Molecular Chaperone-like Properties of an Unfolded Protein,
s-Casein*
Jaya
Bhattacharyya
and
Kali P.
Das§
From the Protein Chemistry Laboratory, Department of Chemistry,
Bose Institute, Calcutta-700 009, India
 |
ABSTRACT |
All molecular chaperones known to date are well
organized, folded protein molecules whose three-dimensional structure
are believed to play a key role in the mechanism of substrate
recognition and subsequent assistance to folding. A common feature of
all protein and nonprotein molecular chaperones is the propensity to
form aggregates very similar to the micellar aggregates. In this paper
we show that
s-casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in
nature as a micellar aggregate, can prevent a variety of unrelated
proteins/enzymes against thermal-, chemical-, or light-induced
aggregation. It also prevents aggregation of its natural substrates,
the whey proteins.
s-Casein interacts with partially
unfolded proteins through its solvent-exposed hydrophobic surfaces. The
absence of disulfide bridge or free thiol groups in its sequence plays
important role in preventing thermal aggregation of whey proteins
caused by thiol-disulfide interchange reactions. Our results indicate
that
s-casein not only prevents the formation of huge
insoluble aggregates but it can also inhibit accumulation of soluble
aggregates of appreciable size. Unlike other molecular chaperones, this
protein can solubilize hydrophobically aggregated proteins. This
protein seems to have some characteristics of cold shock protein, and
its chaperone-like activity increases with decrease of temperature.
 |
INTRODUCTION |
From the time of synthesis through their entire lifetime in the
cell, proteins are under constant threat to structural destabilization because of misfolding, stress, or other unfavorable interactions. Molecular chaperones recognize these unstable nonnative conformers of
proteins and bind to them instantly, preventing their aggregation in
the cell (1-8). Molecular chaperones comprise several structurally unrelated protein families and assist not only in folding of other proteins but also in subcellular transport, oligomeric assembly, and
degradation of undesirable proteins (1-2, 4, 7-8). It is not
understood at present whether the chaperone function of a protein is
because of the presence of a particular sequence or three-dimensional
structure, although the role of higher levels of organization of GroEL,
TriC,
-crystallin, etc. have been emphasized for their chaperone
function (9-12). Although no common sequence has been identified among
chaperones of different family of proteins, some common features
emerges. Chaperones have distinct hydrophilic and hydrophobic domains
to enhance solubility and to bind lipophilic molecules (13-15). Many
of them have a characteristic micelle-like-associated structure. GroEL
exits as an associated 14-mer (7, 9). TriC has a ring-like structure of
8 to 9 subunits (10). Tubulin, reported recently by us (16) to act like
a chaperone, associates to form microtubules.
-Crystallin, which
belongs to small heat shock protein (sHSP) family (17), has been
proposed to have a micellar architecture (11, 12). Even nonprotein
biological molecules such as ribosomal RNA (18, 19) and phospholipid (20), which can form micelle-type aggregates, were shown to function as
molecular chaperones.
It is long known that casein in bovine skim milk remains as large
(40-300 nm) stable micelles (21). Bovine milk contains about 78%
casein, of which 65% is
-casein. More than 75% of
-casein is
Ca+2-sensitive and called
s-casein (22). The
rest is Ca+2-insensitive and is mainly
-casein.
s-Casein has a molecular mass of 23.6 kDa (23). It is
present in the milk of all mammals as a random coil protein (24, 25)
and is the major protein constituent of casein micelle (21). In the
absence of Ca+2 ions, it is a highly soluble protein.
Despite its unorganized secondary and tertiary structure, there are
similarities between
s-casein and other known chaperones
in their tendency to self-associate into micelle-like aggregate. This
prompted us to test if
s-casein possessed any
chaperone-like behavior. In this paper we report for the first time
that a random coil protein
s-casein can prevent in
vitro the thermal aggregation of whey proteins from milk as well
as the aggregation of a variety of unrelated proteins/enzymes caused by
thermal-, chemical-, and light-induced stress. We also show that unlike
other chaperones,
s-casein can solubilize
hydrophobically aggregated proteins and possesses some features similar
to the cold shock proteins
(CSP).1
 |
EXPERIMENTAL PROCEDURES |
Materials--
s-Casein, bovine serum albumin
(BSA), rhodanese, insulin, carbonic anhydrase, alcohol dehydrogenase
from equine lever, and citrate synthase were purchased from Sigma. Whey
protein isolate (WPI) was obtained from Le Sueur Isolates (Le Sueur,
MN). Dithiothreitol (DTT) was purchased from Sisco Research Laboratory,
India. Reagents used for SDS-polyacrylamide gel electrophoresis were
from Fisher. All reagents used for making buffer solutions were of
analytical grade.
