Whale shark rhodopsin adapted to deep-sea lifestyle by a substitution associated with human disease

Abstract

Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with secondarily expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter feeding, was previously shown to possess the ‘blue-shifted’ rhodopsin (RHO), which is a signature of deep-sea adaptation. In this study, our spectroscopy of recombinant whale shark RHO mutants revealed that this blue shift is caused dominantly by an unprecedented spectral tuning site 94. In humans, the mutation at the site causes congenital stationary night blindness (CSNB) by reducing the thermal stability of RHO. Similarly, the RHO of deep-diving whale shark has reduced thermal stability, which was experimentally shown to be achieved by site 178 and 94. RHOs having the natural substitution at site 94 are also found in some Antarctic fishes, suggesting that the blue shift by the substitution at the CSNB site associated with the reduction in thermal stability might be allowed in cold-water deep-sea habitats.

The whale shark Rhincodon typus (Fig. 1A), known as the largest extant fish, dives into the deep sea of 2,000 meters while it forages by filter feeding near the surface (1). Because of its exceptionally wide vertical habitat range, it is of great interest to investigate its visual ecology. Visual capability is achieved by photoreception primarily with visual pigments composed of a photoreceptor protein opsin and a retinal (2, 3). Among those pigments, spectral tuning of rhodopsins (RHOs) is responsible for adaptation to dim-light vision (4).

Fig. 1.

Characterization of whale shark RHO. (A) Photograph of the whale shark. (B) Amino acid residues unique to the whale shark (red). The phylogenetic relationships among sharks and their relatives are depicted based on ref. 5, with the amino acid residues at 94 and 178 as well as previously documented spectral tuning sites. The black rockcod, an Antarctic fish, is also included (SI Appendix and Dataset S3). (C) Absorption spectra of the wild type (WT, black), the A94T mutant (blue), the F178Y mutant (orange), the A94T/F178Y mutant (magenta), and the A94I mutant (green; Inset) of the whale shark RHO. Each absorption spectrum was normalized to the maximum absorbance in the visible light region. (D) Absorption spectrum of the wild type (WT; magenta), the T94A mutant (orange), the Y178F mutant (blue), and the T94A/Y178F mutant (black) of the zebra shark RHO. (E) Time course thermal decay for the whale shark RHO. The colors correspond to those for the constructs in A. The vertical axis shows relative amounts of residual RHO quantified with the average absorbance of individual RHO samples at the five points (λmax-2, λmax-1, λmax, λmax+1, and λmax+2 nm), assuming that the absorbance immediately after heating was 1 and the absorbance after light irradiation was 0. (F) Time course thermal decay for the zebra shark RHO. The colors correspond to those for the constructs in D, and the vertical axis is shown as in E.

Our spectroscopic analysis of the whale shark RHO, performed in vitro, uncovered a remarkable shift of the wavelength of the maximum absorbance (λmax) to 478 nm from the presumable ancestral condition of 500 nm observed in its close relatives, zebra shark and brown-banded bamboo shark (6). As the light of approximately 480 nm is the least attenuated in the deep sea, this finding suggests the reliance of the whale shark on vision in the deep sea via the tuned RHO, but the mechanism that allows this so-called blue shift has remained unknown. We scrutinized the sequence alignment of RHO orthologs of these sharks and their relatives. This analysis revealed two amino acid residues 94 and 178 substituted exclusively in the whale shark (Ala94 and Phe178) and no substitution at previously characterized spectral tuning sites (7, 8) (Fig. 1B). We examined the effect of each of these substitutions with site-directed mutagenesis, which resulted in the shifts of the λmax of whale shark RHO A94T and F178Y mutants toward longer wavelengths by 19 and 3 nm, respectively, compared with that of the wild type (Fig. 1C). Conversely, mutants with substitutions at these sites (T94A and Y178F) of zebra shark recapitulated the blue shift by 19 and 3 nm (Fig. 1D). This result shows a dominant effect of Ala94 accounting for the blue shift of whale shark RHO. At site 94 of RHO, no case of natural substitution has been reported as responsible for spectral tuning, but interestingly, the T94I mutation is known to cause a human disease congenital stationary night blindness (CSNB) (ref. 9; reviewed in ref. 10). The molecular basis of CSNB by the T94I mutation is explained by the substitution from a hydrophilic to hydrophobic residue, which leads to lower thermal stability via thermal isomerization of the retinal and hydrolysis of the Schiff base (SB) (11).

