Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
Correspondence: E-mail: kawamura{at}k.u-tokyo.ac.jp.
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Abstract |
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Key Words: zebrafish RH2 opsins visual pigments gene duplication spectral differentiation ancestral sequence
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Introduction |
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Vertebrate visual opsins can be classified into five phylogenetic groups: RH1 (rod opsin or rhodopsin), RH2 (RH1-like, or green, cone opsin), SWS1 (short wavelengthsensitive type 1, or UV-blue, cone opsin), SWS2 (short wavelengthsensitive type 2, or blue, cone opsin), and M/LWS (middle to long wavelengthsensitive, or red-green, cone opsin) (Yokoyama 2000). RH1 has been a major subject of study for biophysical and biochemical properties of visual pigments. Approximately 20 amino acid (aa) residues responsible for RH1 spectral tuning have been identified (Takahashi and Ebrey 2003). M/LWS and SWS1 pigments have also been well investigated for spectral tuning mechanisms by site-directed mutagenesis.
The max values of the M/LWS pigments are mostly determined by 5 aa residues at positions 164, 181, 261, 269, and 292 (Yokoyama and Radlwimmer 1998, 1999, 2001). In typical "red" opsins, these sites are occupied by serine, histidine, tyrosine, threonine, and alanine, respectively. The aa replacements from serine to alanine at 164 (denoted S164A), H181Y, Y261F, T269A, and A292S shift the
max values toward the shorter wavelength by 7, 28, 8, 15, and 27 nm, respectively (Yokoyama and Radlwimmer 2001). Effects of these aa replacements on the
max shift can be detected separately and are nearly additive.
As for SWS1, spectral differentiation between the UV (max
360 nm) and violet (
max
410 nm) pigments is achieved by combinations of 10 aa replacements, but individual spectral effects appear to be negligible and are largely dependent on background aa sequences, with combined effects being nonadditive and synergistic (Wilkie et al. 2000; Yokoyama, Radlwimmer, and Blow 2000; Shi, Radlwimmer, and Yokoyama 2001; Fasick, Applebury, and Oprian 2002; Shi and Yokoyama 2003; Parry et al. 2004). For example, when 7 of the 10 aa residues of the mouse UV pigment (
max 358 nm) are individually replaced to the corresponding aa residues of the orthologous human blue pigment (
max 414 nm) (F46T, F49L, T52F, F86L, T93P, A114G, and S118T), they do not cause any
max shift, but when all mutations are introduced simultaneously, the
max shifts by 50 nm (Shi, Radlwimmer, and Yokoyama 2001).
Compared to RH1, M/LWS, and SWS1 pigments, much less is known about the spectral tuning mechanisms of RH2 pigments. Two aa replacements, E122Q and M207L, result in 13- and 6-nm blueshift, respectively, to the coelacanth RH2 pigment, and the two effects are nearly additive (Yokoyama et al. 1999). This is the only report where the effect of aa replacements on the absorption spectra was experimentally tested in RH2 pigments. We previously reported that zebrafish (Danio rerio) have four tandemly duplicated RH2 opsin genes (RH2-1, RH2-2, RH2-3, and RH2-4) (Chinen et al. 2003). The reconstituted photopigments for the four genes with 11-cis retinal showed a wide variety of absorption spectra, with the max being 467, 476, 488, and 505 nm, respectively (fig. 1). Gene expression patterns differ among the four zebrafish RH2 opsin genes in quantity, temporal order, and locality in the retina. In adult retina, expression of RH2-2 is most abundant among the four RH2 opsin genes (Chinen et al. 2003). RH2-1 and RH2-2 are expressed in the central to dorsal area of the adult retina, whereas RH2-3 and RH2-4 are expressed in the peripheral area, especially in the ventral side, and in the larval retina RH2-1 is the first to be expressed (M. Takechi and S. Kawamura, unpublished data). In the present study, we aimed to clarify the evolutionary process of the
max differentiation among the four zebrafish RH2 pigments and to identify the relevant aa substitutions to the spectral shifts by inferring ancestral aa sequences of the zebrafish RH2 opsins with likelihood-based Bayesian statistics and by applying site-directed mutagenesis to the reconstituted photopigments.
