Cold-stable eye lens crystallins of the Antarctic nototheniid toothfish Dissostichus mawsoni Norman
1 Department of Animal Biology, University of Illinois at Urbana-Champaign,
Urbana, Illinois, 61801, USA
2 Centre for Biophysics and Computational Biology, University of Illinois at
Urbana-Champaign, Urbana, Illinois, 61801, USA
3 Department of Chemical and Biomolecular Engineering, University of
Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA
* Author for correspondence (e-mail: c-cheng{at}uiuc.edu)
Accepted 22 September 2004
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Summary |
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Key words: lens crystallins, chaperone, Antarctic toothfish, Dissostichus mawsoni, bigeye tuna, cold adaptation, cold cataract, dynamic light scattering, alpha crystallin, gamma crystallin
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Introduction |
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The model system for studies of the vertebrate lens has been that of the
cow. Bovine lens consists of a small insoluble albuminoid fraction, and a
large soluble crystallin fraction
(Mörner, 1864), which
comprises three groups of proteins:
, ß and
crystallins
(Bloemendal, 1986
). In mammals,
the most abundant protein is
crystallin, a large oligomeric structure
composed of two polypeptides,
A and
B, which belong to the small
Heat Shock Protein (sHSP) family with chaperone-like ability to protect
unrelated proteins from heat or chemical-induced protein denaturation
(Horwitz, 2003
;
Narberhaus, 2002
). The ß
and
crystallins are members of the ß
gene superfamily of
crystallins, which all possess a Greek-key fold protein motif, but whose
non-refractive functions remain undetermined
(Liaw et al., 1992
;
Slingsby and Clout, 1999
;
Wistow, 1993
). Depending on
the species, between four and six of the known ß and
crystallin
genes are expressed (Chiou et al.,
1986
; Norledge et al.,
1997
).
When the cow lens is cooled from the body temperature of 37°C to about
19°C, it begins to develop an opacity, a phenomenon known as
cold-cataract, which has been attributed to the cold instability of some of
the constituent crystallins, resulting in a liquidliquid phase
transition within the lens (Delaye et al.,
1982
; Gulik-Krzywicki et al.,
1984
; Norledge et al.,
1997
; Siezen et al.,
1985
). Cold-cataract has also been reported in a few other
endothermic mammals such as the rat (Zigman and Lerman,
1964
,
1965
). There are very few
studies on the temperature response of the lens crystallins of ectothermic
vertebrates, but one study indicates that for the sub-tropical striped mullet
Mugil cephalus, a cold-insoluble precipitate appears to form when the
lens homogenate is cooled from 24°C to 7°C
(Ferguson et al., 1971
). In
marked contrast to these warmer bodied species, polar fishes, particularly the
Antarctic notothenioids that live in perennially freezing temperatures of
2°C, maintain complete lens transparency, suggesting a high degree
of cold stability of their constituent lens proteins.
In this study, we report the first detailed characterisations of the whole lens and the lens crystallin proteins of the giant Antarctic nototheniid fish Dissostichus mawsoni, in terms of thermal response, protein composition, and various physical and biochemical properties. For comparison, we also examined the thermal, physical and biochemical properties of the crystallin proteins of the equally large lens of a mesophilic fish, the bigeye tuna Thunnus obesus and the endothermic cow Bos taurus, which collectively span the vertebrate body temperature range. This comparative analysis has allowed us to gain insights into the adaptive changes in protein properties that may have ensured transparency of the Antarctic notothenioid eye lens in constantly freezing seawater temperatures.
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Materials and methods |
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Cold-cataract formation
The effect of temperature on the transparency of the lens was determined
using fresh lenses. Bovine lenses, either within or removed from the eye
capsule, were wrapped in plastic and embedded in a bucket of wet ice
(0°C). Lenses of the tropical blackbar soldierfish M. jacobus
were placed in test tubes with a small amount of vitreous humour and incubated
at 15°C or in wet ice (0°C). Lenses of the Antarctic toothfish D.
mawsoni were kept at 2°C (body temperature of toothfish and
ambient temperature of Antarctic seawater) or at 12°C for a period
of 48 h in a vial of High Temperature Silicon Oil (Aldrich 17,563-3). The
appearance of each lens after these various temperature incubations was
assessed and photographed with a digital camera
(Fig. 1).
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Size-exclusion chromatographic separation of the soluble crystallin proteins
Size-exclusion chromatography (SEC) using Sephacryl 200 High Resolution
(S200HR) resin (Sigma, St Louis, MO, USA) was performed to fractionate the
soluble lens protein components of D. mawsoni, T. obesus and B.
taurus. Fresh or frozen lenses were first solubilised by stirring
overnight at 8°C in three volumes of solubilisation buffer (10 mmol
l1 potassium phosphate, 100 mmol l1 KCl,
0.05% NaN3, pH 7.6). The potassium phosphate buffer pH was adjusted
as per the method of Gomori
(1955) and the complete buffer
suction filtered through a Buchner funnel with No. 54 Whatman (hardened)
filter paper. Solubilised lens homogenate was centrifuged at 20 198
g (13 000 revs min1) in an SS-34 rotor
(Sorval RC-5B, Asheville, NC, USA) for 20 min set at 4°C to sediment the
insoluble components. Between 0.5 and 1.0 g of soluble lens proteins
(supernatant) were loaded (maximum volume 5 ml) on a S200HR column (2.5 cm
i.d. x116 cm length) equilibrated with the solubilisation buffer, and
protein elution was carried out with the same buffer at room temperature with
a flow rate of
36 ml h1. Tube fractions (5 ml) were
collected, and protein absorbance at 280 nm of each fraction was measured.
Integration of the area under protein absorption peaks in the elution
chromatogram was performed using the programme ORIGIN 6.0.4 (OriginLab,
Northampton, MA, USA).
SDS-polyacrylamide gel electrophoresis and immunoblot detection of , ß and
crystallin polypeptides
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using
BioRad Mini-PROTEAN 3 system (BioRad Laboratories, Hercules, CA, USA).
Crystallin protein (1020 µg per sample) was loaded on a
discontinuous SDS-polyacrylamide gel (4% stacking gel, 15% resolving gel) and
electrophoresed for 1 h and 15 min at a constant voltage of 200 V. Gels were
stained with 0.1% Coomassie R-250 in 10% acetic acid, 40% methanol, and
destained with 10% acetic acid, 40% methanol.
For immunoblotting, SDS-PAGE separated proteins were transferred
electrophoretically (BioRad Mini-Trans Blot Electrophoretic Cell) from
un-stained gels to nitrocellulose membrane (Hybond ECL, Amersham Biosciences,
Corp., Piscataway, NJ, USA) at 90 V for 3.5 h at 8°C. Membranes were
incubated for 1 h at room temperature with the blocking solution 1 x TBS
(20 mmol l1 Tris-HCl, pH 7.5, 150 mmol l1
NaCl) containing 5% non-fat milk powder (Carnation, Nestlé USA Inc.,
Solon, OH, USA) and 0.05% Tween-20, and then probed with polyclonal rabbit
anti-bovine crystallin primary antibodies (gift of Dr J. Sam Zigler, National
Eye Institute, NIH, USA) in antibody (Ab) buffer (1 x TBS, 5% non-fat
milk powder, 0.25% Triton-X100). The anti-bovine crystallin (, ß,
) antisera were able to react with fish crystallins in this study.
Bovine crystallins were detected colorimetrically, while the fish crystallins
required more sensitive chemiluminescence detection. For colorimetric
detection, the primary Ab stock (1 mg ml1) was diluted 1:500
for
, ß and
, and for chemiluminescence detection, 1:2000
for
, 1:2500 for ß, and 1:5000 for
crystallin,
respectively. Unbound primary Ab was removed by washing in 1 x TBS with
0.05% Tween-20. Secondary antibody, horseradish peroxidase (HRP)-conjugated
goat anti-rabbit IgG (H+L) (1.5 mg ml1, ZYMAX 81-6120,
Zymed, San Francisco, CA, USA) was diluted 1:5000 in Ab buffer for
colorimetric detection, and 1:50 000 forchemiluminescence detection. Bovine
crystallin blots were incubated in colormetric reagent (BioRad HRP Conjugate
Substrate Kit) for approximately 10 min, washed with distilled water and
air-dried. Fish crystallin blots were developed with chemiluminescence reagent
(Pierce SuperSignal WestPico Chemiluminescent Substrate, Pierce, IL, USA) per
manufacturer's instructions and autoradiographed on Kodak BioMax film for 10 s
to 5 min, as appropriate.
Determination of upper limits of thermal stability of crystallins
The upper limit of thermal stability or TS is defined
as the maximum temperature that a crystallin fraction can be subjected to
without precipitation of the protein. TS values for the
SEC purified and
crystallin fractions from toothfish, bigeye
tuna and cow were empirically determined to establish the appropriate range of
test temperatures for the chaperone-like activity assays (next section). The
protein concentrations of the crystallin fractions were calculated from their
A280 values (cow) using published
(Siezen et al., 1986
) and our
gravimetrically determined extinction coefficients (fish). To determine these
gravimetrically,
, ß and
fish crystallin solutions were
collected from the SEC separations (separately), diluted down below 1.0
A280 units, and the absorbance at 280 nm recorded. Exactly 10 ml
was lyophilised for 24 h in a pre-weighed, pre-lyophilised clean glass vial.
