(Received for publication, April 17, 1995; and in revised form, July 27, 1995)
From the
Human PDI was expressed to the Escherichia coli periplasm, by using a plasmid encoded ompA-PDI fusion under the
control of the trp promoter. Periplasmic extracts were shown
to contain active PDI using the scrambled ribonuclease assay. PDI
activity was also demonstrated by complementation of two phenotypes
associated with a dsbA mutation. Alkaline phosphatase
activity, which is reduced in dsbA cells, was restored to wild
type levels by PDI. PelC, a pectate lyase from Erwinia
carotovora, was shown to be DsbA dependent in E. coli.
PDI was able to restore its activity to that seen in wild type cells.
Increased expression of PDI was found to increase the yield of active
PelC above that seen in wild type cells. PDI also enhanced the yield of
PelC in DsbA cells but only in the presence of
exogenous oxidized glutathione. PDI is thus able to functionally
substitute for DsbA in the folding of disulfide-bonded proteins in the
bacterial periplasm and to enhance the yield of highly expressed
protein when the ability of the E. coli periplasm to fold
protein may be saturated. However, our results suggest that the
activities of DsbA and PDI in vivo may be different.
Protein folding in vivo is assisted by two general
classes of proteins. Chaperones, such as GroEL and BiP, are thought to
interact noncovalently with substrates preventing or reversing
interactions that can lead to incorrect folding and aggregation.
Folding enzymes, such as PDI ()and
peptidyl-prolyl-cis-trans-isomerases, catalyze
rate-limiting steps in the folding of proteins (for a general review
see (1) ). The effects of chaperones and folding enzymes on
protein folding have been extensively studied in vitro (for
reviews see (2) and (3) ). However, studying assisted
folding of a protein in vivo is more difficult, partly because
of its subcellular location. For example, the major site for folding,
disulfide bonding, and modification of secretory polypeptides in
eukaryotes is the endoplasmic reticulum lumen, the composition of which
is not easily manipulable.
PDI is one of the major proteins of the
endoplasmic reticulum lumen. It was first shown to oxidatively refold
reduced RNase (4) and was subsequently shown to be the
-subunit of prolyl-4-hydroxylase(5) . It is thought to be
the main agent for formation and isomerization of disulfide bonds in
proteins folding in the endoplasmic reticulum lumen (for reviews see (6) and (7) ). However, this cellular function has
largely been demonstrated by indirect methods; PDI has been shown to be
physically associated with folding proteins in
vivo(8, 9) , has restored defective
co-translational disulfide bond formation in PDI-deficient
microsomes(10) , and has been shown to assist the folding of
proteins in vitro(11, 12, 13) .
Here, we have studied PDI-mediated protein folding by expressing human
PDI to the periplasm of Escherichia coli and assaying its
effects on model proteins. PDI is amenable to study in the E. coli periplasm because it is highly soluble, is not glycosylated, and
does not require ATP for its function.
The periplasm is an oxidizing
compartment and is the site of disulfide bond formation in E. coli proteins. A number of proteins have been identified in E. coli that are involved in disulfide bond formation, including DsbA,
DsbB, and
DsbC(14, 15, 16, 17, 18, 19) .
These proteins possess the motif, NH-CXXC-COOH,
containing cysteines that are important for catalytic activity. PDI
also possesses this motif. Purified DsbA acts as a disulfide
oxido-reductase and also acts weakly as a disulfide
isomerase(20, 21) . The relative importance of the two
activities in vivo is not known. Strains lacking DsbA show a
variety of defects in disulfide bond formation (see (22) for a
review). We demonstrate here that human PDI can complement two dsbA-dependent phenotypes, confirming the idea that PDI and
DsbA are functionally similar. We also show that when one particular
disulfide-bonded protein (PelC) is overexpressed, the folding capacity
of wild type E. coli cells can be saturated. The co-expression
of PDI overcame this deficiency resulting in increased levels of active
PelC. Increased levels of PelC could be produced from dsbA cells expressing PDI, but only when oxidized glutathione (GSSG)
was added to the growth media. This suggests that although DsbA and PDI
are functionally similar in complementation studies, their activities in vivo have some significant differences.
