(Received for publication, March 22, 1995; and in revised form, June 22, 1995)
From the
Protein disulfide isomerase in isolated rat hepatocytes was
present at a concentration of 7 µg/mg cell protein, representing a
2-fold enrichment compared to isolated hepatic non-parenchymal
cells. Though localized mainly in microsomal fractions of hepatocytes,
direct immunofluorescence and cell surface radioiodination followed by
immunoprecipitation revealed the presence of M
57,000 disulfide isomerase at the cell surface. Electrostatic
interaction of the protein with the cell surface was suggested by
susceptibility to carbonate washing. Metabolic radiolabeling and
immunoprecipitation studies also indicated that some of the newly
synthesized M
57,000 disulfide isomerase was
secreted. Treatment of cells with colchicine markedly reduced the
recovery of disulfide isomerase from the media, indicating
microtubular-directed secretion of the protein. Partial staphlococcal
V8 proteolytic digestion of the secreted protein revealed a peptide
pattern similar to that of the cellular protein. Immunoprecipitation
with antibody specific to the -KDEL peptide retention sequence
confirmed the presence of this sequence in the secreted protein.
Studies of the turnover of disulfide isomerase revealed a half-life of
approximately 96 h. Treatment of cells with tunicamycin or heat shock
resulted in an increased recovery of newly synthesized disulfide
isomerase from cell lysates but diminished recovery from the media. The
secretion and cell surface distribution of disulfide isomerase in
hepatocytes may be important for the pathogenesis of immune mediated
liver injury.
Protein disulfide isomerase (PDI) ()is a cellular
protein which was first postulated to catalyze thiol-disulfide
interchange reactions in vivo almost 30 years ago(1) .
Since then a great deal has been learned about the structure and
function of the protein (reviewed in Refs. 2, 3) and its potential
substrate specificities(4) . PDI has also been identified as a
subunit of prolyl hydroxylase (5) and of the microsomal
triglyceride-transfer protein complex(6) . Recently it has been
suggested that PDI also serves as a molecular
chaperone(7, 8, 9) . PDI is reported to be
present in all eukaryotic cells in proportion to the secretory capacity
of the specific cell type(2) . It is particularly abundant in
liver where it is reported to constitute 0.4% of the total
protein(10) . Consistent with its proposed function, the main
subcellular localization of PDI in liver and other cells examined is
the endoplasmic reticulum (ER), as determined by its immunodetection in
intact hepatocytes (11) and purification from liver
microsomes(12) . The localization of PDI to the ER is presumed
due to the presence of a carboxyl terminus -KDEL retention
sequence(13) .
Two studies of the distribution of immunodetectable PDI in rat hepatocytes noted the presence of a small amount of cell surface and secretory vesicle reactivity along with abundant reactivity of the microsomes and nuclear membrane(11, 14) . In another study utilizing Chinese hamster ovary cells, disulfide reduction was found to be catalyzed by a surface-associated PDI(15) . The first evidence for the secretion of PDI came from studies by Yoshimori et al.(16) who demonstrated that while the majority of PDI in a rat pancreatic exocrine cell line, ARIP, was concentrated in the ER, there was also PDI present on the cell surface and PDI which was secreted in a form containing the -KDEL retention sequence. No studies to date have described the turnover or secretion of PDI in primary cells.
In the following studies we have utilized isolated primary rat hepatocytes to better define the subcellular localization and metabolism of PDI in liver. We present new evidence for the cell surface localization of PDI and its secretion and report on the distribution of newly synthesized PDI following treatment with tunicamycin or heat shock.
Affinity-purified anti-KDEL antibody (16) was kindly provided from Drs. Yoshimori and Yamamoto (Kansai Medical University, Japan), and purified diferric transferrin was the kind gift of P. Aisen, Albert Einstein College of Medicine.
For sequential immunoprecipitation,
the first immunoprecipitates were eluted in 20 µl of PBS containing
2% SDS and 1% -mercaptoethanol by heating to 90 °C for 10 min,
diluted to 1 ml in PBS, and incubated with 0.4 µg of anti-KDEL
antibody for 18 h at 4 °C. The final immunoprecipitate was
recovered as described above.
All immunoprecipitates were subjected to SDS-PAGE under reducing conditions and prepared for fluorography by fixation in 10% acetic acid and 50% methanol followed by incubation in Amplify or for autoradiography by fixation in 10% acetic acid and 50% methanol. Gels were then dried under vacuum and exposed to Bio-Max film at -70 °C.
Two 60-mm dishes of cultured primary hepatocytes were washed twice with PBS at 4 °C and then incubated on ice. One dish was incubated for 5 min with 0.1 M sodium carbonate, pH 9, and washed twice with ice-cold PBS. One ml of PBS at 4 °C was added to each dish followed by 3 µg of freshly iodinated sulfo-SHPP, prepared as above, and incubated for 30 min on ice. The labeling medium was removed, and the cells were washed with PBS at 4 °C and harvested by scraping in ice-cold PBS containing 1 mg/ml of both potassium iodide and lysine. Cell pellets obtained by centrifugation were utilized for immunoprecipitation of PDI as described above.
