of these methods was used to create input for the Viseur program [30], which produced the two-dimensional plot. The Viseur two-dimensional plot was annotated and re-colored utilizing Adobe Illustrator.
A three-dimensional model of the Xenopus SWS2 sequence was made with the MODELLER program [31]. The template was the 1HZX PDB file [32].
Antibodies
A polyclonal antibody, XGN, was raised against a peptide consisting of the first 14 amino acids from the N-terminus of the Xenopus SWS2, P434 pigment, MSKGRPDLRMEMPD. The peptide (0.3 mg) conjugated to Keyhole Limpet Hemacyanin was emulsified in Complete Freund's Adjuvant (GIBCO-BRL, Gaithersburg, MD) and injected subcutaneously into rabbits. Rabbits were boosted intramuscularly with 0.3 mg of the same emulsion at 3-week-intervals until a significant immune response developed at which time the rabbits were euthanized and their whole serum was collected. The antibody was purified by passing the serum through a column of the epitope peptide coupled to AminoLink beads (Pierce, Rockford, IL), according to the manufacturer's protocol.
The 4D2 monoclonal mouse anti-bovine rhodopsin N-terminal antibody, a generous gift from Robert Molday, was used to identify Xenopus red rods [33]. Texas Red-conjugated wheat germ agglutinin (WGA) was used to label all cone cells [34].
Immunohistochemistry
Immunohistochemistry was performed as described previously [27]. Briefly, the eyes were enucleated, lens-attached retinas were dissected, then transferred to 4% formaldehyde in PBS (pH 7.4) for 2 h at 4 °C. Tissues were then washed three-times, 15 min each, with PBS at room temperature and blocked with 1% BSA in PBS for 30 min. All subsequent steps were carried out at 4 °C unless otherwise specified. The primary XGN (rabbit) antibody, along with 4D2 monoclonal mouse anti-mouse rhodopsin antibody, were added for the next 4 h incubation. After washing with PBS (3-times, 10 min each) the secondary donkey anti-rabbit FITC-conjugated (Jackson Immuno Research Laboratories, Inc., West Grove, PA) antibody along with goat anti-mouse Texas-Red-conjugated antibody (Jackson Immuno Research Laboratories, Inc., West Grove, PA) or wheat germ agglutinin (WGA)-conjugated to TRITC, (Sigma) were added for 4 h. All antibodies and WGA were diluted 1:100. Finally, the retina was washed three-times in PBS and flattened on a slide. One drop of Prolong anti-fade solution (Molecular Probes, Eugene, OR) was added followed by a coverslip.
Samples were analyzed using an Axioplan 2 research microscope (Carl Zeiss, Inc., Jena, Germany) equipped with a 100 W mercury light source, a 100x plan-neofluar N.A. 1.3 and 20x objectives, and an AxioCam black/white camera (Carl Zeiss, Inc., Jena, Germany). Images were captured with AxioVision 3.1 software (Carl Zeiss, Inc., Jena, Germany).
Expression, purification, and absorption spectra of SWS2, P434
The cDNA for the complete SWS2, P434 gene was inserted into a pMT3 expression vector [35] as an EcoRI-NotI cassette. Two additional internal modifications were made to facilitate construction of the expression plasmid and subsequent protein purification. First, a silent mutation was made to destroy the endogenous EcoRI restriction enzyme site. Second, codons for the last eight amino acid residues of bovine rhodopsin (anti-rhodopsin 1D4 epitope) were inserted between the last amino acid codon of the Xenopus SWS2, P434 gene and the stop codon. This Xenopus SWS2, P434 plasmid was transiently transfected into COS cells as previously described [36]. A pigment was generated with 11-cis retinal and purified by 1D4 immunoaffinity chromatography [27,37-39]. The purified pigment was in 0.1% dodecyl maltoside in PBS (pH 7).
The spectrum of the eluted pigment was acquired on a Varian-Cary 300 dual beam or HP-8452A diode array spectrophotometer. The spectrum of the photobleached pigment was acquired after illuminating the sample for 2 min with a 30 W white light source. Hydroxylamine sensitivity was determined by the addition of 50 mmol/L hydroxylamine, pH 7.0, to unphotolyzed pigment; spectra were subsequently recorded at several time points. The hydroxylamine-treated sample was kept in the dark or manipulated under dim red light. The temperature in the spectrophotometer was maintained at 20 °C.
