(Received for publication, June 22, 1995; and in revised form, August 8, 1995)
From the
-Inhibitor 3 (
I
)
is a rodent-specific proteinase inhibitor of about 190 kDa belonging to
the
-macroglobulin family. It consists of five
globular domains, three of which are connected by disulfide bridges,
and contains an intramolecular thiol ester which can react with
attacking proteinases. To explore the folding of newly synthesized
I
, we have used rat hepatocytes and
pulse-chase experiments. In one of the analyses, the radiolabeled
protein was isolated from cell lysates by immunoprecipitation and its
Asp-Pro bonds cleaved by treatment with formic acid. The size of the
major fragment, as assessed by electrophoresis under nonreducing
conditions, was found to increase from 100 to 150 kDa upon the chasing.
This result, together with knowledge of the positions of the cleavage
sites and the disulfide arrangement, indicates that one of the
interdomain disulfide bonds is formed after the synthesis of the
polypeptide. Analysis of the same material by limited proteolysis and
by velocity centrifugation showed that the folded regions became larger
and that the protein became more compact; the thiol ester was found to
be formed after these conformational changes. These results suggest
that the domains of
I
are only partially
developed directly after the synthesis of the polypeptide and that they
acquire their final structure as the protein condenses and the domains
interact with one another.
All proteins are synthesized as linear polypeptides which
subsequently fold into their unique three-dimensional structure.
Studies of the renaturation of isolated proteins have shown that the
information required for proper folding of a nascent polypeptide
resides in the amino acid sequence alone (Anfinsen, 1973). However,
folding in a living cell is faster and more efficient because it is
mediated by several proteins (Lorimer, 1992). So far, the in vivo folding of only a small number of proteins has been investigated
in any detail, but the mechanisms of this process are presently the
subject of intense studies (Jaenicke, 1991; Gething and Sambrook,
1992). Secretory proteins fold in the lumen of the endoplasmic
reticulum (ER) ()where they also undergo various kinds of
modifications (Hurtley and Helenius, 1989). In particular, most of
these proteins acquire disulfide bonds, a reaction mediated by protein
disulfide isomerase (Bulleid and Freedman, 1988), one of the most
abundant proteins in the lumen of the ER (Freedman, 1984). The rate at
which different proteins fold varies greatly; some acquire their final
conformation more or less cotranslationally (Peters and Davidson,
1982), whereas others may require up to an hour (Lodish and Kong, 1991)
facilitating analysis of the reaction.
In this study we have
analyzed the folding of -inhibitor 3
(
I
), a 180-200 kDa proteinase
inhibitor secreted by rodent hepatocytes (Esnard and Gauthier, 1980;
Geiger et al., 1987; Sjöberg et
al., 1991).
I
shows sequence homology
with
-macroglobulin and the complement components 3
and 4 (Braciak et al., 1988; Sottrup-Jensen, 1989). All of
these proteins have two characteristic features: they contain an
intramolecular thiol ester and have a sequence which is particularly
sensitive to proteolysis, the bait region (Esnard et al.,
1985; Sottrup-Jensen et al., 1989). When the polypeptide is
cleaved in the bait region, the protein undergoes a conformational
change, and the thiol ester becomes exposed to the exterior
(Sottrup-Jensen, 1989); in this position the thiol ester may react with
hydroxyl or amino groups on adjacent proteins or carbohydrates forming
a covalent link (Enghild et al., 1989).
I
appears to consist of five globular
domains (Rubenstein et al., 1991), and electron microscopic
analysis has revealed a ring-like structure (Ikai et al.,
1990). In a previous study we found that some disulfides of
I
are formed after the synthesis of the
polypeptide, indicating that part of the folding occurs
posttranslationally (Sjöberg et al.,
1991). In the present paper we have characterized this process further.
Our results indicate that the newly synthesized protein undergoes a
gross conformational change upon which the globular domains acquire
their final structure.
Figure 1:
Posttranslational formation of
disulfides in I
. Hepatocytes were labeled
with [
S]methionine for 10 min and chased for
different times.
I
was then isolated from
the solubilized cells and media by immunoprecipitation and detected by
SDS-PAGE under nonreducing conditions followed by fluorography. The
electrophoretically different forms of
I
are indicated to the left.
