From the Department of Pharmaceutical Sciences,
School of Pharmacy, University of Colorado Health Science Center,
Denver, Colorado 80262, § Department of Chemical
Engineering, University of Colorado, Boulder, Colorado 80309, ¶ Biosciences Division, Argonne National Laboratory,
Argonne, Illinois 60439, and
Human Immunology and Cancer
Program, University of Tennessee Graduate School of Medicine,
Knoxville, Tennessee 37920
Received for publication, August 24, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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In primary (light chain-associated) amyloidosis,
immunoglobulin light chains deposit as amyloid fibrils in vital organs,
especially the kidney. Because the kidney contains high concentrations
of urea that can destabilize light chains as well as solutes such as
betaine and sorbitol that serve as protein stabilizers, we investigated
the effects of these solutes on in vitro amyloid fibril
formation and thermodynamic stability of light chains. Two recombinant
light chain proteins, one amyloidogenic and the other nonamyloidogenic,
were used as models. For both light chains, urea enhanced fibril
formation by reducing the nucleation lag time and diminished protein
thermodynamic stability. Conversely, betaine or sorbitol increased
thermodynamic stability of the proteins and partially inhibited fibril
formation. These solutes also counteracted urea-induced reduction in
protein thermodynamic stability and accelerated fibril formation.
Betaine was more effective than sorbitol. A model is presented to
explain how the thermodynamic effects of the solutes on protein state
equilibria can alter nucleation lag time and, hence, fibril formation
kinetics. Our results provide evidence that renal solutes control
thermodynamic and kinetic stability of light chains and thus may
modulate amyloid fibril formation in the kidney.
Primary systemic antibody light chain
(AL)1 amyloidosis is a plasma
cell disorder in which immunoglobulin light chains deposit pathologically as amyloid fibrils in the body, leading to progressive organ failure and eventual death (1-3). Amyloid fibrils consist of the
variable domain, VL, or the VL and a contiguous
portion of the constant domain of It has been hypothesized that a partially unfolded conformation, the
aggregation-competent species, fosters nucleation and subsequent
fibrillization of proteins (5, 6, 8, 10-12). Certain mutations can
dispose benign proteins to amyloid fibril formation by enhancing
formation of this partially unfolded state (5-8, 11, 13-15). However,
mutations are not always necessary for amyloidosis (e.g.
with The kidney is the major target organ for light chain deposition, which
can be found in various forms: fibrillar (AL-amyloidosis), punctate
(light chain deposition diseases), crystalline (acquired Fanconi's
syndrome), and amorphous (tubular cast neuropathy) (1, 3, 23). For
AL-amyloidosis, about one-third of patients have amyloid fibrils in the
kidney (24). Amyloid is found in all compartments of the kidney, with
the glomerulus being a primary site of fibril deposition (1, 23). In
about 10% of the cases, amyloid is restricted to nonglomerular
regions, especially the inner renal medulla (25). Urea is found at high
concentrations (0.4 M-1.5 M) in the inner renal
medulla, especially during antidiuresis (26). A common feature in
AL-amyloidosis patients is Bence Jones proteinuria, in which high
levels of monoclonal However, the kidney also contains high concentrations of stabilizing
osmolytes that can counteract destabilization by urea, such as betaine,
glycerophosphocholine, sorbitol, and inositol (19-21, 27, 28).
Counteraction of urea-induced protein structural and functional
perturbations by osmolytes has only been demonstrated with a few
enzymes (19-21, 27, 28). We hypothesize that this phenomenon will also
occur with immunoglobulin light chains, because the mechanisms for the
effects of urea and stabilizing osmolytes are nonspecific (28). Urea
destabilizes the compact, native state of proteins because it binds
preferentially to the protein backbone, and protein species with
greater solvent exposure are thermodynamically favored (28, 29).
Stabilizing osmolytes are preferentially excluded from the surface of
proteins, which shifts the unfolding equilibrium to favor compact
states (28, 30-33). Osmolytes (e.g. glycerol and sucrose),
in the absence of urea, have been found to inhibit amyloid fibril
formation from scrapie prion protein (34) and immunoglobulin light
chains (8) by stabilizing the native states and reducing the levels of
the aggregation-competent species.