Vials containing the lyophilised crystallins were weighed immediately on an
analytical scale with 0.001 mg resolution. All measurements for fish
crystallin were made twice. All extinction coefficients are expressed as
A280 in 1 cm path length of a 1% solution
(Table 1). Samples of each
crystallin (800 µl at 1 mg ml1) were incubated at various
temperatures in a UV-transparent polystyrene microcuvette placed in a
spectrophotometer equipped with a water-jacketed 7-cuvette holder (Helios
Gamma, Spectronic Instruments Inc., Rochester, NY, USA). The incubation
temperature was controlled using a circulating water bath (HaakeFS) and the
actual sample temperature was monitored using a digital thermocouple
thermometer equipped with a fine wire probe (Cole-Parmer, Vernon Hills, IL,
USA) in an isothermal adjacent microcuvette containing sample buffer.
Heat-induced protein aggregation results in static light scattering that can
be followed by monitoring turbidity (increased absorbance) at 360
nm(Jaenicke and Seckler,
1997
). A360 of the sample was automatically measured at
5 min intervals by a computer interfaced with the spectrophotometer using the
program HYPERTERMINAL (Hilgraeve, Monroe, MI, USA). The highest incubation
temperature at which A360 remained at or very near zero for at
least 1 h was taken as the TS.
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Assays for chaperone-like activity of crystallin
Lens crystallins are known sHSPs and have the chaperone-like
ability to protect other proteins from stress-induced denaturation
(Horwitz et al., 1998
). The
chaperone-like function of the fish and bovine
crystallins in
protecting against heat-induced aggregation of
crystallins, and from
chemically induced (by DL-dithiothreitol) aggregation of lysozyme
was assayed. Since SEC fractionation of crystallins does not achieve perfect
separation of the individual classes of crystallins (see Results), the
crystallin fractions for these chaperone assays were chosen as follows. The
SEC
crystallin peak from SEC separation of crystallins of all three
species (see Results and Fig.
2AC, peak III) was predominantly
crystallin, based
on immunoblots (see Results and Fig.
3), and thus the five highest A280 fractions of this
peak were used in the assays. The
S fraction
(Fig. 2C, peak IIIa) was not
used in chaperone assays or dynamic light scattering (DLS) studies. For bovine
crystallin, the first 23 fractions of the ascending slope of
the SEC peak I (Fig. 2C, peak
I) are near homogeneous for
crystallin (see Results), so these were
used for the assays. For fish, none of the fractions from the SEC
crystallin peak (Fig. 2A,B,
peak I) are homogeneous for
crystallin, but contain various amounts of
ß crystallins (see Results). We found that the first 23 fractions
of the ascending slope of the elution peak have the highest and most
consistent chaperone activity, indicating the greatest concentration of
crystallins, and these were selected for all subsequent chaperone
assays. Thus, the terms
crystallin and
crystallin in the
described chaperone experiments refer to these particular SEC elution
fractions. Because of this protein heterogeneity, the stated concentrations of
crystallin used in the assays represent the upper bound values. In
other words, the observed chaperone activity is achieved by
crystallin
concentrations at or lower than the stated values. Beta crystallins are not
known to be molecular chaperones (or sHSPs) and therefore their presence in
the
crystallin fractions is not expected to contribute to the observed
chaperone activity in the assays.
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|
Assays of chaperone protection against heat aggregation were performed
similar to previously published methods
(Horwitz, 1992;
Liao et al., 2002
). Gamma
crystallin (final concentration of 1 mg ml1) of each species
was mixed with different amounts of
crystallin (final concentration of
62.5 µg ml1 to 1.0 mg ml1) of the same
species in a total assay volume of 800 µl. The incubation temperature was
47°C, 55°C and 60°C for toothfish, tuna and cow, respectively, and
5 min A360 measurements were carried as described above.
TS of
crystallin is lower than
TS of
crystallin (see Results), thus no increase
in A360 at these incubation temperatures was interpreted to be the
result of chaperone protection by
crystallin against thermal
aggregation of
crystallin. All assays were repeated three times.
The ability of crystallin to protect DTT-induced aggregation of
lysozyme at 37°C, a common approach used for testing the chaperone-like
activity of
crystallins (Abgar et
al., 2000
; Vanhoudt et al.,
2000
), was used for the three species in this study. Various
amounts (0.22.2 mg) of
crystallin fraction were added to 200
µg of chicken egg white lysozyme (EC 3.2.1.17, Sigma) in a final volume of
1 ml. Freshly made 1 mol l1 DTT (Sigma) was added to a final
concentration of 20 mmol l1 to initiate lysozyme unfolding,
and A360 was measured at 5 min intervals. Three replicates of each
condition were performed for each species.
Cross-species chaperone assays
To assess whether crystallins from cold-adapted and warm-adapted organisms
could interact, and
crystallins of toothfish, tuna and cow,
representing three disparate thermal environments (polar, 2°C;
subtropical, 18°C; endothermal, 37°C), were used in pairwise
cross-species chaperone protection assays similar to the same-species assays
described above. Pairwise combinations of
and
crystallin from
D. mawsoni, T. obesus and B. taurus at a 1:1 mass ratio (1
mg:1 mg in 0.8 ml), were incubated at the TS of the
crystallin component, and A360 recorded at 5 min intervals.
For the combination of the bovine
crystallin and toothfish
crystallin, an additional two-part assay was performed as follows. Bovine
crystallin (final concentration
mg ml1) and
toothfish
crystallin (final concentration 1 mg ml1)
were incubated for 1 h at 60°C (final volume of 0.8 ml). Aggregated
protein that was observed was then sedimented by centrifugation at 21 000
g for 10 min at 4°C. The supernatant was combined with
bovine
crystallin (final concentration 1 mg ml1) in
a total volume of 1 ml or less, and incubated for a second 1 h period at
60°C. A360 was recorded at 5 min intervals for all assays.
Estimation of effective crystallin size by dynamic light scattering
The effective molecular size in solution of individual crystallins of cow
and fish lens, and of interacting -crystallin complexes, were
measured by dynamic light scattering (DLS) with the Brookhaven Instruments
goniometer system (BI-200SM, Brookhaven Instruments Corp., Holtsville, NY,
USA) equipped with a Lexel argon-ion laser (model 95, Cambridge Lasers Lab.,
Fremont, CA, USA) operating at 514 nm. Individual
and
crystallin fractions, freshly purified by SEC, were first centrifuged at 18
400 g at 4°C for 20 min and the supernatant was filtered
through 0.050 µm SPI-Pore polycarbonate filters (SPI Supplies, West
Chester, PA, USA) to remove dust and other particulates before sizing. We
found that the filtration step was necessary to obtain reproducible DLS sizes,
especially for
crystallins, which form increasingly larger high
molecular mass (>1000 kDa) oligomers with storage time. For unheated
samples, the filtered crystallin fraction was sized in a clean DLS cell at
room temperature. Heated samples (1 h incubation at the desired temperature)
were first cooled to room temperature, centrifuged to pellet any
heat-aggregated protein, and the supernatant was transferred to a clean DLS
cell and sized. Light intensity at 90° angle was measured by a
photomultiplier tube and the output signal was processed by a Brookhaven
9000AT correlator. Crystallin size was calculated by Brookhaven Instruments
DLS Software Version 2.17 and expressed as the hydrodynamic diameter in
nm.
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Results |
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Cold-cataract formation
Fig. 1el illustrates
the effect of temperatures below normal body temperature on the transparency
of the lens of cow and fish. Fig.
1e is a freshly dissected bovine lens at room temperature
(25°C) showing complete transparency. Cold temperature induced
opacity or cold-cataract in a cow lens after cooling in ice (0°C) for
1.5 h (Fig. 1f). When the
cooled lens was retrieved for photographing at room temperature, the slightly
opaque outer cortex rapidly clarified, while the nucleus remained opaque, as
indicated by the arrow (Fig.
1e). The nucleus completely clarified later, after 2 h at room
temperature. Lenses from the small tropical (24°C) marine fish, the
blackbar soldierfish Myripristis jacobus
(Fig. 1gi), are
approximately 6 mm in diameter and have the same structural morphology as the
D. mawsoni and T. obesus lenses. Transparency of the lens
was maintained on cooling to 15°C (Fig.
1g). A second lens incubated at 0°C (in ice) for 6 hbecame
less transparent (Fig. 1h) than
the lens at 15°C. Further incubation at 0°C (48 h total) resulted in
further opacity throughout the lens. Retrieval from ice for photography at
room temperature partially clarified the cortex, but the nucleus remained
opaque, as indicated by the arrow (Fig.
1i). Further equilibration at room temperature for several hours
did not clarify the nucleus.
