Kinetics Control Preferential Heterodimer Formation of
Platelet-derived Growth Factor from Unfolded A- and B-chains*
Carsten
Müller
,
Susanne
Richter§, and
Ursula
Rinas¶
From the GBF National Research Center for Biotechnology,
Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany
Received for publication, December 4, 2002, and in revised form, February 26, 2003
 |
ABSTRACT |
The folding and assembly of
platelet-derived growth factor (PDGF), a potent mitogen involved in
wound-healing processes and member of the cystine knot growth factor
family, was studied. The kinetics of the formation of disulfide-bonded
dimers were investigated under redox reshuffling conditions starting
either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of
disulfide-bonded dimers is a very slow process occurring in the time
scale of hours with a first-order rate-determining step. The formation
of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed
identical kinetics, indicating that both monomeric forms as well as the
dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three
times more rapidly compared with the formation of the homodimers. As
both monomeric forms revealed similar renaturation kinetics, it can be
concluded that the first-order rate-determining folding step does not
occur during monomer folding but must be attributed to conformational
rearrangements of the dimerized, not yet disulfide-bonded protein.
These structural rearrangements allow a more rapid formation of
intermolecular disulfide bonds between the two different monomers of a
heterodimer compared with the formation of the disulfide bonds between
two identical monomers. The preferential formation of disulfide-bonded
heterodimers from an equimolar mixture of unfolded A- and B-chains is
thus a kinetically controlled process. Moreover, similar activation
enthalpies for the formation of all different isoforms suggest that
faster heterodimerization is controlled by entropic factors.
 |
INTRODUCTION |
Platelet-derived growth factor
(PDGF)1 is a potent mitogen
for cells of mesenchymal origin, i.e. smooth muscle cells,
connective tissue cells, or blood cells (1
3). It is released by
platelets upon wounding and plays an important role in stimulating
adjacent cells to grow and thereby heal the wound (4). PDGF is a
non-glycosylated protein that belongs to the family of dimeric cystine
knot growth factors (5). The PDGF family consists of different gene
products. The most prominent and long known members of this
family are PDGF-A and -B. More recently, two new less abundant members,
PDGF-C and -D, have been discovered (6, 7).
The two different homologous monomers of PDGF, denoted as A- and
B-chains, are known to exist in the three natural occurring dimeric
isoforms PDGF-AA, -AB, and -BB (8, 9). These different isoforms have
apparently distinct biological functions indicated, e.g. by
their different binding affinities to the two different types of PDGF
receptors (10). However, the majority of PDGF purified from human
platelets is the disulfide-bonded heterodimeric growth factor (11),
suggesting that heterodimerization is favored when both genes are
coexpressed. Also, PDGF-AB disulfide-bonded heterodimers are almost
exclusively formed from an equimolar mixture of unfolded and reduced A-
and B-chains when renaturation is carried out under conditions that
allow disulfide bond reshuffling (12
14).
From all potential isoforms of the PDGF dimer, only the structure of
the BB homodimer has been determined so far (Fig.
1; Ref. 15). PDGF-BB is an all-
sheet
protein of about 30 kDa and composed of two very flat subunits arranged
head-to-tail and linked together by two intermolecular disulfide bonds
(5, 15). In addition to intermolecular disulfide bonds, each monomer
contains an unusual knot-like arrangement of three intramolecular
disulfide bridges where one disulfide bond threads through a loop
formed by the two other disulfide bonds (5, 15). As all members of the
cystine knot growth factor family share strong structural homology, it
is most likely that the other PDGF isoforms are of almost identical
structure as the BB isoform.

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Fig. 1.