To examine the possible roles of renal solutes in AL amyloidosis and to
gain further insight into the relationships between the thermodynamic
stability of light chains and their propensity to form amyloid fibrils,
we modulated protein stability by varying concentrations of renal
solutes, i.e. urea, betaine, and sorbitol. The proteins
used, recombinant VL SMA and VL LEN (6,
10), represent prototype amyloidogenic and nonamyloidogenic proteins, based on previous in vivo and in vitro
observations (6, 10). It has previously been established that
recombinant SMA forms fibrils in vitro that are
indistinguishable from in vivo light chain fibrils, and
recombinant LEN does not form fibrils in vitro in buffer
alone (6, 10).
Materials--
Betaine, sorbitol, and guanidine hydrochloride
were purchased from Sigma, and ultrapure urea was purchased from ICN.
Before use, concentrated urea solutions were treated with a chelating resin (C-7901, Sigma) for 1 h and then with a mixed-bed ion
exchange resin (AG501-X8, Bio-Rad) for 1 h to remove ions (21, 27, 29). The solution was then filtered using 0.2-µm nylon filters (Fisher) and finally freeze-dried in an FTS Durastop lyophilizer (Stone
Ridge, NY). The urea concentration was determined by measuring the
refractive index of the solution (35). Other chemicals were purchased
from Sigma and were of reagent or higher grade.
Proteins Expression and Purification--
The recombinant
VL protein SMA has the same sequence as the VL
that originated from lymph node-derived amyloid fibrils of a patient
(SMA) who had AL amyloidosis (10). The recombinant VL
protein LEN has the same sequence as a VL isolated from the urine of a patient (LEN) with multiple myeloma who, despite excretion of up to 50 g of this protein daily, had no renal dysfunction or
evidence of amyloidosis (9, 10). Both proteins were products of same
germline gene family In Vitro Fibril Formation and Sample Analyses--
Fibrils were
produced in vitro with incubation at 37 °C and agitation
at 250 rpm as described by Kim et al. (8). 1 mg/ml protein
was incubated in PBS buffer (pH 7.4, with 10 mM potassium phosphate plus 100 mM NaCl), PBS plus various urea
concentrations (0.5, 1, and 1.5 M), PBS plus 0.5 M betaine or sorbitol (for SMA), and PBS plus urea (0.5 and
1 M) containing either 0.5 M betaine or
sorbitol. All buffer solutions contained 0.1% sodium azide to inhibit
microbial growth. Samples were analyzed at designated time points for
soluble protein using size exclusion high performance liquid
chromatography (HPLC) and for fibrils, using a Thioflavin T (ThT) assay
(36), as described by Kim et al. (8). Size exclusion HPLC
analysis documented that before incubations, the proteins (1 mg/ml)
were dimers, free of higher molecular weight light chain aggregates.
Fibril formation kinetics were analyzed by fitting
time-dependent changes in ThT fluorescence of incubated samples to the following nonlinear, least squares sigmoidal equation.
Guanidine Hydrochloride (GdnHCl) Unfolding--
The free energy
of denaturation, Intrinsic Fluorescence--
Intrinsic fluorescence emission
spectra were measured with an Aviv model ATF105 spectrofluorometer at a
sample temperature of 25 °C. The samples were excited at 295 nm, and
the emission was monitored from 300 to 400 nm. Excitation and emission
slit widths were set at 5 and 10 nm, respectively. Protein solutions (10 µg/ml) were prepared in PBS and in 0.5, 1.0, and 1.5 M urea in PBS and equilibrated overnight at room
temperature before measurement of fluorescence emission spectra. Two
scans of each solution were averaged, and the appropriate spectrum of
the buffer solution was subtracted from this average.
Infrared (IR) Spectroscopy and Transmission Electron Microscopy
of Fibrils--
IR spectra were measured with a Bomem MB-series
spectrometer (Bomem) purged with dry air from a Balston dryer
(Balston) to remove water vapor. Soluble proteins (20 mg/ml) and
fibrils (~5 mg/ml) were measured in a BioTools liquid sampling cell
equipped with CaF2 windows that provided a 6-µm path
length. Before measurement, fibrils formed in various solution
conditions were separated from unaggregated protein and washed by two
rounds of centrifugation and resuspension with PBS buffer. Spectra were
analyzed according to the previously established criteria (38, 39).
Transmission electron microscopy on fibrils was performed by the method
of Raffen et al. (6).
