From the
Plants synthesize several classes of small heat shock proteins
ranging in size from 15 to 30 kDa. Two conserved classes, designated
class I and class II, are localized to the cytosol. Recombinant HSP18.1
and HSP17.7, representing class I and class II proteins from pea,
respectively, were expressed in Escherichia coli and purified.
Non-denaturing polyacrylamide gel electrophoresis and electron
microscopy demonstrated that the purified proteins formed discretely
sized, high molecular weight complexes. Sedimentation equilibrium
analytical ultracentrifugation revealed that the HSP18.1 and HSP17.7
complexes were composed of approximately 12 subunits. Both proteins
were able to enhance the refolding of chemically denatured citrate
synthase and lactate dehydrogenase at stoichiometric levels in an
ATP-independent manner. Furthermore, HSP18.1 and HSP17.7 prevented
aggregation of citrate synthase at 45 °C and irreversible
inactivation of citrate synthase at 38 °C. HSP18.1 also suppressed
aggregation of lactate dehydrogenase at 55 °C. These findings
demonstrate that HSP18.1 and HSP17.7 can function as molecular
chaperones in vitro.
Plants synthesize numerous small heat shock proteins
(smHSPs)
Little is known about the cellular
function of smHSPs, although evidence from mammalian and Drosophila systems supports the theory that smHSPs play a role in
thermotolerance
(6, 7, 8) . Recently, it has
been demonstrated that mammalian smHSPs and the related
Small HSPs from diverse
organisms typically form multimeric complexes ranging in size from 200
to 800 kDa
(1, 4) . The complexes from plant sources are
small and uniform, from 200 to 240 kDa
(5, 16, 17) . Mammalian smHSPs generally form
larger complexes from 500 to 800 kDa
(18, 19) , but
their oligomeric structure appears to be dynamic in response to
phosphorylation
(20, 21) . Consequently, the actual size
of mammalian smHSP complexes has been a matter of controversy.
Phosphorylation of plant smHSPs, however, has not been observed
(22) .
Based on sequence alignments, all plants synthesize
two distinct classes of cytosolic smHSPs, designated class I and class
II
(1) . HSP18.1, a class I protein, and HSP17.7, a class II
protein, both from Pisum sativum (pea), share 60% amino acid
similarity and 38% identity
(15) . These two proteins, with
derived molecular masses of 18,086 and 17,732 Da for HSP18.1 and
HSP17.7, respectively, are normally absent from vegetative tissues in
plants unless induced by heat at temperatures above 30 °C
(15) . Maximum induction of HSP18.1 occurs at 38 °C, during
which class I smHSP levels can reach up to 1% of the total protein in
roots and somewhat lower levels in leaves
(15) . Following heat
stress, accumulated HSP18.1 remains stable with a half-life of 38 h
(15) and may therefore function during both heat stress and
recovery. HSP18.1 and HSP17.7 share marginal overall sequence homology
with mammalian smHSPs. For example, HSP18.1 shares only 25% identity
with human HSP27 at the amino acid level
(23) . However, like
all smHSPs and
HSP18.1
and HSP17.7 expression is not strictly limited to heat stress
conditions. Both proteins have been identified in developing embryos at
levels roughly equivalent to those observed in moderately heat-stressed
tissues
(24) . Cytosolic smHSPs have also been shown to be
expressed during pollen development
(25, 26) , and
corresponding mRNAs have been identified in ripening fruit
(27) . How these other patterns of smHSP expression are related
to their presence during and after heat stress is not understood, but
developmentally regulated patterns of smHSP expression have also been
observed in Drosophila and mammalian cells (reviewed in Ref.
4).
Because of the abundance and diversity of smHSPs in plants, we
have been particularly interested in understanding and comparing the
structural and functional properties of plant smHSPs. Here, we have
characterized recombinant HSP18.1, a class I smHSP, and HSP17.7, a
class II smHSP, with regard to oligomeric structure and in vitro molecular chaperone activity.