This observation in humans prompted us to investigate the thermal stability of whale shark RHO. Similar to the above-mentioned case of T94I, the A94I mutant of whale shark RHO rapidly decayed at 37 °C, indicating that site 94 also affects its thermal stability (Fig. 1E and SI Appendix and Dataset S2). In fact, the wild-type whale shark RHO (Ala94) also showed faster decay with a half-life (t_1/2) of 99.0 min than its A94T mutant (t_1/2 = 173.3 min) (Fig. 1E), indicating a decrease in the thermal stability by the T94A substitution. The thermal stability of whale shark RHO A94T mutant is higher than that of the wild type but still lower than that of zebra shark RHO (Thr94, t_1/2 = 346.6 min). This result is consistent with the observation that the zebra shark RHO T94A mutant has higher thermal stability (t_1/2 = 231.0 min) than the wild-type whale shark RHO (Ala94, t_1/2 = 99.0 min) (Fig. 1 E and F). The mutant F178Y at site 178, the other site substituted exclusively in the whale shark RHO, exhibited comparable thermal stability (t_1/2= 231.0 min) to the A94T mutant, and introducing both A94T and F178Y substitutions into the whale shark RHO recovered high thermal stability (t_1/2 = 346.6 min) close to that of the wild-type zebra shark (Fig. 1E). A parallel effect was observed in the zebra shark RHO; mutations at both sites (T94A/Y178F) resulted in a severe decrease in the thermal stability than either mutation (Fig. 1F). Altogether, substitutions at the two sites and their cumulative effect account for the lower thermal stability of whale shark RHO (SI Appendix and Dataset S2).

Our study revealed a commonality of the substitution at site 94 in the blue-shifted spectrum and lowered thermal stability between the whale shark RHO and the human RHO with a CSNB-causing mutation (T94I). Similar to the case of T94I mutation of mammalian rhodopsin (11), Ala94 in the whale shark RHO could impair the hydrogen-bonding network surrounding the protonated Schiff base (SB) found in mammalian rhodopsins having Thr94 to interfere the counterion Glu113-SB activation switch, one of the hallmarks of rhodopsin activation, leading to destabilization of the dark state. In general, high thermal stability is a critical feature of RHO to achieve a high signal-to-noise ratio by reducing dark noise, inherently enabling dim-light vision (12). This nature of RHO constraining dark noise usually does not permit site 94 or 178 to contribute to its spectral tuning. The substitutions do not co-occur in any visual opsins orthologous to RHO (SI Appendix and Datasets S3 and S4).

In our experiment at 37 °C, the thermal stability of whale shark RHO is higher than that of the T94I mutant (Fig. 1E and SI Appendix and Dataset S2) but is significantly lower than that of zebra shark (Fig. 1 C and D). In the deep sea, however, the functionality of whale shark RHO to reduce dark noise would be maintained at low temperatures, as shown for RHO mutants with persistent functionality at low temperatures (13). This speculation is compatible with the blue shift of the whale shark RHO that is thought to facilitate dim-light sensing in the deep sea, which is supported by our observation that RHOs having the natural substitution at site 94 are also found in some Antarctic fishes including black rockcod Notothenia coriiceps (Fig. 1B and SI Appendix and Datasets S1–S4), suggesting that the blue shift by the substitution at the CSNB site associated with a reduction in the thermal stability might be allowed in cold-water deep-sea habitats.

Interestingly, an in silico study of the whale shark RHO has failed to predict candidate residues that account for the blue shift (ref. 14; also see ref. 15), and our mutagenesis with no animal sacrifice uncovered the pivotal contribution of Ala94 to the blue shift. This solution, which has never been reported for other sharks including deep-sea species (7), could be associated with the unique lifestyle of this elusive, endangered animal including wide-ranging vertical migration. Our findings also imply possible advantages of thermally unstable RHO, like cone opsins, in visual ecology of this animal, considering that they partially inhabit the bright, warmer environment near the sea surface.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Data, Materials, and Software Availability

The nucleotide sequence of the zebra shark RHO is deposited in NCBI GenBank under the accession ID MT625929. All study data are included in the article and/or supporting information.