Schematic presentation of the
three-dimensional structure of PDGF-BB. A, the
two monomers (depicted in green and blue) are
connected head-to-tail via two intermolecular disulfide bonds. Each
monomer contains a knot-like arrangement of three intramolecular
disulfide bonds, where two disulfide bonds connect two -strands and
form a ring structure, and the third disulfide bond threads through
this ring and connects two additional -strands. The positions of the
cysteines are indicated in yellow. The disulfide bonds are
shown in red. The single tryptophan is indicated in
violet. The gray rectangle underlays the area
occupied by one subunit and the gray circle the position of
the cystine knot in the other subunit. B, bowllike structure
of a single subunit with the positions of the prolines indicated in
gray. C, close-up of the local backbone conformation in the
vicinity of the conserved cysteine (Cys43) involved in
intermolecular disulfide-bonding. All solved structures of members of
the cystine knot growth factor family show evidence for the presence of
a conserved cis-proline (Ref. 15: PDGF-BB,
Trp39-Pro40-cis-Pro41-Cys43;
Refs. 30 and 31). The same motif is also present in the PDGF-A chain
(PDGF-A,
Trp35-Pro36-Pro37-Cys38),
although the structures of PDGF-AA or PDGF-AB have not yet been
determined.
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The folding pathways of oligomeric proteins frequently
exhibit very complex profiles, since unimolecular folding reactions and
bimolecular association steps are involved (16, 17). In case of PDGF,
the folding and association process becomes even more complex through
the additional requirement for the formation of the unusual knot-like
arrangement of the three intramolecular disulfide bridges and the
formation of the two intermolecular disulfide bonds. Although many
members of the cystine knot growth factor family are of enormous
medical importance (e.g. PDGFs, transforming growth
factors, bone morphogenetic proteins), almost no knowledge
exists about the mechanisms governing their folding and assembly. Some
more detailed studies have been carried out on the folding and
association kinetics of brain-derived neurotrophic factor (18) and
nerve growth factor (19). Both factors belong to a subgroup of the
cystine knot growth factor family where the subunits are not connected
by intermolecular disulfide bonds in the dimeric protein. Kinetic
studies on the folding and assembly of those growth factors of the
cystine knot family where the subunits are connected by disulfide bonds
in the native protein are missing so far.
It was commonly accepted that the presence of the cystine knot is a
prerequisite for the dimerization of proteins belonging to the cystine
knot growth factor family (5). However, recent studies on the structure
and stability of vascular endothelial growth factor, a member of PDGF
superfamily of cystine knot growth factors, revealed that cystine
deletion mutants lacking one of the two disulfide bonds forming the
outer ring of the knot motif are still able to form disulfide-bonded
dimers (20). Surprisingly, these mutants even revealed an increased
thermodynamic stability although their thermal stability was severely
reduced (20). However, the formation of the cystine knot appears to be
indispensable for the biological activity of PDGF (21), while the
intermolecular disulfide bridges have a stabilizing but non-essential
effect on the biological activity (21, 22). PDGF is very prone to aggregation when renaturation is initiated by diluting unfolded and
reduced monomers into a buffer, which allows refolding and disulfide
bond reshuffling (14). Once folded, however, PDGF is a very stable
protein withstanding temperatures of up to 100 °C (23).
Previously, we have presented a renaturation method based on the
utilization of size exclusion chromatography, which circumvents aggregation during refolding and allows renaturation of PDGF at high
protein concentrations (14). In this study, we present a kinetic
analysis of the formation of disulfide-bonded dimers and propose a
model for the folding pathway of the different isoforms of PDGF. The
unfolded and reduced monomers of PDGF were subjected to size exclusion
chromatography under renaturing conditions and the formation of
disulfide-bonded dimers starting either from pure A- or B-chains or an
equimolar mixture of both monomeric isoforms was followed in the eluate fraction.
 |
EXPERIMENTAL PROCEDURES |
PDGF-BB Structure Visualization--
PDGF-BB structure data were
obtained from the Protein Data Bank (www.rcsb.org/pdb/; accession
number 1pdg) and visualized using the program RasMol, Version 2.7.2.1 (RasMol Molecular Renderer; R. Sayle, Glaxo Research and Development
Greenford, Middlesex, UK).