Structure of HSP18.1 and HSP17.7
Recombinant smHSPs were purified from E. coli cells to
greater than 95% homogeneity as soluble, high molecular weight
complexes (Fig. 1). One liter of cell culture yielded
approximately 6-10 mg of purified protein. Analysis of the
protein preparations by non-denaturing pore exclusion PAGE indicated
that the recombinant proteins, similar to those from heat-stressed pea
roots
(15) ,
From this and other recent studies of a variety of plant
smHSPs, it appears that smHSPs from plant sources generally form
oligomeric complexes ranging in size from 200 to 240 kDa
(4, 15, 16, 17) . Here, we have
characterized two representatives of plant cytosolic smHSPs from P.
sativum, HSP18.1 and HSP17.7. Both behave solely as oligomers
composed of 12 subunits. Since most other plant smHSPs have native
molecular sizes similar to those of HSP18.1 and HSP17.7, as judged by
non-denaturing PAGE, it is likely that other plant smHSPs are also
composed of 12 subunits. A structural model consisting of an inner core
of 12 subunits has been previously proposed for
Although HSP18.1 and HSP17.7 share common
features such as 60% amino acid similarity
(15) , similar
subunit size, and native molecular weight as determined by analytical
ultracentrifugation, their different mobilities on non-denaturing pore
exclusion PAGE suggest that other structural differences may
distinguish class I and class II smHSPs. While the relative migration
of HSP18.1 (compared to globular standards) was consistent with that
predicted by analytical ultracentrifugation, the migration of HSP17.7
was inconsistent. Based on these findings, it is likely that the shape
of the HSP17.7 complex deviates from a globular structure. This
observation would be consistent with the triangular structures seen
exclusively in HSP17.7 preparations by electron microscopy. Such a
large structural difference between HSP18.1 and HSP17.7 may account for
the fact that mixed complexes between the two species are not observed
in vivo and will not form in vitro.
Indicative of a molecular chaperone function for HSP18.1 and
HSP17.7, both proteins were able to influence the folding and
aggregation properties of model substrates during several in vitro assays. As has been previously reported for mammalian smHSPs and
The ability of HSP18.1
and HSP17.7 to prevent thermal aggregation of model substrates provides
further support for their putative role in thermotolerance. Complete
suppression of CS aggregation by HSP18.1 at 45 °C was observed at
an HSP18.1 complex-to-CS monomer ratio of 0.5:1, although HSP17.7 was
far less efficient. By comparison, similar, maximal protection by
murine HSP25 was observed at a ratio of 0.67:1
(11) . Since
under these conditions CS dimers would be expected to prevail, the true
complex-to-substrate stoichiometry would be approximately 1:1 for both
HSP18.1 and HSP25. Less efficient protection by HSP18.1 or lack of
protection by HSP17.7 imparted to LDH at 55 °C suggests that this
temperature may exceed the functional temperature range for these
smHSPs. However, the temperatures necessary to induce insolubilization
of CS (45 °C) and LDH (55 °C) exceed the temperature of maximum
smHSP synthesis in plants
(15) as well as the heat stress
temperatures generally experienced by plants under field conditions
(44) .
We sought to evaluate the protective effects of
HSP18.1 and HSP17.7 at 38 °C, the temperature at which these smHSPs
are maximally induced
(15) . By employing a more physiologically
relevant elevated temperature of 38 °C followed by a return to 22
°C, we found that both HSP18.1 and HSP17.7 displayed similar
behavior in protecting CS from irreversible heat inactivation. The
predominant effect of the smHSPs was to increase the potential of
heat-inactivated CS to properly refold at the permissive temperature of
22 °C. These findings suggest that in vivo the presence of
HSP18.1 and HSP17.7 may have important consequences during both heat
stress and recovery and are consistent with the observation that
HSP18.1 and other smHSPs remain abundant in tissues during and after
heat stress
(15) . The fact that the protective effects of the
smHSPs could only be observed if the smHSPs were present during the 38
°C incubation indicates that the smHSPs must be present during the
time of thermal denaturation when irreversible misfolding and/or
aggregation are most likely to occur. These findings are similar to
those described for heat-inactivated firefly luciferase, which can be
subsequently reactivated by incubation at room temperature in the
presence of the chaperones DnaK, DnaJ, and GrpE
(45) . At
minimum, the presence of DnaJ is required during thermal inactivation
to prevent aggregation of luciferase, whereas reactivation requires the
addition of DnaK and GrpE.