Figure 1:
Western blot demonstration of the
presence of PDI in E. coli periplasmic extracts. Periplasmic
extracts prepared by osmotic shock were separated by 12.5% reducing
SDS-polyacrylamide gel electrophoresis and then electroblotted to
nitrocellulose. Lanes 1-4, periplasmic extracts from
JCB570 containing pDPH5 (lanes 1-3) or pCT54 (lane
4). Lanes 5-8, periplasmic extracts from JCB571
containing pDPH5 (lanes 5-7) or pCT54 (lane 8).
Cultures were grown overnight with 0 µg ml IAA (lanes 1 and 5), 20 µg ml
IAA (lanes 2 and 6), and 80 µgml
IAA (lanes 3, 4, 7, and 8).
The antibody used was a rabbit polyclonal antibody raised against
purified bovine PDI. Each well was loaded for an equal amount of total
periplasmic protein.
Figure 2:
Activity of PDI in E. coli periplasmic extracts assayed by the sRNase method. The units of
PDI activity are A
min
min
mg
periplasmic extract,
and [IAA] is in µg ml
. The means and
standard deviations of three independent experiments are
shown.
Fig. 3shows that dsbA cells are deficient in
alkaline phosphatase activity measured relative to the wild type
(compare columns 1 and 2). Inclusion of the PDI
expression plasmid, pDPH5, under noninduced conditions gave nearly full
restoration of alkaline phosphatase activity. The control plasmid pDPH6
had no effect on alkaline phosphatase activity. This shows clearly that
PDI can functionally substitute for DsbA. Further investigation showed
that the level of alkaline phosphatase activity in the DsbA cells (JCB570) was not enhanced by overexpression of PDI from
either pDPH5 or pDPH13 (data not shown).
Figure 3:
Restoration of dsbA-dependent alkaline
phosphatase activity in vivo with periplasmic PDI. Column
1, JCB570; column 2, JCB571; column 3, JCB571
with uninduced pDPH5; column 4, JCB571 with uninduced pDPH6.
Alkaline phosphatase activity units are A
min
A
. The means and
standard deviations of three independent experiments are
shown.
Induction of PelC expression from pDPH9 with 0.2 mM IPTG resulted in a protein with a molecular mass of 39.8 kDa present in the periplasmic fraction, in agreement with the predicted mass(27) . PelC activity was shown to be dsbA-dependent in E. coli by assaying crude periplasmic extracts in vitro by following the degradation of polygalacturonic acid spectrophotometrically. The results are shown in Fig. 4. There was a significant reduction in the PelC activity recovered from dsbA cells, as shown by comparing columns 1 and 2. When PDI was supplied by including the compatible plasmid pDPH5, the activity of PelC recovered was restored to near wild type levels.
Figure 4:
Restoration of dsbA-dependent
PelC activity in E. coli peripasmic extracts with PDI. Column 1, JCB570 + pDPH9; column 2, JCB571
+ pDPH9; column 3, JCB571 + pDPH9 + pDPH5; column 4, JCB571 + pDPH9 + pDPH6. All cultures were
grown in the presence of 0.2 mM IPTG. PelC activity units are
A
min
mg
periplasmic extract. The means and standard deviations of three
independent experiments are shown.
This demonstrates clearly that PelC activity, and therefore presumably PelC folding, is dsbA-dependent in vivo in E. coli. PDI is able to act on PelC protein and restore its activity to near wild type levels in a dsbA background. pDPH5 was uninduced for PDI expression in this experiment. Under these conditions, there is only a very small amount of PDI in the periplasm, which is only easily visualized by immunoblotting of such periplasmic extracts separated on SDS-polyacrylamide gel electrophoresis gels (data not shown). However, the level of PelC activity recovered from the JCB571 + pDPH9 + pDPH5 cells appeared to be slightly greater than that from wild type cells. This led us to investigate whether increased PDI expression could give enhanced PelC yield.
Figure 5:
Overexpression of PelC in E. coli in the presence of increasing expression of PDI. Column
1, JCB570 + pDPH9; column 2, JCB571 + pDPH9; columns 3-7, JCB570 + pDPH9 + pDPH5 with 0,
10, 20, 40, and 80 µg ml IAA, respectively; column 8, JCB570 + pDPH9 + pCT54 with 80 µg
ml
IAA. All cultures were grown in the presence of
0.4 mM IPTG. PelC activity units are
A
min
mg
periplasmic extract. The means and standard deviations of three
independent experiments are shown.