Figure 2:
Domain structure and disulfide arrangement
of I
. (Data from Rubenstein et
al.(1991) and Braciak et al.(1988), respectively.) The
domains, identified by limited proteolysis, are shown as rectangels numbered I to V. The disulfide bond arrangement
(represented by vertical brackets) is based on that of
-macroglobulin and the amino acid sequence deduced
from the sequence of the cDNA of
I
.
Whether the two cysteine residues between domains II and III (denoted
by vertical lines) are linked or not is unknown; the
corresponding residues in
-macroglobulin are part of
two interchain disulfides. The triangles show the positions of
the amide bonds that are susceptible to cleavage by formic acid. The
positions of the bait region and the thiol ester (TES) are
also indicated.
For the
experiment shown in Fig. 3A, I
was isolated by immunoprecipitation from the medium of
hepatocytes labeled with [
S]methionine or from a
lysate of pulse-labeled cells. Coprecipitating proteins were then
separated from
I
by electrophoresis in a
short polyacrylamide gel. The gel strips containing the different
electrophoretic lanes were cut out and treated with formic acid. They
were then placed along the upper edge of an electrophoresis gel, and
the protein fragments were separated. When the electrophoresis was
performed under nonreducing conditions, secreted
I
(lane 1) yielded two major
bands: the intact protein of 200 kDa and a 150-kDa fragment (arrow). Cellular, pulse-labeled
I
(lane 2) yielded both these fragments as well as smaller
ones, in particular in the range 90-110 kDa (bracket).
Under reducing conditions, identical fragments were obtained from both
secreted and pulse-labeled
I
(lanes 3 and 4, respectively); the major bands had apparent
molecular masses of about 95 and 55 kDa. As judged from the amino acid
sequence of
I
, the largest fragments
formed by mild acid hydrolysis should be of 43 and 44 kDa (cf. Fig. 2). The presence of the 95 kDa band therefore implies that
the cleavage reaction was incomplete; longer incubation was not
beneficial, however, because it resulted in substantial loss of
fragments. In a time course study we found that the relative amount of
polypeptides larger than 110 kDa increased upon chasing (Fig. 3B); densitometric analysis showed that this
amount increased from 38 to 86% during 40 min of chase, the
half-maximal increase occurring after 10-20 min.
Figure 3:
Chemical fragmentation of
I
. Hepatocytes were pulse labeled with
[
S]methionine and chased for different times.
I
was then isolated from the lysed cells
and from the media by immunoprecipitation and run on a 10%
polyacrylamide gel under reducing or nonreducing conditions. The gel
strips containing the different lanes were cut out and treated with
formic acid as described under ``Experimental Procedures.''
They were then placed along the upper edge of a polyacrylamide gel with
a 10-15% gel gradient, and the formed fragments were
electrophoretically separated in the presence of SDS. A shows
the pattern obtained with
I
isolated from
the medium of cells chased for 120 min (Med) and from
pulse-labeled cells (Cell) analyzed either in the presence or
absence of the reducing agent dithiothreitol (DTT). B shows
I
from cells chased for
different times which was analyzed under nonreducing conditions. The
positions of molecular mass standards are shown to the left.
Figure 4:
Limited proteolysis of
I
. Hepatocytes were pulse-labeled with
[
S]methionine and chased for different times.
I
was isolated by immunoprecipitation,
treated with chymotrypsin, and the resulting fragments were separated
by SDS-PAGE under reducing conditions. The apparent molecular masses of
the fragments are shown to the right (mean of two
experiments).
Figure 5:
Velocity centrifugation of
I
. Two cultures of hepatocytes were
pulse-labeled with [
S]methionine, and one was
additionally chased for 20 min. Intracellular proteins were then
extracted from both cultures by saponin treatment (Wassler et
al., 1987), and a mixture of both extracts was loaded on a sucrose
gradient. After centrifugation, fractions were collected from the
bottom of the gradient.
I
was then
isolated by immunoprecipitation and detected by SDS-PAGE under
nonreducing conditions. The lane labeled S shows the
applied sample. The different bands are denoted as described in the
legend to Fig. 1.
Figure 6:
Disulfide formation of
I
in cell-free systems. Hepatocytes were
pulse-labeled and microsomes prepared. Aliquots of the suspended
microsomes were incubated for the times indicated either alone (Microsomes), with detergent (TX), with detergent and
reduced/oxidized glutathione (TX+G), or with detergent,
reduced/oxidized glutathione, and protein disulfide isomerase (TX+G+PDI).