Unlike the HSP60, HSP70, and TriC classes
of molecular chaperones
(12) , the in vitro smHSP
functional cycle does not require ATP or other factors that regulate
the release of substrates bound to the chaperone. Thus, it is probable
that smHSPs recognize non-native substrates but bind them reversibly.
By this mechanism, smHSPs may lower the effective concentration of
free, unfolded intermediates and reduce off-pathway processes such as
aggregation or misfolding. Pools of free substrates could then
spontaneously refold under permissive conditions. This relatively
passive mechanism, which requires neither an energy source nor
coordination with other proteins, may explain why refolding of
chemically denatured CS is relatively less efficient in the presence of
the smHSPs than in the presence of GroEL, GroES, and ATP. For example,
up to 80% CS reactivation has been observed in the presence of
saturating amounts (6-fold molar excess) of the GroE system
(35) , compared with 45% in the presence of saturating
(stoichiometric) amounts of smHSPs. However, it is highly unlikely that
the enhancements in refolding of chemically denatured CS or LDH
observed in the present study resulted from nonspecific solute effects
since excesses of the control proteins catalase or lysozyme did not
stimulate refolding. These results imply that the smHSPs were not
simply reducing the number of molecular collisions between
aggregation-prone folding intermediates.
The failure of the smHSPs
to facilitate reactivation of CS incubated at 45 °C may be
indicative of irreversible CS misfolding that cannot be prevented even
by association with the smHSPs at this higher temperature. Under such
conditions, however, smHSP-promoted solubilization of permanently
damaged proteins, like that observed with CS in the presence of
HSP18.1, may facilitate degradation by proteolytic systems. Such a role
has been proposed for other molecular chaperones
(46, 47) .
The in vivo function of HSP18.1
and HSP17.7 is not understood at present; nor have any endogenous
substrates of these smHSPs been identified. The present study suggests
that both HSP18.1 and HSP17.7 can recognize and interact with different
foreign proteins that possess non-native structures as the common
feature. In this light, HSP18.1 and HSP17.7 may function as nonspecific
chaperones. However, in vivo, more specific protein-protein
interactions may occur and may be highly specific for either class I or
class II proteins. Such specialization may account for the strong
evolutionary conservation of these two classes of cytosolic smHSPs in
higher plants.
We thank Jörg Thierfelder for providing the
Pshsp18.1 and Pshsp17.7 cDNAs subcloned into pJC20 and Dr. Paul
Matsudaira for performing the electron microscopic analysis of the
smHSPs. We also thank James Grille for expert photographic assistance
and Dr. Katherine Osteryoung for critical reading of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
ranging in size from 15 to 30 kDa that
are related to the evolutionarily conserved family of smHSPs and
-crystallins of the vertebrate eye lens
(1, 2) . In
contrast to mammalian smHSPs, plant smHSPs constitute the most abundant
and diverse group of proteins synthesized in response to heat stress.
For example, more than 25 individual smHSP polypeptides can be observed
by two-dimensional PAGE from a variety of heat-stressed plants
(3) . Only one species of smHSP has been identified in human,
mouse, and yeast, and each is typically present in the cytosol
(4) . At least four major classes of plant smHSPs have been
identified based on sequence alignments and immunological
cross-reactivity; two are found in the cytosol, one is localized to the
chloroplast, and another is localized to the endoplasmic reticulum
(1) . Recently, a plant smHSP localized to mitochondria has also
been identified
(5) .
-crystallins possess molecular chaperone activity in vitro(9, 10, 11) . In contrast to the
ATP-dependent molecular chaperones of the HSP60 and HSP70 classes
(12) , the smHSPs were able to refold chemically denatured
proteins in an ATP-independent manner
(9, 10, 11) . More recent data have suggested
that mammalian smHSPs may regulate actin filament dynamics
(7, 13, 14) .
-crystallins, the regions of greatest homology
occur in the C-terminal halves of the proteins
(23) .
