James Bellingham

Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors.

Neuropsin (Opn5) : a novel opsin identified in mammalian neural tissue

We have cloned and characterised the expression of a new opsin gene, neuropsin (Opn5), in mice and humans. Neuropsin comprises seven exons on mouse chromosome 17. Its deduced protein sequence suggests a polypeptide of 377 amino acids in mice (354 in humans), with many structural features common to all opsins, including a lysine in the seventh transmembrane domain required to form a Schiff base link with retinaldehyde. Neuropsin shares 25–30% amino acid identity with all known opsins, making it the founding member of a new opsin family. It is expressed in the eye, brain, testis and spinal cord.

Differential Expression of Two Distinct Functional Isoforms of Melanopsin (Opn4) in the Mammalian Retina

Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra‐retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod‐opsins from the retina of these species. By PCR, we have determined that the novel rod‐like opsin is not expressed in the salmon retina, and the retinal rod‐opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod‐like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod‐like opsins, their extra‐retinal sites of expression, and phylogenetic position we have termed these novel opsins ‘extra‐retinal rod‐like opsins’ (ERrod‐like opsins). We speculate that the differences between retinal rod‐opsins and ERrod‐like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.

Expression of opsin genes early in ocular development of humans and mice.

We have compared the onsets of expression of the classical visual opsins with those of the non-rod, non-cone opsins in foetal and post-natal eye tissue from mice and humans. Mouse Rgr-opsin, peropsin, encephalopsin and melanopsin are all expressed in foetal development by E11.5, unlike the murine rod and cone opsins that exhibit post-natal expression, e.g. P1 for ultraviolet cone opsin and P5 for rod opsin. Human non-rod, non-cone opsins are also all expressed early, by 8.6 weeks post-conception. The implications of these observations are discussed with regard to the possible functions of these opsins at early stages of ocular development.

Sequence, genomic structure and tissue expression of carp (Cyprinus carpio L.) vertebrate ancient (VA) opsin

We report the isolation and characterisation of a novel opsin cDNA from the retina and pineal of the common carp (Cyprinus carpio L.). When a comparison of the amino acid sequences of salmon vertebrate ancient opsin (sVA) and the novel carp opsin are made, and the carboxyl terminus is omitted, the level of identity between these two opsins is 81% and represents the second example of the VA opsin family. We have therefore termed this C. carpio opsin as carp VA opsin (cVA opsin). We show that members of the VA opsin family may exist in two variants or isoforms based upon the length of the carboxyl terminus and propose that the mechanism of production of the short VA opsin isoform is alternative splicing of intron 4 of the VA opsin gene. The VA opsin gene consists of five exons, with intron 2 significantly shifted in a 3′ direction relative to the corresponding intron in rod and cone opsins. The position (or lack) of intron 2 appears to be a diagnostic feature which separates the image forming rod and cone opsin families from the more recently discovered non‐visual opsin families (pin‐opsins (P), vertebrate ancient (VA), parapinopsin (PP)). Finally, we suggest that lamprey P opsin should be reassigned to the VA opsin family based upon its level of amino acid identity, genomic structure with respect to the position of intron 2 and nucleotide phylogeny.

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65–85 Myr ago.

Opsins and melanopsins.

What are opsins? Opsins are generally considered members of the superfamily of G-protein coupled receptors. But not all opsins activate a G-protein. Their distinguishing features are a 7 transmembrane α-helical structure, and an ability to bind a vitamin A chromophore, retinaldehyde, using a lysine in the 7th α-helix. The range of amino acid identity among opsin families is 22–40%, and they have ∼17% identity to other hepta-helical receptor families.How do they work? Two well established functions are those of photosensor and photoisomerase (Figure 1Figure 1). In the photosensory opsins the chromophore, 11-cis-retinal, is located in a cage formed by the α-helices. Light is absorbed by 11-cis-retinal which is photoisomerised to all-trans. This conformational change allows the opsin to bind the α-subunit of the G-protein transducin. In the rods and cones of the retina G-protein activation leads to hyper-polarisation of the photoreceptor through cGMP-gated cation channels. The photoisomerase function is exhibited by retinal G-protein coupled receptor (RGR) opsin, expressed in the retinal pigment epithelium. It binds all-trans-retinal and uses light to convert it to the 11-cis configuration. Photoisomerisation is not thought to activate a G-protein but supplies the rod and cone opsins with 11-cis chromophore.Figure 1Vertebrate opsins are known to act as photosensors, where light induces a conformation change in the chromophore from the 11-cis-retinal to the all-trans configuration. This changes the opsin shape and results in the activation of a G-protein. Opsins that act as photoisomerases use light energy to convert all-trans-retinal into the 11-cis configuration, and supply photosensory opsins with chromophore.View Large Image | View Hi-Res Image | Download PowerPoint SlideHow many opsin families are there in vertebrates? On the basis of sequence similarity there are 14:7 are photosensory, including the 4 cone and 1 rod-opsin families, pineal-opsins, and vertebrate ancient (VA) opsins, first isolated from retinal horizontal and amacrine cells of teleost fish. Four other families, exorhodopsin, parapinopsin, tmt-opsin and encephalopsin, share relatively high levels of identity with the photosensory opsins (30–40%) but functional data are lacking. Other opsin families include RGR-opsin, melanopsin, and peropsin, a presumed photoisomerase.Why look for more photosensory and photoisomerase opsins? Photoreception is not limited to the rods and cones, but can occur in cells of the inner retina, pineal, brain or skin. Action spectra for these photoresponses have implicated opsin/vitamin A based photopigments, but the photosensory genes and proteins remain unknown. It is also unclear how photopigment chromophore is regenerated in photoreceptors that lack an retinal pigment epithelium-like structure. The assumption has been that there will be a ‘local’ photoisomerase to perform this task, but none has been identified.What is melanopsin and where is it expressed? Melanopsin was first isolated from the photo-sensitive melanophores of Xenopus. Orthologues have been found in most vertebrate classes, including mammals. All show little homology to the photosensory opsins and to each other. In mammals, melanopsin is expressed in a subset of photosensitive retinal ganglion cells, and in non-mammals, in photoreceptive structures such as the pineal and hypothalamus.Are melanopsins photosensors or photoisomerases? The expression of melanopsin in photoreceptors could indicate either a photosensory or photoisomerase function, but their homology to the known opsins predicts neither. If the melanopsins are novel photosensors then it will be critical to show that melanopsin not only binds retinal to form a photopigment with a sensitivity maxima predicted by action spectra, but that the melanopsin–chromophore complex can activate a phototransduction cascade. It is also possible that the melanopsins act as both photosensors and photoisomerases, and in this respect resemble the invertebrate photopigments.

Gene structure and tissue expression of human selenoprotein W, SEPW1, and identification of a retroprocessed pseudogene, SEPW1P

We have determined that the human SEPW1 (selenoprotein W) gene maps to chromosome 19q13.3, spans approximately 6.3 kb and comprises six exons, in contrast to the previously published five exons. The gene lacks canonical TATA and CAAT boxes, but has numerous Sp1 consensus binding sites upstream of multiple transcription start sites. SEPW1 is expressed in all of the 22 tissues assayed, and shows highest expression in skeletal muscle and heart. Additionally, we have also identified a retroprocessed SEPW1 pseudogene, SEPW1P, which maps to chromosome 1p34-35.

Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii

SUMMARY Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λmax around 500 nm when reconstituted with an A1 chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A2 absorbance spectra corrected for skin/skull absorbance indicates that the A2 versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.