Fibril Formation in PBS--
Fig. 1
shows time courses for soluble native protein concentrations and ThT
fluorescence, which is indicative of fibril formation (6, 7, 36). In
PBS buffer alone, the amount of soluble SMA started to decrease after 3 days of incubation, and soluble protein was undetectable 1 day later
(Fig. 1A). During this period, there was a concomitant
increase in ThT fluorescence, indicative of fibril formation (Fig.
1B). Conversely, LEN remained soluble and did not form
fibrils (Fig. 1, C and D) in PBS buffer until 15 days (data not shown) of incubation. The fibril formation profile for
SMA showed a characteristic nucleation-dependent
polymerization pattern, having an initial lag phase followed by a rapid
growth phase (7, 13, 40, 41). SMA in PBS buffer had a lag time of
66 h and a fibril formation rate constant of 0.32 h Effects of Urea on Fibril Formation--
Before assessing effects
of urea on fibril formation, we determined whether physiological
concentrations of urea (i.e. up to 1.5 M) are
sufficient to cause structural perturbation of SMA and LEN. Unfolding
curves were constructed by measuring intrinsic fluorescence intensity
(8) as a function of urea concentrations (data not shown). The onset of
unfolding was at 2.9 and 4.1 M urea for SMA and LEN,
respectively. The midpoint of the unfolding transition was at 4.1 and
5.2 M urea, respectively, for SMA and LEN. Thus, even at
the highest urea concentration tested (1.5 M) in the fibril
formation experiments, both proteins were native. Furthermore,
fluorescence emission spectra for SMA and LEN in 0.5, 1.0, and 1.5 M urea were unperturbed relative to the respective spectrum
for each protein in PBS (data not shown). For both proteins, neither
the wavelength of the fluorescence emission maximum nor the emission
spectrum width at half maximum intensity was affected by urea.
When SMA and LEN were incubated in physiological concentrations of urea
(0.5, 1, and 1.5 M), both proteins formed fibrils (Fig. 1).
Urea dramatically decreased the lag time compared with that in PBS
buffer alone (Table I), with the
highest urea level resulting in the shortest lag times. At 0.5 and 1 M urea concentration, SMA displayed much shorter lag phases
than LEN. In 1.5 M urea, both proteins had similar lag
times (Table I).
Effects of Stabilizing Osmolytes on Fibril Formation--
To test
the hypothesis that stabilizing osmolytes can counteract the effects of
urea on fibril formation, incubations were conducted in betaine or
sorbitol and mixtures of urea and betaine or sorbitol. In the absence
of urea, 0.5 M betaine or sorbitol partially inhibited
fibril formation by SMA compared with that in PBS alone (Fig.
2). The lag phase was much longer, and
the fibril growth rates were slower than those for the protein in PBS
alone (Table I). Betaine and sorbitol were not tested on LEN in PBS
alone because the protein did not form fibrils over the 5-day time
course in just PBS. When both proteins were incubated in mixtures of
urea and betaine or sorbitol, the stabilizing osmolytes partially
counteracted the acceleration of fibril formation induced by urea. The
lag times were lengthened relative to values measured in urea alone.
However, there were not significant differences in fibril growth rates
between the mixtures of urea and betaine or sorbitol and urea alone
(Table I). Mixtures with 1:1 molar ratios of osmolytes to urea were
more effective at counteracting urea-mediated fibril formation than
those with 1:2 molar ratios. Betaine was much more effective at
inhibiting fibril nucleation than sorbitol, either in the presence or
absence of urea.
Characterization of Fibrils--
Fibrils formed under all of the
various buffer conditions showed the typical characteristics of amyloid
fibrils, based on transmission electron microscopy (Fig.
3) and IR spectroscopy (data not shown).
Fig. 3 shows representative electron photomicrographs of SMA fibrils
formed in PBS alone and in 1 M urea. Both sets of fibrils
consist of linear, unbranched structures. Based on transmission
electron microscopy, the fibrils formed from each of the two proteins,
under all of the various buffer conditions, were morphologically
indistinguishable (data not shown). IR spectra of fibrils of SMA and
LEN showed characteristic intermolecular Effect of Urea and Stabilizing Osmolytes on the Free Energy of
Denaturation--
Prompted by previous studies (2, 5-8) that
demonstrated a strong inverse relationship between thermodynamic
stability of light chains and fibril formation propensity, free
energies of denaturation for SMA and LEN were measured under the same
solution conditions used for the fibril formation studies.