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Materials and Methods |
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Given the tree topology in figure 2 (determined in Chinen et al. 2003), the ancestral opsin sequences at each phylogenetic node were inferred by using PAML computer program with a likelihood-based Bayesian method (http://abacus.gene.ucl.ac.uk/software/paml.html) (Yang, Kumar, and Nei 1995; Yang 1997). In the computation, the empirical substitution matrix of Jones, Taylor, and Thornton (1992) (JTT model) and that of Dayhoff, Schwartz, and Orcutt (1978) (Dayhoff model) were used as mathematical models of a substitution. The certainty of each of the inferred aa was expressed as a posterior probability.
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Results |
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We then introduced point mutations into Ancestor 2 or A2_Q122E to further locate the residues responsible for spectral differentiation. Among the 32 aa that are different, 23 are located in the putative TM regions and 1 is in the second extracellular (E2) loop (fig. 3). These are the domains which contain aa positions that together constitute the retinal-binding pocket (Palczewski et al. 2000). All aa replacements involved in spectral tuning have been identified in the TM and E2 regions (Yokoyama 2002; Takahashi and Ebrey 2003), and these regions were subjected to mutagenesis (fig. 3).
In region 199, there are 9 aa differences in the TM domains between Ancestors 1 and 2 (see fig. 3). All of these mutations were introduced into Ancestor 2 singly or doubly when the two sites were in close proximity (table 3). Maximum max shift by these mutations was only +3 nm (by F88C). Double mutations C97S/S98A resulted in no recognizable absorbance peak. When these mutations were introduced to the A2_F88C, the
max shift from A2_F88C was negligible (1 nm). Simple addition of these single- or double-replacement effects is only +1 nm (or 0 nm if effect of C97S/S98A is counted as 1 nm) (table 3). This is at odds with the results of the segment swapping of region 199, which showed a +6 nm
max shift. Amino acid difference (P/T at residue 27; fig. 3) was not considered for the mutagenesis, is located in the N-terminal tail, and is unlikely to have any spectral effect. These results suggest that individual spectral effects of aa replacements in this region are not additive but rather synergistically cooperate together.
In the region 100234, there are 9 aa differences in the TM domains other than the E/Q difference at residue 122 and one difference in the E2 region between Ancestors 1 and 2 (see fig. 3). All of these mutations were introduced into A2_Q122E. Maximum max shift from A2_Q122E was +4 nm (by N151S), followed by +3 nm (by S209I) and +2 nm (by T218I) (table 3). A mutation in the E2 region, T185C, resulted in an opposite spectral shift, 4 nm, though the pigment's absorption peak was low and not clearly discernable (table 3). Simple addition of these effects and other minor ±1-nm shifts count +5 nm, which is close to the +6-nm difference between A2_Q122E and A2(99)A1(234)A2. However, when the mutations causing +2- to +4-nm shifts were introduced together (A2_Q122E/N151S/S209I/T218I), combined effect for the
max shift from A2_Q122E was only 2 nm (table 3). These results suggest that individual spectral effects in this region are neither additive nor synergistic but are partly regressive. The net effect of them could vary depending on the physicochemical background of the residues making up the region.
In the region 235349, there are 5 aa differences in the TM domains between Ancestors 1 and 2 (see fig. 3), all of which were tested. Individual mutation effects were all minor, +1 or +2 nm (table 3). Simple addition of the effects adds up to +9 nm, which is greater than the +4-nm difference between the max values of Ancestor 2 and A2(234)A1. When mutations with +2-nm effect were introduced together (V266T/F271V/A287F/A297S), the spectral shift was only +3 nm and the combined effect appeared to be regressive. This total effect is, however, close to the segment-swapping effect of 4 nm, and the regressive spectral effects can explain the spectral shift by this segment from these sites.
Mutations in Branch B
To explore aa replacements that explain the 7-nm max difference between RH2-1 and Ancestor 2, we first constructed two chimeric pigments, where aa 1144 and 145349 of Ancestor 2 were replaced with those of RH2-1 using HindIII restriction site (fig. 3) (the resulting chimeric pigments designated RH2-1(144)A2 and A2(144)RH2-1, respectively). Whereas RH2-1(144)A2 pigment was successfully reconstituted, functional A2(144)RH2-1 pigment with a recognizable absorption peak was not formed. However, the
max value of RH2-1(144)A2 was identical to that of RH2-1 (467 nm) (table 4), suggesting that aa replacements responsible for the
max difference between RH2-1 and Ancestor 2 pigments lie in the 1144 region. We therefore introduced point mutations to the TM domains in the 1144 region.
