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PubMed2006

Spectral tuning of the long wavelength-sensitive cone pigment in four Australian marsupials.

The molecular basis for the spectral tuning of longwave-sensitive (LWS) visual pigments in mammals have been described in a wide range of placental species, including the primates. However, little is known about the molecular mechanisms in marsupial LWS pigments. Here, we have studied and compared the LWS opsins in four Australian marsupials: two diprotodonts and two polyprotodonts. Phylogenetic analysis establishes that all LWS marsupial sequences form a distinct clade from the placental mammals that is subdivided into diprotodont and polyprotodont groups. Amino acid sequences reveal that substitutions at sites 277 and 285 are largely responsible for the spectral shifts in marsupial LWS pigments and species comparison indicates that the ancestral gene most likely encoded Tyr277 and Ala180. Amino acid substitutions are discussed in the context of spectral shifts in marsupial LWS and in relation to the mechanisms in primate pigments.

Amino Acid Sequence↗
PubMed2006

Recent and simultaneous origins of eusociality in halictid bees.

Eusocial organisms are characterized by cooperative brood care, generation overlap and reproductive division of labour. Traits associated with eusociality are most developed in ants, termites, paper wasps and corbiculate bees; the fossil record indicates that each of these advanced eusocial taxa evolved in the Late Cretaceous or earlier (greater than 65 Myr ago). Halictid bees also include a large and diverse number of eusocial members, but, in contrast to advanced eusocial taxa, they are characterized by substantial intra- and inter-specific variation in social behaviour, which may be indicative of more recent eusocial evolution. To test this hypothesis, we used over 2400 bp of DNA sequence data gathered from three protein-coding nuclear genes (opsin, wingless and EF-1a) to infer the phylogeny of eusocial halictid lineages and their relatives. Results from relaxed molecular clock dating techniques that utilize a combination of molecular and fossil data indicate that the three independent origins of eusociality in halictid bees occurred within a narrow time frame between approximately 20 and 22 Myr ago. This relatively recent evolution helps to explain the pronounced levels of social variation observed within these bees. The three origins of eusociality appear to be temporally correlated with a period of global warming, suggesting that climate may have had an important role in the evolution and maintenance of eusociality in these bees.

Animals↗
PubMed2005

Convergent loss of an anciently duplicated, functionally divergent RH2 opsin gene in the fugu and Tetraodon pufferfish lineages.

We describe the complete opsin gene families from the sequenced fugu and Tetraodon pufferfish genomes. We report the convergent loss of function of an anciently duplicated, functionally divergent RH2 or "green-sensitive" opsin gene in both pufferfish lineages, designated RH2-2. In fugu, RH2-2 apparently ceased to function very recently following a transposon-induced deletion that truncated the N-terminal 115 amino acids from the translated protein. Although a lack of frameshift or nonsense mutations in the fugu RH2-2 pseudogene suggests that the gene was lost very recently in this lineage, we were unable to detect any evidence of a selective sweep associated with the fixation of the truncated allele from population data. Interspecific comparison of the remaining fugu RH2-2 coding sequence paradoxically indicates that the gene was under strong purifying selection until the truncation occurred.

Amino Acid Sequence↗
PubMed2003

Co-localization of mesotocin and opsin immunoreactivity in the hypothalamic preoptic nucleus of Xenopus laevis.

The purpose of the present investigation was to provide a detailed description of the encephalic photoreceptors of Xenopus laevis at the light microscopic level and to determine their relationship with the neurosecretory cells of the hypothalamus in order to further our understanding of photoperiodic regulation of seasonal rhythms. Numerous opsin-positive neurons were found in the hypothalamic magnocellular preoptic nucleus and their axonal processes were seen to run laterally towards the basal regions of the brain, reaching the neural lobe of the hypophysis. Analysis of labelling with different antisera in adjacent sections, as well as double-immunolabelling carried out on the same section, revealed that mesotocin immunoreactivity was present in most of the opsin-positive neurons; however, no evidence for opsin and vasotocin coexpression was found in any of the sections analysed. The close localization of LHRH and opsin/mesotocin fibers in some regions of the brain, such as the median eminence, suggests that some interaction between these two systems might exist. In conclusion, in this study we provide the first strong evidence that the hypothalamic mesotocinergic neurons, which have been proved to be connected to the GnRH system in other species, are directly involved in photoreception in Xenopus laevis. These findings represent a novel contribution to our understanding of how light influences the seasonal reproductive cycles in lower vertebrates.

Animals↗
PubMed2002

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein.

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.

Animals↗
PubMed2003

Diurnality and cone photopigment polymorphism in strepsirrhines: examination of linkage in Lemur catta.

