©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Vitamin A Bound to Cellular Retinol-binding Protein as Ultraviolet Filter in the Eye Lens of the Gecko Lygodactylus picturatus(*)

(Received for publication, February 13, 1996)

Beate Röll (1)(§) Reinout Amons (2) Wilfried W. de Jong (3) (4)

From the  (1)Lehrstuhl für Tierphysiologie, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany, (2)Department of Medical Biochemistry, Leiden University, P. O. Box 9503, NL-2300 RA Leiden, The Netherlands, (3)Department of Biochemistry, University of Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands, and (4)Institute of Systematics and Population Biology, University of Amsterdam, P. O. Box 94766, NL-1090 GT Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yellow eye lenses of the diurnal gecko Lygodactylus picturatus contain, in addition to the usual crystallins, a monomeric protein with a molecular mass of 16 kDa. It comprises 6-8% of the total water-soluble lens proteins. We here identify it as a novel type of crystallin, most closely related with cellular retinol-binding protein I (CRBP I). Because of its tiny size, we designate it as -crystallin. The typical endogenous ligand of CRBP is all-trans-retinol. In the gecko lens, however, the ligand of -crystallin turns out to be 3-dehydroretinol (vitamin A(2)), which causes the yellow color of this lens. The -crystallinbullet3-dehydroretinol complex absorbs shortwave radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against ultraviolet damage. Whereas other crystallins have been recruited from stress proteins and metabolic enzymes, -crystallin represents a completely new class of taxon-specific lens proteins. Also, its ligand 3-dehydroretinol represents a novel type of lens pigment.


INTRODUCTION

The transparent vertebrate eye lens essentially consists of elongated fiber cells, which contain high concentrations of soluble proteins called crystallins(1, 2, 3) . The alpha- and beta/-crystallins, ubiquitous in vertebrate lenses, are (more or less distantly) related to stress response proteins. In addition there are taxon-specific crystallins, of which 10 different types have been described up to now in various vertebrate groups(3, 4, 5) . All known taxon-specific crystallins are related with or even identical to enzymes, mostly pyridine nucleotide binding oxidoreductases. A major question is why and how these additional crystallins have arisen during evolution of specific vertebrate lineages(3, 6, 7, 8) . Are these the result of neutral evolutionary events, tolerated as long as they are compatible with normal lens functioning? Or has the recruitment of such taxon-specific crystallins been favored because they confer some selective advantage upon the species involved? This problem can best be approached by comparing taxa that are phylogenetically related yet exposed to different selective pressures with regard to visual requirements. The lizard family of geckos is promising in this respect, comprising both nocturnal and diurnal genera. Nocturnal geckos are supposed to be descended from a diurnal lizard ancestor, but different genera have reverted from nocturnal to diurnal habits again(9, 10) . These decisive changes in living habits resulted in morphological modifications of the visual cells (9, 11) and alterations in absorption maxima of the visual pigments (12) and coincided with changes in crystallin composition of the lenses(13) .

In lenses of diurnal and diurno-nocturnal geckos, two new taxon-specific crystallins have recently been identified. A 38-kDa crystallin in the genus Phelsuma(12, 13) was named -crystallin and is identical to glyceraldehyde-3-phosphate dehydrogenase(4) . The 37-kDa B-crystallin of Lepidodactylus lugubris was found to be most similar to aldose reductase(5) . In the lenses of the strictly diurnal Lygodactylus picturatus another major crystallin has been observed(13) . We here report the identification of this protein and its ligand, which provides a convincing example of positive Darwinian evolution. Being the smallest crystallin yet observed, we designate it as -crystallin.


MATERIALS AND METHODS

Preparation of Lens Extracts

Freshly isolated lenses of L. picturatus were homogenized in various buffers, depending on the different experiments. Insoluble fractions were removed by centrifugation at 4 °C for 15 min at 15,000 times g. Protein concentrations of the different lens extracts were determined using bovine serum albumin as standard(12) .

Gel Electrophoresis

Samples for SDS-electrophoresis gels were prepared in 50 mM Tris-HCl buffer, pH 8.8. After determination of the protein concentration, the samples were denatured in a solution containing 5% SDS, 2% mercaptoethanol, and 10% glycerol with bromphenol blue and boiled for 3-5 min. Aliquots were run on 14% polyacrylamide gels containing 0.1% SDS. Protein bands were stained with Coomassie Brilliant Blue R-250 and scanned densitometrically.

