From the
The isolated cDNAs were cloned into the pET8c vector, resulting in
pET8c
To see
whether the quaternary structure of
Tryptophan
fluorescence spectroscopy can be used to study the conformational
stability of tertiary protein structures. The insert peptide of
Our data demonstrate that the presence of 23 inserted
residues in
However, the insert peptide of
Unlike for
Our results support the idea that
Alternative splicing allows formation
of various related protein isoforms from a single gene (for a review
see
(54) ). From an evolutionary point of view, alternative
splicing might be an important mechanism to generate possibly
advantageous variants of essential gene products in the presence of the
normally functioning original gene products. It has been shown that
Although the chaperone-like activity of
Determinations were made with
and without prior heating of the proteins at 58 °C. Molecular mass
determinations were performed at least in duplicate using high
molecular mass standards as reference (see Fig. 2). Diameters were
determined by measuring at least 25 particles (see Fig. 4) using the PC
Image software package. Volume calculations are based on the assumption
that the particles are perfectly spherical.
We thank Dr. N. H. Lubsen for providing the rat lens
cDNA library. We also would like to thank Dr. M. A. M. van Boekel and
P. S. G. Overkamp for advice and technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Crystallin is a multimeric protein complex which is
constitutively expressed at high levels in the vertebrate eye lens,
where it serves a structural role, and at low levels in several
non-lenticular tissues. Like other members of the small heat shock
protein family,
-crystallin has a chaperone-like activity in
suppressing nonspecific aggregation of denaturing proteins in
vitro. Apart from the major
A- and
B-subunits,
-crystallin of rodents contains an additional minor subunit
resulting from alternative splicing,
A
-crystallin.
This polypeptide is identical to normal
A-crystallin except for an
insert peptide of 23 residues. To explore the structural and functional
consequences of this insertion, we have expressed rat
A- and
A
-crystallin in Escherichia coli. The
multimeric particles formed by
A
are larger and more
disperse than those of
A, but they are native-like and display a
similar thermostability and morphology, as revealed by gel permeation
chromatography, tryptophan fluorescence measurements, and electron
microscopy. However, as compared with
A, the
A
-particles display a diminished chaperone-like
activity in the protection of heat-induced aggregation of
-crystallin. Our experiments indicate that
A
-multimers have a 3-4-fold reduced substrate
binding capacity, which might be correlated to their increased particle
size and to a shielding of binding sites by the insert peptides. The
structure-function relationship of the natural mutant
A
-crystallin may shed light on the mechanism of
chaperone-like activity displayed by all small heat shock proteins.
-Crystallins are members of the small heat shock protein
family and are highly expressed as multimeric structural proteins in
the vertebrate eye lens
(1) . There are two types of homologous
20-kDa subunits,
A- and
B-crystallin, which are 173 and 175
amino acids in length, respectively
(2) .
A-crystallin is
the more lens-specific one, occurring only in trace amounts in some
other tissues, including brain, spleen, liver, and
retina
(3, 4, 5) . In contrast,
B-crystallin
is constitutively expressed at considerable levels in many non-lens
tissues, notably in heart and striated muscle
(6) , and is
induced in various neurodegenerative diseases and brain
tumors
(7, 8, 9, 10) .
-Crystallins
and other small heat shock proteins (hsps) have chaperone-like
properties, suppressing heat- or chemically induced aggregation of
other proteins
(11, 12) . Like for other small hsps,
increased expression of
A- and
B-crystallin make cells
thermotolerant
(13, 14) .
-Crystallins and other
small hsps normally occur as large complexes, both homo- and
heteromultimeric, with molecular masses of up to 800 kDa. The tertiary
structure of these proteins is unknown, although experimental evidence
supports the proposal by Wistow
(15) that the subunits consist
of two similarly folded domains and a short exposed and flexible
C-terminal arm (16-18). Likewise, the arrangement of subunits in
the multimeric complex is still unsolved, probably due to its
polydisperse, noncompact, and dynamic nature
(19, 20) .
Conflicting models for the quaternary structure of
-crystallin
have been postulated, including three-layered
structures
(21, 22) , a micelle-like
arrangement
(23) , a combination of these two
(24) , a
rhombododecahedric structure
(25) , and most recently a
cylindrical structure
(26) . This lack of structural information
seriously hampers the understanding of their chaperone-like behavior.
