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PubMed2006

Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina.

Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal-ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone beta2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRbeta2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development.

Animals↗
PubMed2002

Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants.

Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.

Animals↗
PubMed2004

Signatures of selection and gene conversion associated with human color vision variation.

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.

Africa↗
PubMed2004

Nonvisual photoreception in the chick iris.

The embryonic chicken iris constricts to light ex vivo, but with characteristics atypical of visual phototransduction. The chick iris was most sensitive to short-wavelength light, demonstrating an action spectrum consistent with cryptochrome rather than with opsin pigments. Pupillary responses did not attenuate after saturating light exposure, but showed paradoxical potentiation. Iris photosensitivity was not affected by retinoid depletion or inhibitors of visual phototransduction. Knockdown of cryptochrome expression, but not of melanopsin expression, decreased iris photosensitivity. These data characterize a non-opsin photoreception mechanism in a vertebrate eye and suggest a conserved photoreceptive role for cryptochromes in vertebrates.

Animals↗
PubMed2001

Characterization of an opsin gene from the ascomycete Leptosphaeria maculans.

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes black-leg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.

Amino Acid Sequence↗
PubMed2002

Combined QM/MM study of the opsin shift in bacteriorhodopsin.

Combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations of bacteriorhodopsin (bR) in the membrane matrix have been carried out to determine the factors that make significant contributions to the opsin shift. We found that both solvation and interactions with the protein significantly shifts the absorption maximum of the retinal protonated Schiff base, but the effects are much more pronounced in polar solvents such as methanol, acetonitrile, and water than in the protein environment. The differential solvatochromic shifts of PSB in methanol and in bR leads to a bathochromic shift of about 1800 cm(-1). Because the combined QM/MM configuration interaction calculation is essentially a point charge model, this contribution is attributed to the extended point-charge model of Honig and Nakanishi. The incorporation of retinal in bR is accompanied by a change in retinal conformation from the 6-s-cis form in solution to the 6-s-trans configuration in bR. The extension of the pi-conjugated system further increases the red-shift by 2400 cm(-1). The remaining factors are due to the change in dispersion interactions. Using an estimate of about 1000 cm(-1) in the dispersion contribution by Houjou et al., we obtained a theoretical opsin shift of 5200 cm(-1) in bR, which is in excellent agreement with the experimental value of 5100 cm(-1). Structural analysis of the PSB binding site revealed the specific interactions that make contributions to the observed opsin shift. The combined QM/MM method used in the present study provides an opportunity to accurately model the photoisomerization and proton transfer reactions in bR.

Amino Acids↗
PubMed1997

Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin. These results show that P. pastoris cells have the capacity to functionally express bovine opsin.

Alcohol Oxidoreductases↗
PubMed1996

Opsin gene expression and regulation in retinal transplants.

In the present study we investigated the expression and regulation of the opsin gene in retinal transplants. Embryonic retinae were transplanted to intracranial locations in neonatal rodents in which they either reliably projected to the superior colliculus, or in locations (such as the cerebral cortex) in which they did not project to subcortical visual nuclei. Our results show that, regardless of the graft location, the developmental schedule of opsin gene expression in the outer nuclear layer was similar to normal, and that it was maintained in transplants for at least 6 months. To test if ambient light affected opsin gene expression, we dark-reared rats containing a retinal transplant for up to 26 days before assaying for opsin transcripts. In situ hybridization experiments showed that opsin gene expression in the transplants of these dark-reared recipients was not different either from transplants in animals reared in cyclic light conditions, or from the retina in situ. These observations support the hypothesis that the opsin gene is activated and maintained by molecular mechanisms intrinsic to the photoreceptor.

Animals↗
PubMed2005

Molecular cloning of cone opsin genes and their expression in the retina of a smelt, Ayu (Plecoglossus altivelis, Teleostei).