Purification and Sequence Analysis of -Crystallin

Lens proteins, dissolved in 100 mM Tris buffer, pH 7.0, were separated by fast protein liquid chromatography using a column of Sephacryl 100. The proteins were eluted with the same buffer, at a flow rate of 60 ml/h, and monitored by absorbance at 280 nm. Peak fractions were pooled, concentrated, and desalted over Centricon 10 filters. Aliquots were analyzed on SDS-PAGE. (^1)The fraction enriched in -crystallin was lyophilized, dissolved in 50% (v/v) acetic acid, and used for sequence analysis on a Hewlett Packard G1000A protein sequencer linked to a 1090 phenylthiohydantoin-derivative analyzer, using the routine 3.0 program.

Extraction of Retinoids and Spectrophotometry

Retinoids of the lenses were extracted with a mixture of dichloromethane and ethanol (90%/10%, v/v) under dim red light. After centrifugation at 14,000 times g the supernatants were investigated, using a Uvikon 810 recording spectrophotometer. Absorption spectra of commercial retinoids (different isomers of retinol, retinal, retinoic acid, and retinyl palmitate; Sigma) were used to characterize the lenticular chromophore. For irradiation of the samples, a halogene projection lamp (Osram, 100 watts) was used. Monochromatic lights were produced with interference filters combined with heat protection filters.


RESULTS

Crystallin Composition of Lygodactylus Lenses

The lens (wet weight about 1 mg) contains about 20% water-soluble proteins. The elution profile of the lens extract shows four peaks (Fig. 1A). The first, very low peak contains alpha-crystallin, apparently composed of only a single type of 22-kDa subunit (Fig. 1B). The major protein of the second peak is -crystallin/argininosuccinate lyase, which has subunits of about 54 kDa. There is in addition some alpha- and beta-crystallin from the neighboring fractions. The third peak contains the monomeric 53-kDa -crystallin/alpha-enolase and the oligomeric beta-crystallin with different subunits between 27 and 35 kDa. The identities of - and -crystallin were confirmed by immunoblotting (data not shown). The fourth peak reveals the monomeric S-crystallin of 23 kDa and the new -crystallin. This is a monomer with a molecular mass of about 16 kDa, comprising 6-8% of the water-soluble lens proteins (Fig. 1C).


Figure 1: Crystallin composition of Lygodactylus lenses. A, elution profile of water-soluble lens proteins (Sephacryl 100 gel permeation). B, analysis of peak fractions by SDS-PAGE. Numbering of peak fractions as in A. Lane M, marker proteins (molecular masses in kDa); lane 1, 2 µg of protein; lanes 2-4, 10 µg of protein. The arrow indicates the -crystallin. C, protein composition of a crude lens extract (SDS-PAGE, 10 µg of protein).



Sequence Analysis of -Crystallin

A sample of 10 µg of protein from peak 4, containing about 30% -crystallin, was subjected to automated Edman degradation. The presence of a major amount of S-crystallin, having a blocked N terminus(2, 14) , should not interfere with the sequence determination of -crystallin if this had a free N terminus. A unique sequence, with an initial yield of 150 pmol, could indeed be assigned for up to 83 steps. A SwissProt data base search revealed the highest similarity with mammalian cellular retinol-binding protein I (CRBP I, Fig. 2). The established sequence of 83 residues corresponds to 63% of the length of CRBP I. Over this sequence, the identity between the two proteins is 56%. The next closest sequence is that of mammalian CRBP II, with 28% identity. These are monomeric 16-kDa proteins, belonging to a family of cytoplasmic fatty acid and retinoid transport proteins(15, 16) .


Figure 2: Sequence alignment of -crystallin with cellular retinol-binding proteins I and II from rat(31) . The total length of the CRBPs is 134 residues. Residues in lowercase are tentatively identified; x is unidentified; dash indicates a gap to maximize identity; dots indicate residues that are identical to the top sequence.