Production and Purification of PDGF Isoforms--
The different
PDGF isoforms were produced as inclusion bodies using the
Escherichia coli strain TG1 carrying temperature-inducible expression vectors encoding either the PDGF A- or B-chain or a bicistronic vector encoding both chains synthesized in a 1:1 ratio upon
induction (13). Production of PDGF was carried out in a high cell
density cultivation procedure that has been described previously (24).
Purification of unfolded and reduced PDGF monomers from solubilized
PDGF containing inclusion bodies was done by size exclusion
chromatography (SEC) under denaturing conditions (14).
Kinetic Analysis of PDGF Dimerization--
Purified, unfolded,
and reduced PDGF monomers (either pure A-, or B-chains, or an equimolar
mixture of the two chains) were subjected to SEC under conditions that
allow refolding and reshuffling of disulfide bridges as described
previously (14). Standard renaturation conditions by SEC were: 1 mol
l
1 Tris-HCl (pH 7.8), 0.5 mol l
1
guanidinium hydrochloride (GdnHCl), 10 mmol l
1
glutathione reduced (GSH), 0.25 mmol l
1 glutathione
oxidized (GSSG). Under these conditions, the eluted PDGF monomers were
able to dimerize in the eluate fraction to yield the dimeric,
disulfide-bonded, and biologically active growth factor (14).
Aggregation of PDGF during the renaturation procedure was not observed
unless otherwise indicated. The formation of disulfide-bonded dimers
was followed in aliquots taken from the reaction mixture in the eluate
fraction through disulfide trapping by irreversible blocking of free
thiol groups and subsequent separation of monomeric and dimeric PDGF by
gel electrophoresis under non-reducing conditions. Blocking of the free
thiol groups by the addition of iodoacetate and gel electrophoresis was
carried out as described previously (14). Gels were stained with
Coomassie Brilliant Blue, and quantification of the monomeric and
dimeric fraction of PDGF was carried out by densitometry (Hirschman
elscript 400).
The putative reaction order for the rate-limiting step during
dimerization of PDGF was determined from the slopes of the linearized kinetic equations assuming either a first-order (Equation 1),
|
(Eq. 1)
|
or a second-order rate-determining reaction (Equation 2),
|
(Eq. 2)
|
where [A]t and
[A]0 are the monomer concentrations at times
t and zero, respectively,
va is the
sum of the stoichiometric factors, and k1 and
k2 are the rate constants for a first- or
second-order reaction, respectively. By rearranging the kinetic
Equations 1 and 2, the monomer turnover U can be simulated assuming either a first-order (Equation 3) or a second-order
rate-determining reaction (Equation 4).
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(Eq. 3)
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(Eq. 4)
|
In case of a unimolecular rate-limiting reaction, the monomer
turnover with time should be independent of the initial monomer concentration (Equation 3), while a second-order rate-limiting reaction
should be reflected by an increased monomer turnover with increasing
initial monomer concentration (Equation 4). Best-fit simulations of the
data from kinetic experiments for the determination of the rate
constants and modeling of the monomer turnover were carried out
using standard software.
The temperature dependence of the rate of dimerization was described by
an Arrhenius relationship, i.e. a plot of lnk
versus 1/T,
|
(Eq. 5)
|
where k is the experimentally determined rate
constant, A is a constant in the activated complex theory,
S# and
H# the
entropy and enthalpy of activation of the reaction, respectively, T the temperature, and R the universal gas
constant. If there is linearity for the temperature dependence of the
rate constant, the enthalpy and entropy of activation can be determined
from the slope and the y intercept of Equation 5, respectively.
 |
RESULTS |
Kinetics of Formation of Disulfide-bonded PDGF-AB Dimers from
Unfolded and Reduced PDGF-A and -B Monomers--
The kinetics of the
formation of disulfide-bonded dimers of PDGF were investigated under
redox reshuffling conditions starting from an equimolar mixture of
completely unfolded and reduced A- and B-chains (Fig.
2A). The kinetic data are well
described either by assuming a first- or a second-order
rate-determining reaction and the putative rate constants extracted
from the slopes of the linearized kinetic equations (cf.
experimental procedures) were determined to be
k1 = 1.5 10
5 s
1 or
k2 = 2.5 mol
1 l s
1,
respectively (Fig. 2B).

