(Received for publication, April 26, 1995; and in revised form, October 3, 1995)
From the
With the aim to improve our understanding of the structural
basis for protein self-association and aggregation, in particular in
relationship to protein refolding and amyloid formation,
folding-related processes for human cystatin C have been studied. Using
NMR spectroscopy together with chromatographic and electrophoretic
methods, a self-association process resulting in dimer formation for
protein samples treated with denaturing agents as well as for samples
subjected to low pH or high temperature conditions could be studied
with amino acid resolution. In all three cases, the dimerization
involves properly folded molecules and proceeds via the reactive site
of the inhibitor, which leads to complete loss of its biological
activity. This dimerization process has potential relevance for amyloid
formation by the brain hemorrhage-causing Leu-Gln variant
of cystatin C. The results also indicate that cystatin C dimerization
and inactivation may occur in acidified compartments in vivo,
which could be relevant for the physiological regulation of cysteine
proteinase activity.
The self-association and aggregation of proteins constitutes one
of the least understood problems in protein chemistry. ()Although some insight has been gained in the kinetic
aspects of the aggregation process and in the field of its general
theory (Glatz, 1992; Honig and Yang, 1995; De Young et al.,
1993), very little is known about its structural aspects and about the
precise nature of the interactions involved. It is generally assumed
that aggregation proceeds via a partially exposed hydrophobic core in a
molten globule, but the very general character of the molten globule
concept seriously limits any practical consequences. For controlled
refolding of recombinant proteins and for stabilization of
pharmaceutical formulations, as well as for the development of new
therapies for aggregation-related disease states including amyloidosis,
more development in this research area clearly is needed.
To add to
our understanding of the molecular aspects of amyloid formation, in
particular of the specific intermolecular interactions leading to
fibril formation, we have started systematic studies of the properties
of amyloidogenic proteins. Human cystatin C provides a good starting
point for such studies. This small size inhibitor of cysteine
proteinases is present in all human body fluids at physiologically
relevant concentrations, being most abundant in the cerebrospinal fluid
and in seminal plasma (Abrahamson et al., 1986). Although
cystatin C in its wild-type form has not been reported to form amyloid in vivo, its Leu
Gln mutated variant
(L68Q-cystatin C) is responsible for the dominantly inherited disorder
called hereditary cystatin C amyloid angiopathy (Ghiso et al.,
1986; Jensson et al., 1987; Palsdottir et al., 1988).
Expression systems for both wild-type and L68Q-cystatin C have been
developed (Dalbøge et al., 1989; Abrahamson and Grubb,
1994), and the three-dimensional structure of the human cystatin C-like
type II cystatin, chicken cystatin, is well characterized by both x-ray
crystallography and NMR spectroscopy (Bode et al., 1988; Engh et al., 1993). The conformation of the 120-residue polypeptide
chain of cystatin C is very similar to that of chicken cystatin, (
)with a proteinase-interacting wedge-shaped side involving
residues Arg
-Gly
of the N-terminal segment and
two loop-forming segments constituting polypeptide turns in the main,
-pleated sheet structure of the molecule (segments
Gln
-Gly
and around the single tryptophan
residue, Trp
). An additional advantage for the study of
cystatin C in the aspect of protein aggregation and amyloid formation
is the fact that it normally exists in a monomeric form, in sharp
contrast to many other amyloid-forming proteins which are found in
multimeric, often heterogeneous, and condition-dependent forms (Brange et al., 1987; Brader and Dunn, 1991; Colon and Kelly, 1992).
Only about 15 of the human body fluid proteins are proven to be amyloidogenic (Stone, 1990; Sipe, 1992). The formation of amyloid is often either directly related to specific point mutations or is enhanced significantly by point mutations (Frangione, 1991; Glenner et al., 1991; Sipe, 1992). As is particularly clear in the case of transthyretin, many different point mutations which do not form a clear pattern can lead to amyloid formation. Therefore, there is a tendency to explain amyloidogenity as being caused by reduced stability of the proteins (Hurle et al., 1994; McCutchen et al., 1993). Earlier results (Abrahamson and Grubb, 1994) suggest that cystatin C and its L68Q variant can follow a similar pattern. In the present investigation, we have undertaken a detailed study of wild-type cystatin C under conditions leading to its folding-related self-association, with the aim to define the molecular background to the events leading to amyloid formation and physiological inactivation of L68Q-cystatin C.