Antarctic toothfish lenses are completely transparent at 2°C, the ambient environmental temperature and normal body temperature of this ectotherm (Fig. 1j). A second lens after 6 h and 48 h incubation at 12°C is shown in Fig. 1k and l, respectively. The lens was immersed in a silicon oil to permit incubation temperature as low as 40°C without the oil freezing or losing optical clarity. In contrast to cold-cataract development in cooled bovine and soldierfish lens, the toothfish lens remained internally clear even after 48 h at12°C (Fig. 1l), with only a surface veneer of slight opacity, probably due to the reaction of the silicon oil with the outer layers of the collagen capsule. The silicon oil did become slightly translucent itself, but the lens remained clear. Cooling the lens below 12°C was not possible as the lens invariably froze. It can be concluded, however, that cold-cataract formation does not occur in the Antarctic toothfish lens at temperatures as low as 12°C.
SEC fractionation of lens crystallins
Fig. 2 shows that the
chromatograms of the crystallins from toothfish, bigeye tuna and cow
fractionated on S200HR size-exclusion resin are all similar in pattern. The
bovine elution chromatogram (Fig.
2C) is similar to that reported in the literature
(Björk, 1961;
Bloemendal, 1986
;
Siezen et al., 1986
;
van Dam, 1966
) using G75
Sephadex and 200HR Sephacryl resins. Prior characterisations of bovine lens
showed that the three predominant peaks I, II and III (or IIIa and III)
containing primarily
, ß and
crystallin, respectively
(Bloemendal, 1986
). Using the
S200HR resin, the high molecular mass
crystallin and
ßH (ßHigh) crystallin both elute in peak I,
which is designated as the `
/ßH' peak in
Fig. 2. (The constituents of
these elution peaks are given here, with the verification of their specific
identities detailed in the next Results section.) Fractionation on Sepharose
6B resin provided better separation of cow
and ßH
crystallins (data not shown), but much poorer separation of ßL
(ßLow) from the
crystallins for all three species.
Therefore, the S200HR resin was used in this study to allow optimal
comparisons of SEC chromatograms and subsequent analyses.
Although similar in pattern, the SEC elution profiles of fish crystallins
differ from the cow in the relative size of the ß and peaks. The
bovine ß crystallins partitioned into ßH (part of peak I,
apex and descending slope; Fig.
2C) and a prominent distinct ßL peak
(Fig. 2C, peak II). While
ßH is present in peak I of tuna and toothfish, there are also
distinct small ßH and ßL peaks in the bigeye
tuna chromatogram (Fig. 2B,
peaks IIa and IIb). The toothfish profile
(Fig. 2A) has no
ßH peak and only a very minor ßL peak
(Fig. 2A, peak II). In
contrast, the
crystallin peak of both fish species
(Fig. 2 A, B peak III) is much
larger than the bovine
crystallin peaks
(Fig. 2C, peaks IIIa and III
combined). Fig. 2C, peak IIIa
contains cow
S, which elutes ahead of the rest of the cow
crystallins in peak III.
Estimations of relative protein abundance based on peak areas
(Fig. 2) for each species are
tabulated in Table 2. Toothfish
(52% /ßH, 5% ßL and 43%
) is
similar to bigeye tuna (47%
/ßH, 6% ßH,
6% ßL and 41%
) in relative percentage abundance of the
three classes of crystallins. Both fish differ from the cow (54%
/ßH, 27% ßL and 19%
) in having
a substantially higher percentage of
crystallins, over 40% as opposed
to 19%, making it the predominant crystallin fraction in the fish lens.
Separate estimates of percentage abundance of
and ß crystallins
are not possible because of their overlapping elution within peak I
(Fig. 2A,B), as revealed by
SDS-PAGE analysis and immunoblotting with
, ß,
antisera
(next section). The SEC
peaks, peak III of fish and peaks IIIa and III
for cow, however, are near homogeneous for
crystallins, based on
immunoblots (next section), and therefore their percentage abundance
estimations and comparisons are reliable.
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SDS-PAGE and immunoblot analyses of crystallin fractions
The identities of the class of crystallins and the extent of protein
heterogeneity in the SEC elution peaks
(Fig. 2) were assessed by
SDS-PAGE and immunoblots (Fig.
3). The lane numbers in the SDS gels and the immunoblots (western
blots) (Fig. 3) correspond to
the number of selected fractions from the SEC elution
(Fig. 2, labelled next to the
peaks). The results of the SEC fractionation, SDS gels and immunoblots
collectively show the following. For all three species, the crystallin
polypeptides range in size from about 18 kDa to 35 kDa. Rabbit antisera
against the three bovine crystallins could crossreact with the respective fish
crystallins. SEC separation achieved near-homogeneous crystallin
fractions in all species (Fig.
3A,E,I,
fractions), but did not adequately resolve
and ßH crystallins from each other in the two fish species,
such that SEC peak I of both fish species encompasses considerable protein and
size heterogeneity (Fig.
3A,E,I,
/ßH fractions).
More specifically, the selected fractions 15 from the bovine peak I
(Fig. 2C) contain A and
B crystallins at
21 and 23 kDa, and an additional
18 kDa band
for fractions 14 (Fig.
3I, lanes 15), which is probably an
A crystallin
truncation product previously reported in the literature
(Augusteyn et al., 1992
;
Horwitz et al., 1998
). These
protein bands are all immunopositive to anti-bovine
crystallin
polyclonal antibodies in the immunoblot
(Fig. 3J, lanes 15), and
of these five fractions, the first two (indicated with arrow at peak I,
Fig. 2C) are predominantly
crystallin, as indicated by strong anti-
immunopositive bands
(Fig. 3J, lanes 1,2), and
little or no immunopositivity to anti-ß
(Fig. 3K, lanes 1,2). The
subsequent fractions 35 of bovine SEC peak I
(Fig. 3I, lanes 35), all
contain ßH crystallins, as indicated by the two sets of
anti-ß crystallin immunopositive bands close to the 31.1 kDa and 28.4 kDa
and markers in the immunoblot (Fig.
3K, lanes 35).
The SEC peak I fractions of the two fish
(Fig. 2A,B) contain
substantially more protein heterogeneity on SDS-PAGE
(Fig. 3A,E, lanes 15)
than their bovine counterpart and none of the selected fractions examined is
homogeneous for crystallin. Chemiluminescence immunodetection using
anti-bovine
crystallin polyclonal antibodies show
crystallins
of
21 kDa in peak I fractions 14 for toothfish
(Fig. 3B, lanes 14), and
peak I fractions 1-4 for bigeye tuna (Fig.
3F, lanes 14, signal weak for lane 4). All five selected
fractions of SEC peak I of both fish species contain two sets of anti-ß
immunoreactive bands, close to the 28.4 kDa and 31.1 kDa markers
(Fig. 3B, F, lanes 15).
These sizes are consistent with the reported values (2635 kDa) for
mammalian ß crystallins (Slingsby and
Clout, 1999
) and known fish ß crystallins
(Wistow, 1995
;
Zigler and Sidbury, 1976
). The
presence of the
31.1 kDa immunopositive bands indicates that
ßH coeluted with
crystallin in peak I of fish. For
both fish and cow, some fractions of the
/ßH peak
contain several weakly staining, large-sized protein bands, close to the 49.9
kDa marker (and above in the toothfish) in the SDS gel
(Fig. 3A,E,I). These
49.9
kDa bands are likely oligomeric ß or
/ß aggregates since they
are weakly immunoreactive to anti-ß
(Fig. 2C,G,K), and to
anti-
(Fig. 2J). The
bands greater than 49.9 kDa of the toothfish
(Fig. 3A) are not
immunoreactive to any antisera, and thus their identity remains to be
investigated.
For both fish and cow, the SEC peak II (peaks IIa and IIb for bigeye tuna) contains only ß crystallin. SEC peak II of toothfish and cow, and peak IIb of the bigeye tuna (Fig. 2AC) appears homogeneous for ßL in immunoblots (Fig. 3C,G, lane 7, and K, lane 6).
For all three species, SEC peak III
(Fig. 2) is homogeneous for
crystallins on immunoprobing (Fig.
3D,H, lanes 8,9, L, lane 8). In the case of the cow,
S (Fig. 3L,
lane 7), formerly ßS in older literature, eluted as a discrete
peak ahead of the other
crystallins
(Fig. 2C, peaks IIIa and III).
Because the SEC
crystallin fractions do not have
or ß
crystallins within them, their relative percentage abundance in the lens,
estimated on the basis of protein elution peak areas
(Table 2), is reliable. Gamma
crystallins constitute the predominant crystallin in fish lens (43% of
toothfish crystallins, 41% of bigeye tuna crystallins), which is over twofold
more abundant than its bovine counterpart (19% of total crystallins).
Upper limit of temperature stability (TS) of and
crystallin from D. mawsoni, T. obesus and B. taurus
Fig. 4 shows A360
versus time for a series of incubations at increasing temperatures
for toothfish crystallin and
crystallin. The highest
temperature at which the A360 remained at or near zero for at least
1 h was defined as the TS value, and was found to be
47°C and 33°C for toothfish
and
crystallins,
respectively. The TS value for the
and
crystallins of bigeye tuna and cow were similarly determined.