Five cone opsin genes of landlocked ayu fish (Plecoglossus altivelis) were cloned, and the expression patterns of these genes were investigated. AYU-LWS, -RH2-1, -RH2-2, -SWS1-1, and -SWS1-2 were isolated and had high (more than 75%) identity with red, green, green, UV, and UV-sensitive opsin, respectively, genes of other fish reported previously. The results of Southern blotting experiments showed that each gene is present as a single copy. Gene expression was measured by RT-PCR using four populations collected from rivers and a lake in spring and summer. The results of the RT-PCR experiment showed that AYU-SWS1-2 was highly expressed, whereas AYU-SWS1-1 was scarce. Two RH2 opsins were expressed simultaneously in the same individual, and the expression ratio between these opsins changed among populations. In situ hybridization revealed that AYU-LWS and -RH2-1 were expressed in the double cones and that AYU-RH2-2 and -SWS1-2 were expressed in the long and short single cones (LSC and SSC), respectively. It was shown that an individual ayu expresses two RH2 opsins simultaneously in different types of cone cells.

Amino Acid Sequence↗
PubMed1980

Aromatic retinal analogues and their interaction with cattle opsin.

The preparation of seven aromatic analogues of retinal, the isolation and characterization of their geometric isomers, and the interaction of these isomers with cattle opsin are reported. Within certain limitations, it has been demonstrated that stable aromatic rhodopsin analogues can be prepared. In general, the stereoselectivity of isomers of these compounds in their interaction with opsin and the absorption properties of the resultant pigments are similar to those of the parent retinal.

Animals↗
PubMed1996

Examining rhodopsin folding and assembly through expression of polypeptide fragments.

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.

Amino Acid Sequence↗
PubMed1984

Cycloheptatrienylidene analog of 11-cis retinal. Formation of pigment in photoreceptor membranes.

In biochemical and electrophysiologic studies employing the bullfrog (Rana catesbeiana) and the rat, the authors examined the interaction of opsin and an 11-cis-locked analog of retinal. In previously bleached preparations of bullfrog receptor outer segments (ROS) and isolated retinas, incubation with the aldehyde form (I) of the analog leads to the appearance of a pigment that is degraded slowly by hydroxylamine but is relatively resistant to photolysis. In the ROS preparation, the analog pigment (lambda max of difference spectrum congruent to 497 nm) also forms on incubation with NADP+ and the alcohol form (II) of the analog. In vitamin A deprived rats possessing only approximately 45% of the normal complement of rhodopsin, intraperitoneal injection of II leads within 1 day to the appearance of the analog pigment in the photoreceptors, at levels representing a major fraction of the opsin initially available for pigment formation. Formation of the analog pigment appears to have no significant effect on the sensitivity of electroretinographic b-wave responses recorded from the rat eye; furthermore, administration of II appears to suppress the sensitizing activity of all-trans retinol injected 1 day later. The data are discussed in relation to other studies examining chromophore-opsin interactions and electrophysiologic changes associated with the formation of rhodopsin in situ.

Animals↗
PubMed1998

Docosahexaenoic acid promotes differentiation of developing photoreceptors in culture.

PURPOSE: The purpose of this work was to study the effects of diverse fatty acids on the composition, metabolism, differentiation, and characteristics of opsin expression in retina photoreceptors. METHODS: Cultures of rat retinal neurons were incubated with or without 22:6 n-3, 22:5 n-3, 20:4 n-6, 18:1 n-9, and 16:0, labeled and unlabeled. RESULTS: In photoreceptor cells incubated with 22:6 n-3 and 22:5 n-3, the proportions of these fatty acids in phospholipids increased four- to sixfold. The remaining fatty acids did not change lipid acyl chain composition. The labeled fatty acids were all actively esterified in neuronal lipids, particularly in phosphatidylcholine. Addition of unlabeled 22:6 n-3 did not affect the distribution among lipids of the other fatty acids but displaced [3H]20:4 n-6 from phosphatidylcholine and phosphatidylethanolamine. These results suggest that retinal neurons have specific mechanisms for processing fatty acids of different lengths and degrees of unsaturation and that 22:6 n-3 incorporation takes priority. Of all fatty acids, 22:6 n-3 was the most effective in promoting photoreceptor differentiation. In 22:6-sufficient photoreceptors, new apical processes formed, the expression of opsin augmented, and its localization improved, concentrating in the apical processes of the cells. CONCLUSIONS: The advancement in differentiation selectively elicited by 22:6 correlates with the fact that 22:6 n-3, but none of the other fatty acids, delays significantly the onset of apoptosis in photoreceptors in culture. The synthesis of 22:6-containing phospholipid molecules could be required for the proper localization of opsin. This could contribute to furthering the differentiation of photoreceptors, preventing their apoptosis, and extending their survival.