-Crystallins of rodents and some other mammals contain a minor
product resulting from alternative
splicing
(27, 28, 29, 30) . This subunit,
A
, is identical to normal
A-crystallin, except
for an insertion of 23 amino acids between residues 63 and 64, at the
junction of the two putative
domains
(28, 31, 32) . This insertion is encoded
by the optional exon 2 of the
A gene, which is only spliced into
10-20% of the mature
A mRNA
(33) . Structural analysis
has demonstrated that
A
is, next to
A and
B, an integral part of the rodent
-crystallin complex,
suggesting that the insert peptide does not seriously perturb the
tertiary or quaternary structure
(32) . It is an intriguing
question how this variant could arise and be maintained for over 70
million years in evolution
(34) and how the insertion influences
the properties of
-crystallin. Information on such a natural
insertion mutant might shed light on the structure-function relation of
-crystallins in general. We therefore report in this paper a
comparison of recombinant rat
A- and
A
-crystallin. We show that both subunits are able to
form homogeneous multimeric particles which display a similar
thermostability and morphology. Furthermore, we describe heat
protection experiments revealing that
A
homomultimers have considerably less chaperone-like activity than
A homomultimers. Our results thus indicate that, in spite of the
structural similarities between
A- and
A
-multimers, the insertion imposes considerable
restraints on the functional potential of the
A
-subunit.
Materials
Restriction enzymes, T4 DNA
polymerase, T4 polynucleotide kinase, and calf intestinal alkaline
phosphatase were from Life Technologies, Inc. Ampicillin,
chloramphenicol, isopropyl-1-thio--D-galactopyranoside,
trypsin, and protease inhibitors were obtained from Sigma.
AatII and
5-bromo-4-chloro-3-indolyl-
-D-galactoside were purchased
from Promega. [
-
P]dCTP was from Amersham
Corp. Oligonucleotides were synthesized by Eurogentec. Escherichia
coli strains used as recipients during cloning and expression
experiments were TG-1 (Amersham Corp.) and B
BL21(DE3)pLysS
(35) .
DNA Probe Preparations
In order to get DNA probes
for cDNA library screening, a Puc18 construct containing the complete
hamster A gene sequence
(29) was digested with
HindIII/BamHI and BamHI/StuI. These
double digestions resulted in a 242-base pair fragment containing exon
1 and a 553-base pair fragment containing exon 2, respectively. Both
fragments were radioactively labeled using a random primers labeling
system (Life Technologies, Inc.).
A rat lens gt11 Library Screening
gt11
library was screened using standard techniques
(36) . Briefly,
plaques were lifted in duplicate onto Hybond-N filters (Amersham
Corp.), which were denatured, neutralized, and exposed to UV light for
3 min. Replicate filters were prehybridized in 6
SSC, 0.05
Blotto, and 100 µg/ml salmon sperm DNA at 68 °C for 1
h. The duplicate filters were hybridized in the same solution with
either the ``exon 1'' probe or the ``exon 2'' probe
at 68 °C overnight. Filters were successively washed at least two
times with each 2
SSC, 0.1% SDS (5 min); 1
SSC, 0.1%
SDS (1 h); and 0.02
SSC, 0.1% SDS (1 h) at 68 °C.
Subsequently, the filters were dried and subjected to autoradiography
on Kodak XAR-5 films. Three clones positive for exon 1 (
A) and
three clones positive for both exon 1 and exon 2 (
A
)
were plaque-purified using secondary and tertiary screening procedures.
DNA Subcloning
phage lysates generated from
tertiary screening dishes were used to obtain sufficient recombinant
phage DNA
(36) . The EcoRI fragments of the cDNA
inserts were isolated from gel using an electro-elution method
(37) and ligated into the multiple cloning site of the
pGEM3Zf(+) vector (Promega). To be sure that the cloned cDNAs were
full length, 5` ends of the coding sequences were verified using the
kilobase sequencing system (Life Technologies, Inc.). To get the
required expression constructs, pGEM3Zf(+)
A and
pGEM3Zf(+)
A
were digested with
AatII/StuI, and the resulting fragments were treated
with T4 DNA polymerase to remove the AatII protruding 3`
termini. The 519- and 588-base pair fragments, containing the
A
and
A
coding sequence, respectively, were isolated
from agarose gel and ligated to a synthetic linker. This linker was
obtained by cooling down a mixture of an unphosphorylated CATGGACGT
oligonucleotide and a T4 polynucleotide kinase phosphorylated ACGTC
oligonucleotide from 75 °C to room temperature in a water bath
during 2 h. The ligation products were purified using low melting
temperature agarose gel electrophoresis
(36) and cloned into the
NcoI site of the pET8c expression vector
(35) .