I
was then
isolated by immunoprecipitation and analyzed by SDS-PAGE under
nonreducing conditions. The different electrophoretic forms of
I
are indicated to the left. The arrow indicates the top of the separating
gel.
To see whether the conformational change of
I
could also occur in free solution, we
isolated microsomes from pulse-labeled cells, solubilized them with
Triton X-100, and incubated at 37 °C; predominantly the
intermediate forms,
1 and
2, were formed (Fig. 6, TX). If, however, the medium was made less oxidizing by the
addition of reduced and oxidized glutathione (both of 0.6 mM),
a minor increase of
3 occurred (Fig. 6, TX+G); the calculated redox potential of this solution is
-0.14 V (Hwang et al., 1992). Addition of protein
disulfide isomerase to the incubation mixture (TX+G+PDI) enhanced the rate of transition several
times, yielding band
3 as the end product.
Figure 7:
Thiol
ester formation of I
in cells and in
cell-free systems. A, hepatocytes were pulse-labeled and
chased for different times and
I
was
isolated from the cells and the media by immunoprecipitation. Thiol
ester-dependent cleavage was then induced by heating the samples, and
cleavage was assessed by SDS-PAGE under reducing conditions, a and a` denoting intra- and extracellular forms of intact
I
. In the cells, only the larger fragment
of
I
(band b) is apparent,
whereas in the medium, both the larger and the smaller cleavage
products can be seen (bands b` and c`, respectively). B, microsomes were prepared from pulse-labeled cells and
incubated at 37 °C for different times either in the presence of
reduced/oxidized glutathione (Microsomes) or with detergent,
reduced/oxidized glutathione, and protein disulfide isomerase (Free
solution).
I
was then isolated by
immunoprecipitation and assayed for thiol ester-dependent cleavage;
only the upper part of the gel is shown.
In this study we obtained evidence that at least one of the
interdomain disulfides of I
is formed
after the synthesis of the polypeptide. This conclusion is based on the
analysis of pulse-chase experiments in which
I
was isolated by immunoprecipitation and cleaved with formic acid.
Subsequent detection of the cleavage products by SDS-PAGE under
nonreducing conditions showed that the size of the major fragment
shifted from 100 to 150 kDa upon chasing (Fig. 3B).
With the assumption that cleavage occurred at the same rate at all
Gly-Pro bonds, the simplest explanation for this finding is that the
disulfide that links domains III and IV (Fig. 2), and which
spans half of the cleavage sites, is formed during the chase.
Another method we used to monitor the folding of
I
is based on the observation that tightly
folded regions or domains are more resistant to proteolytic degradation
than extended polypeptide segments (Porter, 1959; Fontana et
al., 1986). This method had earlier been used to identify the
domains of mature, secreted
I
(Rubenstein et al. 1991; Fig. 2). In that investigation, the
protein was isolated from rat serum and treated with increasing
concentrations of chymotrypsin. The cleavage products were then
separated by SDS-PAGE and the obtained polypeptide bands identified by
amino acid sequencing. The initial cleavage products were two bands of
about 100 kDa, which were found to be formed by scission of the bait
region (see Fig. 2), the upper and lower bands representing the
C- and N-terminal halves, respectively. At higher chymotrypsin
concentrations, the C-terminal fragment gave rise to two bands of 64
and 43 kDa (domains IV and V, respectively; see Fig. 2).
In
the present study we found that when radiolabeled
I
from cells chased for 20 min or more was
treated with a low concentration of chymotrypsin, three major fragments
of 86/84, 65, and 37 kDa were obtained (Fig. 4). The similarity
of the sizes of these fragments to those obtained from secreted
I
suggests that they represent the
N-terminal half of the molecule and domains IV and V, respectively.
(Intracellular forms usually have lower apparent molecular masses due
to incomplete carbohydrate processing.) Chymotrypsin treatment of
pulse-labeled
I
also yielded the 37-kDa
band, but the other major fragments, of 77 and 50 kDa, were clearly
different from those obtained from chased cells; however, comparison of
the peptide patterns of the 77- and the 86/84-kDa fragments after
cleavage with formic acid indicated that these fragments were partially
identical. (
)A simple explanation for these results is that
the C-terminal region of
I
(domain V) is
fully folded in the pulse-labeled protein, whereas the central region
is not and is therefore partially degraded upon treatment with
chymotrypsin. The 77- and 50-kDa fragments would then represent
truncated forms of domains I-III and domain IV, respectively.