Materials
Pig heart citrate synthase, bovine
muscle lactate dehydrogenase, bovine liver catalase, bovine IgG, Tris,
and HEPES were obtained from Sigma. Ultrapure
(NH)
SO
and ultrapure urea were
obtained from Baker. Ultrapure guanidine hydrochloride was obtained
from Life Technologies, Inc.
Construction of Expression Vectors
The Pshsp18.1
and Pshsp17.7 cDNAs
(15, 28) , which encode pea HSP18.1
and HSP17.7, respectively, were subcloned into the expression vector
pJC20
(29) as follows. The EcoRI fragment of Pshsp18.1
was end-filled with Klenow fragment and inserted by blunt-end ligation
into the BamHI site of pJC20 that had also been end-filled.
The SmaI/ ApaI fragment of Pshsp17.7 was inserted into
the similarly prepared pJC20. To eliminate N-terminal fusion proteins
encoded by these pJC20 derivatives, polymerase chain reaction
mutagenesis was used to introduce NdeI sites at the
translation start sites of the Pshsp18.1 and Pshsp17.7 cDNAs. The PCR
products were subcloned into vector pCR using the TA cloning system
(Invitrogen). The NdeI/ NcoI fragment from the
modified Pshsp18.1 cDNA and the NdeI fragment from the
modified Pshsp17.7 cDNA were exchanged for the corresponding wild-type
fragments in the pJC20 derivatives to yield pAZ316 and pAZ317,
respectively. The engineered mutations were confirmed by dideoxy
sequencing
(30) of the regions derived from the polymerase
chain reaction.
Expression and Purification of Recombinant HSP18.1 and
HSP17.7
Escherichia coli BL21(DE3) cells were
transformed with pAZ316 or pAZ317 for T7 RNA polymerase-directed
expression of the target proteins
(31) . Exponentially growing
cells were induced with 1 mM isopropyl
-D-thiogalactopyranoside for 6 h and then harvested.
Cells were washed with 50 mM Tris-HCl, 1 mM EDTA, pH
7.5 (buffer A), broken by sonic lysis, and centrifuged at 17,500
g for 30 min to yield soluble crude extracts. Crude
extracts were then fractionated by ammonium sulfate precipitation. The
60-95% (for HSP18.1) and 40-70% (for HSP17.7) ammonium
sulfate fractions were dialyzed against 25 mM Tris-HCl, 1
mM EDTA, pH 7.5 (buffer B) and separated on 0.2-0.8
M linear sucrose gradients in buffer B for 2 h, 45 min at
50,000 rpm in a Beckman VTi50 rotor. Fractions containing the smHSPs
were pooled and applied to a diethylaminoethyl-Sepharose Fast Flow
column equilibrated with buffer B supplemented with 3 M urea.
Small HSPs were eluted with a 0-0.2 M NaCl gradient in
buffer B supplemented with 3 M urea and then dialyzed
extensively against buffer B. SDS-PAGE indicated that the smHSP
preparations were at least 95% pure (see Fig. 1).
Figure 1:
Polyacrylamide gel electrophoresis of
purified HSP18.1 and HSP17.7 in the presence ( A) or absence
( B) of SDS. In panelA, 3 µg of
protein/ lane was run on a 12.5% acrylamide gel. Lane1, HSP18.1; lane2, HSP17.7. In
panelB, 7 µg of protein/ lane was run
for 48 h on a 4-22.5% gel. Lane1, HSP18.1;
lane2, HSP17.7. Both gels were stained with
Coomassie Blue. The positions of markers with molecular sizes expressed
in kDa are indicated in the
margins.
Polyacrylamide Gel Electrophoresis
SDS-PAGE was
performed using a 12.5% acrylamide gel as described
(32) .
Non-denaturing pore exclusion PAGE was performed on a 4-22.5%
acrylamide gel according to the method of Helm et al.(15) .
Electron Microscopy
Small HSPs were applied to a
carbon film, rinsed with phosphate-buffered saline, and negatively
stained with 1% uranyl acetate. The samples were viewed in a Phillips
EM410 electron microscope operating at 80 kV. Micrographs were taken at
55,000 magnification.