TS values of the
crystallins of bigeye tuna and
cow were 55°C and 68°C, respectively, and TS of
their
crystallins were 39°C and 50°C respectively
(Table 3). Thus the
crystallins are much more heat-labile relative to
crystallins, by
14°C, 16°C and 18°C for toothfish, tuna and cow, respectively.
Additionally, there was a direct correlation between the
TS of the crystallins and the body temperature of each
animal, highest for the endothermic cow (mean body temperature 38.3°C;
Piccione et al., 2003
),
followed by the sub-tropical ectothermic bigeye tuna (mean body temperature
18°C; Holland et al.,
1992
), and lowest for the ectothermic Antarctic toothfish, which
inhabits the perennially freezing seawater of the Southern Ocean (mean body
temperature 1.9°C; Hunt et al.,
2003
).
|
|
Chaperone-like activity of crystallin from D. mawsoni, T. obesus and B. taurus
In the absence of crystallin, heat aggregation of Antarctic
toothfish
crystallin occurred rapidly, after 10 min at 47°C.
Turbidity as measured by A360 increased sharply to 0.8 in the first
30 min, and reached a plateau of 1.0 in the next 30 min
(Fig. 5A). At mass ratios of
1:1 and 1:2 of
:
crystallin, aggregation of the
crystallin was prevented during the 1 h assay time
(Fig. 5A). Protection decreased
as
crystallin concentration was decreased, but even with 1:16 ratio of
to
crystallin, there was still significant protection compared
to unchaperoned
crystallin (Fig.
5A).
|
For comparison, chaperone assays were performed using crystallin of
the bigeye tuna T. obesus and the cow B. taurus and
same-species
crystallin, at 55°C for bigeye tuna and at 60°C
for the cow (Fig. 5B,C). By
itself,
crystallin from either species increased in turbidity rapidly
on heating, while mass
to
ratios of 1:1, 1:2 and 1:4 displayed
effective protection of
crystallin
(Fig. 5B,C). At the lower
to
mass ratios of 1:8 and 1:16, very effective chaperone
protection of
crystallin was observed in the case of the tuna, while
much less chaperone protection was observed in the case of cow
(Fig. 5AC).
Chaperone protection of lysozyme by crystallin from D. mawsoni, T. obesus and B. taurus
In the absence of crystallin, lysozyme rapidly unfolded in 20 mmol
l1 DTT at 37°C, with A360 kinetics similar to
heat denaturation, plateauing at A360
1.1 over 1 h
(Fig. 5DF). Protection
against DTT-induced denaturation of lysozyme by
crystallin (calculated
by dividing the reduction in A360 between the unchaperoned and
chaperoned assays by the A360 of the unchaperoned assay at the 1 h
end point) increased with increasing amounts of
crystallin added. A
10:1 mass ratio of D. mawsoni
crystallin to lysozyme resulted
in 79% protection, 66% at a 5:1 mass ratio, and 25% at a 2.5:1 mass ratio
(Fig. 5D). For T.
obesus
crystallin, an 11:1 mass ratio provided 88% protection,
75% at a 7.5:1 mass ratio, but only 9% at a 5:1 mass ratio of T.
obesus
crystallin to lysozyme
(Fig. 5E). The B.
taurus
crystallin exhibited much greater chaperone ability than
either fish, achieving 95% protection of lysozyme at a mass ratio of only 3:1
crystallin to lysozyme (Fig.
5F).
Dynamic light scattering (DLS) sizing of and
crystallins
To determine whether the chaperone effect is related to a stable
interaction between the and
crystallins, the sizes of
individual crystallins, and of
and
together before and after
incubation at the TS of the
crystallin of each
species, were measured by DLS (Table
4). The hydrodynamic diameters of
crystallin at room
temperature were 27.6 nm, 29.1 nm and 31.7 nm for toothfish, bigeye tuna and
cow, respectively, while that of
crystallin was about 45 nm for
all three species. The major bovine
crystallin fraction from Sepharose
6B fractionation (data not shown) was found to be 19.4 nm in diameter, which
is smaller than the 31.7 nm of the S200HR bovine
crystallin fraction.
This discrepancy is probably due to the presence of residual high molecular
mass aggregate in the S200HR
crystallin fraction, which partitioned as
a small peak in Sepharose 6B elution ahead of the major
peak (data not
shown). Except for the cow, the
crystallin increased in size after 1 h
incubation at its TS, consistent with `activation' of the
crystallin chaperone complex
(Putilina et al., 2003
), and
perhaps also due to association of
with the ß crystallin present
in the heterogeneous
crystallin fraction used. A mixture of
and
crystallin (1:1 mass ratio) at room temperature has a similar size
to
crystallin alone, probably because there is no molecular
interaction between
and
crystallin before heating, and thus
the DLS size of the mixture essentially reflects that of the much bigger
crystallins. After a 1 h incubation at the respective
crystallin TS, the hydrodynamic diameter of the of
and
crystallin mixture increased by 94% (27.453.4 nm) for
D. mawsoni, 142% (24.960.3 nm) for T. obesus, and 55%
increase (from 30.3 nm to 47.2 nm) for B. taurus
(Table 4), indicative of
molecular association of the two crystallins forming a larger size
complex.
|
Cross-species chaperone protection of crystallin by
crystallin
The ability of crystallin of each species to protect the
crystallin of a different species from heat aggregation was evaluated
(Fig. 6). The chaperone assays
were carried out at the TS of the
crystallin, for
pairwise combinations in which the
crystallin has a lower
TS than that of the
crystallin. The combination of
toothfish
plus cow
at 47°C, though not meaningful as a
chaperone assay since cow
with a TS of 50°C is
more heat stable, was included as a `non-interacting' control. Protection was
observed for bigeye tuna
crystallin plus bovine
at 55°C,
toothfish
crystallin plus tuna
at 47°C, and bovine
plus tuna
at 68°C, with A360 reaching only
0.05,
0.10 and 0.25 (respectively) at the 1 h end point. Tuna
crystallin did
not completely protect toothfish
at 55°C, and an A360
of 0.75 was reached at 1 h; however, the slope of the turbidity increase was
much less steep (
tenfold) than that of toothfish
alone at
55°C, indicative of partial interaction of the two crystallins leading to
partial protection. The most notable and unexpected result is that cow
crystallin offered no protection at all to toothfish
at 68°C,
resulting in a rate of turbidity increase identical to that of toothfish
alone at 55°C, indicating that all or most of the toothfish
crystallins had precipitated (Fig.
6).
|
DLS measurements of heated heterologous combinations showed
that chaperone protection was associated with a size increase of the
crystallins, indicating the formation of a
complex
(Table 5). Assuming the
pre-incubation DLS size of the pairwise
+
combinations is
similar to that of the
component,
2029 nm
(Table 4), the post-incubation
DLS size of the cross-species
+
complex increased significantly,
to
4373 nm for the combinations that exhibited chaperone
protection (Table 5). In
contrast, the hydrodynamic diameter of the heated cow
and toothfish
combination (the supernatant after removal of protein aggregates) was
the smallest, at 38.5 nm, indicating the least extent of interaction.
|
The extent of non-interaction of cow with toothfish
was
assessed in a two-part chaperone assay
(Fig. 7).
Fig. 7A shows the
A360 of a 1 h incubation of bovine
plus toothfish
at 60°C, and the control incubation of bovine
plus bovine
,
both at mass ratio
mg
to 1 mg
in 1 ml final volume
(
mg of cow
crystallin is the estimated minimal amount that
would completely protect 1 mg of bovine
crystallin;
Fig. 5). The 60°C
incubation of bovine
crystallin plus toothfish
crystallin
resulted in rapid turbidity increase, with the same kinetics and peak
A360 as the 68°C incubation
(Fig. 6), while bovine
was fully protected by bovine
, as expected
(Fig. 7A). After
centrifugation, additional bovine
crystallin was added to the
supernatant of both incubations to the same final concentration (1 mg
ml1) as the first 1 h incubation, for a second 1 h
incubation at 60°C (Fig.