Figure 6:
Expression of PelC in dsbA E. coli in the presence of increasing expression of PDI. Column
1, JCB570 + pDPH9; column 2, JCB571 + pDPH9; columns 3-5, JCB571 + pDPH9 + pDPH5 with 0,
10, and 80 µg ml IAA, respectively; column
6, JCB571 + pDPH9 + pCT54 with 80 µg
ml
IAA. All cultures were grown in the presence of
0.4 mM IPTG. PelC activity units are
A
min
mg
periplasmic extract.
The means and standard deviations of three independent experiments are
shown.
We tested if this limitation in
DsbA cells could be reversed by supplying GSSG in the
media. It is thought that the relatively oxidizing environment of the
endoplasmic reticulum lumen is maintained by a mixed glutathione
buffer(34) , and it has been shown that use of glutathione
buffers in bacterial media affects the redox state of the
periplasm(35, 36, 37, 38) . Fig. 7shows that in the presence of GSSG, PDI can enhance the
yield of PelC above that seen in wild type cells even in a dsbA
background. The yield of PelC seen with
5 mM GSSG was approximately half that seen with an equivalent
level of PDI induction in wild type cells as seen in Fig. 5but
is still approximately twice the yield seen in wild type cells without
PDI.
Figure 7:
Functional replacement of DsbA by oxidized
glutathione in overexpression of PelC. Column 1, JCB570 +
pDPH9; column 2, JCB571 + pDPH9; columns 3, 5, and 7, JCB571 + pDPH9 + pDPH5 with 0, 1,
and 5 mM GSSG, respectively; columns 4, 6,
and 8, JCB571 + pDPH9 + pCT54 with 0, 1, and 5
mM GSSG, respectively. All cultures were grown in the presence
of 0.4 mM IPTG, and cultures 3-8 also had 20
µg ml IAA. PelC activity units are
A
min
mg
periplasmic extract. The means and standard deviations of three
independent experiments are shown.
In this paper we present direct evidence for the ability of human PDI to assist the oxidative folding of proteins in vivo in E. coli. We show for two different proteins that PDI can functionally substitute for DsbA. We also show that high levels of co-expression of PDI result in increased yields of active PelC above that achieved by wild type cells. This overproduction is itself DsbA-dependent, but the provision of GSSG in media overcame this dependence.
The rate of folding of alkaline phosphatase in dsbA strains is lower than that of wild type cells(14) , resulting in lower overall alkaline phosphatase activity in dsbA cells. Co-expression of low levels of PDI restores the measured alkaline phosphatase activity to near wild type levels, suggesting that PDI is influencing the rate of folding of alkaline phosphatase.
We
used PelC as a model folding protein and by assaying crude periplasmic
extracts in vitro showed that the amount of active PelC
recovered from dsbA cells is significantly
reduced compared with that recovered from dsbA
cells. We interpret this as demonstrating that the folding of
PelC is DsbA-dependent in vivo. Because E. carotovora is known to contain Dsb proteins similar to those in E.
coli, we would expect PelC folding to be DsbA-dependent in its
native host also, as suggested previously(19) . Co-expression
of low levels of PDI from a plasmid compatible to the one carrying the
PelC gene resulted in a restoration of the recovered PelC activity to
near wild type levels, demonstrating again that PDI can functionally
substitute for DsbA.
While doing PelC assay experiments on the phoR strains JCB570 and JCB571, we observed
that the total protein concentration of periplasmic extracts from dsbA
JCB571 was consistently about 30% lower
than that from dsbA
JCB570. Low levels of PDI
expression restored protein concentrations in dsbA
cells to normal levels. (
)PhoR
strains constitutively express several periplasmic proteins (for
example, alkaline phosphatase, phosphate-binding protein, and glycerol
3 phosphate-binding protein) (for reviews see (39) and (40) ). The lower rate of folding in the periplasm of dsbA cells probably results in a greater proteolytic susceptibility for
these proteins, similar to that seen with alkaline phosphatase,
-lactamase, OmpA, and BPTI(14, 36) , and hence a
lower final protein concentration. This suggests that PDI can act on
several different E. coli proteins.
