Figure 1.
Elephant shark melanopsin sequences.
An alignment of the four melanopsin polypeptides (opn4m1, opn4m2, opn4xlong and opn4xshort) expressed in the elephant shark (Callorhinchus milii, Cm) compared to the rod opsin (rh1) protein sequence from the same species (GenBank accession number: ABU84865) and the human (Homo sapiens, Hs) melanopsin (opn4m) polypeptide sequence (GenBank accession number: AAI43689). Identical amino acids (*) and gaps (–) present within the alignment are shown. Intron positions are shown as vertical lines or circles, where a red vertical line represents phase 0 introns (i.e. between two codons); a red circle marks the codons that are split in phase 1 (i.e. between the first and second bases of the codon); and a blue circle denotes codons that are split in phase 2 (i.e. between the second and third bases of the codon). Seven putative transmembrane (TM) domains for each elephant shark melanopsin protein were predicted using TMHMM Server Version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and indicated by green shading. The TM domains for the elephant shark rod opsin were taken from [10]. Additional residues identified as being critical for correct opsin protein conformation and function are shown (highlighted in black or boxed) and discussed elsewhere [6], [16]. These include: (i) two putative glycosylation sites (Asn, N) that adhere to the consensus motif (Asn-X-Ser/Thr, N-X-S/T; X, any amino acid) for N-linked glycosylation present in the extracellular (EC) amino-termini of opn4m1 and opn4m2, but not in either isoform of opn4x (predicted online using NetNGlyc Server 1.0, http://www.cbs.dtu.dk/services/NetNGlyc/); (ii) two conserved cysteine (Cys, C) residues at positions 110 (TMIII) and 187 (EC2) that are involved in disulfide bond formation; (iii) a conserved tyrosine (Tyr, Y) at position 113 (TMIII), as found in many non-cone, non-rod opsins, instead of the glutamate (Glu, E) residue that serves as the negative counterion to the proton of the Schiff base for many visual opsins; (iv) a conserved glutamate (Glu, E) at position 134 (TMIII), located within a conserved Asp/Glu-Arg-Tyr (D/ERY) motif (134–136), that provides a negative charge to stabilise the inactive opsin molecule; (v) a conserved charged residue at site 181 that may serve as a counterion in many non-cone, non-rod opsins or affect spectral tuning; (vi) a conserved lysine (Lys, K) at position 296 (TMVII) that is covalently linked to a retinal chromophore via a Schiff base; (vii) a conserved Asn-Pro-X-X-Tyr-X5,6-Phe (NPxxY(x)5,6F) motif (302–313, dotted box) that assists in maintaining structural integrity upon photopigment activation; (viii) a small number of palmitoylation sites in both “long” and “short” isoforms of opn4x (and rh1), but absent in opn4m1 and opn4m2, that may be important in the tethering of the carboxyl-termini to the membrane (predicted online using CSS-Palm Server 3.0, http://csspalm.biocuckoo.org/online3.php [69]); and (ix) numerous potential serine (Ser, S), threonine (Thr, T) and tyrosine (Tyr, Y) phosphorylation sites in the carboxyl-terminal tails of all four protein isoforms that may be important in the regulation of phototransduction via the action of opsin-specific kinases (predicted online using NetPhos Server 2.0, http://www.cbs.dtu.dk/services/NetPhos/[70]). All amino acids are conventionally numbered based on the bovine rod opsin protein (GenBank: accession number: NP001014890).
Figure 2.
Phylogeny of melanopsin in C. milii and other vertebrates.
Phylogenetic analyses based on a codon-matched nucleotide alignment of elephant shark opn4 cDNA sequences compared to the melanopsin sequences of representative chordates and the published visual and non-visual photosensory pigments of the zebrafish (Danio rerio), showing the relative positioning of elephant shark opn4m1, opn4m2 and opn4x photopigments (arrow) within the two main clades (m-class (purple) and x-class (blue)) of melanopsin. (A) A Bayesian Probabilistic Inference (BPI) method, performed with a Metropolis Markov chain Monte Carlo (MCMC) algorithm [43], [44] and incorporating a general time-reversal (GTR) model [45] with posterior probability values (represented as a percentage) indicated at the base of each node. (B) Maximum Composite Likelihood (MCL) [47] and (C) Kimura 2-Parameter (K2P) substitution [48] matrix methods, both generating bootstrapped, Neighbour-Joining (NJ) phylogenetic trees [49], with the degree of internal branching expressed as a percentage. The scale bar indicates the number of nucleotide substitutions per site. The human GPR21 (GenBank accession number: NM005294) and GPR52 (GenBank accession number: NM005684) nucleotide sequences were used as outgroups. The opsin sequences and their GenBank accession numbers used for generating the tree are as follows: (i) exorhodopsin (exorh): zebrafish (Danio rerio), NM131212; (ii) rod opsin (rh1): zebrafish (Danio rerio), NM131084 (rh1.1), HM367062 (rh1.2); (iii) rod opsin-like 2 (rh2): zebrafish (Danio rerio), NM131253 (Rh2.1), NM182891 (Rh2.2), NM182892 (Rh2.3), NM131254 (Rh2.4); (iv) short-wavelength-sensitive 2 (sws2): zebrafish (Danio rerio), NM131192; (v) short-wavelength-sensitive 1 (sws1): zebrafish (Danio rerio), NM131319; (vi) long-wavelength-sensitive/middle-wavelength-sensitive (lws/mws): zebrafish (Danio rerio), NM131175 (lws1), NM001002443 (lws2); (vii) vertebrate ancient (va) opsin: zebrafish (Danio rerio), AB035276 (va1), AY996588 (va2); (viii) panopsin (opn3): zebrafish (Danio rerio), NM001111164; (ix) teleost multiple tissue (tmt) opsin: zebrafish (Danio rerio), BC163681; (x) retinal pigment epithelium-specific rhodopsin homolog (rrh) (peropsin): zebrafish (Danio rerio), NM001004654; (xi) retinal G protein-coupled receptor (rgr): zebrafish (Danio rerio), NM001017877; (xii) mammalian-like melanopsin (opn4m): human (Homo sapiens), NM033282 (OPN4V1); cat (Felis catus), AY382594; mouse (Mus musculus), EU303118 (Opn4mlong); rat (Rattus norvegicus), NM138860; hamster (Phodopus sungorus), AY726733; mole-rat (Spalax ehrenbergi), AM748539; dunnart (Sminthopsis crassicaudata), DQ383281; chicken (Gallus gallus), EU124632 (OPN4Mlong); catfish (Ictalurus punctatus), FJ839437 (opn4m1), FJ839438 (opn4m2); roach (Rutilus rutilus), AY226847; cichlid (Astatotilapia burtoni), EU523855; zebrafish (Danio rerio), GQ925715 (opn4m1), GQ925716 (opn4m2), GQ925717 (opn4m3); elephant shark (Callorhinchus milii), JQ172797 (opn4m1), JQ172798 (opn4m2); (xiii) xenopus-like melanopsin (opn4x): chicken (Gallus gallus), EU124630 (OPN4Xlong); lizard (Podarcis siculus), DQ013043; African clawed frog (Xenopus laevis), AF014797; cod (Gadus morhua), AF385823 (opn4xa), AY126448 (opn4xb); zebrafish (Danio rerio), GQ925718 (opn4x1), GQ925719 (opn4x2); elephant shark (Callorhinchus milii), JQ172799 (opn4xlong); (xiv) chordate melanopsin (opn4): lancelet (Branchiostoma belcheri), AB205400; and (xv) outgroup (not shown): human (Homo sapiens), NM005294 (GPR21), NM005684 (GPR52). The gene nomenclature used follows the guidelines adopted by the Entrez Gene database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene). In brief, the genes of all terrestrial species are in uppercase, except for the mouse and rat, where only the first letter is capitalized. The genes of all aquatic species, including amphibians, are in lowercase.
Figure 3.
Expression of opn4 genes in elephant shark tissues.
(A) Relative expression of opn4m1, opn4m2 and opn4x genes in a tissue panel of samples derived from the brain (without the hypothalamus), eye, fin, gills, hypothalamus, kidney, liver, snout, skin and testis of an adult elephant shark. Actin was included as a control for cDNA quality. (B) A summary table showing the relative patterns of melanopsin expression shown in the upper panel. In both panels, opn4m1 transcripts were shown to be limited to the eye, whereas both opn4m2 and opn4x (“long” and “short”) mRNAs exhibited broad expression across all the main tissue types investigated, including the adult eye.
Figure 4.
Functional analysis of elephant shark melanopsin photopigments.
Light-evoked responses of neuro-2a cells expressing elephant shark melanopsins. Whole-cell recordings from cells expressing (A) opn4m1, (B) opn4m2 or (C) opn4xlong, preincubated with 9-cis (blue), all-trans (red) or no retinal (black) and exposed to a 10 s light pulse (8×1014 photons.cm−2.s−1) revealed that elephant shark melanopsins encode functional photopigments in vitro with cis-isomers of retinal chromophore, whereas only opn4m1 and opn4m2 exhibit light-evoked currents in the presence of all-trans retinal. (D) Quantification of melanopsin-dependent light-evoked currents with 9-cis retinal (n = 4–8 cells) (blue), all-trans retinal (n = 4–7 cells) (red) and no retinal (n = 4 cells) (black), respectively. Controls using untransfected cells in the presence of retinal were not light-responsive, with background currents comparable with no retinal experiments. Light-evoked currents (mean ± SEM) observed in cells expressing melanopsin preincubated with 9-cis retinal were significantly larger compared with cells with no retinal (p<0.05; Student’s t-test). Recordings with all-trans retinal showed that light-evoked responses of opn4m1- and opn4m2-transfected cells were larger (p<0.01; Student’s t-test) than those with no retinal. All possible permutations between two individual experimental values yielded significant differences (p<0.05; Student’s t-test), except for a comparison between the light-evoked currents determined for opn4xlong-transfected cells with or without all-trans retinal (0.05<p<0.5; Student’s t-test; denoted by n.s. (not significant)).