Results
Cloning and sequence analysis of the cDNA encoding Xenopus SWS2, P434
The cDNA of the Xenopus SWS2, P434 consists of 1135 nucleotides including the full-length coding region of 1089 bp, 12 nucleotides of the 5'-untranslated region (UTR) and 34 bp of the 3'-UTR sequence (Figure 1). The first ATG (at position 13) is in a favorable position for translation initiation with A at position -3 and was designated as the translation initiation codon. The stop codon, TAA, was identified at position 1099. The largest open-reading frame in the SWS2, P434 cDNA was translated to 362 amino acids.
The deduced amino acid sequence shows high identity with the SWS2 opsins (Figure 2), and thus this pigment is named Xenopus SWS2, P434. Xenopus SWS2, P434 has conserved cysteine residues at 119 and 196 (which correspond to bovine sequence 110 and 187) that are thought to form a disulfide bridge. The putative Schiff-base counter-ion glutamic acid is present at 122 (113 bovine), and the retinal binding lysine 305 (296 bovine) is conserved as well. The conserved glycine 130 (121 bovine) is present. At position 131 (bovine 121) members of the SWS2 clade have a methionine, whereas the Xenopus SWS2, P434 pigment has an isoleucine at this position. However, Xenopus SWS2, P434 is still cone-like in this property as the red rod opsins all possess glutamic acid at position 122 (bovine). The computed isoelectric point of Xenopus 434 is 8.12 (Accelrys PEPTIDESORT). This computed isoelectric point for Xenopus SWS2, P434 is consistent with the observation that cone opsins tend to be more basic than their rod opsin counter parts [40]. In conclusion, the molecular properties of Xenopus SWS2, P434 indicate that it is most similar to other SWS2 cone opsins.
Sequence comparison with other visual pigments
The Xenopus SWS2, P434 exhibits moderate sequence similarity to Xenopus opsin genes, but relatively higher sequence similarity to other amphibian SWS2 opsins. Xenopus SWS2 shares 41%, 53%, and 50% amino acid sequence identity with Xenopus red and violet cone opsins, and rhodopsin, respectively. In comparison, 73%, 80%, and 89% identity was observed when comparing Xenopus SWS2 to newt, tiger salamander, and bullfrog SWS2 opsins, respectively. Gene tree reconstructions always placed Xenopus SWS2 within a group of SWS2 sequences that were supported in 100% of bootstrap replicates, using either parsimony or distance based methods (Figure 3). These data strongly support the idea that Xenopus SWS2 is an SWS2 ortholog.
Location and distribution in Xenopus retina
Xenopus retinas were separately labeled with the XGN antibody and pre-immune serum. The XGN antibody labeled a type of rod cell with a long outer-segment in the flat-mounted Xenopus retina (Figure 4). The control staining with pre-immune serum was negative (data not shown), demonstrating the specificity of the antibody. The green rod cells stained with XGN antibody have typical rod morphology (i.e., a long cylindrical outer-segment shape).
To confirm that the labeled rods were not the red rods, Xenopus retinas were double-labeled with the antibodies XGN and 4D2, which specifically recognizes red rod opsins. As shown in Figure 5A-C, the XGN-positive rods are distinct from the red rods labeled by 4D2. Furthermore, the XGN-labeled rods are at a low density and have a different morphology than the red rods, with slim outer-segments.
Expression in rods and not cones
To determine whether this SWS2 pigment is also expressed in the cone photoreceptor cells, as has been shown for the tiger salamander [27], Xenopus retinas were double-labeled with WGA and XGN antibody. WGA has previously been shown to label all Xenopus cones [34]. As seen in Figure 5D-F, Xenopus cone cells were labeled with WGA while only the green rods were labeled with XGN. There was no cone labeling observed by the XGN antibody in any of the Xenopus (n = 5) retinas studied.
Absorbance spectral analysis
The SWS2, P434 opsin was expressed in COS cells. After regeneration and purification, the recombinant Xenopus SWS2, P434 pigment generated with 11-cis retinal shows an absorption maximum at 434 nm (Figure 6A), confirming that the cloned cDNA indeed encodes a SWS2, P434 pigment. As are all other visual pigments, this pigment is light sensitive (Figure 6A). The Xenopus SWS2 pigment is also sensitive to hydroxylamine attack in the dark and the pigment bleaches with a time constant of approximately 20 min in the dark (Figure 6B,C). Cone pigments are known to be unstable in the presence of hydroxylamine, unlike the red rod pigment, rhodopsin [11]. The loss of pigment in the dark in the presence of hydroxylamine occurs with cone pigments, but not with rhodopsin. The blue cone/green rod pigment from the tiger salamander, which belongs to the SWS2 class of visual pigments, was likewise shown to bleach in the dark in the presence of hydroxylamine [27].