7B). The supernatant from the cow
crystallin plus
toothfish
crystallin incubation showed substantial chaperone
protection of added cow
crystallin in the second hour, reaching an
A360 of only 0.486. This result indicates that most of the cow
crystallin did not complex with toothfish
crystallin during
the first 1 hincubation, and therefore was free to interact with the added cow
crystallin in the second 1 h incubation. In contrast, the supernatant
from cow
crystallin plus cow
crystallin provided no protection
of added cow
crystallin in the second hour, reaching an
A360 of 1.5 at 50 min. This indicates that little or no free cow
crystallin was available to chaperone the added cow
crystallin, probably because most or all of the cow
crystallin was
saturated or complexed to cow
crystallin during the first 1 h
incubation.
|
![]() |
Discussion |
---|
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---|
Cold-cataract formation in bovine lens is attributed to a
liquidliquid phase separation of one or more of the cold-labile
crystallin isoforms (Broide et al.,
1991
; Siezen et al.,
1985
). Fish lenses are much denser than mammalian (bovine) lenses
(Jagger and Sands, 1996
,
1999
;
Pierscionek and Augusteyn,
1995
), thus a plausible hypothesis might be that the greater cold
stability of fish lens could be due to protein density-constrained mobility of
the crystallin molecules, reducing the propensity for the kind of cold-induced
structural rearrangement of the small
crystallins that occurs in the
much softer cow lens, leading to cold-cataract
(Gulik-Krzywicki et al.,
1984
). However, while the increased density could account for the
slower onset of the cold-cataract in the blackbar soldierfish, the toothfish
lens is as compact and protein-dense as other teleost fish lens (slightly less
dense than tuna), but is far more cold stable
(Ferguson et al., 1971
;
Smith, 1972
). Therefore, the
basis of cold stability in ectothermic fish lens is likely to lie in the
adaptive protein properties of the constituent crystallins for function in
lower habitat temperatures (synonymous with body temperatures for ectotherms)
than the warm-bodied mammals. The extraordinary cold stability of Antarctic
toothfish lens exemplifies the preservation of normal protein function at the
coldest known extreme of marine ectothermic vertebrate life, the freezing
point of seawater. Moreover, initial results showing that the toothfish
crystallin fraction at concentration of 58 mg ml1
remains clear to temperatures as low as 10°C, which is 14°C
lower than similarly concentrated cow
crystallin fraction
(Siezen et al., 1985
), further
implicates the cold-adapted
crystallins as the basis of toothfish
whole lens cold stability in vivo.
In order to examine the thermal, biochemical and physical properties of the individual classes of lens crystallins of Antarctic toothfish, we first fractionated whole lens homogenate by Sephacryl 200HR size-exclusion chromatography (SEC) (Fig. 2). This was also performed for the subtropical bigeye tuna T. obesus and the endothermic cow in parallel, to allow comparison of the relationship between crystallin protein properties and organismal body temperature. The three species have lenses of comparable size, and collectively they span the range of vertebrate body temperatures the very cold-bodied toothfish (2°C), the cool-bodied bigeye tuna (18°C), and the warm-bodied cow (37°C).
Incomplete SEC separation of individual classes of fish crystallin,
especially between and ß crystallin, is a commonly reported
difficulty (Chiou et al.,
1987
; Posner et al.,
1999
; Wistow,
1995
), which also occurred for Antarctic toothfish and bigeye tuna
in this study (Figs 2,
3). Sepharose 6B, which
provided good separations of bovine crystallins
(Horwitz et al., 1998
), is
even less adequate than Sephacryl 200HR for fish crystallins separation (data
not shown). While S200HR could not achieve complete separation of
crystallin from ß crystallins, we documented in detail the identity of
the crystallins in fractions spanning the heterogeneous
/ß peak as
well as in the ß and
elution peaks, with good resolution SDS gels
and immunoblots (Figs 2,
3). This allowed us to perform
the downstream experiments and interpret the results in a logical manner. The
S200HR SEC produced near-homogeneous separation of the
crystallin for
all three species (Figs 2,
3), which permitted a reliable
estimation of their percent abundance in the lens
(Table 2). Gamma crystallins of
the toothfish and bigeye tuna comprise over 40% of the lens crystallins, and
are therefore the predominant class of crystallin in the fish lens. This
relative abundance is comparable to the reported 52% in carp Cyprinus
carpio lens (Chiou et al.,
1987
) and the 35.3% in bluefish Pomatomas saltatrix
(Zigler and Sidbury, 1976
).
The percentage abundance of
crystallins in toothfish (43%) and bigeye
tuna (41%) lens is twofold higher than in the bovine (19%) lens
(Table 2). The predominance of
crystallin in fish lens and the predicted ability of
crystallins to pack at high density
(Summers et al., 1986
) is
consistent with the necessary refractive index differential required in an
aquatic medium where little to no refractive benefit occurs between the water
and the fish cornea, unlike in the terrestrial mammals that benefit from a
substantial refractive index change between air and cornea
(Jagger and Sands, 1996
;
Wistow, 1993
;
Wolken, 1995
).
Examination of the thermal response of individual crystallin ( and
) from the three species showed that
crystallin is a more
heat-labile component relative to
, and there is a direct correlation
between organismal body temperature and heat stability
(TS) of the
crystallins (
fraction is
near-homogeneous), with bovine being the most heat stable, followed by the
tuna, and toothfish the least heat stable
(Table 3). While the
TS of the
crystallin fraction of the two fish may
be influenced to some extent by the presence of some ßH (Figs
2,
3), the clear relationship
between TS and organismal body temperature for the
crystallins from these species support the increasing and disparate
TS values of
from toothfish, tuna and cow
(47°C, 55°C, 68°C) as reflecting the same relationship. The
thermal stabilities of the individual crystallin components agreed with the
findings in our whole lens cold stability experiments, with low organismal
body temperature correlated with the greatest cold stability
(Fig. 1). The
TS values for the
crystallins of the three species
are 3150°C higher than the body temperature, and
TS of the
crystallins are 1335°C
higher. Our heat tolerance findings of the individual crystallin classes are
consistent with a previous whole lens study that showed lens transparency was
maintained at temperatures up to 20°C higher than the normal body
temperature of a number of vertebrate animals
(McFall-Ngai and Horwitz,
1990
).
Vertebrate crystallin is a small heat shock protein
(Horwitz, 1992
;
Ingolia and Craig, 1982
), thus
a common approach in the studies of lens crystallin properties is to examine
the thermal response of the different crystallins, and the chaperone-like
protection by
crystallins of the heat-labile crystallin components, in
assessing the mechanism of molecular interaction between them. Chaperone
assays in the bovine system have focused on protection of the ß
crystallin fractions, the most abundant non-chaperone crystallin in cow lens
(Augusteyn et al., 2002
;
Horwitz, 1992
). In teleost
fish lens, the
crystallins are the major heat-labile constituents
(Fig. 2, Table 2). Sequence similarity
between fish and mammalian
crystallins indicated that the former are
also sHSPs and would have chaperone-like activity
(Posner et al., 1999
;
Runkle et al., 2002
), which
has indeed shown to be so for recombinant
A crystallin from zebrafish
in chaperone assays with DTT-denatured lysozyme
(Posner, 2003
), and for
recombinant
B from carp with DTT-denatured insulin
(Yu et al., 2004
). Here we
demonstrate that native
crystallin isolated from the lens of two other
fish, Antarctic toothfish and bigeye tuna, are also effective molecular
chaperones, with the ability to protect the same species heat-labile
crystallins from aggregation induced by heat
(Fig. 5AC), and chemical
denaturation of non-lens protein (lysozyme) by DL-dithiothreitol
(Fig. 5DF). The relative
effectiveness of the
crystallins of the two fish species as a
chaperone may not be directly compared to each other or to the cow, because of
uncertainty over the actual amounts of pure
crystallin in the SEC
crystallin fraction used in these assays, the presence of some
ßH crystallin in these fractions that may also interact with
the
crystallin during heat or DTT exposure reducing the amount of
crystallin available for binding to the test protein, and the
different species-specific assay temperatures (TS) and
thermal properties of the test
crystallin. Notwithstanding, the
chaperone-like nature of native fish
crystallins with the hallmark
ability to prevent stress-induced aggregation of unrelated proteins
(Narberhaus, 2002
) was clearly
established.
Chaperone-like heat protection assays are commonly carried out at high
in vitro temperatures (Augusteyn
et al., 2002; Horwitz,
1992
; Liao et al.,
2002
). While such temperatures are not physiological, the
chaperone protection has its mechanistic basis in molecular interactions
between
crystallins and labile molecules, which can be manifest upon
heating, and thus this experimental approach is instructive for understanding
intercrystallin molecular interactions in vivo. The chaperone-like
ability of
crystallin was deduced to require a heat-activated
structural modification that was necessary for association with other
crystallins, based on the pronounced size increase observed in bovine
crystallins at 60°C, with a further increase at 66°C, by small angle
X-ray scattering (SAXS; Putilina et al.,
2003
). Our DLS measurements showed that the fish
crystallins heated at their respective TS for 1 h resulted
in a larger hydrodynamic diameter (Table
4), consistent with heat-activated formation of a larger
oligomeric structure. The fish
crystallin fractions contained various
amounts of ß crystallins; thus the heat-induced size increase could be
partly due to formation of an
/ß heterocomplex. However, the
further increase in size after incubation of
crystallin fraction with
the same-species
crystallin (Table
4) supports the molecular association of
crystallin
molecules with a heat-activated
crystallin oligomeric structure,
relevant to the observed chaperone activity. In the bovine system, a similar
increase in
crystallin size when heated in the presence of heat-labile
ßL crystallin has recently been reported when measured by SAXS
(Krivandin et al., 2004
).