Preparation of
- and
-Crystallin--
Freshly excised
bovine eyes were obtained from a local slaughterhouse. The lenses were
surgically removed and homogenized in 10 mM Tris-HCl
buffer, pH 8.0, containing 0.1 M NaCl and 0.02% (w/v)
NaN3. The homogenate was centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was then loaded to
a Sephacryl S-300 column (1.5 cm × 90 cm). Five distinct peaks
corresponding to high molecular weight
(
H-), low
molecular weight
(
L-), high molecular weight
(
H-), low molecular weight
(
L-), and
-crystallin were obtained. Pooled fractions were stored at
20 °C until use. Only
L- and
-crystallin were
used in the present study.
Assay of Aggregation of Protein--
Thermally induced
aggregation of proteins was measured in a Shimadzu UV-2401PC
spectrophotometer fitted with thermostatic cell holder assembly with
electronic temperature control. Protein solution and buffer with or
without
s-casein were mixed in the cuvette at room
temperature and then placed in the thermostatic cell holder, and the
apparent absorbance at 400 nm was monitored as a function of time.
Aggregation of insulin was assayed as described by us earlier (26).
Briefly, insulin was dissolved in a minimum volume of 0.02 M NaOH and immediately diluted in 10 mM
phosphate buffer, pH 7.0. The aggregation of insulin B-chain was
initiated by adding to insulin solution (0.35 mg/ml) DTT (from a 500 mM stock solution) to a final concentration of 20 mM. The extent of aggregation was followed by measuring
apparent absorbance because of light scattering at 400 nm. The
ultraviolet light-induced aggregation of
-crystallin (27, 28) was
followed in a Hitachi F-4500 spectrofluorometer by setting both
excitation and emission wavelengths at 295 nm with excitation and
emission slits of 10 and 5 mm, respectively.
Gel Filtration--
The gel filtration of the samples was
performed using a prepacked column (1 × 30 cm) of Suparose 12 attached to a fast protein liquid chromatography system of Amersham
Pharmacia Biotech. Protein samples with or without
s-casein were first heated at the given temperature and
immediately put on ice to bring the temperature to 25 °C. The
solution was filtered through a 0.22-µm filter before 100 µl of it
was loaded onto the column equilibrated with 20 mM sodium
phosphate buffer of pH 7.0. Elution was carried out at a flow rate of
1.0 ml/min. Samples were monitored by their absorbance at 280 nm.
 |
RESULTS AND DISCUSSION |
Prevention of thermal aggregation of substrate proteins by
molecular chaperones is a commonly used method for in vitro
assay of their activity. When solutions of substrate proteins such as alcohol dehydrogenase, carbonic anhydrase, and
-crystallin are heated at 40, 60, and 60 °C, respectively, the solutions get turbid because of the formation of large aggregates. Fig.
1 shows the kinetic traces of the
apparent absorbance at 400 nm of these proteins in the presence and
absence of
s-casein. In the absence of
s-casein, the substrates at the respective temperatures
undergo denaturation followed by aggregation. However in the presence
of
s-casein, aggregation was suppressed. Approximately
92% protection was found at a 1:1 (w/w) ratio of alcohol
dehydrogenase:
s-casein (Fig. 1A). Complete
protection occurred at a 1:1.5 (w/w) ratio of alcohol dehydrogenase:
s-casein, corresponding to a mole ratio of
1:4. In the case of
-crystallin (Fig. 1B), complete
suppression of aggregation required a
-crystallin:casein weight
ratio 1:0.3 (1:0.6 molar ratio). At 1:3.5 weight ratio between carbonic
anhydrase and
s-casein, 90% protection was obtained
(Fig. 1C), and complete protection required approximately a
1:5 molar ratio (data not shown).
s-Casein was also
effective in preventing thermal aggregation of a number of other
proteins, including citrate synthase,
-crystallin, and rhodanese
(data not shown).