Expression and Purification of Recombinant
Proteins
The A and
A
expression
constructs were transformed in the host E. coli B
BL21(DE3)pLysS. Induction, cell lysis, and fractionation were
essentially performed as described by Merck et
al.(17) . The water-soluble fraction of the
A cell
lysate was dialyzed against DEAE starting buffer (50 mM NaCl,
1 mM EDTA, 20 mM Tris/HCl, pH 7.5) and applied onto a
Fast Flow DEAE-Sepharose column (Pharmacia-LKB). A linear gradient from
50 to 500 mM NaCl at pH 7.5 was used to elute the proteins at
a linear flow rate of 0.8 cm/min at 4 °C. The peak fractions
containing
A were pooled and successively dialyzed against
demineralized water and DE52 starting buffer (6 M urea, 0.02%
-mercaptoethanol (v/v), 5 mM Tris/HCl, pH 8.0). Isolation
of pure
A was carried out by ion-exchange chromatography under
denaturing conditions on a DE52 column (Whatman)
(38) . With
regard to the purification of
A
, the DEAE-Sepharose
step used for
A was omitted. The water-insoluble fraction of the
A
cell lysate was stirred directly in DE52 starting
buffer at 4 °C for 1.5 h, centrifuged at 15,000
g for 30 min, dialyzed briefly against DE52 starting buffer, and
applied onto the DE52 column. Selected DE52 fractions of
A and
A
were pooled, dialyzed against demineralized water,
and lyophilized.
Reconstitution
For structural and functional
studies, A and
A
were refolded under identical
conditions: 6 mg of lyophilized protein was dissolved in 1 ml of
denaturing buffer (6 M urea, 0.1 M
Na
SO
, 0.02%
-mercaptoethanol (v/v), 20
mM NaP
, pH 6.9), stored for 2 h at 4 °C, and
diluted with phosphate buffer (0.1 M
Na
SO
, 20 mM NaP
, pH 6.9)
(39) to a concentration of 1 M urea and 1 mg/ml of
protein. Subsequently, the solution was dialyzed against phosphate
buffer and stored at -20 °C.
Gel Permeation Analysis
An LKB Bromma
HPLC(
)
system was used in conjunction with a
Superose 6 HR 10/30 prepacked size exclusion column (Pharmacia-LKB) for
analysis of aggregate sizes. Samples contained 100 µg of protein in
1 ml of phosphate buffer (0.1 M Na
SO
,
20 mM NaP
, pH 6.9). The mobile phase was phosphate
buffer at a flow rate of 0.5 ml/min. Absorbance was monitored at 225
nm. High molecular mass standards (Pharmacia-LKB) were used for
calibration.
Tryptophan Fluorescence
Samples containing 100
µg of protein in 1 ml of phosphate buffer (0.1 M
NaSO
, 20 mM NaP
, pH 6.9)
were centrifuged for 15 min at 15,000
g to reduce
scattering from possible insoluble particles. Fluorescence spectra were
measured as a function of temperature on a Hitachi F-3000
spectrofluorometer equipped with a four-cuvette holder accessory and a
thermostated circulating water bath. The excitation wavelength was set
to 295 nm with a 5 nm band pass. Fluorescence emission was detected
over a range of 320-380 nm with a 3 nm band pass at right angles
to the incident beam after passing through a 10-mm quartz cuvette.
Sample temperature was measured by a thermometer present in one of the
four cuvettes.
Electron Microscopy
A small drop of
-crystallin in phosphate buffer (0.1 M
Na
SO
, 20 mM NaP
, pH 6.9)
was applied onto a copper grid coated with collodion and carbon. After
5-10 min, excess liquid was removed using filter paper, and the
sample was fixated by incubation with 1% formaldehyde for 5 min. The
grid was extensively washed with 0.1% ammonium acetate and negatively
stained with 1% uranyl acetate for 5 min. Dried grids were examined in
a Jeol 1210 electron microscope at an acceleration voltage of 80 kV.