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Fig. 2.
Kinetics of formation of disulfide-bonded
PDGF-AB dimers from unfolded and reduced PDGF-A and -B monomers.
A, time course data of the relative concentrations of PDGF-A
and -B monomers ( ) and disulfide-bonded dimers ( ) after
subjecting an equimolar mixture of unfolded and reduced PDGF-A and -B
chains to SEC under renaturing conditions. Time zero indicates the
start of the kinetic experiment in the eluate fraction immediately
after elution of monomeric PDGF-A and -B from the SEC column.
B, determination of the rate constants assuming either a
first-order ( ) or a second-order rate-determining step ( ) as
described under "Experimental Procedures." The experimental
conditions were as follows: 0.1 mol l 1 Tris-HCl (pH 7.8),
0.5 mol l 1 GdnHCl, 10 mmol l 1 GSH, 0.25 mmol l 1 GSSG, 8.9 µmol l 1 (110 µg
ml 1) PDGF-A and -B monomers; T = 25 °C.
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To discriminate between a first- and a second-order rate-limiting
reaction controlling the formation of disulfide-bonded PDGF dimers,
renaturation experiments were carried out with varying initial monomer
concentrations (Fig. 3). The kinetic
analysis revealed an independence of the monomer turnover at given time points on the initial monomer concentration (Fig. 3A), thus
clearly excluding a second-order rate-limiting step in the renaturation of PDGF-AB. In addition, kinetic modeling revealed that the formation of the disulfide-bonded dimer is best described by a first-order rate-determining reaction (Fig. 3B). Deviation of the
predicted monomer turnover from the experimental data originates from
the formation of soluble off-pathway products, which are not able to
form native PDGF-AB. To account for the incomplete monomer turnover,
the kinetic model was further refined by including an additional step
leading to the irreversible formation of misfolded off-pathway products
(Fig. 4A). The experimental
data are now well described by the productive first-order reaction with
the rate constant of k1 = 1.5 10
5
s
1 yielding the native disulfide-bonded PDGF-AB dimer and
an unproductive reaction with a rate constant of
k1' = 7.5 10
6 s
1
leading to non-native off-pathway products (Fig. 4B).

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Fig. 3.
Analysis of the monomer turnover for the
determination of the reaction order of the rate-limiting step during
the formation of disulfide-bonded PDGF-AB. A,
determination of the monomer turnover at different time points with
varying initial monomer concentrations: 1 h ( ), 5 h ( ),
10 h ( ), and 20 h ( ) after eluting monomeric PDGF-A and
-B from the SEC column. B, time course data of the monomer
turnover of PDGF-A and -B starting with initial monomer concentrations
of 1.9 µmol l 1 ( ), 4.6 µmol l 1
( ), 6.2 µmol l 1 ( ), 7.4 µmol l 1
( ), 8.0 µmol l 1 ( ), 8.9 µmol l 1
( ), and 47 µmol l 1 ( ) are shown. In addition,
best fit simulations are depicted assuming a first-order with
k1 = 1.5 10 5 s 1
(thick line) or a second-order rate-limiting step with
k2 = 2.5 mol 1 l s 1
for the renaturation of PDGF-AB: 1.9 µmol l 1 (23.5 µg
ml 1), · · ·; 4.6 µmol l 1 (56.9 µg ml 1), -··-; 7.4 µmol l 1 (91.5 µg ml 1), thin line; 8.9 µmol
l 1 (110 µg ml 1), - - -; and 47 µmol L 1 (581 µg ml 1), .
Experimental conditions were the same as described in the legend to
Fig. 2 except for the initial concentrations of PDGF-A and -B
monomers.
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Fig. 4.