Regardless of whether this interpretation is correct or not, our
results show that the proteinase resistant regions become larger after
the synthesis of the polypeptide. Furthermore, they indicate that the
protein occurs in two distinct conformations, since the fragments of
I
obtained from pulse-labeled and chased
cells were markedly different and there seemed to be no intermediates.
Concurrent with the shift in the sizes of the proteinase-resistant
fragments and the formation of new disulfides, I
became more compact as shown by the fact that its sedimentation
rate increased (Fig. 5). It is possible that these
conformational changes reflect a transition from an open, extended
structure to the ring-like shape of the mature protein (Ikai et
al., 1990). We also found that these changes preceded the
appearance of the thiol ester, suggesting that the formation of this
structure requires an almost completely folded protein. This notion is
supported by the finding that mutated forms of complement component 3,
in which the thiol ester could not be formed because one of the
necessary amino acid residues was missing, had a native-like
conformation (Isaac and Isenman, 1992). When the bait region of
I
and related proteins is proteolytically
cleaved, the thiol ester becomes exposed to the exterior of the
molecule making linkage to proteases possible (Enghild et al.,
1989). Simultaneously, the conformation of the C-terminal part changes
so that the proteins will bind to cell surface receptors effecting
their removal from the blood stream (Van Leuven et al., 1986;
Law and Dodds, 1990). Under the electron microscope,
I
appears to consist of one large and two
small elements forming an assymmetric ring; upon cleavage of the bait
region, the molecule acquires a more open, C-like shape (Ikai et
al., 1990). Presumably, the bait region is located in the larger,
central domain, and upon cleavage, this part of the molecule changes
its conformation so that the flanking structures are forced apart.
Clearly, this reaction must require a close association between
neighboring domains; the presence of interdomain disulfides (Fig. 2) indicates that this is indeed the case.
The redox
potential of the interior of the ER has been found to be intermediate
between that of the cytoplasm and the extracellular space (Hwang et
al., 1992). The physiological significance of this observation has
been borne out by studies on the folding of proteins in cell-free
systems which have shown that the formation of correct disulfides is
optimal under weakly oxidizing conditions (Scheele and Jacoby, 1982;
Huth et al., 1993; Lilie et al., 1994). Consistent
with these observations, we found that when pulse-labeled
I
in free form was incubated in the
presence of a mixture of reduced and oxidized glutathione, a
substantial part of the protein acquired the correct disulfides as
judged by its mobility upon SDS-PAGE under nonreducing conditions (Fig. 6, TX+G); under more oxidizing conditions,
in the absence of glutathione, the formation of the disulfides was
incomplete and/or incorrect (Fig. 6, TX). In agreement
with previous studies on other proteins (Bulleid and Freedman, 1988;
Huth et al., 1993; Creighton et al., 1993), we found
that the efficiency of the folding reaction was greatly enhanced in the
presence of protein disulfide isomerase (Fig. 6, TX+G+PDI); in addition to mediating the formation of
disulfides, this protein may also act as a chaperone (Noiva and
Lennarz, 1992). We also measured the formation of the thiol ester of
I
in the cell-free systems. This analysis
showed that the in vitro folding was only partially complete (Fig. 7B). It is possible that this result is due to
our incubation mixture being too oxidizing. This explanation is
supported by the fact that the folding rate, as judged by the rate of
disulfide formation, was higher in vitro than in vivo and that the redox potential of our incubation medium was higher
(-0.14 V) than what has been reported to be optimal for other in vitro folding systems (-0.18 to -0.31 V; Huth et al. 1993; Marquardt et al., 1993). It should also
be noted that in the living cell, the thiol ester formation is less
efficient than the formation of the disulfides (cf. Fig. 1and Fig. 7A).
In summary, our results
suggest that I
directly after its
synthesis has an only partially developed domain structure. It then
undergoes a conformational change that appears to bring the domains
together, whereby they acquire their final structure. Such an
interactive folding process has previously been described for
oligomeric proteins in which the conformation of the subunits has been
found to change upon assembly (Huth et al., 1992; Hurtley and
Helenius, 1989).