Identification of the Lenticular Chromophore

After homogenization of the yellow lenses and subsequent centrifugation, all of the pigment is present in the soluble fraction. The chromophore elutes with fraction 4 from the Sephacryl 100 column (Fig. 1A) and is retained after Amicon 10 ultrafiltration. Thus, the chromophore is apparently associated either with S- or -crystallin. The UV spectrum of the concentrated fraction 4 (Fig. 3B, curve 3) is identical with that of crude lens extract. The spectrum has two major absorption maxima, due to the protein ((max) = 280 nm) and the protein-bound chromophore ((max) = 377 nm). The peak at 377 nm has a definite secondary peak at 398 nm and a shoulder at about 362 nm. The absorption maximum of the free chromophore, extracted in dichloromethane/ethanol, is shifted to 354 nm (Fig. 3B, curve 4). Additionally, there are two minor peaks at 287 and 277 nm.


Figure 3: A, ultraviolet absorption spectra of CRBP I and 3-dehydroretinol (after (17) ). 1, CRBP from rat liver complexed with 3-dehydroretinol; 2, 3-dehydroretinol in ethanol. B, ultraviolet absorption spectra of -crystallin and isolated lenticular chromophore from Lygodactylus. 3, aqueous solution of concentrated fractions of peak 4 (Fig. 1A); the high protein peak at 280 nm is due to the presence of large amounts of S-crystallin (Fig. 1B, lane 4). 4, isolated lenticular chromophore in dichloromethane/ethanol. All curves in A and B are normalized to the highest non-protein peaks.



To identify the chromophore, its spectral properties were compared with those of various retinoids. Retinol (vitamin A(1)), the typical endogenous ligand of CRBP I and II, has an absorption maximum at 330 nm in dichloromethane/ethanol, and its bandwidth at 50% absorbance is considerably smaller than that of the extracted lens chromophore. Retinol can thus be excluded. Both CRBP I and II can also bind 3-dehydroretinol (3,4-didehydroretinol, vitamin A(2)) (17) . The absorption spectrum of pure 3-dehydroretinol shows a major peak at 352 nm and two very characteristic minor peaks at 288 and 278 nm (Fig. 3A, curve 2)(17) . This spectrum is virtually identical to that of the lens chromophore (Fig. 3B, curve 4). All other retinoids tested are unlikely candidates because their spectral characteristics do not match those of the lens chromophore.

Only the binding of retinol and 3-dehydroretinol to either type of CRBP induces a marked fine structure into their UV spectra (e.g.(17) and (18) ). The non-protein peak then shows a secondary peak on the bathochromic (red) side and a shoulder on the hypsochromic side. This fine structure is clearly seen in the chromophorebullet-crystallin complex (Fig. 3B, curve 3). It is indeed nearly identical with the absorption spectrum of the 3-dehydroretinolbulletCRBP I complex, which is characterized by a major peak at 380 nm, a prominent shoulder at about 360 nm, and a definite secondary peak at about 400 nm (Fig. 3A, curve 1)(17) . The spectral characteristics of the retinolbulletCRBP I complex are quite different (major peak at 350 nm, shoulder at 335 nm, secondary peak at 367 nm). Not being a retinoid binding protein, S-crystallin is extremely unlikely to mimic these spectral characteristics.

It thus can be concluded that the chromophore of the Lygodactylus lens is 3-dehydroretinol, bound to -crystallin. This is further supported by the fact that holo--crystallin and its isolated chromophore are non-fluorescent upon excitation with (ultra)violet light (336, 382, and 413 nm) at room temperature. This is also the case for 3-dehydroretinol (17) but not for retinol(19) . It is estimated that one molecule of 3-dehydroretinol is bound to one molecule of -crystallin. The chromophore associated with -crystallin and the isolated chromophore are completely photostable under prolonged successive irradiation with monochromatic light of 413 and 382 nm (10 + 10 min). Clearly, no release of the chromophore from the crystallin occurs upon irradiation. In contrast, the CRBP-retinol association seems to be photosensitive(18) .