Simplified model for the formation of
disulfide-bonded PDGF-AB dimers from unfolded and reduced PDGF-A and -B
monomers. A, simplified model for the formation of
disulfide-bonded PDGF-AB dimers additionally including the formation of
non-native off-pathway folding products Mmisfolded
incompetent for the formation of native disulfide-bonded PDGF-AB
dimers. B, modeling of the kinetics assuming a first-order
rate-limiting step with k1 = 1.5 10 5 s 1 for the formation of the
disulfide-bonded PDGF-AB dimer. Formation of Mmisfolded
is described assuming a first-order rate-limiting step with
k1' = 7.5 10 6 s 1. In
addition, the experimental time course data of the relative
concentrations of PDGF-A and -B monomers ( ) and disulfide-bonded
dimers ( ) are shown. Experimental conditions were the same as
described in the legend to Fig. 2.
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A variation of the GdnHCl concentration between 0.25 and 1.5 mol
l
1 in the renaturation buffer revealed a strong decrease
in the rate of the formation of the disulfide-linked dimer with
increasing concentrations of the chaotropic agent (Fig.
5). The graphic representation of the
rate constants in a "chevron plot" revealed a linear dependence on
the GdnHCl concentration by anticipating a first-order rate-limiting step for the generation of the disulfide-linked PDGF-AB dimer (Fig.
5B). Final yields of disulfide-linked PDGF-AB dimers
increased from 13 to 75% by decreasing the concentration of GdnHCl
from 1.5 to 0.25 mol l
1. At 2 mol l
1
GdnHCl, formation of disulfide-linked dimers was not detectable (data
not shown).

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Fig. 5.
GdnHCl dependence of formation of
disulfide-bonded PDGF-AB dimers from unfolded and reduced PDGF-A and -B
monomers. A, time-dependent monomer
turnover of PDGF-A and -B at GdnHCl concentrations ranging from 0.25 to
1.5 mol l 1: 0.25 mol l 1 GdnHCl ( ), 0.5 mol l 1 GdnHCl ( ), 0.8 mol l 1 GdnHCl
( ), 1.2 mol l 1 GdnHCl ( ), and 1.5 mol
l 1 GdnHCl ( ). B, GdnHCl dependence of the
kinetic rate constant k1 assuming a first-order
rate-limiting reaction. Experimental conditions were the same as
described in the legend to Fig. 2, except for the GdnHCl
concentrations.
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Kinetics of Formation of Disulfide-bonded Dimers of the
Different PDGF Isoforms--
A unimolecular rate-limiting reaction
during the formation of the disulfide bonded PDGF dimer could either
indicate a rate-limiting folding reaction on the level of the monomeric
chain or structural rearrangements on the level of the dimeric not yet
disulfide-bonded growth factor. To discriminate between these two
possibilities, a kinetic study of the formation of disulfide-bonded
dimers was carried out using either the purified A- or B-chains or an
equimolar mixture of both chains (Fig.
6). The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, disulfide-bonded heterodimers were formed three times more rapidly
(k1 = 1.5 10
5 s
1,
experimental conditions, cf. Fig. 6) compared with the
formation of the two different disulfide-bonded homodimers from either
pure A- or B-chains (k1 = 0.5 10
5
s
1, experimental conditions, cf. Fig. 6) when
renaturation was started from an equimolar mixture of both chains.

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Fig. 6.
Kinetics of formation of disulfide-bonded
PDGF dimers of the different PDGF isoforms from unfolded and reduced
PDGF monomers. The time-dependent monomer turnover of
the three different PDGF isoforms PDGF-A, PDGF-A and -B, and PDGF-B are
shown starting with initial monomer concentrations of: 0.8 µmol
l 1 PDGF-A ( ), 9.25 µmol l 1 PDGF-A and
-B ( ), and 3.5 µmol l 1 PDGF-B ( ). Experimental
conditions were the same as described in the legend to Fig. 2, except
that the temperature was 35 °C.
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At all temperatures ranging from 4 °C to 45 °C heterodimerzation
occurred more rapidly compared with the formation of homodimers (Fig. 7
and Table I), suggesting a general
preference for the formation of the heterodimer. Also,
the kinetics of homodimerization of the
two different PDGF-AA or -BB isoforms did not show any significant
difference in the temperature range studied, supporting the conclusion
that homodimerization of either PDGF-AA or -BB follows most likely
similar pathways.