Trichromatic color vision is routine among catarrhine primates, but occurs only as a variant form of color vision in some individuals in most platyrrhine genera. This arises from a fundamental difference in the organization of X-chromosome cone opsin genes in these two lineages: catarrhines have two opsin genes specifying middle- and long-wavelength-sensitive cone pigments, while platyrrhines have only a single gene. Some female platyrrhine monkeys achieve trichromacy because of a species polymorphism that allows the possibility of different opsin gene alleles on the two X-chromosomes. Recently, a similar opsin gene polymorphism was detected in some diurnal strepsirrhines, while at the same time appearing to be absent in any nocturnal genera. The aim of this study was to assess whether cone pigment polymorphism is inevitably linked to diurnality in strepsirrhines. Cone photopigments were measured in a species usually classified as diurnal, the ring-tailed lemur (Lemur catta), using electroretinogram flicker photometry, a noninvasive electrophysiological procedure. Each of 12 animals studied was found to have the same middle-wavelength cone pigment, with peak sensitivity at about 547 nm. In conjunction with earlier results, this implies that cone pigment polymorphism is unlikely to exist in this species and that, accordingly, such variation is not a consistently predictable feature of vision in diurnal strepsirrhines.

Animals↗
PubMed1992

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro.

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.

Animals↗
PubMed1999

State-dependent disulfide cross-linking in rhodopsin.

In previous studies, we developed a new method for detecting tertiary interactions in rhodopsin using split receptors and disulfide cross-linking. Cysteines are engineered into separate fragments of the split opsin, the disulfide bond can be formed between the juxtaposed residues by treatment with Cu(phen)3(2+), and then disulfide cross-links can be detected on the gel by an electrophoretic mobility shift. In this study, we utilized this method to examine the cross-linking reactions between native cysteines in the ground state and after photoexcitation of rhodopsin. In the dark, Cys140 on transmembrane segment (TM) 3 cross-links to Cys222 on TM5. After photobleaching, Cys140 cross-links to Cys316 and Cys222, and the rate of the cross-linking reaction between Cys140 and Cys222 significantly increases.

Amino Acid Sequence↗
PubMed2000

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism.

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.

Acylation↗
PubMed2001

Characterization of a novel human opsin gene with wide tissue expression and identification of embedded and flanking genes on chromosome 1q43.

As part of an ongoing search to identify novel mammalian photopigments that may mediate nonvisual tasks such as circadian entrainment and acute suppression of pineal melatonin levels, a number of recently cloned nonvisual opsin sequences were used to search dbEST. panopsin (OPN3) was one of the clones identified using this approach. Expression analysis detects two transcripts of approximately 2.1 and 2.5 kb, in a wide range of tissues including brain, liver, and retina, which encode a predicted protein of 403 amino acids. The gene was localized to the region of chromosome 1q43 also encompassing the kynurenine monooxygenase (KMO) and choroideremia-like Rab escort protein 2 (CHML) genes. KMO and panopsin overlap at their 3' ends but are transcribed in opposite directions. CHML, an intronless gene, lies in intron 1 of panopsin.

Amino Acid Sequence↗
PubMed2002

Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals.

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.

Chromatography, High Pressure Liquid↗
PubMed1991

In vitro expression of bovine opsin using recombinant baculovirus: the role of glutamic acid (134) in opsin biosynthesis and glycosylation.

Expression levels of functional bovine opsin in the insect cell line IPLB-Sf9 using recombinant baculovirus were shown not to depend on the use of novel transfer vectors (pAcRP23, pAcDZ1) that were reported to improve biosynthesis levels of other proteins in this system. A production of 5 micrograms opsin per 10(6) cells (approx. 1.5% of total cell protein) was achieved by batch fermentation of infected cells in spinner cultures. Infection of the cells in the presence of the glycosyltransferase inhibitor tunicamycin led to the synthesis of the complete protein, which, however, now migrated with a substantially lower Mr. This demonstrates that opsin in insect cells also undergoes N-linked glycosylation and allowed partial purification (10-fold) of the resulting rhodopsin by affinity chromatography over Concanavalin A-Sepharose. Through site-directed mutagenesis (rhod)opsin mutants have been obtained allowing dissection of functional domains of opsin. Amino acid substitutions that involved Glu-134 and/or Arg-135 affected the normal biosynthetic process leading in part to nonglycosylated, to a small extent even incomplete, protein. A number of mutations, that involve other charged residues within the second and third transmembrane domain of the protein, had no effect on the biosynthetic processing of the protein. We therefore suggest that the charge-pair Glu-134-Arg-135 is part of an important internal signal sequence and that alterations in this region may result in incorrect membrane translocation and/or folding of the protein.