GdnHCl-induced unfolding curves are shown in Fig.
4, and Table
II lists the calculated denaturation
midpoints (Cm), differences in Cm relative to values in PBS alone ( Our results document that urea decreases the thermodynamic
stability of both SMA and LEN and decreases the lag time for nucleation of fibril formation. Betaine and sorbitol counteract the thermodynamic destabilization caused by urea, explaining why these solutes are able
to offset, at least in part, the enhanced fibril formation induced by
urea. To understand the mechanism(s) for these effects one must
consider the different nonspecific interactions of urea versus betaine or sorbitol with proteins (cf.
Ref. 28).
Urea is a destabilizer of native protein structure and generally an
inhibitor of enzyme function (19, 29, 35). Destabilization of the
native conformation is due to preferential binding of urea to protein
backbone and other polar groups (28, 29). Urea binding decreases
protein chemical potential and is directly proportional to
solvent-exposed protein surface area. Thus, urea shifts the equilibrium
away from the native state, favoring partially or fully unfolded states
with greater solvent exposure than the native state. Urea also favors
dissociation of higher order assemblies (e.g. native
oligomers or fibril nuclei), because the total solvent-exposed surface
area of the constituent monomers is greater than that for the assembled
state (28).
Conversely, both betaine and sorbitol have been shown to increase the
stability of several proteins against stresses such as high temperature
(19, 21, 30) due to preferential exclusion from the protein surface
(28, 30, 31, 33). Preferential exclusion of solutes increases the
protein chemical potential in direct proportion to the protein surface
area. The magnitudes of preferential exclusion and increase in chemical
potential are greater for the denatured state and partially folded
species than for the native state. Thus, the free energy barrier
between the states increases, thereby stabilizing the native state (28, 31-33). Concomitantly, preferentially excluded solutes also
thermodynamically favor higher order protein assemblies, which have a
reduced surface area relative to the total for the constituent monomers
(28).
Preferential interactions of urea and osmolytes with protein surfaces
are nonspecific. Therefore, in mixtures of urea and osmolytes, the two
thermodynamic effects should be compensatory and approximately additive
(19, 21, 27-29). We found that the thermodynamic parameters,
Betaine is a more effective stabilizer than sorbitol, either in the
absence or presence of urea, both in terms of thermodynamic stability
and its effect on the lag time for fibril nucleation (Tables I and II).
In addition to the chemical nature of the protein surface, the chemical
structure and physical properties of a solute determine its
interactions (attractive or repulsive) with the protein and itself
(28). The magnitude of protein chemical potential change caused by
solute addition is determined by two parameters according to the
following equation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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or
light chains (1-4). Light
chains that are amyloidogenic in vivo are less
thermodynamically stable than those that are nonamyloidogenic (5-8).
In vitro there is a strong inverse relationship between the
intrinsic thermodynamic stability of light chains and their propensity
to form fibrils (2, 5-8). Furthermore, thermodynamic stability and
propensity to form fibrils have been modulated extrinsically by
employing solutes such as urea and sucrose (6, 8, 9).
2-microglobulin in dialysis patients and with transthyretin
during senile systemic amyloidosis) (11, 16, 17), presumably because in
certain cases the wild-type protein population contains
aggregation-competent species. In addition, properties of the aqueous
environment such as pH, temperature, ionic strength, and the presence
of chaotropic agents may also be important parameters affecting levels
of partially unfolded species and, hence, in amyloidosis (5, 6, 11, 13,
18). In vivo, partially denaturing environments can be
found, for example, in the lysosome (acidic pH) and in the kidney,
which has high intra- and extracellular concentrations of urea (11,
19-21). Lysosomal extracts can convert amyloidogenic immunoglobulins
into amyloid fibrils in vitro (22). In vitro
exposure of transthyretin to acidic pH, mimicking that of the
lysosome, causes partial unfolding, which in turn promotes
fibril formation (17). The conversion of VLs into amyloid
fibrils under acidic conditions proceeds through at least one partially
unfolded intermediate (5). Thus, both intrinsic stability of proteins
and effects of local solution environments are important criteria for
the determination of whether light chains will form amyloid fibrils.
or
light chains are excreted in the urine,
although urinary concentrations of light chains do not necessarily
correlate with pathogenesis (24). As a component of the glomerular
filtrate, light chains are exposed to various concentrations of urea
during processing by the kidney. Exposure to urea is expected to
enhance fibril formation by thermodynamically favoring the population
of aggregation-competent, partially unfolded conformations of light
chains (6-9). As a result of this effect, for example, a light chain
variant with an intrinsic thermodynamic stability that is just
sufficient to avoid amyloid fibril deposition in other areas of the
body, might form fibrils in the renal medulla.