Analytical Ultracentrifugation
Molecular weights
of HSP18.1 and HSP17.7 complexes were determined at 4 °C by
sedimentation equilibrium in a Beckman Optima XLA analytical
ultracentrifuge equipped with absorbance optics. A Beckman An60Ti rotor
was used with six-sector equilibrium centerpieces at speeds ranging
from 7,000 to 15,000 rpm. Centrifugation of HSP18.1 was carried out in
buffer B, and centrifugation of HSP17.7 was carried out in buffer B
supplemented with 50 mM NaCl. All samples were clarified at
40,000 g before centrifugation. Solvent densities were
calculated from buffer and protein composition, respectively, as
described by Laue et al.(33) . Multiple data sets of
A
versus radial position were
simultaneously fit to the Yphantis equation using a nonlinear least
squares fit with the program HID4000
(34) . The residuals were
examined for aggregation and/or non-ideality. Analysis of the residuals
for HSP18.1 indicated a homogenous solution, whereas analysis of the
residuals for HSP17.7 indicated aggregation in some of the samples.
These HSP17.7 data sets were deleted from the pool, and the remainder
of the data sets was reanalyzed, resulting in better residuals. The
molecular weights calculated with the edited and unedited data sets
were close to each other (less than 5% difference), but the error was
smaller with the edited sets.
Chemical Denaturation and Renaturation
Experiments
CS or LDH was denatured at a concentration of 1.5
µM in 6 M guanidine hydrochloride, 2 mM
dithiothreitol, 100 mM Tris-HCl, pH 8.0 (for CS) or pH 7.5
(for LDH) for 1.5 h at 25 °C. Refolding was initiated by diluting
2.5 µl of denatured CS or LDH into 247.5 µl of rapidly
vortexing refolding buffer in 1.5-ml microcentrifuge tubes as described
(35) . The final refolding conditions were 150 nM CS or
LDH in 100 mM Tris-HCl, pH 8.0 (for CS) or pH 7.5 (for LDH),
20 µM dithiothreitol, 2 mM
(NH)
SO
carried over from the
commercial enzyme stocks, and variable amounts of HSP18.1, HSP17.7,
catalase, or lysozyme at 25 °C. At the times indicated, 25-µl
aliquots were assayed for CS or LDH activity.
Thermal Aggregation Experiments
150 nM CS
or LDH was combined with varying amounts of HSP18.1 or HSP17.7 in 50
mM HEPES-KOH, pH 7.5 (total volume, 1 ml) in covered quartz
cuvettes at 25 °C. Where indicated, catalase or IgG was added at
the concentrations indicated. Samples were incubated in a water bath at
45 °C (for CS) or 55 °C (for LDH) and then monitored for light
scattering at 320 nm
(36) at the times indicated.
Thermal Inactivation Experiments
150 nM
CS was incubated in the absence or presence of 150 nM HSP18.1,
HSP17.7, 32 µg/ml catalase, or 50 µg/ml lysozyme in 50
mM HEPES-KOH, pH 8.0 in covered glass tubes (total volume, 0.5
ml ) at the indicated temperatures. At various time points 25 µl
aliquots were removed and measured for CS activity.
CS and LDH Activity Assays
All assays were
performed at 25 °C. CS activity was measured according to the
method of Ochoa
(37) in which the disappearance of acetyl CoA
is monitored by the decrease in absorbance at 233 nm. LDH activity was
measured as described
(38) .
Protein Determination
The concentrations of smHSPs
and LDH were determined using the Bio-Rad protein assay with bovine
serum albumin as the standard. The concentration of CS was determined
spectrophotometrically at 280 nm
(39) . The concentrations of
smHSPs indicated in the text refer to the complexes composed of 12
subunits. Therefore, 150 nM smHSP is equivalent to
approximately 32 µg/ml. The concentrations of CS and LDH refer to
monomers.
Data Analysis
Errorbars in
figures represent standard errors generated from at least three
replicate trials.