Animals↗
PubMed1985

Biochemical properties of 9-cis- and all-trans-retinoylopsins.

The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.

Animals↗
PubMed1999

The evolution of trichromatic color vision by opsin gene duplication in New World and Old World primates.

Trichromacy in all Old World primates is dependent on separate X-linked MW and LW opsin genes that are organized into a head-to-tail tandem array flanked on the upstream side by a locus control region (LCR). The 5' regions of these two genes show homology for only the first 236 bp, although within this region, the differences are conserved in humans, chimpanzees, and two species of cercopithecoid monkeys. In contrast, most New World primates have only a single polymorphic X-linked opsin gene; all males are dichromats and trichromacy is achieved only in those females that possess a different form of this gene on each X chromosome. By sequencing the upstream region of this gene in a New World monkey, the marmoset, we have been able to demonstrate the presence of an LCR in an equivalent position to that in Old World primates. Moreover, the marmoset sequence shows extensive homology from the coding region to the LCR with the upstream sequence of the human LW gene, a distance of >3 kb, whereas homology with the human MW gene is again limited to the first 236 bp, indicating that the divergent MW sequence identifies the site of insertion of the duplicated gene. This is further supported by the presence of an incomplete Alu element on the upstream side of this insertion point in the MW gene of both humans and a cercopithecoid monkey, with additional Alu elements present further upstream. Therefore, these Alu elements may have been involved in the initial gene duplication and may also be responsible for the high frequency of gene loss and gene duplication within the opsin gene array. Full trichromacy is present in one species of New World monkey, the howler monkey, in which separate MW and LW genes are again present. In contrast to the separate genes in humans, however, the upstream sequences of the two howler genes show homology with the marmoset for at least 600 bp, which is well beyond the point of divergence of the human MW and LW genes, and each sequence is associated with a different LCR, indicating that the duplication in the howler monkey involved the entire upstream region. [The sequence data described in this paper have been submitted to GenBank under accession nos. AF155218, AF156715, and AF156716.]

Animals↗
PubMed1997

Spectroscopic mimicry for the protonated retinal Schiff base in vivo with modified amphiphilic clay interlayers as a possible model of opsin environment.

We have found that clay acts as a novel model matrix for the amphiphilic protein-opsin to mimic the visible absorption spectrum of a protonated retinal Schiff base (RSB) in vivo. Without strong acids at ambient temperature, a visible broad absorption spectrum with a lambda max at 530 nm covering the range from 400 to 680 nm was achieved for the protonated RSB with cationic surfactant-modified montmorillonite clay. The interlayers of the dimethyloctadecylamine (DOA) modified clay were found to provide amphiphilic space allowing the amphiphilic RSB to be intercalated easily and sequentially and protonated by the DOA. It is proposed that the visible absorption spectrum at lambda max 530 nm was attributable to electrostatic effects, permitting the appropriate distance between the nitrogen of the protonated RSB and the negatively charged clay interlayers and also to the anisotropic orientation of the RSB molecules in the interlayers.

Humans↗
PubMed1997

Early emergence of photoreceptor mosaicism in the primate retina revealed by a novel cone-specific monoclonal antibody.

We have examined the emergence of the photoreceptor mosaic in fetal macaque monkeys by using a novel monoclonal antibody, 7G6, that recognizes all cones in the adult primate retina. In the fetal retina, however, between embryonic (E) day 80 and E130, some opsin-positive cones were not labeled by 7G6. Double-labeling experiments revealed that though long and middle wavelength-sensitive fetal cones are 7G6-positive, a subset of short wavelength-sensitive cones are delayed in their acquisition of 7G6 immunoreactivity. Heterogeneity in cone labeling with 7G6 was evident at all fetal ages. The onset of 7G6 immunoreactivity, surprisingly, precedes both the expression of the cone opsins and the formation of synaptic contacts in the outer plexiform layer. Specifically, a population of cones was labeled in the periphery of the E65 retina, concomitant with ongoing cone genesis. Moreover, early-differentiating 7G6-positive cones are organized into a regular array in immature, peripheral regions of the fetal retina indicating that the spatial arrangement of cones is initiated either during active cone proliferation or the initial differentiation of these cells. These results suggest that the periodic spacing of cones in the primate retina emerges autonomously within the photoreceptor layer, prior to the formation of synaptic connections within the retina or with the brain.

Animals↗
PubMed1987

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail.

The retinal proteins opsin, alpha-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocytochemistry we have employed antibodies directed against these "photoreceptor proteins" in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and alpha-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina, in the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.

Animals↗
PubMed2003

Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes.

G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65-/- mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.

Animals↗