NMR
experiments were run in both regular and heavy water. From the NH
signals observed in water, NH of the Trp residue is
particularly important (see below). In heavy water, as NH signals are
exchanged to ND, many aromatic signals can be interpreted clearly. To
give an overview of advantages given by both methods and to avoid
repetitions, results for the GdnHCl experiments will be presented only
for the H
O solutions, and results for temperature and pH
experiments only for the D
O solutions.
Figure 1: Agarose gel electrophoresis under partially unfolding conditions. Isolated recombinant cystatin C was incubated at 75 mM concentration. a, for 30 min at various temperatures in 50 mM sodium phosphate buffer, pH 6.7, containing 0.1 M NaCl; b, for 17 h in varying concentrations of GdnHCl (GuHCl) in 50 mM sodium phosphate buffer, pH 6.0, at room temperature; c, for 11 h at various pD values. The incubation mixtures were analyzed by electrophoresis in 1% (w/v) agarose gels. The direction of the electric field is indicated to the left. The points of sample application are marked by arrows.
In the experiments with increasing concentrations of guanidine hydrochloride, self-associated cystatin C started to appear at 0.3-0.4 M GdnHCl, and the amount of trapped self-associated form started decreasing at 1.2 M GdnHCl. Similar results were obtained at low pH values, with maximum amounts of self-associated cystatin C observed in the pH range 3.0 to 4.4. The relative amount of self-associated cystatin decreased rapidly when pH was lowered further (Fig. 1c).
Figure 2: Size exclusion chromatography of cystatin C dimers. Dimer-containing cystatin C samples obtained under conditions described in the legend to Fig. 1were trapped by rapid transfer to a 50 mM phosphate buffer, pH 6.7, containing 0.1 M NaCl. a, monomeric cystatin C; b, dimer produced by an incubation in 0.8 M GdnHCl for 16 h at room temperature; c, dimer produced at pD 3.0 (16-h incubation at room temperature); d, dimer formed by incubation at 68 °C for 30 min. Retention times for the calibration standards bovine serum albumin (A), ovalbumin (O), chymotrypsinogen (C), and ribonuclease A (R) are indicated.
Figure 3:
H NMR spectra of cystatin C in
various concentrations of GdnHCl. Spectra were run in 50 mM phosphate buffer, pH 6.0, containing between 0.0 and 2.0 M GdnHCl at 30 °C after overnight (17 h) incubation. A,
downfield region; B, upfield
region.
Figure 4: Downfield (A) and upfield (B) regions of NMR spectra of cystatin C as a function of pD. Spectra were run in 0.1 M sodium acetate buffer in heavy water. Titrating histidine signals are marked with asterisks.
Figure 5:
The upfield region of the NMR spectra of
monomeric cystatin C. A, fragment of the two-dimensional TOCSY
spectrum at 40 °C (mixing time 114 ms) showing assignments for
methyl group signals used as markers in one-dimensional spectra. B, temperature dependence of one-dimensional spectra. Spectra
were run in sodium phosphate buffer containing 0.1 M NaCl at
pD 6.7, with data collection time of 2 h (4000 accumulations). T and T
denote two unassigned threonine methyl group signals. Signals of
residues Leu
, Leu
, and Leu
were
assigned using the aliphatic region of the same TOCSY spectrum, and the
methyl group signals of three methionines using a two-dimensional NOESY
spectrum (see text).
Figure 6:
Comparison of the aromatic (A)
and aliphatic (B) regions of H NMR spectra of
monomeric and trapped dimeric cystatin. Spectra were run at 62 °C
in 50 mM sodium phosphate buffer containing 0.1 M NaCl, at pD 6.7 in heavy water, using 4000 accumulations for each
sample. a, monomeric cystatin C; b, dimer formed by
an incubation at 62 °C for 32 h (notice temperature-induced
exchange of histidine signals); c, dimer formed by an
incubation for 16 h in 0.8 M GdnHCl solution; d,
dimer formed at pD 3.0 for 16 h. Three histidine hydrogens which
exchanged to deuterium during incubation at high temperature (spectrum b) are labeled with asterisks.
As can be seen in Fig. 6, A and B, NMR spectra of the monomeric
and dimeric cystatin C are very similar. In particular, most of the
characteristic signals which could be assigned easily and followed at
high temperature were essentially not changed during dimerization. That
includes amino acids in the -sheet (Tyr
,
Leu
, Leu
, Met
, and
Phe
) as well as in the
-helix (Tyr
and
Ala
). Also unchanged are Tyr
and Met
in the loop between the
-helix and the
-sheet,
Leu
in the loop area of the
-sheet, as well as all
histidines and most of the alanine methyl groups. The overall envelope,
especially of the aromatic and
-protons of the
-sheet, also
remains the same. That is in contrast to Trp
,
Tyr
, and Val
signals, which undergo
relatively large (above 0.2 ppm) changes. These latter changes develop
slowly, on the time scale parallel to the formation of dimers (data not
shown). Similarly, time-dependent changes were detected for the
Ala
methyl group signal.