There are 11 aa differences in the TM domains in the region 1144 between Ancestor 2 and RH2-1 (see fig. 3). All of these mutations were introduced into RH2-1, except at residue 88 for which Ancestor 2 was used as a template (A2_F88I) (table 2). Unexpectedly, many mutations resulted in opposite spectral shifts (K36Q, L46F, I49C, V60L, L108T, and M112I causing a short-wave shift and F88I to Ancestor 2 a long-wave shift) (table 4). S94T had no spectral effect, and V111A showed only a 1-nm long-wave shift. A double mutation L99I/V100N resulted in no recognizable peak. Simple addition of the spectral effects by these single point mutations counts as a 16-nm shift from RH2-1 (with I88F assumed to cause 2-nm shift from RH2-1), contrasting with the +7-nm shift caused by the segment swapping of the entire 1144 region. When some mutations were introduced together (RH2-1_V111A/M112I, RH2-1_S94T/V111A/M112I and RH2-1_L46F/I49C/S94T/V111A/M112I; see table 4), the amount of spectral shift markedly differed from their sum, e.g., +4-nm shift in RH2-1_L46F/I49C/S94T/V111A/M112I, while the sum of the individual effect was 7-nm. It has been reported that residues 46 and 49 are part of the spectral tuning sites of SWS1 opsins which show individually unrecognizable shifts, but large spectral effects occur when combined together (Shi, Radlwimmer, and Yokoyama 2001). These results indicate that the spectral effect of these aa replacements can vary depending on their physicochemical environment as shown for mutations in branch A.
Mutations in Branch C
In the evolutionary branch C between Ancestor 3 and RH2-3, there are 18 aa replacements in the TM and E2 domains (see fig. 3). However, a majority of the spectral difference (14/18) was explained by a single aa replacement E122Q (fig. 5, right panel). E122Q was estimated to have occurred independently in branches A and C (fig. 3). Such a parallel substitution was also inferred at residues 50 (L50F in branches A and C), 60 (L60V in B and C), 151 (S151N in A and C), 162 (V162F in B and V162I in C), 165 (M165S in A, S165C in B, and M165L in C), 209 (I209S in A and I209C in C), 214 (V214I in B and L214F in C), 218 (I218T in A and C), 270 (T270S in A and C), and 300 (L300V in B and L300I in C) (fig. 3). Among these, 2 nm or more of spectral shift was experimentally verified with an expected direction in branch A mutations N151S, S209I, and T218I introduced to Ancestor 2 (table 3). We then tested the spectral effect of a combined mutation Q122E/N151S/C209I/T218I introduced to RH2-3. The observed spectral shift from RH2-3 was +16 nm (table 4) and was close to the actual spectral difference of Ancestor 3 from RH2-3 (+18 nm), although contributions from the other 14 aa substitutions cannot be ruled out.
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Discussion |
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Figure 6 summarizes the evolutionary changes that occurred in the phylogenetic clade derived from Ancestor 1. In branch A, 25 aa changes occurred in the TM + E2 regions, and we tested all of them for their spectral effects (table 3). The E122Q accounted for about half (15 nm) of the spectral difference (32 nm) between A1 and A2 pigments. The 32-nm total difference was nearly perfectly explained by linear addition of the spectral effects of three segmental replacements (table 3). However, within the segments, such additivity no longer held for individual aa mutations (table 3). There are 9, 11, and 5 mutations in TM + E2 regions in segments 199, 100234, and 235349, respectively (fig. 3). These effects were individually small and not additive and may have been even partly regressive. To clarify the aa replacements, other than E122Q, that explain the spectral shift, it is necessary to examine all the combinations of aa mutations in each segment. The same holds true for mutations in branch B. We verified that segment 1144 is responsible for the spectral difference of 7 nm between A2 and RH2-1 (table 4). There are 11 mutations in the TM regions in the segment (fig. 3). We tested all of their spectral effects and showed that their individual effects were small and many of them were oriented to the opposite direction (table 4). It is necessary to explore the combinations of the mutations that account for the spectral difference between A2 and RH2-1.
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Figure 6 also indicates that spectral differentiation of zebrafish RH2 opsins occurred independently of that of goldfish RH2 opsins. The ancestral opsin of the two goldfish opsins (GFgr-1 and GFgr-2; Johnson et al. 1993) was designated GF and that of GF and Ancestor 3 was designated ZG. The ZG was inferred to have an identical aa sequence with Ancestor 3 and must have a max of 506 nm as well. The
max of GF would also be 506 nm because those of ZG and GFgr-2 are both 506 nm and because the 6 aa changes in the TM + E2 regions in the ZG-GF branch are all conservative and outside the retinal-surrounding sites (sequences not shown).