Heat Protection Assay
Various concentrations of
recombinant A- and
A
-crystallin were
preincubated in a 4
10-mm quartz cuvette for 3 min at 58
°C. A constant amount of bovine
-crystallin (234
µg in 150 µl) was added, and scattering was monitored at 360 nm
as a function of time using a Perkin-Elmer Lambda 2 UV/VIS
spectrophotometer equipped with a thermostated circulating water bath
and a thermocouple to register the sample temperature. Total volume was
1.0 ml, and all solutions were in degassed phosphate buffer (0.1
M Na
SO
, 20 mM
NaP
, pH 6.9). After 30 min of analysis, the content of the
cuvette was centrifuged at 15,000
g for 30 min,
resulting in a soluble and an insoluble fraction. Proteins in the
soluble fraction were precipitated using 72% trichloroacetic acid.
Protein precipitates obtained from both fractions were dissolved in an
equal volume of SDS-sample buffer and analyzed by SDS-PAGE,
immunoblotting, and densitometry. The reasons for us to use a
heterogeneous substrate-like
-crystallin were first
of all the natural occurrence of this protein complex in lens, and
secondly, suppressing the aggregation of
-crystallin
required relatively small amounts of highly purified recombinant
-crystallins. The reproducibility of our results was checked by
performing the assay three times with different batches of purified
A- and
A
-crystallin.
Saturation Assay
Samples containing 50 µg of
A- or
A
-crystallin in 425 µl of phosphate
buffer (0.1 M Na
SO
, 20 mM
NaP
, pH 6.9) were preincubated for 3 min at 58 °C.
Various concentrations of
-crystallin (40-200
µg in 75 µl of phosphate buffer) were added, and the mixtures
were incubated for 30 min at 58 °C. Subsequently, the mixtures were
centrifuged for 30 min at 15,000
g resulting in a
soluble and an insoluble fraction. Proteins in the soluble fraction
were precipitated using 72% trichloroacetic acid. Protein precipitates
obtained from both fractions were dissolved in an equal volume of
SDS-sample buffer and analyzed by SDS-PAGE, Western blotting, and
densitometry. The experiments were performed several times to check
reproducibility.
Miscellaneous Methods
Bovine
-crystallin was isolated from the water-soluble
fraction of calf lens cortex by gel permeation chromatography over
Ultrogel AcA-34 (Pharmacia-LKB)
(40) . Bovine
B2-crystallin
was isolated from the
-fraction following the method
described by Horwitz et al.(41) . Native rat
-crystallin was isolated from the water-soluble fraction of rat
lens using a Superose 6 HR 10/30 column (see ``Gel Permeation
Analysis''). Peptide mapping of tryptic digestions was performed
on a C-18 reverse phase column (Merck). Amino acid compositions were
determined on an LKB Alpha plus amino acid analyzer. Isoelectric
focusing was performed as described by van den Oetelaar et
al.(42) . SDS-PAGE was performed according to standard
procedures
(43) . Protein concentrations were determined in
triplicate
(44) . Immunoblot analysis was performed using a
monoclonal antiserum raised against rat
A
-crystallin
(34) at a 1:50,000 dilution. Positive bands were visualized
using an alkaline phosphatase-conjugated second antibody according to
the manufacturer's instructions (Promega). Scanning of
A-
and
A
-crystallin on immunoblot was performed using a
Bio-Rad GS-670 imaging densitometer.
Cloning, Expression, and Isolation of
A rat lens cDNA library
in A- and
A
-Subunits
gt11 was screened for
A- and
A
-crystallin using as probes two DNA fragments
isolated from a hamster
A gene construct
(29) . The first
probe contained exon 1, encoding residues 1-63 of hamster
A
crystallin, and the second one contained the optional exon 2, encoding
the 23 amino acids of the insert peptide. Screening with these probes
allowed discrimination between
A and
A
cDNAs:
clones hybridizing only to the exon 1 probe contained
A cDNA,
whereas clones hybridizing to both probes contained
A
cDNA. The ratio of identified
A
to
A
clones was about 5-10%, apparently reflecting the proportion of
both types of mature mRNA present in the rat lens. To confirm that the
isolated cDNAs were full length, we have sequenced their 5`-coding
region corresponding to exon 1 and exon 2. The obtained nucleotide
sequences showed 96% identity to the hamster sequence
(29) and
were in complete agreement with the rat protein sequence
(32) .