Animals↗
PubMedDate not supplied

Morphology and visual pigment content of photoreceptors from injured goldfish retina.

Adult teleost fish retinas can regenerate neurons following either surgical or pharmacological injury. The cellular milieu of the damaged retina within which regenerated neurons are produced might be different in these two model systems of retinal injury, and thus the phenotypic attributes of regenerated neurons in the two model systems might also differ. To determine if the phenotypic attributes of photoreceptors, and by extension the recovery of vision, are different between these two model systems, we compared the visual pigment content and morphology of photoreceptors derived from goldfish retinas of both models with control retina. Visual pigments-which consist of a protein moiety (opsin) and a chromophore--were analyzed in single, isolated photoreceptors using microspectrophotometric techniques. We report that visual pigments and photoreceptor morphologies in the surgical model closely matched those of native retina. In contrast, neither photoreceptor morphology nor visual pigment content matched closely in the pharmacological model. The results indicate that phenotypic attributes of photoreceptors can differ significantly between the two model systems of retinal regeneration, but that in both systems, rod- and cone-mediated visual functions can potentially be reestablished.

Animals↗
PubMed2002

Synthesis of 11-cis-locked bicyclo[5.1.0]octanyl retinal and an enantioselective binding to bovine opsin.

Both enantiomers of 13-(E) and 13-(Z) isomers of 11-cis-locked bicyclo[5.1.0]octanyl retinal were prepared by an improved synthesis and incubated with bovine opsin. The synthesis also establishes the absolute configuration of the enantiomers. Only one of the enantiomers binds to opsin, thus showing the steric restrictions regarding the middle polyene moiety of the retinoid molecule; this is in sharp contrast to the known leniency of the ring moiety binding site of retinoids. However, although one enantiomer is incorporated into the pigment, the circular dichroic spectrum of the pigment incorporating the bound enantiomer yields only a very weak Cotton effect, showing that, once incorporated, the bicyclo[5.1.0]octanyl chromophore is flattened by the opsin binding site. The titled retinoid was synthesized for study of the absolute conformation of the retinal pigment in rhodopsin.

Animals↗
PubMed2002

Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors.

The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.

Amino Acid Sequence↗
PubMed1992

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) >> wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.

Animals↗
PubMed2002

A pharaonis phoborhodopsin mutant with the same retinal binding site residues as in bacteriorhodopsin.

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption maximum (500 nm) relative those of other archaeal rhodopsins such as the proton-pump bacteriorhodopsin (BR; 570 nm). Among the 25 amino acids that are within 5 A of the retinal chromophore, 10 are different in BR and ppR, and they are presumed to be crucial in determining the color of their chromophores. However, the spectral red shift in a multiple mutant of ppR, in which the retinal binding site was made similar to that of BR (BR/ppR), was smaller than 40% (lambda(max) = 524 nm) than expected. In the paper presented here, we report on low-temperature Fourier transform infrared (FTIR) spectroscopy of BR/ppR, and compare the infrared spectral changes before and after photoisomerization with those for ppR and BR. The C[bond]C stretch and hydrogen out-of-plane (HOOP) vibrations of BR/ppR were similar to those of BR, suggesting that the surrounding protein moiety of BR/ppR becomes like BR. However, BR/ppR exhibited a unique IR band regarding the hydrogen bond of the protonated Schiff base. It has been known that ppR has a stronger hydrogen bond for the Schiff base than BR as judged from the frequency difference between their C[double bond]NH and C[double bond]ND stretches. We now find that replacement of the 10 amino acids of BR with ppR (BR/ppR) does not weaken the hydrogen bond of the Schiff base. Rather, the hydrogen bond in BR/ppR is stronger than that in the native ppR. We conclude that the principal factor of the smaller than expected opsin shift in BR/ppR is the strong association of the Schiff base with the surrounding counterion complex.

Amides↗