There was an apparent anomalous decrease of the cow crystallin DLS
size of 31.7 nm to 22.0 nm upon heating
(Table 4). The large size of
31.7 nm of unheated cow
crystallin can be attributed to the presence
of a small amount of large
crystallin oligomers in the S200HR SEC
crystallin fraction. The presence of this large
crystallin
component was confirmed by Sepharose 6B chromatography of bovine lens
homogenate, in which it eluted as a small distinct peak, ahead of the dominant
crystallin peak that contains the remaining and more abundant smaller
crystallins (data not shown) that gave a 19.4 nm on DLS sizing. The
DLS signal of a heterogeneous size mixture tends to be dominated by that of
the large size component even if it is present in smaller amounts, thus the
presence of the smaller
crystallins in the S200HR
crystallin
fraction was masked by that of the much larger
oligomers. Upon
heating, the large
oligomers likely increased rapidly in size, and
sedimented when the sample was spun after heating and prior to DLS sizing,
leaving the soluble `activated'
crystallin that gave the 22.0 nm size.
The 19.4 nm bovine
crystallin from the major
crystallin peak
of the Sepharose 6B separation probably represents the true `unactivated'
crystallin prior to heating, and agrees more closely with previously
reported results (Abgar et al.,
2001
; Putilina et al.,
2003
; Vanhoudt et al.,
2000
).
The cross-species chaperone heat protection assays to evaluate the ability
of warm-adapted crystallins to interact with cold-adapted crystallins produced
an important and unexpected result; the total lack of protection of toothfish
crystallin by cow
crystallin, which is very likely to be
related to adaptive changes in protein properties that evolved in disparate
thermal environments (Fig. 6). For the combinations of cow
plus tuna
at 68°C, tuna
plus cow
at 55°C, and toothfish
plus tuna
at 47°C, the
component could fully or nearly fully protect the
heat-labile
component, regardless of the body temperatures of the
species from which the
crystallin was derived, and despite the
disparity between the body temperatures of the species pair. This attests to
the small heat shock protein ancestry of the vertebrate
crystallins
(de Jong et al., 1993
), with
their intrinsic chaperone-like ability to provide stress protection of other
protein molecules regardless of ectothermic or endothermic organismal origin.
However, while tuna
could fully protect cow
, it only partly
protected toothfish
crystallin, and the cow
crystallin
completely failed to protect the toothfish
(Fig. 6). Since both cow and
tuna
crystallins have proven chaperone-like function, the lack of
protection of the toothfish
crystallin is likely a result of
particular biochemical properties of the latter. Conceivably, the changes in
the
crystallins that have occurred in the frigid Antarctic environment
over evolutionary time to maintain the toothfish lens transparency may be the
basis for the observed weak interaction with the
crystallin from the
warmer bodied bigeye tuna, and complete non-interaction with the
crystallin from the warm-bodied cow.
That the non-protection of toothfish crystallin by cow
is a
result of non-interaction between the two crystallins was supported by the
results of the two-part chaperone assay
(Fig. 7). In the first 1 h
incubation, the kinetics of the turbidity (A360) increase
(Fig. 7A) were the same as
observed when heating toothfish
crystallins alone
(Fig. 6), indicating that the
heat-precipitated protein was predominantly toothfish
crystallins,
presumably due to their failure to associate with cow
crystallin to
gain protection. The reasonable expectation then, is that after removing the
heat-precipitated protein the supernatant should consist primarily of
functionally intact cow
crystallin, which was confirmed by the
significant protection of added cow
crystallins for an additional hour
of heat incubation (Fig. 7B).
Thus the non-protection in the first hour of incubation must indeed be due to
non-interaction between the two crystallin components. To ensure that any
protection in the second 1 h test incubation is not due to excess cow
crystallins, in the first 1 h incubation we only used
mg of cow
crystallins, which is estimated as the minimal amount of cow
crystallins that will provide full protection of 1 mg of cow or tuna
crystallins (Fig. 5). The
supernatant from the first 1 h incubation of control cow
+
crystallin did not protect added cow
crystallin in the second 1 h
incubation (Fig. 7B), which
confirmed that the
mg cow
crystallins was indeed the
appropriate minimum, and that they must have fully complexed to the bovine
in the first 1 h incubation.
The protection by the supernatant from the cow plus toothfish
incubation of added cow
crystallin in the second 1 h test
incubation is not complete, for two reasons. It is difficult to determine the
amount of cow
crystallins remaining in the supernatant to accurately
achieve the minimum
to
mass ratio of
:1. Secondly, a
portion of the cow
crystallins may have been complexed with some
toothfish
crystallins that are stable at 60°C in the first 1 h
incubation. Stepwise incubation of a toothfish
crystallin sample
showed that the majority of the isoforms (77%) were heat-labile at 47°C,
but a small percentage (4.4%) remained soluble at 60°C
(Table 6), and presumably these
could complex with cow
crystallins in the first 1 h incubation.
Regardless, the results of the two-part incubation assay strongly support the
majority of Antarctic toothfish
crystallins being sufficiently
biochemically different that they do not associate with cow
crystallins, resulting in the lack of chaperone protection when heat
stressed.
|
The structural basis for the non-interaction between the majority of
toothfish crystallin polypeptides with cow
crystallins, or
weak interaction with tuna
crystallins, is currently unknown. It is
conceivable that the pertinent structural properties of toothfish
crystallin responsible for this lack of interaction with
crystallins
of other warmer-bodied species may be the same ones that contribute to the
extraordinary cold stability of the Antarctic toothfish lens, such that it
does not suffer cold-cataract even at 12°C (and probably lower).
The Antarctic toothfish has an estimated evolutionary age of 714
million years (Chen et al.,
1997a
), and its lens has clearly evolved to preserve transparency
at the freezing Antarctic seawater temperature of 2°C. Because of
the important role of
crystallins in teleost fish lens in achieving
the high protein density and refractive index gradient required for aquatic
light diffraction, it is reasonable to expect that key structural adaptations
would occur in the
components of the lens when subjected to
environmental selection. In addition, studies of the bovine system have
conclusively demonstrated that it is one or more of the
crystallins
that display a cold-lability and are responsible for mammalian whole lens
cold-cataract (Broide et al.,
1991
; Clark and Benedek,
1980b
; Siezen et al.,
1985
; Thomson et al.,
1987
). The adaptive amino acid changes that have occurred in the
Antarctic toothfish
crystallins may be ascertained by cDNA cloning for
comparative sequence analyses with
crystallins of different taxa (work
in progress). It has been demonstrated that non-polar or so-called
`hydrophobic' residues are more easily solvated by water at low temperatures,
which diminishes the importance of these `hydrophobic' interactions as the
predominant intramolecular stabilising factor at low temperatures
(Privalov, 1990
;
Tsai et al., 2002
). Previous
reports indicate that the association of protein subunits in the
polymerisation of actin filaments in the relatives of the Antarctic toothfish,
Pagothenia borchgrevinki and Gymnodraco acuticeps, as well
as in the stabilisation of myosin in the related Notothenia rossii,
are found to rely less on hydrophobic interactions
(Hochachka and Somero, 2002
;
Johnston et al., 1975
).
Moreover, chaperone function of the bovine
crystallin is believed to
involve complexing with the unfolding labile protein largely through
hydrophobic interactions (Raman and Rao,
1997
). Thus, an evolutionary decrease in the hydrophobicity of the
toothfish
crystallins for adaptation in the cold would be consistent
with diminished interaction of toothfish
crystallin with the
crystallins of other species.
A reduced hydrophobicity in the toothfish crystallin, however, has
structural implications for toothfish
crystallin if the primary basis
for
chaperone function is via hydrophobic interactions with
the labile protein. Because attempts to identify chaperone essential aspects
from mammalian
crystallin polypeptides have been less than convincing,
suggesting that the chaperone process is multifaceted and complex and not
dependent on one `key' element (Derham et
al., 2001
; Kokke et al.,
2001
; Pasta et al.,
2002
,
2003
;
Posner, 2003
), the specific
effect of reduced hydrophobicity remains to be determined. In other words, the
toothfish
crystallin may have co-evolved to achieve the structural
requisites for interacting with the less hydrophobic toothfish
crystallin. It is not known if there is reduced hydrophobic residue content in
all the toothfish
crystallins, but this can be ascertained by
comparative sequence analyses with homologues from different ectothermic and
endothermic taxa. Any resultant higher order structural characteristics of the
crystallin proteins that may be responsible for cold stability are testable
with comparative tertiary and quarternary structure analyses of native and
site-directed mutants.
An alternative to the hypothesis of adaptive changes in the toothfish
crystallin proteins as the primary basis for whole lens cold stability
is the possibility that toothfish lens is largely composed of a homologue of
the bovine
S crystallin, which has recently been shown to be
extremely cold-stable, with a theoretical liquidliquid phase separation
temperature of 28°C (Annunziata
et al., 2003
). However, our SEC fractionation of fish lens
homogenates did not show an elution peak corresponding to the bovine
S (Fig. 2).