Despite vast amounts of experimental data obtained in the
last 2 decades about protein folding, rather little is known about
specific intermolecular interactions, competing with the intramolecular
interactions in the process of folding. To a large extent, that
situation follows methodological approaches of using techniques that
characterize proteins in a global way (such as CD, fluorescence, light
scattering, hydrodynamic, and enzymatic methods). Results of the
present work show that with the addition of the high resolution NMR
spectroscopy small proteins with a limited degree of self-association
or aggregation can be characterized in great detail, especially in
quite common cases where small oligomers are formed. NMR spectroscopy
in such a case can provide a picture of simultaneous folding and
self-association or aggregation with a resolution at the amino acid
level. Two other methods, agarose gel electrophoresis and size
exclusion chromatography, were selected to complement the NMR studies
by giving direct global information about the presence and size of the
self-associated forms. Standard spectroscopic methods used to follow
protein folding such as fluorescence and CD were avoided; as in the
case of cystatin C, they give a complex blend of information about both
self-association and folding down to micromolar concentrations.
In the present work, cystatin C properties were examined under
conditions close to those leading to unfolding of the protein. Three
different ways of denaturing the protein were used, i.e. by
decreasing pH, adding a denaturing reagent (guanidine hydrochloride),
and raising the temperature. In all three approaches, agarose gel
electrophoresis demonstrated broad pretransitional regions, in which
self-association in the form of a dimerization takes place. Although
some precipitation was observed, especially at high temperatures and
concentrations above 1 mg/ml, no stable intermediate-sized
self-associated forms were detected by either electrophoresis or SEC
experiments. Pretransitional regions were examined in a more detailed
way using NMR spectroscopy. In the experiments with varying pH and
GdnHCl concentrations, NMR spectra directly revealed under which
conditions unfolding of the protein was occurring. All NMR results
clearly indicated that the pretransitional region observed for cystatin
C is completely different from the molten globule type of an
intermediate state, which is quite common in protein folding paths and
is often associated with an increased aggregation (Dolgikh et
al., 1981; Kuwajima, 1989; Ptitsyn et al., 1990;
Ghélis and Yon, 1982). NMR spectra of proteins in
a molten globule state show features characteristic of a loss of the
tertiary structure, as was well characterized for -lactoglobulin
(Dolgikh et al., 1985; Baum et al., 1989). Such
spectra are more similar to those of the unfolded than to the native
protein. On the contrary, NMR spectra of the dimeric cystatin C are
very similar to those of the native monomeric form, with the majority
of the tertiary structure clearly preserved. Although at the current
stage of the spectral analysis there is a possibility of changes in
other parts of the molecule, the majority of observed shifts in proton
signals clusters around the proteinase binding site. Therefore, one can
postulate that in approaching the unfolding point, the structure of the
protein becomes more loose, which leads to conformational or hydration
changes in the loops forming the proteinase binding site. The most
evident differences in one-dimensional NMR spectra between the
monomeric and dimeric form of cystatin C are in the second hairpin loop
(for residues Tyr
, Val
, and
Trp
), which according to computer docking of chicken
cystatin with papain (Bode et al., 1988) should be within van
der Waals contact with the proteinase. Either conformational changes in
the active site directly, or dimer formation via the binding site
loops, can explain the observed loss of the activity of cystatin C as
an inhibitor of cysteine proteinases upon dimerization. The K
value for cystatin C interacting with papain is
in the picomolar range (Abrahamson et al., 1986), and the
equilibrium constant for the self-association of cystatin C is in the
micromolar range according to our present results. Still, dimerized
cystatin C was unable to inhibit papain even after prolonged
incubations, which must be explained by the cystatin being trapped in a
dimeric form that is separated by a high energy barrier from the
monomeric state (details about the barriers will be presented
elsewhere).