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DISCUSSION
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IV (10). The VL sequence of SMA differs from that of LEN by only eight amino acid residues (6, 10).
Recombinant VLs SMA and LEN were expressed in
Escherichia coli and purified by the methods of
Wilkins-Stevens et al. (10) and Raffen et al. (6)
with the following modifications. Instead of 5-ml prepacked Econo-Pac Q
and S cartridges, High Q and S resins (Bio-Rad) were packed in 1 × 50-cm glass columns (Bio-Rad). Fractions were eluted with 8× volume
(400 ml), 0-900 mM NaCl gradient for SMA and 8× volume
(400 ml), 0-150 mM NaCl gradient for LEN. Fractions were
collected and assayed by SDS-polyacrylamide gel electrophoresis. The
fractions containing VLs were pooled and stored at 4 °C
in PBS buffer (10 mM potassium phosphate, pH 7.4, with 100 mM NaCl). The purity of the VLs exceeded 99%
(based on SDS-polyacrylamide gel electrophoresis). Protein
concentration was determined using extinction coefficients of 1.71 and
1.82 (mg ml
1 cm
1)
at 280 nm for SMA and LEN, respectively, which were calculated from the
amino acid sequence (6).
where FThT is the fluorescence intensity
of ThT, A is the ThT fluorescence intensity in the
post-transition plateau, ti is the inflection point,
i.e. the midpoint of the transition region, B
(h
(Eq. 1)
1) is the fibril growth rate constant, and
t is the time in h. The lag time
(tlag) of fibrillogenesis was calculated by
extrapolation of the linear region of the sigmoidal transition phase of
ThT fluorescence assay to the abscissa intercept (7).
G, was measured using tryptophan
fluorescence at 25 °C after samples were equilibrated with various
concentrations of GdnHCl overnight at room temperature (8). Protein
concentration was 10 µg/ml. GdnHCl concentrations were determined
using the refractometer (35). The Cm value, the
midpoint of unfolding transition region, was calculated by complex
sigmoid nonlinear analysis (8). The m and
G values were
calculated as the slope and ordinate intercept, respectively, of a
linear regression of
G versus GdnHCl
concentration (35, 37).
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1.
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Fig. 1.
Effects of urea on soluble protein levels
measured by size exclusion HPLC and fibril formation measured by ThT
fluorescence for SMA (A and B) and
LEN (C and D) during incubation at
37 °C with agitation. The symbols represent the following
buffer conditions: , PBS buffer;
, 0.5 M urea;
, 1 M urea;
, 1.5 M urea. In B and
D, the solid lines were drawn by nonlinear, least
square fits of the data using Equation 1. Data points represent the
mean ± S.D. for triplicate incubated samples.
Calculated kinetic parameters for the fibril formation of SMA and LEN
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Fig. 2.
Effects of betaine or sorbitol with or
without urea on soluble protein levels and fibril formation for SMA
(A and B) and LEN (C
and D) during incubation at 37 °C with
agitation. The symbols represent the following buffer conditions:
, 0.5 M betaine; (
),0.5 M sorbitol;
,
0.5 M urea plus 0.5 M betaine;
, 0.5 M urea plus 0.5 M sorbitol;
, 1 M urea plus 0.5 M betaine;
, 1 M
urea plus 0.5 M sorbitol. In B and
D, the solid lines were drawn by nonlinear, least
square fits of the data using Equation 1. Data points represent the
mean ± S.D. for triplicate incubated samples.
-sheet bands,
i.e. peaks around 1625 and 1695 cm
1 (8, 39, 42), in the amide I region (data
not shown).
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Fig. 3.
Transmission electron microscopy of
negatively stained SMA fibrils produced in PBS buffer
(A) and in 1 M urea buffer
(B). Scale bars represent 100 nm.