The recent X-ray diffraction analysis of bovine rod opsin revealed its three-dimensional crystal structure at 2.8-Å resolution (Palczewski et al. 2000). Residue 122 is one of the 27 aa forming the retinal-binding pocket which are located within 4.5 Å from 11-cis retinal (residues 43, 44, 47, 94, 113, 117, 118, 120, 121, 122, 178, 181, 186, 187, 188, 189, 191, 207, 211, 212, 261, 265, 268, 269, 293, 295, and 296) (Palczewski et al. 2000). In addition, a total of 38 aa sites, including the 27 sites, were identified as surrounding 11-cis retinal (Menon, Han, and Sakmar 2001; Takahashi and Ebrey 2003) (the sites indicated in red in fig. 3). Residue 122 interacts directly with ß-ionone ring of 11-cis retinal, and its replacement has been verified to result in significant spectral shifts in various visual pigments (Takahashi and Ebrey 2003). In general, there are two factors controlling the opsin shift: (1) electrostatic interaction between the glutamic acid counterion at residue 113 and the protonated Schiff base of retinal at lysine residue 296 and (2) electrostatic interaction between the retinal body and charged or polar groups of aa surrounding it (Takahashi and Ebrey 2003). In both cases, aa replacements surrounding retinal can directly influence absorption spectra of visual pigments, and hence their spectral effects tend to be additive as manifested in the cases of M/LWS pigments (Yokoyama and Radlwimmer 2001) and coelacanth RH2 and RH1 pigments (Yokoyama et al. 1999). The 5 aa sites 164, 181, 261, 269, and 292, which are known to exert major contributions to tuning of the M/LWS opsins, the two sites 122 and 207 in the coelacanth RH2 opsin, and the two sites 122 and 292 in the coelacanth RH1 opsin are all in the retinal-surrounding sites (see fig. 3).
On the other hand, spectral effects of aa replacements outside the retinal-surrounding sites would be generally indirect, small, nonadditive, and dependent on the molecular environment of the aa sites concerned. These replacements possibly work through alteration of conformation or hydrophobicity of TM domains. The spectral tuning mechanism of the violet/uv (SWS1) pigments appears to be a typical example of such an indirect interaction mechanism, where combinations of 10 aa sites (46, 49, 52, 86, 90, 93, 97, 114, 116, and 118) are involved in the spectral differentiation and seven of them (sites other than 90, 114, and 118) are located outside the retinal-surrounding sites (Wilkie et al. 2000; Yokoyama, Radlwimmer, and Blow 2000; Shi, Radlwimmer, and Yokoyama 2001; Fasick, Applebury, and Oprian 2002; Shi and Yokoyama 2003; Parry et al. 2004). The tuning sites possibly affect trafficking of water molecules at the retinal-binding pocket by altering net hydrophobicity of TM domains with other residues and control protonation and deprotonation status of the Schiff base (Shi, Radlwimmer, and Yokoyama 2001). Whereas spectral effects of the aa replacements in the retinal-surrounding region are likely to be universal beyond differences of opsin groups or animal species because of their directness of the interaction with retinal, spectral effects of the aa replacements outside the region found in a system may be of little general implication to another system because of their indirectness of the interaction with retinal.
All the mutations, other than E122Q tested in this study, fall outside the retinal-surrounding region (fig. 3) (site 94 is in the region but see the following explanations). These mutations resulted in only minor spectral effects with 0 to 4-nm shift (tables 3 and 4). S94A is known to cause a 14-nm blueshift in newt SWS2 pigment (Takahashi and Ebrey 2003), but the substitution in our case was between the same polar residues with a hydroxyl group, S94T, and resulted in no recognizable spectral shift (table 4). Characteristically and consistently with their locations relative to retinal, combined effects of these minor sites were not additive and were, in some combinations, even opposite in direction of spectral shift from the shift direction in individual mutations. However, this nonlinear nature of cumulative effects from multiple aa sites with individual minor effects does play an important role in the spectral differentiation of the zebrafish RH2 opsins.
In zebrafish RH2 opsins, spectral diversification is achieved by direct and indirect interaction mechanisms with retinal, the former by residue 122 in the retinal-surrounding region with a large spectral effect and the latter by multiple residues outside it with individually minor and collectively nonadditive effects. This pattern of spectral differentiation could represent a general paradigm to account for the spectral diversity of vertebrate RH2 pigments, considering that a range of 470510 nm is covered by the four zebrafish pigments.
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Acknowledgements |
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Footnotes |
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