(
)
migrated exclusively as
complexes with apparent molecular masses of 240 and 320 kDa for HSP18.1
and HSP17.7, respectively. Analysis of HSP18.1 by sedimentation
equilibrium analytical ultracentrifugation yielded a molecular mass of
216,418 ± 6,601 Da, consistent with the apparent molecular size
derived from non-denaturing PAGE. This molecular mass corresponds to
11.97 ± 0.37 HSP18.1 subunits, indicating that the native
HSP18.1 oligomer consists of 12 subunits. Similar analysis revealed
that HSP17.7 had a molecular size of 200,183 ± 8,208 Da, which
corresponds to 11.28 ± 0.46 subunits, suggesting that HSP17.7,
like HSP18.1, may also be composed of 12 subunits. Since migration of
proteins on non-denaturing pore exclusion PAGE is dependent on both
size and shape
(40) , it appears that HSP17.7 may differ
structurally from HSP18.1 by virtue of shape. Indeed, electron
microscopy of negatively stained HSP18.1 complexes at a magnification
of 55,000 suggested that HSP18.1 forms uniformly globular particles
with approximate diameters of 10 nm (Fig. 2 A). In
contrast, similar analysis of HSP17.7 complexes revealed both round and
triangular structures (Fig. 2 B), which may represent
different orientations of the complexes. In Vitro Function of HSP18.1 and HSP17.7
Figure 2:
Electron microscopy of purified HSP18.1
( A) and HSP17.7 ( B) negatively stained with 1% uranyl
acetate. Micrographs were taken at 55,000 magnification.
Bar, 100 nm.
Small HSPs Enhance Refolding of Chemically Denatured Citrate
Synthase and Lactate Dehydrogenase
Both HSP18.1 and HSP17.7
increased the folding yields of CS (homodimer of 50-kDa subunits) and
LDH (tetramer of 35-kDa subunits), which had been denatured in 6
M guanidine hydrochloride. When 150 nM HSP18.1 or 240
nM HSP17.7 was added to 150 nM denatured CS monomers
under conditions that stimulated refolding, greater than 40% (relative
to an equivalent amount of non-denatured CS) attained the native state
after 60 min (Fig. 3 A). In contrast, only 17% of the CS
activity was spontaneously reactivated in the absence of the smHSPs.
Although the smHSPs increased the yield of refolded CS, no rate
enhancements were observed in their presence. Similar results were
obtained when LDH was used as the target protein
(Fig. 3 B). As a control, bovine catalase, a protein with
a similar native molecular mass and isoelectric point (247 kDa, pI
= 5.8) to HSP18.1 (217 kDa, pI = 5.96
(15) ) was
substituted for the smHSPs. Addition of 32 µg/ml catalase, the
equivalent weight-to-volume concentration of 150 nM smHSP,
resulted in less than 18% reactivation of either CS or LDH,
demonstrating that the enhancement in refolding was specific to the
smHSPs (see Fig. 5). Adding 50 µg/ml lysozyme or further
increasing the concentration of catalase to 128 µg/ml did not
increase refolding yields (not shown).
Figure 3:
Kinetics of CS and LDH refolding. 15
µM CS ( A) or LDH ( B) was denatured in 6
M guanidine hydrochloride and then diluted 100-fold into
solutions lacking smHSPs () or supplemented with 150 nM
HSP18.1 (
) or 240 nM HSP17.7 (
). At the times
indicated, CS or LDH enzymatic activity was determined. Amounts of
reactivation are expressed as percentages relative to the activity of
an equivalent amount of non-denatured CS or
LDH.
Figure 5:
ATP or GTP does not affect smHSP-enhanced
CS or LDH refolding. CS ( A) or LDH ( B) was denatured
and allowed to refold as described in the legend to Fig. 4. Refolding
reactions were carried out in the absence of any additions
( Unassisted), in the presence of 32 µg/ml catalase, or in
the presence of 150 nM HSP18.1 or HSP17.7, as indicated. Where
indicated, reactions included 3 mM ATP or GTP supplemented
with 6 mM MgCl and 50 mM
KCl.
The refolding of CS and LDH
was next examined as a function of the amount of smHSP in the refolding
reactions. For both CS and LDH, refolding yields saturated at an smHSP
complex-to-CS or -LDH monomer ratio of approximately 1 (Fig. 4).
The observed enhancement of refolding by the smHSPs occurred in a
nucleotide triphosphate-independent manner; addition of MgATP or MgGTP
did not further stimulate refolding yields (Fig. 5).