Cm), free
energies of denaturation (
G), and differences in free
energy of denaturation relative to that in PBS alone
(
G). In PBS alone, Cm and
G values, respectively, were 1.2 M GdnHCl and
5.5 kcal/mol for SMA and 1.8 M GdnHCl and 7.2 kcal/mol for
LEN (Table II), confirming previous studies showing that SMA is
thermodynamically less stable than LEN (6). Urea decreased the
thermodynamic stability of both proteins as measured by
Cm and
G (Fig. 4 and Table II).
Betaine and sorbitol increased the values of Cm and
G for SMA and counteracted the effects of urea on both
proteins (Fig. 4 and Table II). Betaine increased Cm and
G more than sorbitol. In mixtures containing 1:1
molar ratios of urea to betaine or sorbitol, the effects of the solutes
on Cm and
G values for SMA were
approximately additive (Table II). For SMA, Cm and
G in the 1:1 molar mixtures were higher than those in PBS
alone because the stabilizing effects of 0.5 M betaine or
sorbitol were greater than the destabilization caused by 0.5 M urea.
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Fig. 4.
Guanidine-HCl unfolding curves in various
solutes conditions for SMA (A) and LEN
(B). The symbols represents the following buffer
conditions: , PBS buffer;
, 0.5 M urea;
, 0.5 M betaine;
, 0.5 M sorbitol;
, 0.5 M urea plus 0.5 M betaine;
, 0.5 urea plus
0.5 M sorbitol;
, 1 M urea. Data points
represent the mean ± S.D. for triplicate samples.
Thermodynamic parameters of SMA and LEN in various solute conditions,
measured by GdnHCl unfolding experiments
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Cm and
G, in mixtures of urea and
betaine or sorbitol were, within error of the determinations, equal to
the sum of the individual values in each solute (Table I).
where µi and mi are the chemical potential and
molal concentration of component i (1 = water, 2 = protein, and 3 = solute), respectively (28).
(Eq. 2)
m3/
m2 is the preferential interaction
parameter, and its negative value indicates preferential exclusion
(28). For bovine serum albumin, betaine has been found to have higher
preferential exclusion (
m3/
m2 =
39.6
mol of betaine per mol of bovine serum albumin) than sorbitol
(
m3/
m2 =
9.8 mol of sorbitol per mol of
bovine serum albumin) (30, 31). The solute self-interaction parameter,
µ3/
m3, is the other major component
that determines the effect of solute on protein chemical potential
(28). Calculation of self-interaction parameter using the activity
coefficient of each solute (28, 33) gives a greater value for betaine
(
µ3/
m3 = 776 cal/mol2) than
for sorbitol (
µ3/
m3 = 591 cal/mol2). From Equation 2, the total effect of the two
parameters shows that
µ2/
m3 is 30.7 kcal/mol for betaine and 5.8 kcal/mol for sorbitol. Thus, betaine
provides a greater increase in the protein chemical potential and,
hence, in free energy of unfolding than sorbitol, and it will be more
effective in counteracting destabilization by urea.
Amyloid fibril formation, including that for light chains (see
"Results" and Ref. 7) is nucleation-dependent (13, 40, 41). To explain our results for the effects of urea and stabilizing osmolytes on light chain fibril formation, we propose a model that
takes into account the effects of solutes on thermodynamic and kinetic
aspects of the fibril formation pathway. Schematically, the native
state, N, is in equilibrium with an aggregation-competent species, A,
that is a partially unfolded form of N (5, 6, 8, 10-12, 43).
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The aggregation-competent species, A, may be a structurally perturbed
monomer formed by dissociation of the native dimer or a dimer with
altered tertiary structure. Because urea both favors protein
dissociation and accelerates the rate of fibril formation and the
stabilizing solutes betaine and sorbitol have the opposite effects, it
seems plausible that light chain monomers could be the
aggregation-competent species. However, if propensity to form fibrils
was governed solely by the levels of monomers in solution, then LEN
should form fibrils more readily than SMA, because the respective
dimerization constants for the proteins are 4 × 105
M1 and 7 × 105
M
1 (10). Even though at a given
protein concentration LEN has a higher level of monomers than SMA, LEN
is much more resistant to fibril formation than SMA (6, 10, Fig. 1).