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Fig. 1.
Prevention of thermal aggregation of proteins
by s-casein. A,
aggregation assay of alcohol dehydrogenase (0.4 mg/ml, 10 mM phosphate buffer, pH 7.0, 40 °C). Curve a,
only alcohol dehydrogenase; curve b, plus 0.1 mg/ml
s-casein. B, aggregation assay of
L-crystallin (0.2 mg/ml, 10 mM phosphate, pH
7.0, 60 °C). Curve a, only L-crystallin;
curve b, plus 0.03 mg/ml s-casein; and curve
c, plus 0.06 mg/ml s-casein. C,
aggregation assay of carbonic anhydrase (0.1 mg/ml, 10 mM
phosphate buffer, pH 7.0, 60 °C). Curve a, only carbonic
anhydrase; curve b, plus 0.35 mg/ml
s-casein.
|
|
Milk contains a number of globular proteins such as
-lactoglobulin,
-lactalbumin, BSA, etc., collectively called whey proteins, which
are highly sensitive to temperature, pH, and other conditions (22, 23,
29). WPI is a mixture of these proteins (about 20% of total milk
proteins), which remain in the milk serum after removal of casein (29).
When 0.5 mg/ml WPI solution in phosphate buffer, pH 6.6, is heated to
70 °C, the solution develops visible turbidity with time because of
aggregation (Fig. 2A, trace
1). The presence of 0.4 mg/ml
s-casein
completely prevents this aggregation (Fig. 2A, trace
2). Similarly, when 0.5 mg/ml BSA solution at pH 6.6 was
heated at 70 °C, a low level of scattering was visible because of
aggregated proteins (Fig. 2B, trace 1). The
presence of 0.5 mg/ml completely prevents the formation of scattering
BSA particles (Fig. 2B, trace 2). Our results
show that
s-casein not only prevents the aggregation of
unrelated proteins but also protects its natural substrates in
vivo against thermal aggregation. Early work by Morr and
co-workers (30, 31) also clearly demonstrated that whole casein
prevented gross heat-induced aggregation of whey proteins through
nonspecific interaction, even in calcium-containing systems.

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Fig. 2.
Prevention of thermal aggregation of whey
proteins by s-casein.
A, aggregation assay of whey protein isolate (0.5 mg/ml, 10 mM phosphate buffer, pH 6.6, 70 °C). Curve 1,
only whey protein isolate; curve 2, plus 0.4 mg/ml
s-casein. B, aggregation assay of bovine
serum albumin (0.5 mg/ml, 10 mM phosphate buffer, pH 6.6, temperature 70 °C). Curve 1, only bovine serum albumin;
curve 2, plus 0.5 mg/ml s-casein.
|
|
Many investigators (32-38) have extensively studied the thermal
aggregation properties of whey proteins. It is well known that the
major whey proteins
-lactoglobulin,
-lactalbumin, and BSA have
disulfide bridges, and the former two have free thiol groups as
well (21-23, 29). It was shown that thermal aggregation of whey
protein was caused by a combination of hydrophobic as well as
thiol-disulfide interchange reactions (34-38).
s-Casein, being a highly hydrophobic protein, interacts
instantly with the exposed hydrophobic groups of denaturing proteins,
preventing aggregation. However we feel that the lack of disulfide
bridges and free thiol groups in
s-casein sequence (39)
is another very important and unique feature that plays a significant
role in inhibiting thiol-disulfide interchange reactions. These
covalent reactions require close contact of appropriate residues, and
s-casein creates a nonreactive barrier by placing itself
between the whey proteins.