Figure 4:
Stoichiometry of smHSP-enhanced CS or LDH
refolding. CS ( A) or LDH ( B) was denatured and
allowed to refold at a concentration of 150 nM monomers for 60
min (for CS) or 90 min (for LDH) as described in the legend to Fig. 3.
Refolding reactions were supplemented with increasing amounts of
HSP18.1 () or HSP17.7 (
). The smHSP/substrate ratio refers
to the molar ratio of smHSP complex to CS or LDH monomers in the
refolding reactions.
Since
HSP18.1 and HSP17.7 are coordinately expressed in various tissues(24) , a mixture of the two proteins was evaluated for its
ability to refold chemically denatured LDH. The amount of LDH
reactivation observed for a mixture of 30 nM HSP18.1 plus 30
nM HSP17.7 was approximately equal to that observed in the
presence of 60 nM (nonsaturating amounts according to
Fig. 4
) of either species alone (Fig. 6). These results
suggest that HSP18.1 and HSP17.7 function independently of one another
rather than synergistically in these assays.
Figure 6:
HSP18.1
and HSP17.7 do not synergistically enhance refolding of LDH. LDH was
denatured and allowed to refold at a concentration of 150 nM
as described in the legend to Fig. 4. Refolding reactions were
supplemented with 30 or 60 nM HSP18.1 or HSP17.7 as indicated.
Mixture, the addition of 30 nM HSP18.1 plus 30
nM HSP17.7 to the same refolding
reaction.
Small HSPs Prevent Thermal Aggregation of CS and
LDH
When heated to 45 °C, CS began to form insoluble
aggregates that could be detected by light scattering (Fig. 7).
Aggregation, however, could be reduced in the presence of the smHSPs
and was completely suppressed at an HSP18.1-to-CS ratio of 0.5
(Fig. 7 A). Catalase added to CS at a concentration of 32
µg/ml had no protective effect; nor did 50 µg/ml lysozyme (not
shown). HSP17.7 also prevented CS thermal aggregation but was less
effective than HSP18.1; only 65% of CS scattering could be prevented
upon addition of HSP17.7 at an HSP17.7-to-CS ratio of 1.6
(Fig. 7 B). Beyond this, increasing amounts of HSP17.7
had no further protective effect. Under these conditions, neither
HSP18.1 nor HSP17.7 alone gave rise to any detectable light scattering
(not shown).
Figure 7:
HSP18.1 and HSP17.7 protect CS from
thermal aggregation. 150 nM CS monomers were incubated at 45
°C in the absence or presence of increasing amounts of HSP18.1
( A) or HSP17.7 ( B) as indicated. Where indicated,
catalase was added at a concentration of 32 µg/ml in the absence of
smHSPs. At the times indicated, samples were monitored for their
apparent absorbance at 320 nm, which is indicative of light scattering
due to CS aggregation. Relative scattering is expressed in arbitrary
units.
Light scattering by LDH could not be detected at 45
°C but was observed when the temperature was increased to 55
°C. At this higher temperature HSP18.1 was less effective against
LDH aggregation as compared with CS aggregation at 45 °C
(Fig. 8). At a concentration of 150 nM LDH monomers, the
protective effect of HSP18.1 saturated at a concentration of 600
nM HSP18.1 complex. However, addition of 128 µg/ml bovine
IgG or lysozyme (not shown), both of which are stable at 55 °C, had
no protective effect, suggesting the level of protection by HSP18.1 was
significant. HSP17.7 aggregated itself at 55 °C, thereby preventing
further analysis (not shown).
Figure 8:
HSP18.1 protects LDH from thermal
aggregation. 150 nM LDH monomers were incubated at 55 °C
in the absence or presence of increasing amounts of HSP18.1 as
indicated. Where indicated, 128 µg/ml bovine IgG was added in the
absence of HSP18.1. Light scattering was monitored as described in the
legend to Fig. 7.