Furthermore, the light chain REC forms fibrils readily and has a
dimerization constant of 2 × 107
M
1 (6, 10). Thus, in comparisons
between light chain variants, the levels of monomers do not correlate
with propensities to form fibrils. Speculatively, within the population
of monomers of SMA and REC there may be many more structurally
perturbed molecules, which are prone to form non-native aggregates
leading to fibrils, than within the population of LEN monomers. If this
were the case, alteration by urea and stabilizing osmolytes of the
total SMA monomer level and the fraction of the monomer population that is aggregation competent could play an important role in governing the
rate of fibril formation. Similarly, urea-induced perturbations of the
LEN monomer structure and urea-induced increase in the total level of
monomers might lead to a sufficient level of aggregation-competent monomers to promote aggregation and fibril formation. Based on this
putative mechanism the critical factor governing propensity to form
fibrils is the level of partially unfolded, aggregation-competent monomers and not the total level of monomers. The conformational perturbation needed to form aggregation-competent light chain monomers
from native monomers is not known. Investigating this structural
transition is an important area for future research.
Even though the structure of the aggregation-competent species, A, has not been elucidated, for our explanation about the effects of solutes on fibril formation, the main property that is assumed is that A has a greater surface area than N (on a per-monomer basis) and that higher temperatures (e.g. 37 versus 4 °C) also favor the more highly solvated expanded state, A.
The kinetics of formation of higher order species such as An
can be highly dependent on the concentration of A, especially if
n is a relatively large number. Thus, at low concentrations of A, the nucleation rate may be negligible, whereas above an apparent
threshold concentration, AC, the rate of nucleation increases
dramatically (Fig. 5A). Thus,
for our Scheme 1 the lag time for fibril formation is dependent on the
time it takes to populate A to the threshold concentration, AC
(Fig. 5B).
|
As depicted schematically in Fig. 5A, urea increases
AC because protein assembly is less favorable in urea than in buffer alone (8, 28). Conversely, betaine or sorbitol favors assembly
and, thus, a lower concentration of A is needed to foster nucleation;
there is a decrease in AC. If solutes were only affecting
AC, then one would predict that urea should slow fibril
formation and betaine or sorbitol should speed the process, the
opposite of our results (Figs. 1 and 2). In an earlier study on fibril
formation from A-peptide, it was found that stabilizing solutes such
as trimethylamine-N-oxide and glycerol accelerated fibril
formation (44). This could be because the solutes reduced AC
and the overall process was not rate-limited by a step leading to the
accumulation of A.
However, according to our proposed pathway, the solutes can also affect the rate at which the aggregation-competent state, A, accumulates in solution (Fig. 5B). First, consider the protein in PBS alone. Upon transferring the sample from 4 to 37 °C, there is an increase in the level of A, as the system approaches the new equilibrium between N and A. The levels of A increase until the threshold concentration is reached and fibril formation is nucleated; the system may never actually reach equilibrium levels of A. The time from the initiation of the exposure to 37 °C to the point at which the threshold concentration is reached is equal to the lag time for fibril nucleation.
In the presence of urea, the equilibrium between N and A will be shifted toward A because of the greater solvent-exposed surface area of this species, and the level of A will increase more rapidly in urea than in PBS alone (Fig. 5B). As a result, even though the threshold concentration for nucleation, AC, is higher in urea than in PBS, the time it takes to reach this concentration of A is less in urea. Thus, to explain our observed reduction in duration of lag time in the presence of urea, the acceleration by urea of accumulation of A must predominate over the urea-induced increase in AC. Note this is probably not the case in extremely high urea concentrations (e.g. 8 M) where AC is so high that fibril formation cannot be nucleated.
Conversely, betaine or sorbitol thermodynamically favors N over A and slows the rate of increase in level of A. Even though AC is lower in these solutes than in PBS, it takes a longer time to achieve this level of A, and the nucleation of fibrils is slowed. In this case the effect of the solutes on the rate of accumulation of A predominates over the solute-induced decrease in AC.