Whey proteins under various conditions are also known to form soluble
aggregates, which apparently cannot be detected by the light-scattering
technique we have employed as the aggregation assay method. To check if
s-casein can prevent formation of soluble aggregates of
appreciable size, we have employed a gel filtration assay using fast
protein liquid chromatography. Although
-lactalbumin does not
aggregate on its own on heating, it is known to form soluble aggregates
in the presence of
-lactoglobulin in the early stages of heat
treatment (36, 37). A mixture of
-lactalbumin (2 mg/ml) and
-lactoglobulin (2 mg/ml) at pH 7.0 was heated to 70 °C for 5 min
and rapidly cooled to room temperature. The sample on gel filtration
showed the presence of aggregated species of molecular mass in excess
of 300 kDa eluting at the void volume, unreacted proteins corresponding
to dimeric
-lactoglobulin (36.5 kDa), monomeric
-lactalbumin
(14.4 kDa), and some intermediate aggregates centered around molecular
masses of approximately 100-120 kDa (Fig.
3, trace 1). In the presence
of
s-casein (4 mg/ml), a considerable reduction in the
high molecular mass species was observed, and most of the proteins was
eluted in a relatively single peak centered around 60 kDa. In presence
of 6 mg/ml casein, no trace of any species of more than ~80 kDa was
observed. This clearly shows that in presence of sufficient quantity of
s-casein formation of soluble aggregates of any
appreciable size is prevented.

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Fig. 3.
Effect of
s-casein on the aggregate size of
heated mixtures of -lactalbumin (2 mg/ml)
and -lactoglobulin (2 mg/ml) in 10 mM phosphate buffer, pH 7.0. Trace 1, only
-lactalbumin and -lactoglobulin heated at 70 °C for 5 min;
trace 2, plus s-casein, (4 mg/ml); trace
3, plus s-casein (6 mg/ml). Arrows
on top show elution peaks of the standards.
|
|
For nonthermal aggregation of substrate proteins, insulin became a
popular choice for as the assay system, because reduction of disulfide
bond by DTT leads to aggregation of its B-chain at room temperature
(16, 26, 40). Like other chaperones,
s-casein also can
prevent disulfide cleavage-induced aggregation of insulin at 27 °C,
requiring a 1:0.35 weight ratio between insulin and
s-casein for complete prevention of aggregation (Fig.
4, inset). Using this assay
system, we investigated the effect of temperature on its chaperoning
efficiency. Fig. 4 shows the bar diagram of the percentage
protection of insulin aggregation with
s-casein (1:0.035
w/w ratio) at 37, 27, 22, and 18 °C. At 37, 27, and 22 °C, the
suppression of aggregation is 39, 52, and 90%, respectively, whereas
there was complete protection against insulin aggregation at 18 °C
at the same ratio. This finding was in sharp contrast to the behavior
of other known chaperones such as
-crystallin, tubulin, etc., whose
activity were generally found to increase with the increase of
temperature (16, 26, 27). Also unlike other chaperones (16, 26, 27),
preheating of
s-casein solution to 50 °C for 30 min
and cooling back to 27 °C did not alter its chaperone efficiency.

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Fig. 4.
Percentage protection of DTT-induced
aggregation of insulin B-chain with
s-casein. Insulin concentration
was 0.25 mg/ml and s-casein concentration was 0.01 mg/ml
at each temperature. The inset shows the aggregation of
insulin and its complete protection by 0.1 mg/ml
s-casein at 27C.
|
|
s-Casein can also prevent other nonthermal aggregation
of proteins as well such as those induced by UV light. The eye lens protein
-crystallin in solution on being exposed to UV light (295 nm) becomes turbid because of aggregation (27, 28). Like the
chaperone-like
-crystallin,
s-casein also can prevent
this aggregation (Fig. 5). Complete
prevention requires a 1:2 weight ratio between
-crystallin and
s-casein, corresponding to a molar ratio of
~1:0.7.

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Fig. 5.