While fish
S may have co-eluted in the only and prominent
crystallin peak, our ongoing work, including ion-exchange separation,
cDNA cloning and sequencing, and two-dimensional PAGE analysis of the
toothfish
crystallin peak, has indicated a richer diversity of unique
individual
isoforms present in the toothfish lens than in bovine lens,
which is inconsistent with the predominance of a single isoform. Continued
structural elucidations of the toothfish
crystallins will help provide
insights into the remarkable cold stability of the toothfish lens, which may
in turn enhance our understanding of the cold sensitivity of their mammalian
counterparts.
![]() |
Acknowledgments |
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![]() |
References |
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Abgar, S., Yevlampieva, N., Aerts, T., Vanhoudt, J. and Clauwaert, J. (2000). Chaperone-like activity of bovine lens alpha-crystallin in the presence of dithiothreitol-destabilized proteins: characterization of the formed complexes. Biochem. Biophys. Res. Commun. 276,619 -625.[CrossRef][Medline]
Abgar, S., Vanhoudt, J., Aerts, T. and Clauwaert, J.
(2001). Study of the chaperoning mechanism of bovine lens
alpha-crystallin, a member of the alpha-small heat shock superfamily.
Biophys. J. 80,1986
-1995.
Annunziata, O., Ogun, O. and Benedek, G. B. (2003). Observation of liquid-liquid phase separation for eye lens {gamma}S-crystallin. Proc. Natl. Acad. Sci. USA 15, 15.[CrossRef]
Augusteyn, R. C., Parkhill, E. M. and Stevens, A.
(1992). The effects of isolation buffers on the properties of
-crystallin. Exp. Eye Res.
54,219
-228.[Medline]
Augusteyn, R. C., Murnane, L., Nicola, A. and Stevens, A. (2002). Chaperone activity in the lens. Clin. Exp. Optom. 85,83 -90.[Medline]
Banh, A. and Sivak, J. G. (2004). Laser scanning analysis of cold-cataract in young and old bovine lenses. Mol. Vis. 10,144 -147.[Medline]
Björk, I. (1961). Studies on
-crystallin from calf lens: I isolation by gel filtration.
Exp. Eye Res. 1,145
-154.[Medline]
Bloemendal, H. (1986). The lens proteins. In Molecular and Cellular Biology of the Eye Lens (ed. H. Bloemendal), pp. 1-47. New York: Wiley and Sons.
Broide, M. L., Berland, C. R., Pande, J., Ogun, O. O. and Benedek, G. B. (1991). Binary-liquid phase separation of lens protein solutions. Proc. Natl. Acad. Sci. USA 88,5660 -5664.[Abstract]
Chen, L., DeVries, A. L. and Cheng, C. H.
(1997a). Convergent evolution of antifreeze glycoproteins in
Antarctic notothenioid fish and Arctic cod. Proc. Natl. Acad. Sci.
USA 94,3817
-3822.
Chen, L., DeVries, A. L. and Cheng, C. H.
(1997b). Evolution of antifreeze glycoprotein gene from a
trypsinogen gene in Antarctic notothenioid fish. Proc. Natl. Acad.
Sci. USA 94,3811
-3816.
Cheng, C. H. and Chen, L. (1999). Evolution of an antifreeze glycoprotein. Nature 401,443 -444.[CrossRef][Medline]
Chiou, S. H., Chang, T., Chang, W. C., Kuo, J. and Lo, T. B. (1986). Characterization of lens crystallins and their mRNA from the carp lenses. Biochim. Biophys. Acta 871,324 -328.[Medline]
Chiou, S. H., Chang, W. C., Pan, F. M., Chang, T. and Lo, T. B. (1987). Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins. J. Biochem. (Tokyo) 101,751 -759.[Abstract]
Clark, J. I. and Benedek, G. B. (1980a). The effects of glycols, aldehydes, and acrylamide on phase separation and opacification in the calf lens. Invest. Ophthalmol. Vis. Sci. 19,771 -776.[Abstract]
Clark, J. I. and Benedek, G. B. (1980b). Phase diagram for cell cytoplasm from the calf lens. Biochem. Biophys. Res. Commun. 95,482 -489.[Medline]
Cossins, A. R., Murray, P. A., Gracey, A. Y., Logue, J., Polley, S., Caddick, M., Brooks, S., Postle, T. and Maclean, N. (2002). The role of desaturases in cold-induced lipid restructuring. Biochem. Soc. Trans. 30,1082 -1086.[CrossRef][Medline]
Davson, H. (1990). Physiology of the Eye. New York, NY: Pergamon Press, Inc.
de Jong, W. W., Leunissen, J. A. and Voorter, C. E. (1993). Evolution of the alpha-crystallin/small heat-shock protein family. Mol. Biol. Evol. 10,103 -126.[Abstract]
Delaye, M., Clark, J. I. and Benedek, G. B. (1982). Identification of the scattering elements responsible for lens opacification in cold-cataracts. Biophys. J. 37,647 -656.[Abstract]
Derham, B. K., van Boekel, M. A., Muchowski, P. J., Clark, J.
I., Horwitz, J., Hepburne-Scott, H. W., de Jong, W. W., Crabbe, M. J. and
Harding, J. J. (2001). Chaperone function of mutant versions
of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding
sites. Eur. J. Biochem.
268,713
-721.
Detrich, H. W., 3rd, Parker, S. K., Williams, R. C., Jr,
Nogales, E. and Downing, K. H. (2000). Cold adaptation of
microtubule assembly and dynamics. Structural interpretation of primary
sequence changes present in the alpha- and beta-tubulins of Antarctic fishes.
J. Biol. Chem. 275,37038
-37047.
DeVries, A. L. (1971). Glycoproteins as biological antifreeze agents in antarctic fishes. Science 172,1152 -1155.[Medline]
Eastman, J. T. (1993). Antarctic Fish Biology: Evolution in a Unique Environment. New York: Academic Press, Inc.
Eastman, J. T. and Lannoo, M. J. (2001). Anatomy and histology of the brain and sense organs of the Antarctic eel cod Muraenolepis microps (Gadiformes; Muraenolepididae). J. Morphol. 250,34 -50.[CrossRef][Medline]
Eastman, J. T. and Lannoo, M. J. (2003). Anatomy and histology of the brain and sense organs of the Antarctic plunderfish Dolloidraco longedorsalis (Perciformes: Notothenioidei: Artedidraconidae), with comments on the brain morphology of other artedidraconids and closely related harpagiferids. J. Morphol. 255,358 -377.[CrossRef][Medline]
Eastman, J. T. and McCune, A. R. (2000). Fishes on the Antarctic continental shelf: evolution of a marine species flock? J. Fish Biol. 57 Suppl. A, 84-102.[CrossRef]
Ferguson, W. E., Calhoun, W. B., 3rd and Koenig, V. L. (1971). Studies on the cold insoluble proteins from the lens of the striped mullet (Mugil cephalus). Comp. Biochem. Physiol. 40B,959 -972.
Fields, P. A. and Somero, G. N. (1998). Hot
spots in cold adaptation: localized increases in conformational flexibility in
lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes.
Proc. Natl. Acad. Sci. USA
95,11476
-11481.
Gomori, G. (1955). Preparation for use in enzyme studies. Meth. Enzymol. 1, 138-146.
Gulik-Krzywicki, T., Tardieu, A. and Delaye, M. (1984). Spatial reorganization of low molecular weight proteins during cold-cataract opacification. Biochim. Biophys. Acta 800,28 -32.[Medline]
Hikida, M. and Iwata, S. (1985). Studies on the eye lens in poikilothermal animals. II. Stimulation of anaerobic glycolysis in rainbow trout lenses incubated with Ca2+-free medium. Exp. Eye Res. 41,179 -182.[CrossRef][Medline]
Hikida, M. and Iwata, S. (1986). Studies of eye lens in poikilothermal animals. III. Long-term incubation of rainbow trout lenses. Jpn. J. Ophthalmol. 30, 43-50.[Medline]
Hochachka, P. W. and Somero, G. (2002). Biochemical Adaptation: Mechanism and Process in Physiological Evolution. New York: Oxford University Press.