This model also helps explain why betaine and sorbitol, at equimolar concentrations to urea, only partially offset the reduced lag time for fibril formation in urea, yet the thermodynamic stabilities of the SMA and LEN proteins were greater under these solution conditions than in PBS alone. Because the free energy of unfolding of the proteins in equimolar betaine or sorbitol plus urea was greater than that in buffer alone, the native state, N, is more favored in the mixed solute system than the denatured state or the A state. Accordingly, the threshold concentration of A and the rate of formation of A are reduced compared with that in PBS (Fig. 5). However, in contrast to the situation in betaine or sorbitol alone, in the mixed solute system the reduction in threshold concentration predominates, and the lag phase is shorter than that in PBS alone.
The arguments presented above can also be used to explain the effects of solutes of fibril formation by other pathways. For example, it may be that A first has to convert via an essentially irreversible step into a prenucleus species, P, which is in equilibrium with the nucleus, Pn. For immunoglobulin light chains this species could be a non-native dimer formed from structurally perturbed monomers (43, 45). For the purposes of our arguments, the key issue is that shifting the equilibrium between N and A toward A will increase the rate at which P appears. Conversely, employing solution conditions that thermodynamically favor N will slow the appearance of P. Alternatively, nucleation may depend on the time-dependent accumulation of covalently modified (e.g. with oxidized methionine residues) protein molecules. If formation of these modified species is also fostered by a protein conformational expansion, then counteracting solutes could modulate the rate of their generation and, hence, the rate of nucleation.
Finally, it is important to address the observations that VL LEN did not form amyloid fibrils in vivo, but it did so in the presence of urea in vitro. In addition to the effects of high concentrations of urea, agitation also accelerates in vitro fibril formation of both LEN and SMA. For example, in a 1 mg/ml solution of SMA, without agitation there were no fibrils detected, even after 17 days of incubation at 37 °C (data not shown). Furthermore, in the absence of agitation, LEN did not form fibrils even after 14 days of incubation at 37 °C in the presence of 1 M urea (data not shown). Solomon et al. (4) developed an in vitro fibrillogenesis assay that uses agitation to accelerate fibril formation of light chains and found a direct correlation between in vivo and in vitro fibril formation by light chains (4, 6-8). Agitation fosters protein aggregation mainly due to adsorption of the proteins at the air-liquid interface (46, 47). Adsorption may lead to both an increase in the local protein concentration and an alteration in protein structure at the interface, which can facilitate protein aggregation, including fibril formation. This physical stress is an important driving force to induce fibril formation of the proteins in in vitro systems, which is most likely not present in vivo.
In addition to properties of the protein itself and local environmental conditions, the effect of host factors, e.g. renal physiology, on fibril formation in vivo must be considered (9, 18, 23). Other constituents of amyloid fibrils such as proteoglycans and apolipoprotein-E may also be relevant (3, 9, 26). Thus, different behaviors could be seen with the same protein, depending on individual-specific factors.
Intrinsic thermodynamic stability, conferred by primary sequence and
tertiary structure, and extrinsic stability, modulated by solution
conditions such as pH, temperature, and stabilizing and destabilizing
solutes, are all important factors for regulating fibril formation
in vivo and in vitro. Kinetic factors, which are
impacted by shifts in equilibria between protein species, are important
as well because accumulation of a sufficiently high level of an
aggregation-competent species is the critical step for nucleation and
fibril formation. Physiological concentrations of urea strongly
accelerate fibril formation of both pathologic and nonpathologic light
chains predominantly by decreasing the time it takes to accumulate a
nucleation-favoring concentration of an aggregation-competent species,
implying that high concentrations of urea in the renal medulla may be
at least one reason why the kidney is the most common site for
AL-amyloidosis and other forms of pathological light chain deposition.
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ACKNOWLEDGEMENTS |
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We thank Drs. Carlos Catalano and Qin Yang for allowing us to use of the fast protein liquid chromatography system.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant BES 9816975 (to J. F. C. and T. W. R.) and United States Public Health Service Grant CA10050 (National Institutes of Health (NCI) (to A. S.)).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.
** An American Cancer Society Clinical Research Professor.
To whom correspondence should be addressed. Tel.: 303-315-6075;
Fax: 303-315-6281; E-mail: John.Carpenter@uchsc.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007766200
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ABBREVIATIONS |
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The abbreviations used are: AL-amyloidosis, light chain-associated amyloidosis; VL, immunoglobulin light chain variable domain; PBS, phosphate-buffered saline; ThT, thioflavine T; GdnHCl, guanidine hydrochloride; HPLC, high performance liquid chromatography.
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