In the complementation experiments PDI was produced from uninduced pDPH5, so the level of expression of PDI is low; rich media are known to repress the trp promoter(41) . This demonstrates that only small amounts of PDI are necessary to complement dsbA phenotypes, suggesting that PDI is acting catalytically and not just as an extra source of a periplasmic redox buffer. The fact that PDI appears to act on many periplasmic proteins suggests that PDI has a relaxed peptide binding specificity in vivo, which is consistent with experiments done in vitro(42, 43) .
We tested if increased
production of PDI would result in increased yields of PelC. The results
in Fig. 5show that indeed higher levels of induction of PDI
gave greatly increased yield of PelC activity above that seen in wild
type cells. The yield of PelC activity increased with increasing levels
of PDI expression, although it reached a plateau in the range of IAA
concentrations tested. This suggests that the yield of overexpressed
PelC in DsbAE. coli is limited by the
cellular folding machinery. This may be due to saturation of the Dsb
system, resulting in increased proteolytic susceptibility for some
molecules of PelC in the periplasm.
We showed that higher levels of
periplasmic PDI only achieved overproduction of PelC in the presence of
DsbA. 1 and 5 mM GSSG partially restored the ability of PDI to
increase the yield of active PelC in a dsbA background. Others
have used redox buffers supplied in culture media while trying to
influence folding of periplasmic proteins directly but with mixed
success(36, 37, 38, 39) . In our
experiments, the addition of 1 and 5 mM GSSG had no effect on
the yield of active PelC in DsbA cells containing no
PDI. Therefore, GSSG alone lacks the ability to increase the yield of
oxidatively folding proteins in the periplasm, which is consistent with
previous work. However, the same concentrations of GSSG in
DsbA
cells in the presence of PDI has a marked
positive effect.
The precise mechanism of complementation of dsbA by PDI is not known. It could simply be a result of PDIs dithiol oxidase activity substituting directly for that of DsbA. If this is true, an unknown oxidant(s) must be responsible for regenerating active PDI. Alternatively, PDI acting as an isomerase may increase the flux through a DsbA-independent pathway, for example by isomerization of disulfide-bonded intermediates that have spontaneously formed in the incorrect conformation. Such a pathway is probably normally catalyzed by DsbC, and it will be of great interest to investigate the ability of PDI to complement dsbC mutants. The fact that overproduction of PelC is only seen in wild type cells but not in dsbA cells suggests that a DsbA-dependent step is rate-limiting, at least for PelC. This can be overcome by provision of oxidized glutathione. GSSG may act either as an oxidant, thereby improving the regeneration of oxidized PDI, or by forming a mixed adducts with PelC and PDI, which may accelerate formation of the active protein, by the mechanisms demonstrated by Darby et al.(44) .
Understanding the exact actions of PDI and
DsbA on alkaline phosphatase and PelC folding are complicated in
vivo by the dynamic nature of the periplasm, which may have
changing needs for oxidation/isomerization activities during cell
growth. Co-expressed PDI was able to effect increases in the yield of
PelC in the absence of any supplied redox buffers. In contrast,
co-expression of DsbA only gave increased yield of native
-amylase/trypsin inhibitor from Ragi (RBI) in the
presence of media redox buffers(36) . This suggests that PDI
and DsbA may have different activities in vivo. We also note
that overexpression of PelC in DsbA
cells in the
presence of PDI and GSSG resulted in only approximately half the yield
of PelC compared with that in the DsbA
cells with PDI
(compare column 7 in Fig. 7with column 5 in Fig. 5). This suggests that the enhancement by GSSG (in
DsbA
cells) was not due to direct replacement of the
DsbA activity missing.
The phoR cells used for these
experiments constitutively translocate at least six periplasmic
proteins, alkaline phosphatase, phosphate-binding protein, glycerol 3
phosphate-binding protein, PelC, PDI, and -lactamase (from pDPH5),
without apparent difficulty. However, overexpression of PelC may have
saturated the Dsb machinery. Perhaps PDI only has a beneficial role to
play in E. coli under Dsb saturated conditions?
Bacterial proteins generally have fewer disulfide bonds than secreted eukaryotic proteins(20) . The ability of PDI to catalyze the folding of more complex proteins in a range of genetic backgrounds is currently being evaluated. Co-expression systems such as this provide a powerful investigative tool. Individual proteins such as PDI can be investigated in an environment closer to that in the cell than can be generated using reconstituted systems in vitro, thereby enabling investigation of other factors that affect the function of PDI, such as redox conditions and PDI-protein interactions.