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Table I
Temperature dependence of the renaturation of the different PDGF
isoforms
Experimental conditions were the same as described in the legend to
Fig. 7.
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Fig. 7.
Arrhenius plots of the rate constants of the
formation of disulfide-bonded PDGF dimers of the different PDGF
isoforms from unfolded and reduced PDGF monomers. The rate
constants of dimerization of the three different PDGF isoforms PDGF-AA,
PDGF-AB, and PDGF-BB were determined at temperatures ranging from 4 to
45 °C assuming a first-order rate-determining reaction. The initial
monomer concentrations were: 0.8 µmol l 1 PDGF-A ( ),
9.25 µmol l 1 PDGF-A and -B ( ), and 3.5 µmol
l 1 PDGF-B ( ). Experimental conditions were the same as
described in the legend to Fig. 2, except for the temperatures.
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During the renaturation of all the three different PDGF isoforms
aggregation was not observed up to temperatures of 35 °C. However at
45 °C, partial aggregation of all PDGF isoforms occurred. A summary
of the results from the renaturation experiments carried out at the
different temperatures is shown in Table I. An estimation of the
activation enthalpies from the Arrhenius plots (Fig. 7; only data from
4 to 35 °C) of either homo- or heterodimerization did not reveal a
significant difference for the different PDGF isoforms. Assuming a
first-order rate-limiting renaturation step, activation enthalpies in
the range of 70-80 kJ mol
1 were estimated for the
formation of PDGF-AA, -AB, or -BB.
 |
DISCUSSION |
Subunit association can be a very fast process with rate constants
in the order of 107 mol
1 l s
1
(e.g. Ref. 25) that are encountered in diffusion-controlled reactions. However, when folding and association is connected with the
formation of intra- and intermolecular disulfide bonds, e.g.
during renaturation of antibody fragments, the regain of the biological
activity can occur in the time scale of hours to days (26).
The formation of disulfide-bonded dimers of PDGF is also a very slow
process occurring in the time scale of hours. Studies on the folding
and association of brain-derived neurotrophic factor (18) and nerve
growth factor (19), growth factors where the subunits are not connected
by intermolecular disulfide bonds, also revealed slow renaturation
kinetics, although, in general, little information exists on the
folding and association pathways of dimeric proteins of the cystine
knot growth factor family.
The rate-limiting step during the renaturation of a dimeric protein can
either be a first-order step resulting from unimolecular conformational
changes or a second-order step originating from the encounter and
assembly of the subunits. The experimental results clearly show that
the formation of the PDGF dimer is a process controlled by a
first-order reaction, thus proving that the encounter of the monomeric
chains to form the dimeric growth factor is not the rate-limiting step
in the renaturation of PDGF. A first-order rate-determining reaction
during the renaturation of a multimeric protein is not unusual. For
example, the kinetic analysis of the renaturation of the homodimeric
mitochondrial malate dehydrogenase revealed a second-order association
reaction (27), whereas the renaturation kinetics of the cytoplasmic
enzyme, also a homodimer, were governed by a first-order rate-limiting
step (28).