A and pET8c
A
expression constructs. As can
be seen from SDS-PAGE, induction of these constructs in E. coli B BL21(DE3)pLysS host cells gave expression of polypeptides up to
50% of total bacterial protein (Fig. 1A, lanes 3 and
6). The identity of the induced proteins was confirmed by
Western blotting with a monoclonal anti-rat
A
antiserum
(34) (Fig. 1B). The bacterial
lysate was divided into a water-soluble fraction and a urea-soluble
fraction. SDS-PAGE showed
A to be predominantly present in the
water-soluble fraction, whereas
A
only appeared in
the urea-soluble fraction, indicating a different behavior of
solubility or aggregation in the E. coli host cell (data not
shown). The expressed proteins were purified by denaturing
anion-exchange chromatography, resulting in 95-98% pure
A
and
A
as estimated by SDS-PAGE (Fig. 1A,
lanes 4 and 7). The primary structure of these purified
products was checked by amino acid analysis, isoelectric focusing, and
peptide mapping (data not shown). To avoid isolation and purification
artifacts, both products were refolded in urea under identical
conditions before further comparison.
Figure 1:
Bacterial expression
and purification of A and
A
-crystallin.
A, Coomassie Brilliant Blue-stained protein pattern after
SDS-PAGE. Molecular mass markers (M in kDa) are indicated.
B, corresponding Western blot probed with a monoclonal
antiserum raised against rat
A
-crystallin. Samples
are: native rat
-crystallin (lane 1, 5.2 µg in A and 1 µg in B), cell lysate of E. coli expressing
A before (lane 2) and after induction
(lane 3), purified recombinant
A (lane 4, 4
µg in A and 0.8 µg in B), cell lysate of
E. coli expressing
A
before (lane
5) and after induction (lane 6), purified recombinant
A
(lane 7, 4 µg in A and 0.8
µg in B).
Multimerization of
The refolded A and
A
subunits
A and
A
subunits were analyzed by gel permeation chromatography in order
to check whether they are able to form native-like multimeric
structures. As can be seen from Fig. 2, A and
B, the elution times for
A and
A
homomultimers, kept at 20 °C, appear to be of the same order
of magnitude as for native
-crystallin isolated from rat lens.
However,
A
displays a clearly smaller elution volume
than
A, revealing its higher molecular mass. Estimated molecular
masses of the various multimers were obtained on basis of calibration
with high molecular mass protein standards (). Although we
are aware of the limitations of this calibration method, it seems that
the estimated difference between the masses of
A and
A
cannot solely be attributed to the mass of the
insert peptide. Therefore, it may be possible that the
A- and
A
-multimers have either a dissimilar number of
subunits or a different hydrodynamic volume
(45) .
Figure 2:
The multimeric size of A and
A
with and without prior heating. Amounts of 100
µg
A and
A
in 1 ml of phosphate buffer pH
6.9 were heated for 30 min at 58 °C and compared with room
temperature controls on a Superose 6 gel permeation column. A,
elution profiles of heated and unheated
A compared with native
-crystallin isolated from rat lens. B, elution profiles
of heated and unheated
A
. Calibration was performed
using the following high molecular mass standards: blue dextran (2000
kDa, 9.1 ml), thyroglobulin (669 kDa, 12.8 ml), ferritin (400 kDa, 15.1
ml), and catalase (232 kDa, 16.3 ml).
Thermostability of
From a structural point of view it is
interesting to know whether the insert peptide of A and
A
Homomultimers
A
may interfere with its heat stability. In order to investigate
the thermal properties of
A and
A
we used gel
permeation analysis and tryptophan fluorescence measurements.
A and
A
undergoes irreversible alteration at high temperatures, we
incubated both proteins for 30 min at 58 °C and analyzed their
multimeric size on an HPLC gel permeation column. As can be seen from
Fig. 2
and , exposure to heat nearly doubles the size
of
A as well as
A
compared with room
temperature controls. However, in spite of this increase, it should be
emphasized that both
A and
A
remain soluble.