DISCUSSION

Our results demonstrate that -crystallin is most closely related to CRBP I and that its ligand is 3-dehydroretinol (vitamin A(2)). CRBP I and the next closest relative CRBP II are members of the family of intracellular lipid-binding proteins (iLBP). CRBP I is present in all rat tissues examined, whereas CRBP II has mainly been found in the small intestine(20) . In the eye, CRBP I is notably present in the retinal pigment epithelium and in Müller cells of neural retina, where it participates in the visual cycle(21) . CRBP I has not been reported in the lens. Interestingly, though, another member of the iLBP family, most closely resembling epidermal fatty acid binding protein, has been found at low level in the bovine lens and is supposed to be involved in differentiation of lens cells (22) . The CRBPs and other retinoid-binding proteins are suggested to function in the stabilization of retinoids in aqueous media, in cytoplasmic transport, and as substrate carriers for enzymatic reactions(21, 23) . The iLBPs are extremely stable proteins(15) . They are characterized by a beta-barrel structure made up of 10 antiparallel strands, with the ligand localized inside the barrel(15, 23) .

Comparison of the sequence of -crystallin, as far as determined, with those of the other iLBPs reveals that residues responsible for correct folding and for ligand binding are generally well conserved (15, 23) . A notable exception is the deletion of two residues and the replacement of three others at positions 64-69 in a very conserved turn between two beta sheets (Fig. 2). Remarkable, too, is that sequence comparison indicates an accelerated rate of change in the -crystallin lineage, after the divergence from its CRBP ancestors. This suggests that structural adaptations may have occurred to make the protein even more suitable for accommodating the unusual ligand 3-dehydroretinol and for functioning in the lens.

3-Dehydroretinol is only rarely found in terrestrial vertebrates, although its aldehyde is a fairly common chromophore of visual pigments in aquatic lower vertebrates. A remarkable exception is the occurrence of vitamin A(2)-based visual pigments in the lizards Anolis carolinensis and Podarcis sicula(24) . All other terrestrial vertebrates examined up to now employ vitamin A(1) for visual pigment generation. This is also true for L. picturatus. (^2)

3-Dehydroretinol as a lens pigment has not been reported earlier. Yellow lenses are common in diurnal terrestrial vertebrates and in fishes. Three other types of lenticular pigments are known. Lenses of man, squirrel, and some fishes contain kynurenin derivatives(25, 26) , while mycosporine-like amino acids occur in the lenses of many marine fishes(26) . These two pigments are not protein-bound, in contrast to the lipid-soluble carotinoids, which are bound to alpha-crystallin in the hatchetfish Argyropelecus affinis(27) . Prerequisites for pigments to be recruited as lenticular chromophores are photostability and low fluorescence in the range of visible light. As is obvious from our results, these conditions are met by -crystallin and 3-dehydroretinol but not if retinol would have been the chromophore (18, 19) .

Lens pigments absorb shortwave radiation and serve as a filter to promote visual quality by decreasing chromatic aberration and glare (10) . They are also supposed to protect the retina against damage resulting from the high energy content of UV and shortwave blue radiation(28) . Elimination of these wavelengths is especially important in animals that live at high ambient light intensities. L. picturatus is a strictly diurnal gecko, which lives predominantly in dry savannah areas in East Africa(29) , where light intensities may reach 100,000 lux(30) . In most geckos the eyelids are fused to a transparent spectacle, as in snakes. As its pupil diameter is virtually constant, Lygodactylus (like several other diurnal geckos) cannot regulate the incident light by closing eyelids or diminishing the aperture of the iris. A good alternative is to reduce potentially harmful wavelengths by absorption in the lens.

In the lens of Lygodactylus this is achieved by a unique and novel type of lens pigment, 3-dehydroretinol, which is poorly soluble and unstable in aqueous media. Therefore, to reach sufficiently high levels to be optically effective, the expression of a binding protein, recruited from the iLBP family, had to be simultaneously up-regulated. This CRBP-like protein, -crystallin, is very stable and thus suitable for functioning as a lens structural protein. It is the first example of a non-enzyme protein identified as a taxon-specific crystallin.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49 234 700-4330; Fax: 49 234 7094-189.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; CRBP, cellular retinol-binding protein; iLBP, intracellular lipid-binding protein.

(^2)
B. Röll, unpublished observations.


ACKNOWLEDGEMENTS

We thank Prof. Dr. K. Hamdorf, Bochum, for the gift of several retinoids and helpful discussions.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.