Discussion
The Xenopus retina contains two rod types (red and green) and at least 3 cone types [41]. Previously, the red rod rhodopsin, red cone and violet cone pigments have been cloned [22-24]. In the present study, we report the cloning and sequencing of a fourth pigment from the Xenopus retina. Sequence analysis indicates that it belongs to the SWS2 class of visual pigments, which are predominantly expressed in cone cells. The Xenopus SWS2 pigment regenerated with 11-cis retinal showed a λ_max_ of 434 nm, similar to that previously reported using physiological methods [9]. This is similar to the λ_max_ of other SWS2 pigments, which range from 430-450 nm [42]. Of interest, even though this pigment is found in a rod cell, it is sensitive to hydroxylamine, a property generally attributed to cone pigments. Although the opsin has many cone-like properties, immunohistochemical analyses show that it is expressed only in rod cells.
The retinal photoreceptor cells (rods and cones) are distinctly different both structurally and functionally. In vertebrate species, this distinction classically is made based on morphological, physiological, molecular, and functional features [43]. Immunohistochemical studies using antibodies specific to various opsins and plant lectins have also been useful in identifying the photoreceptor cell types [41]. Immunohistochemical studies in the bullfrog have shown that the SWS2 pigment is expressed only in rod cells [17]. In the tiger salamander, SWS2 pigment is expressed in both rods and cones [21,27], while in the newt (which has no green rods), SWS2 pigment is expressed in cones [21]. In other non-amphibian species, the SWS2 pigment is only expressed in cone cells. We report here the cloning and expression of another SWS2 opsin in an anuran, Xenopus. Figure 4 and Figure 5 demonstrate, however, that the expression of the Xenopus SWS2 opsin is localized only to the thinner, narrower type of rods, probably "green" rod cells. Our in situ observations are consistent with the proposal that the ability of SWS2 pigments to be expressed in cone-like photoreceptors has been lost in anurans [21].
Physiologic studies, such as microspectrophotometry and electrophysiology, contributed to the spectral identification of rods and cones. In Xenopus, these studies identified a green rod absorbing at 445 nm [9,44]. In this report, the SWS2, P434 cDNA was expressed in COS cells and identified at an absorbance maximum of 434 nm when regenerated with 11-cis retinal (A1). Naturally, Xenopus visual pigments are formed from 11-cis-dehydroretinal (A2), which leads to a red shift in the absorption maximum, accounting for the difference seen in this study.
Sequence analyses and spectral tuning experiments have further identified key properties that distinguish rods and cones. (1) A common property of cone visual pigments is that they have many basic amino acid residues whereas rod visual pigments have more acidic amino acid residues. (2) Residue 122 (bovine) has been shown to regulate the intramolecular signal transduction in visual pigments. At this position, all rod opsins have glutamic acid, whereas cone opsins do not. (3) Studies in intact retina and extracts have shown that the green rod pigment is sensitive to hydroxylamine, a property common to cone opsins but not to red rod opsins [11]. In this study we have shown that for all three of these molecular properties, the Xenopus SWS2, P434 opsin appears to be a cone photoreceptor.
The Xenopus SWS2 pigment described in this paper has many cone-like properties, although it is localized in rod cells. These cone-like properties include: it is an SWS2 class opsin; it is sensitive to hydroxylamine; it has a basic pI; and it does not have a glutamic acid at position 122 (bovine). Molecular studies in the tiger salamander identifying individual molecules in the signal transduction pathway have shown that although rod and cone cells may contain the same opsin molecule, the transducins are different in the two cell types, which contributes to the functional differences between the two cell types [27]. Xenopus laevis provides another example of a species in which a cone-like opsin is expressed in a rod cell.
Acknowledgements
This work was supported by grants from the National Institutes of Health/National Eye Institute (EY04939 and EY12231); joint grants from the National Science Foundation/EPSCoR under Grant Number EPS-0132573 and National Institutes of Health/BRIN under Grant Number 8-P0RR16461A; The Citadel Foundation, Charleston, SC; an unrestricted grant to Storm Eye Institute from Research to Prevent Blindness (RPB), NY; and the Charleston Scientific and Cultural Education Fund, Charleston, SC. Rosalie Crouch is an RPB Senior Scientific Investigator. We thank Robert Molday for the 4D2 antibody. We would like to acknowledge the technical assistance of Brandy Perry and Jandi Withrow, and the critical reading of the manuscript by Luanna Bartholomew.
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