Some other studies have revealed the same behavior for native
-crystallin
(46, 47) , but there are also conditions
known which preserve the complex size upon heating (48).
A
contains a tryptophan (position 69) in addition to
the single one present in the N-terminal domain of
A (position
9)
(32) . Fig. 3shows the fluorescence emission maximum
(
) of
A-,
A
-, and
B2-crystallin (positive control) as a function of temperature. The
for
A and
A
remains
essentially unchanged, whereas for
B2 a red shift from 333 to 348
nm is observed at about 55 °C. The sigmoidal transition found for
B2 agrees with previous results
(49) and indicates a
conformational change of the tertiary structure. In general, the
for tryptophan residues can vary from about 330 nm
(completely buried) to about 353 nm (completely exposed)
(50) .
Therefore, the emission of
A and
A
at 337 nm
suggests all tryptophan residues of these subunits to be partially
buried. Importantly, the relative constancy of this emission upon
heating, which was found earlier for native
-crystallin
(49) , reveals that at least the N-terminal
domains of
A and
A
, and the insertion of
A
, retain their local conformation at high
temperatures.
Figure 3:
Tryptophan emission maximum of A,
A
, and
B2 as a function of temperature.
Proteins were dissolved in phosphate buffer pH 6.9 to a concentration
of 100 µg/ml (see ``Experimental Procedures''). The
excitation wavelength was set to 295 nm, and fluorescence emission
spectra were recorded at increasing temperatures over a range of
320-380 nm.
Electron Microscopy
Electron microscopical
analysis after negative staining (Fig. 4) reveals that
recombinant A and
A
as well as freshly purified
native rat
-crystallin form similar more or less globular
particles. Although these kind of spherical structures have been
observed for many years (2, 51, 52), some recent reports indicate that
-crystallin can also display a torus-like
morphology
(5, 53) . The reason for this variation is
unclear. The
A
-particles have a larger diameter than
the
A-particles (). The
A/
A
spherical volume ratio, calculated from the measured diameters,
is in agreement with the molecular mass ratio as determined by gel
permeation chromatography (). Heating
A and
A
at 58 °C does not result in formation of
differently shaped particles (data not shown). However, the electron
micrographs of these heated samples do show a clear increase of the
particle diameters corresponding to our gel permeation data.
Figure 4:
Electron micrographs of A,
A
, and native rat
-crystallin. Samples of
A (A),
A
(B), and freshly
purified native
-crystallin (C) were negatively stained
and shown at a magnification of
200,000. The bars are
50 nm.
Chaperone Activity of
Using an in vitro assay, it has
been shown that A and
A
Homomultimers
-crystallin from lens as well as reconstituted
A and
B homomultimers can prevent thermal aggregation of
other proteins
(11) . To determine whether the insert peptide of
A
influences this chaperone-like activity, we
performed heat protection experiments with bovine
-crystallin as a substrate. Fig. 5, A and B, show how the typical aggregation of
at 58 °C (curve 1) is influenced by the presence of
increasing amounts of
A and
A
, respectively
(curves 2-6). It is immediately clear that
A
displays a greatly reduced and altered chaperoning
behavior. As can be seen from Fig. 5A, aggregation of
can be prevented almost completely when the
A
to
mass ratio is increased to 86%. In contrast,
Fig. 5B shows that an
A
to
mass ratio of up to 94% indeed delays aggregation,
but eventually even leads to an increase of the final level of
aggregation.
Figure 5:
Ability of increasing amounts of A
and
A
to prevent heat-induced aggregation of
-crystallin. A, aggregation curves of 234
µg of
in the presence of increasing amounts of
recombinant
A-crystallin: curve 1, none; curve
2, 22 µg (
A to
mass ratio =
9.6%); curve 3, 67 µg (29%); curve 4, 90 µg
(38%); curve 5, 135 µg (58%); curve 6, 202 µg
(86%). Curve 7 shows the behavior of 202 µg of
A
without
. B, aggregation curves of 234
µg of
in the presence of increasing amounts of
recombinant
A
-crystallin: curve 1, none;
curve 2, 25 µg (
A
to
mass ratio = 10%); curve 3, 74 µg (31%);
curve 4, 98 µg (42%); curve 5, 147 µg (63%);
curve 6, 221 µg (94%). Curve 7 shows the behavior
of 221 µg
A
without
. All
protein concentrations were determined in triplicate using the Bradford
assay. The proteins were dissolved in a total volume of 1 ml of
phosphate buffer, pH 6.9 (see ``Experimental Procedures'')
and the incubation temperature was 58 ± 1 °C. The dashed
lines represent the average of an irregular pattern caused by
large insoluble aggregates disturbing the light path of the incident
beam.
