(Received for publication, February 13, 1996)
From the
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
), which causes the yellow color of this lens. The
-crystallin
3-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.
The transparent vertebrate eye lens essentially consists of
elongated fiber cells, which contain high concentrations of soluble
proteins called crystallins(1, 2, 3) . The
- and
/
-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.
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).
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.
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), 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
) (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 chromophore-crystallin complex (Fig. 3B, curve 3). It is indeed nearly
identical with the absorption spectrum of the
3-dehydroretinol
CRBP 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
retinol
CRBP 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) .
Our results demonstrate that -crystallin is most closely
related to CRBP I and that its ligand is 3-dehydroretinol (vitamin
A
). 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
-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
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-based visual pigments in the lizards Anolis carolinensis and Podarcis sicula(24) .
All other terrestrial vertebrates examined up to now employ vitamin
A
for visual pigment generation. This is also true for L. picturatus. (
)
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
-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.