The aggregation and precipitation during refolding of proteins produced as inclusion bodies constitutes one of the major obstacles in efficient production of recombinant proteins in bacterial systems (Cleland, 1991; Mitraki and King, 1989). These problems were studied extensively (Ghélis and Yon, 1982; Mitraki et al., 1987; Brems, 1988; Lehrman et al., 1991; Cleland and Wang, 1990), with the general conclusions that folding intermediates with a partially exposed hydrophobic core are responsible for the aggregation and precipitation (Wetzel, 1992; Mitraki and King, 1989). The dimerization of cystatin C (leading to precipitation at higher concentrations) resembles other systems by exhibiting a characteristic trough (Ghélis and Yon, 1982) under conditions directly preceding unfolding. However, there is a distinct difference, as both the tertiary and secondary structure of cystatin C are conserved under such conditions. As mentioned above, the unusual large hydrophobic patch on the protein surface shows large changes in the NMR signals and, likely, participates directly in the self-association event.
It is clear from the results of the present study that cystatin C can undergo dimerization under conditions when the protein seemingly has a ``normal'' conformation, quite far from those leading to unfolding. It is possible that other proteins which self-associate or display problems during refolding may follow this case directly. Potentially pretransition-related changes were reported recently for the reverse transcriptase from HIV (Wright et al., 1994), and, in the past, many other proteins were reported having predenaturational changes (Privalov, 1979). Similar mechanisms of interaction could play a role in folding of multidomain or oligomeric proteins. Further studies are needed to characterize such a way of self-association, and cystatin C seems to be an ideal model case for such work.
A mutated
variant of cystatin C, L68Q-cystatin C, has been identified as cause
for the genetic disease that is known as hereditary cystatin C amyloid
angiopathy or cerebral hemorrhage with amyloidosis, Icelandic type. The
Leu
Gln substitution in cystatin C results in
massive systemic deposits of the protein, in particular in brain blood
vessels, leading to hemorrhages and early death (reviewed by Jensson et al.(1987)). Recently, L68Q-cystatin C has been produced by E. coli expression (Abrahamson and Grubb, 1994), and it was
shown that the mutated variant precipitates rapidly at human body
temperatures, with transitory formation of dimers. Although more work
on folding of the L68Q variant of cystatin C is needed, most likely
formation of its dimers is similar to that observed in the
pretransitional region for the wild-type protein. However, dimers of
L68Q-cystatin C are formed at temperatures nearly 30 degrees lower than
needed for the wild-type cystatin (Abrahamson and Grubb, 1994).
Therefore, one can postulate that the Leu
Gln
substitution lowers the transition temperature for the unfolding. That
would be expected, as it is common that a substitution of an amino acid
in the hydrophobic core of a protein decreases its stability (Pacula
and Sauer, 1989; Alber, 1989). Results presented in this work suggest
that cystatin C provides another system where decreased stability of a
mutant protein correlates with its amyloidogenic nature. Increased
propensity for the self-association in the broad range before unfolding
may have direct relevance for the formation of the amyloid. The
wild-type protein should be quite stable under physiological
conditions; however, the L68Q variant should be in the self-association
prone, pretransitional region. It remains to be determined if partial
unfolding of the protein is necessary for the nucleation step and if
dimers are indeed on the path to the fibril formation. Since we have
found similarities between the behavior of L68Q and wild-type cystatin
C under partially denaturing conditions, the next relevant question to
address must be a structural comparison between the dimers and
exploration of the role of these dimers as potential intermediates in
the amyloid formation.
It also remains to be clarified if dimer
formation has any physiological significance for wild-type cystatin C,
especially in pathological states. The most likely possibility for a
physiological dimerization would be in acidic compartments in cells,
involved in the endo- and exocytosis. For example, pH in lysosomes is
in the 4.6-5.0 region, which corresponds to the range where
cystatin C dimerizes to some extent according to our in vitro results. Even lower pH values, corresponding to those resulting in
extensive dimerization and parallel inactivation of cystatin C in
vitro have been observed beneath adherent macrophages and
osteoclasts (Silver et al., 1988). It is important to keep in
mind that our in vitro experiments demonstrate that cystatin C
is capable of ``remembering its history,'' as the dimeric
inactive form is very easily trapped for amounts of time practically
infinite on the physiological time scale. In this context, it is
intriguing that inactive cystatin C dimers quite likely similar to
those described in this paper are present in lysates of human
neuroendocrine cells in which cystatin C is stored in secretory
granules together with neuropeptides. ()Also, cathepsins B
and L have been reported to be responsible for the extracellular
process of bone resorption (Delaisséet
al., 1984; Kakegawa et al., 1993), even though cystatin C
is ubiquitous extracellularly and a potent inhibitor of cathepsin B and
L activity (Abrahamson et al., 1986, 1990). This apparent
conflict could be explained by a dimerization-caused inactivation of
cystatin C in the sealed off and acidified compartment under the
bone-degrading osteoclasts.