To determine the nature of the obtained aggregates, the
heat protection samples were centrifuged at the end of the experiment
and soluble and insoluble fractions were analyzed by SDS-PAGE
(Fig. 6, A and C). As can be seen from
Fig. 6C, the A
band coincides with
the lower
-crystallin bands. Therefore, we have quantified
relative amounts of
A and
A
using Western
blotting followed by densitometry (Fig. 6, B and
D). Fig. 6B shows
A to shift from the
insoluble to the soluble fraction when its concentration is increased,
whereas Fig. 6D shows
A
to remain in
the insoluble fraction. This different behavior must be the result of
interaction with
-crystallin because neither
A
nor
A
precipitate significantly when incubated at 58
°C without a denaturing substrate (Fig. 5, curve 7).
Therefore, the data presented in Fig. 5and Fig. 6indicate
that
A
has a much lower capacity than
A to
remain soluble when binding unfolding
-chains. To confirm this
observation, we heated
A and
A
with increasing
amounts of
-crystallin and tried to make a rough
estimate at which
-concentration
A and
A
become insoluble using SDS-PAGE, Western blotting,
and densitometry (Fig. 7). As can be seen from Fig. 7,
B and D,
A
precipitates at a much
lower molecular mass ratio than
A. Taking into account the
difference in molecular mass between
A and
A
,
we estimate that
A
has a three to four times lower
substrate binding capacity than
A.
Figure 6:
Insolubilization of A and
A
during the heat protection assay as a function of
the
A
to
mass ratio. The heat
protection samples of which the aggregation profiles are shown in Fig.
5 were centrifuged and soluble and insoluble fractions were subjected
to SDS-PAGE and densitometric Western blot analysis. Left:
Coomassie Brilliant Blue-stained SDS-gels of
incubated with
A (A) and
A
(C). The numbers refer to
A
to
mass ratios. Native
-crystallin
(
) and heat-exposed
A
(
A
) are shown as references.
Right, volume analysis of corresponding immunoblots to
quantify relative amounts of
A (B) and
A
(D) present in the soluble and insoluble
fraction.
Figure 7:
Insolubilization of A and
A
with increasing amounts of
-crystallin. Amounts of 50 µg of
A and
A
in 500 µl of phosphate buffer, pH 6.9, were
incubated with increasing amounts of
for 30 min at
58 °C. The reaction mixtures were centrifuged and soluble, and
insoluble fractions were subjected to SDS-PAGE and densitometric
Western blot analysis. Left: Coomassie Brilliant Blue-stained
SDS-gels of
A (A) and
A
(C)
incubated with
. The numbers refer to the
to
A
mass ratios. Native
-crystallin (
) is shown as a reference. Please
note that
A
in C, bottom row,
coincides with the lower
-crystallin bands in the soluble
fraction. Right: volume analysis of corresponding immunoblots
to quantify relative amounts of
A (B) and
A
(D) present in the soluble and insoluble
fraction.
A
-crystallin does not notably influence
its multimerization, heat stability, and particle morphology. The
A
complexes are larger than those of
A and
appear to be somewhat more disperse. Furthermore, the size of both the
A and
A
complexes increases approximately
2-fold at 58 °C.