A first-order rate-limiting reaction can result from folding events on
the monomer level or from structural rearrangements of an already
dimerized protein. The comparative analysis of homo- and
heterodimerization revealed identical kinetics for the formation of the
disulfide-bonded AA or BB homodimers, suggesting that their renaturation pathways do not differ significantly, e.g. that
folding of the two different monomeric chains into association
competent molecules, association of these monomers into homodimers,
and, finally, formation of intermolecular disulfide bridges do not exhibit significantly different pathways when renaturation was started
either from the unfolded and reduced pure A- or B-chains. In contrast,
the AB heterodimers were formed three times more rapidly compared with
the formation of the homodimers. Assuming a rate-determining
first-order step on the level of monomer folding and observing
identical homodimerization kinetics is not contradictive if both
monomeric isoforms exhibit identical folding kinetics prior to the
formation of the dimer. But a rate-determining first-order step on the
level of monomer folding should also result in the same kinetics for
homo- and heterodimerization if both monomeric isoforms exhibit
identical folding kinetics. However, we observe that heterodimerization
of PDGF-AB occurs three times more rapidly than homodimerization. These
results clearly exclude the possibility that the rate-determining
first-order step can be assigned to conformational folding steps on the
level of the not yet dimerized monomer but point clearly to structural
rearrangements on the level of the dimerized but not yet
disulfide-bonded dimer as the pace maker of renaturation. In this case,
the different monomers can fold on identical pathways as the
experimental results strongly suggest; however, once dimerized but not
yet disulfide-bonded, the formation of the cystine bonds between the
different chains must be facilitated between the PDGF-A and -B monomer
compared with disulfide-bonding between identical chains. The results
also show that a statistically and diffusion controlled encounter of the different monomers must be reversible, i.e. that
non-covalent monomer association and dissociation must occur prior to
the formation of intermolecular disulfide bonds under the conditions
studied. Once connected by intermolecular disulfide bonds, however,
there are no indications for subunit exchange processes (data not
shown). The results from the kinetic studies on the folding and
assembly of PDGF are summarized in the model depicted in Fig.
8.

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Fig. 8.
Basic model for the formation of
disulfide-bonded PDGF dimers from unfolded and reduced PDGF
monomers. In this scheme Munfolded represents the
unfolded and reduced PDGF monomers, which are subjected to SEC under
conditions allowing refolding and disulfide bond reshuffling. M*
signifies the PDGF monomer recovered directly after SEC in the eluate
fraction. The reaction includes the transformation of M* into
association competent monomers Mfolded, the subsequent
encounter of Mfolded with another Mfolded into
a non-covalently associated dimer (MM)*, the first-order rate
determining structural rearrangements yielding the not yet
disulfide-bonded dimer D*, which can be transformed through
intermolecular disulfide-bonding into the native growth factor
Dnative. The model additionally includes the formation of
non-native off-pathway folding products Mmisfolded
incompetent for the formation of native disulfide-bonded PDGF
dimers.
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The kinetically preferred formation of the heterodimeric growth factor
should also affect the isoform distribution. Statistically an isoform
distribution of 1:2:1 is expected when refolding occurs from an
equimolar mixture of A- and B-chains. However, three times faster
heterodimerization compared with homodimerization should result in an
isoform distribution of 1:6:1, which accounts for 12.5% PDGF-AA, 75%
PDGF-AB, and 12.5% PDGF-BB. A similar isoform distribution was reached
when Chinese hamster ovary cells were used for coexpression of the
genes encoding the PDGF-A and -B chains (19% AA, 69% AB, and 12% BB;
Ref. 29).
The strong dependence of the kinetic constant of the rate-limiting
renaturation step on the concentration of the denaturant GdnHCl
strongly suggests that general structural rearrangements on the level
of the non-covalently associated but not yet disulfide-bonded dimer
determine the speed of renaturation. Finally, similar activation enthalpies of hetero- and homodimerization indicate that preferential heterodimerization must be controlled by entropic factors resulting in
a more favorable positioning of the cysteines from different chains for
the formation of the intermolecular disulfide bonds compared with
disulfide bond formation between identical chains.
 |
ACKNOWLEDGEMENT |
We are grateful to F. X. Schmid for
useful comments concerning the interpretation of the Arrhenius plot data.
 |
FOOTNOTES |
*
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.
Current address: Aventis Pharma Deutschland GmbH, 65926 Frankfurt, Germany.
§
Current address: Cytos Biotechnology AG, Wagistrasse 25, 8952 Zürich-Schlieren, Switzerland.
¶
To whom correspondence should be addressed. Tel.:
49-531-6181-126; Fax: 49-531-6181-111; E-mail:
URI@gbf.de.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M212317200
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ABBREVIATIONS |
The abbreviations used are:
PDGF, platelet-derived growth factor;
GdnHCl, guanidinium
hydrochloride;
SEC, size exclusion chromatography.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.