Holland, K. N., Brill, R. W., Chang, R. K. C., Sibert, J. R. and Fournier, D. A. (1992). Physiological and behavioral thermoregulation in bigeye tuna (Thunnus obesus). Nature 358,410 -412.[CrossRef][Medline]
Horwitz, J. (1992). Alpha-crystallin can function as a molecular chaperone. Proc. Natl. Acad. Sci. USA 89,10449 -10453.[Abstract]
Horwitz, J. (2003). Alpha-crystallin. Exp. Eye Res. 76,145 -153.[CrossRef][Medline]
Horwitz, J., Huang, Q. L., Ding, L. and Bova, M. P. (1998). Lens alpha-crystallin: chaperone-like properties. Meth. Enzymol. 290,365 -383.[Medline]
Hunt, B. M., Hoefling, K. and Cheng, C.-H. C. (2003). Annual warming episodes in seawater temperatures in McMurdo Sound in relationship to endogenous ice in notothenioid fish. Antarctic Sci. 15,333 -338.[CrossRef]
Ingolia, T. D. and Craig, E. A. (1982). Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin. Proc. Natl. Acad. Sci. USA 79,2360 -2364.[Abstract]
Jaenicke, R. and Seckler, R. (1997). Protein misassembly in vitro. Adv. Protein Chem. 50, 1-59.[Medline]
Jagger, W. S. and Sands, P. J. (1996). A wide-angle gradient index optical model of the crystallin lens and eye of the rainbow trout. Vision Res. 36,2623 -2639.[CrossRef][Medline]
Jagger, W. S. and Sands, P. J. (1999). A wide-angle gradient index optical model of the crystallin lens and eye of the octopus. Vision Res. 39,2841 -2852.[CrossRef][Medline]
Johnston, I. A., Walesby, N. J., Davison, W. and Goldspink, G. (1975). Temperature adaptation in myosin of Antarctic fish. Nature 254,74 -75.[Medline]
Kokke, B. P., Boelens, W. C. and de Jong, W. W. (2001). The lack of chaperonelike activity of Caenorhabditis elegans Hsp12.2 cannot be restored by domain swapping with human alphaB-crystallin. Cell Stress Chaperones 6, 360-367.[CrossRef][Medline]
Krivandin, A. V., Muranov, K. O. and Ostrovsky, M. A. (2004). Heat-induced complex formation in solutions of alpha- and beta L-crystallins: a small-angle X-ray scattering study. Dokl. Biochem. Biophys. 394,1 -4.[CrossRef][Medline]
Liao, J. H., Lee, J. S. and Chiou, S. H. (2002). Distinct roles of alphaA- and alphaB-crystallins under thermal and UV stresses. Biochem. Biophys. Res. Commun. 295,854 -861.[CrossRef][Medline]
Liaw, Y. C., Chiou, S. H., Chang, T. and Chang, W. C. (1992). Predicted secondary and tertiary structures of carp gamma-crystallins with high methionine content: role of methionine residues in the protein stability. J. Biochem. (Tokyo) 112,341 -345.[Abstract]
Loewenstein, M. A. and Bettelheim, F. A. (1979). Cold-cataract formation in fish lenses. Exp. Eye Res. 28,651 -663.[CrossRef][Medline]
McFall-Ngai, M. J. and Horwitz, J. (1990). A comparative study of the thermal stability of the vertebrate eye lens: Antarctic ice fish to the desert iguana. Exp. Eye Res. 50,703 -709.[CrossRef][Medline]
Mörner, C. T. (1864). Untersuchung der Proteïnsubstanzen in den leichtbrechenden Medien des Auges I. Zeit. Physiol. Chemie 18, 61-106.
Narberhaus, F. (2002). Alpha-crystallin-type
heat shock proteins: socializing minichaperones in the context of a
multichaperone network. Microbiol. Mol. Biol. Rev.
66, 64-93.
Norledge, B. V., Hay, R. E., Bateman, O. A., Slingsby, C. and Driessen, H. P. C. (1997). Towards a molecular understanding of phase separation in the lens a comparison of the X-ray structures of two high T-C Gamma-crystallins, Gamma-E and Gamma-F, with two low T-C Gamma-crystallins, Gamma-B and Gamma-D. Exp. Eye Res. 65,609 -630.[CrossRef][Medline]
Pasta, S. Y., Raman, B., Ramakrishna, T. and Rao, C. M.
(2002). Role of the C-terminal extensions of alpha-crystallins.
Swapping the C-terminal extension of alpha A-crystallin to alpha B-crystallin
results in enhanced chaperone activity. J. Biol. Chem.
277,45821
-45828.
Pasta, S. Y., Raman, B., Ramakrishna, T. and Rao, C. M.
(2003). Role of the conserved SRLFDQFFG region of
alpha-crystallin, a small heat shock protein. Effect on oligomeric size,
subunit exchange, and chaperone-like activity. J Biol
Chem 278,51159
-51166.
Piccione, G., Caola, G. and Refinetti, R. (2003). Daily and estrous rhythmicity of body temperature in domestic cattle. BMC Physiol. 3, 7.[CrossRef][Medline]
Pierscionek, B. K. and Augusteyn, R. C. (1995). The refractive index and protein distribution in the blue eye trevally lens. J. Am. Optom. Assn. 66,739 -743.
Posner, M. (2003). A comparative view of alpha crystallins: The contribution of comparative studies to understanding function. Int. Comp. Biol. 43,481 -491.
Posner, M., Kantorow, M. and Horwitz, J. (1999). Cloning, sequencing and differential expression of alphaB-crystallin in the zebrafish, Danio rerio. Biochim. Biophys. Acta 1447,271 -277.[Medline]
Privalov, P. L. (1990). Cold denaturation of proteins. Crit. Rev. Biochem. Mol. Biol 25,281 -305.[Abstract]
Putilina, T., Skouri-Panet, F., Prat, K., Lubsen, N. H. and
Tardieu, A. (2003). Subunit exchange demonstrates a
differential chaperone activity of calf alpha-crystallin toward beta low- and
individual gamma-crystallins. J. Biol. Chem.
278,13747
-13756.
Raman, B. and Rao, C. M. (1997). Chaperone-like
activity and temperature-induced structural changes of alpha-crystallin.
J. Biol. Chem. 272,23559
-23564.
Romisch, K., Collie, N., Soto, N., Logue, J., Lindsay, M.,
Scheper, W. and Cheng, C. H. (2003). Protein translocation
across the endoplasmic reticulum membrane in cold-adapted organisms.
J. Cell Sci. 116,2875
-2883.
Runkle, S., Hill, J., Kantorow, M., Horwitz, J. and Posner, M. (2002). Sequence and spatial expression of zebrafish (Danio rerio) alphaA-crystallin. Mol. Vis. 8, 45-50.[Medline]
Siezen, R. J., Fisch, M. R., Slingsby, C. and Benedek, G. B. (1985). Opacification of gamma-crystallin solutions from calf lens in relation to cold-cataract formation. Proc. Natl. Acad. Sci. USA 82,1701 -1705.[Abstract]
Siezen, R. J., Anello, R. D. and Thomson, J. A. (1986). Interactions of lens proteins. Concentration dependence of beta-crystallin aggregation. Exp. Eye Res. 43,293 -303.[CrossRef][Medline]
Slingsby, C. and Clout, N. J. (1999). Structure of the crystallins. Eye 13,395 -402.[Medline]
Smith, A. C. (1972). Lens iso-precipitin in yellowfin tuna (Thunnus albacares). Comp. Biochem. Physiol. 42B,497 -499.
Summers, L. J., Slingsby, C., Blundell, T. L., den Dunnen, J. T., Moormann, R. J. and Schoenmakers, J. G. (1986). Structural variation in mammalian gamma-crystallins based on computer graphics analyses of human, rat and calf sequences. 1. Core packing and surface properties. Exp. Eye Res. 43, 77-92.[Medline]
Thomson, J. A., Schurtenberger, P., Thurston, G. M. and Benedek, G. B. (1987). Binary liquid phase separation and critical phenomena in a protein/water solution. Proc. Natl. Acad. Sci. USA 84,7079 -7083.[Abstract]
Tsai, C. J., Maizel, J. V., Jr and Nussinov, R.
(2002). The hydrophobic effect: a new insight from cold
denaturation and a two-state water structure. Crit. Rev. Biochem.
Mol. Biol. 37,55
-69.
van Dam, A. F. (1966). Purification and composition studies of ßs-crystallin. Exp. Eye Res. 5,255 -266.[Medline]
Vanhoudt, J., Abgar, S., Aerts, T. and Clauwaert, J. (2000). Native quaternary structure of bovine alpha-crystallin. Biochemistry 39,4483 -4492.[CrossRef][Medline]
Williams, R. C., Jr, Correia, J. J. and DeVries, A. L. (1985). Formation of microtubules at low temperature by tubulin from Antarctic fish. Biochemistry 24,2790 -2798.[Medline]
Wistow, G. (1993). Lens crystallins: gene recruitment and evolutionary dynamism. Trends Biochem Sci 18,301 -306.[CrossRef][Medline]
Wistow, G. (1995). Peptide sequences for beta-crystallins of a teleost fish. Mol. Vis 1, 1. http://www.molvis.org/molvis/vl/al/>.[Medline]
Wolken, J. J. (1995). Bird and fish eyes. In Light Detectors, Photoreceptors and Imaging Systems in Nature, vol. 1 (ed. J. J. Wolken), pp.259 . New York: Oxford University Press.
Yu, C. M., Chang, G. G., Chang, H. C. and Chiou, S. H. (2004). Cloning and characterization of a thermostable catfish alphaB-crystallin with chaperone-like activity at high temperatures. Exp. Eye Res. 79,249 -261.[CrossRef][Medline]
Zigler, J. S., Jr and Sidbury, J. B., Jr (1976). A comparative study of the beta-crystallins of four sub-mammalian species. Comp. Biochem. Physiol. 55B, 19-24.
Zigman, S. and Lerman, S. (1964). A cold precipitable protein in the lens. Nature 203,662 -663.[Medline]
Zigman, S. and Lerman, S. (1965). Properties of a cold-precipitable protein fraction in the lens. Exp. Eye Res. 159,24 -30.[Medline]
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