A
has a major impact on its chaperoning behavior. From the combined
data presented in Figs. 5-7, we can deduce the following sequence
of events for the interaction of normal
A with
during thermal denaturation. We assume that denaturing of the
subunits of
-crystallin is a stochastic process, so
they do not unfold all at once. The first unfolding
-chains will
noncovalently bind to an
A-particle forming an unsaturated
A-
complex. The formation of this soluble complex prevents
the unfolded
-chains from aggregation between themselves,
explaining the delay of aggregation shown in Fig. 5A (curves 2-6): the more
A-particles are present
the more soluble
A-
complexes can be formed and the longer
aggregation of
is delayed. However, at a certain
moment the
A-particles become saturated with unfolding
-chains, and further interaction with more unfolding
-chains
leads to insolubilization. At this stage, also precipitation of
-chains will occur, because there are no unsaturated
A-
complexes left to prevent their aggregation. Therefore, oversaturated
A-
complexes as well as insoluble
-aggregates are
probably responsible for the aggregation profiles shown in
Fig. 5A. When the
A to
mass
ratio is increased to 86% (Fig. 5A, curve 6), the level
of oversaturation is not reached, which makes
A to remain almost
totally in the soluble fraction (Fig. 6B). At this ratio
there is apparently an excess of
A-particles for which the amount
of unfolding
-chains is insufficient to form insoluble
oversaturated particles. The above postulated mechanism of
oversaturation for our recombinant
A, largely corresponds with
that recently proposed for bovine
-crystallin by Wang and
Spector
(48) .
A, increase of the
A
to
mass ratio enlarges the
final level of aggregation (Fig. 5B) and does not
promote a shift of
A
to the soluble fraction
(Fig. 6D). Apparently, an excess of
A
, necessary to prevent aggregation, is not reached
under our conditions. On the contrary, addition of
A
contributes to the formation of insoluble particles, because even
high concentrations of
A
precipitate as a result of
oversaturation. Therefore, it seems that
A
has a
lower capacity to bind unfolding
-chains than
A. Titration of
A and
A
with increasing
amounts of
-crystallin (Fig. 7) indeed
confirms this conclusion because
A
precipitates at a
significantly lower
-concentration than
A.
-crystallin complexes have a
more or less defined number of binding sites for denaturing proteins.
As long as these sites are available, the complex can prevent
aggregation by acting as a scavenger for hydrophobic surfaces presented
by unfolding proteins. Applying these concepts to native-like
A
-multimers, it appears that the increased complex
size of
A
, with a concomitant relative decrease of
accessible surface area, may already account to some extent for its
reduced substrate binding capacity. In addition it can be envisaged
that the insert peptides interfere with the accessibility of at least
some of the potential binding sites. Such an intervention might be
caused by steric hindrance or by an actual interaction of hydrophobic
insert residues with the binding sites. Alternatively, the insert
peptides might induce a subtle conformational change, which is
unfavorable for the substrate binding capacity but not for
multimerization and stability.
A
is present in various unrelated mammalian groups,
suggesting that it has independently disappeared in most mammalian
orders after radiation from a common
A
-expressing
ancestor
(30, 34) . The question then is why
A
has been silenced again in most groups whereas in
others, like the rodents, it has been retained for at least 70 million
years. It appears that
A
, in spite of its insertion,
still is a viable structural lens protein because it is very
thermostable and it can form native-like multimers. Furthermore, it
should be realized that
A
is only a minor component
(less than 20%) of native
-crystallin complexes in all
investigated species where it occurs. This might be expected to result
in a proportionally small decrease in the chaperone-like capacity of
the complex. We observed indeed that heteromultimeric complexes,
reconstituted from equal amounts of
A- and
A
-subunits, display an intermediate chaperoning
behavior as compared with the respective homomultimers (data not
shown). The fact that
A
has not become the sole or
major
A-like subunit in any species may suggest that high levels
of expression of
A
are indeed selectively
disadvantageous. However, as long as the proportion of
A
is small, there is apparently a sufficiently great excess of
chaperoning capacity in the lens so that a small decrease can be
tolerated. Low levels of expression of
A
are thus
likely to be a selectively neutral character, which may disappear
without adverse effects by fortuitous mutations, like in
human
(55) , but can also be maintained for tens of millions of
years, as has been demonstrated for other redundant gene
products
(56) .
-crystallin and small hsps currently is a major issue in the field
of protein folding and interaction
(57) , insight into the mode
of interaction with denaturing substrate proteins, is still very
limited. This is a direct consequence of our poor knowledge of the
arrangement of subunits in the multimeric complex of these proteins,
making it difficult to design straightforward ``interaction
experiments.'' Therefore, studying the structural localization and
functional influence of the large insertion in the unique
A
mutant might provide some general clues about the
chaperone-like mechanism displayed by all members of the small heat
shock protein and
-crystallin family.
Table:
Size parameters of A and
A
with and without prior heating as determined by gel permeation
chromatography and electron microscopy
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.