HSP18.1 and HSP17.7 Prevent Thermal Inactivation of
CS
When CS was incubated at 38 °C alone or in the presence
of 32 µg/ml catalase, less than 5% of the original CS activity
remained after 60 min (Fig. 9). Upon temperature shift of the
samples to 22 °C, less than 15% of the original CS activity
remained after 60 min in the presence or absence of 32 µg/ml
catalase. Similar results were obtained when 50 µg/ml lysozyme was
substituted for catalase (not shown). In contrast, when CS was treated
similarly but in the presence of stoichiometric amounts of HSP18.1 or
HSP17.7, CS activity was minimally protected at 38 °C, but
approximately 65-70% of the original activity was subsequently
regained after 60 min at 22 °C. However, the thermal protective
effect of the smHSPs was only observed if the smHSPs were present
during the initial high temperature incubation; addition of smHSPs at
the time of temperature shift to 22 °C did not result in CS
reactivation (Fig. 9).
Figure 9:
HSP18.1 and HSP17.7 prevent irreversible
thermal inactivation of CS at 38 °C. 150 nM CS monomers
were incubated at 38 °C in the absence () or presence of 32
µg/ml catalase (
), 150 nM HSP18.1 (
), or 150
nM HSP17.7 (
). Where indicated, samples were shifted to
22 °C.
, samples in which 150 nM CS monomers were
incubated alone for 60 min at 38 °C and then supplemented with 150
nM HSP18.1 or HSP17.7 at the time of temperature shift to 22
°C. CS enzymatic activity was determined at the times
indicated.
Although smHSPs protected CS against
irreversible inactivation at 38 °C, essentially no protection of
activity was observed at 45 °C in the presence of either HSP18.1 or
HSP17.7 (Fig. 10) despite the fact that HSP18.1 efficiently
suppressed CS aggregation at this temperature (Fig. 7).
Furthermore, in the presence or absence of the smHSPs, no CS
reactivation could be detected after shifting the samples from 45 to 22
°C (not shown).
Figure 10:
HSP18.1 and HSP17.7 do not prevent
thermal inactivation of CS at 45 °C. 150 nM CS monomers
were incubated at 45 °C in the absence () or presence of 150
nM HSP18.1 (
) or 150 nM HSP17.7 (
). CS
activity was determined at the indicated times. After the 60-min
incubation period at 45 °C, samples were shifted to 22 °C, but
no CS reactivation could be detected in any of the samples (not
shown).
-crystallin high
molecular weight complexes
(41) . In contrast, smHSP complexes
from mammalian sources have been found to exist in larger, more dynamic
complexes. For example, the murine smHSP, HSP25, has been characterized
as a 32-mer with a native molecular mass of 730 kDa
(19) .
Recent evidence suggests that the subunit composition of mammalian
smHSPs can be highly variable in response to phosphorylation state
(20, 21) and that the control of smHSP oligomerization
may regulate their interaction with actin
(21) . At present
however, phosphorylation of plant small HSPs has not been detected
(22) , and the recognition sites for phosphorylation observed in
mammalian smHSPs
(41, 42) have not been conserved in
plant smHSPs
(23) .
-crystallins
(9, 11) , refolding of chemically
denatured proteins could be enhanced with the addition of
stoichiometric levels of HSP18.1 or HSP17.7 in an ATP-independent
manner. Whereas murine HSP25 maximally enhanced the refolding of
chemically denatured CS at an HSP25 complex-to-CS monomer ratio of 1:3,
the enhancement by the plant smHSPs tested here saturated at a ratio of
approximately 1:1. Since the native molecular mass of HSP25 has been
estimated at 730 kDa and those for HSP18.1 and HSP17.7 have been
estimated between 200 and 216 kDa, the plant smHSPs appear to be as
efficient as the mammalian smHSPs on a per weight basis. An additional
consideration is that in the present study, we have utilized 6
M guanidine HCl as the protein denaturant, as opposed to 8
M urea, based on the finding that 8 M urea is
insufficient to produce CS monomers
(43) . Therefore, a precise
stoichiometric comparison between the previous and the present study
may be difficult based on the different oligomerization and folding
states adopted by CS after guanidine versus urea denaturation.
Nonetheless, it appears that the functional properties among plant and
mammalian smHSPs have been conserved despite the fact the two groups
share only limited overall sequence homology.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.