Photo-aggregation of 0.2 mg/ml
-crystallin in absence (curve A)
and presence (curve B) of 0.4 mg/ml
s-casein. 295 nm wavelength light
from a spectrofluorometer was used to irradiate the sample, and
emission due light scattering at the same 295-nm wavelength was
measured to monitor aggregation.
|
|
It is known that molecular chaperones bind only aggregation-prone
conformers of the substrate protein but do not interact with native
proteins or proteins that have already aggregated (41). To test if
s-casein acted similarly, we started an insulin aggregation reaction by adding DTT to it, and when nearly 50% aggregation occurred, we added
s-casein to the reaction
mixture. Our results show that
s-casein not only
prevented further aggregation of insulin, but unlike other known
chaperones, it also slowly solubilized the already-aggregated insulin
(Fig. 6). It has also been observed that
GroEL can prevent aggregation of substrate proteins both on the
unfolding and refolding pathway (42, 43) (e.g. on dilution
from 6 M guanidine hydrochloride solution). However, unlike
GroEL but like
-crystallin (42),
s-casein can
effectively prevent aggregation in the unfolding pathway but fails to
prevent aggregation completely on the refolding pathway (data not
shown).

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Fig. 6.
Effect of addition of casein on partially
aggregated insulin in 10 mM phosphate buffer, pH 7.0. The kinetics of DTT-induced aggregation of 0.35 mg/ml insulin was
followed at 400 nm. 0.7 mg/ml s-casein was added at
2900 s, and kinetics were continued for 12,000 s.
|
|
We have thus identified
s-casein, which exits in nature
as an unfolded random coil protein, as having chaperone-like functions. Like all other known chaperones, it can prevent irreversible
aggregation of proteins induced by thermal as well as nonthermal stress
by providing hydrophobic surfaces to unfolding proteins. The important features responsible for its chaperone-like activity are (i) its high
hydrophobicity characterized by Bigelow's parameter of 1170 (44) along
with high estimated net negative charge of about 22 at pH 6.5 (39),
(ii) its highly flexible nature because of the relatively high amount
(8.5%) of proline residues distributed uniformly throughout the chain
(29, 39), (iii) its lack of a cystine residue in the sequence (39).
Interestingly
s-casein possesses a few features similar
to that of the CSP. Generally CSPs in bacteria have higher levels of
expression following a cold shock (45) and play a major regulatory role
in the activation (46) and physiology of adaptation to low temperature
(45). Similarly greater amounts of
s-casein are found in
mammals of the low temperature zones such as reindeer (47), moose, yak, etc. compared with mammals living in other temperature zones (21). CSPs
are known to be devoid of disulfide bonds in their sequence (48) and so
is
s-casein (39). These similarities between
s-casein and CSPs of prokaryotes might suggest that
s-casein act as a CSP in eukaryotes.
It is now recognized that molecular chaperones, all of which are known
to date, are folded protein molecules and provide guidance to newly
synthesized peptides through the folding process, and their presence is
essential for cell survival (1-3, 5-8). How the chaperones themselves
get folded, however, remains a mystery. It is too early to say if
s-casein in vivo may help fold other proteins. It has very recently been shown that cyclophilin was able to
refold properly from the unfolded state in the presence of
-casein
(43). The discovery of RNA chaperones is considered to be of vital
importance because their early appearance in the evolution is believed
to have made the transition from RNA to a protein/RNA world by rescuing
the RNA from possible kinetic trap (49). It has recently been
hypothesized that proteins in the early stages of evolution of life
were unfolded proteins, which through long evolutionary process
ultimately became folded (50). Our observation for the first time that
a commonly occurring unfolded protein
s-casein can
function as a molecular chaperone may be significant in understanding
this aspect.
 |
FOOTNOTES |
*
This work was supported by the Council of Scientific and
Industrial Research CSIR grant No. 37 (0943)/97/EMR-II (to K. P. D.)
and Department of Biotechnology (DBT), Government of India.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Research associate under the Umbrella Program of the Department of Biotechnology.
§
To whom correspondence should be addressed: Dept. of Chemistry,
Bose Institute, Main campus, 93/1 A.P.C. Rd. Calcutta-700 009, India.
Tel.: 91 33 350 6619; Fax: 91 33 350 6790; E-mail: kalipada{at}boseinst.ernet.in.
 |
ABBREVIATIONS |
The abbreviations used are:
CSP, cold shock
proteins;
BSA, bovine serum albumin;
DTT, dithiothreitol;
WPI, whey
protein isolate.
 |
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