Figure 1:
Comparison of PDI content in isolated
hepatocytes and hepatic non-parenchymal cells by Western blot analysis.
Shown are the chemiluminescent blots of equivalent amounts of protein
(20 µg) from total isolated liver cells (lanes 1 and 4), isolated primary hepatocytes (lanes 2 and 5), and isolated hepatic non-parenchymal cells (lanes 3 and 6) subjected to 10% SDS-PAGE, transfer to Immobilon
polyvinyldifluoride membranes, and probing with anti-rat PDI IgG (lanes 1-3) or with anti-rat ASGR antisera (lanes
4-6) as described under ``Experimental
Procedures.'' Molecular mass markers shown are kDa
10
.
Western blot analysis of equal amounts of protein from isolated subcellular fractions from rat primary hepatocytes for PDI indicated an enrichment of PDI in the microsomal fraction compared to the whole cell homogenate (Fig. 2). Only a very small amount of PDI was detected in the cytosol (Fig. 2), its presence there likely due to disruption of a small fraction of the microsomes during their isolation.
Figure 2:
Detection of PDI in subcellular fractions
of primary rat hepatocytes by Western blot analysis. Shown is the
chemiluminescent blot of equivalent amounts of protein samples (20
µg) obtained from primary rat hepatocyte homogenates (lane
1), and microsomal fractions (lane 2), cytosol (lane
3), and from normal rat serum (lane 4) subjected to 10%
SDS-PAGE, transfer to Immobilon polyvinyldifluoride membranes, and
probing with anti-rat PDI IgG as described under ``Experimental
Procedures.'' Purified rat liver PDI (0.2 µg) is shown for
comparison (lane 5). Molecular mass markers shown are kDa
10
.
To quantitate the degree of microsomal enrichment, serially diluted protein samples from the microsomal fraction and cell homogenates were subjected to slot-blot analysis and compared with known amounts of serially diluted purified PDI standard. PDI concentrations in the microsomal fraction and in the homogenate of primary hepatocytes were estimated by densitometric scanning to be 10.5 and 7 µg/mg of protein, respectively.
Figure 3:
PDI synthesis and secretion by primary rat
hepatocytes. Primary rat hepatocytes were radiolabeled with
[S]cysteine and methionine for 4 h and chased
for various periods as described. PDI (monomer at 57 kDa, presumed
homodimer 90-110 kDa) recovered by immunoprecipitation from cell
lysates (panel A) and media (panel B) was resolved by
10% SDS-PAGE and visualized by fluorography as described under
``Experimental Procedures.'' Chase periods were as follows: 0
min (lane 1); 15 min (lane 2); 30 min (lane
3); 60 min (lane 4); 120 min (lane 5); 240 min (lane 6). Radiolabeled PDI was also recovered by
immunoprecipitation from a cell lysate incubated for 2 h with the 2-h
chase medium containing radiolabeled secreted proteins (panel
A, lane 7). Panel C, fluorogram showing the
effect of addition of non-radiolabeled cell lysate on the recovery of
radiolabeled secreted PDI. PDI recovered by immunoprecipitation from
media of radiolabeled cells after a 4-h chase period following the
addition of non-radiolabeled cell lysate obtained from one 60-mm
culture dish was markedly reduced (lane 1) compared to PDI
recovered from the media alone (lane 2). PDI is indicated by
the arrow. Molecular mass markers shown are kDa
10
.
Since the amount of radiolabeled PDI recovered from the media was much greater than that of cell lysates, we assumed that the specific activity (radioactivity/total protein) of intracellular PDI was much lower. To confirm this, non-radiolabeled cell lysates were added to the media containing radiolabeled secreted proteins following a 4-h chase period and then PDI was recovered by immunoprecipitation. As a result of the addition of the cell lysate, recovery of radiolabeled PDI from the media was reduced to 8.7% of the radiolabeled PDI recovered from media alone (Fig. 3C). This indicated that the reduced recovery of radiolabeled PDI from cell lysates compared to media was due to competition with unlabeled intracellular PDI.
To confirm that PDI recovered from the media was
secreted and did not result from cell rupture, cells were treated with
colchicine prior to and during labeling and chase periods. Results of
densitometric analysis of fluorography of PDI immunoprecipitates
revealed that the cellular content of newly synthesized PDI increased
by almost 50% following colchicine treatment (control 0.031 ± 0,
colchicine 0.047 ± 0.007, n = 2), while the
amount of PDI in the media was reduced by 50% (control 0.124
± 0.020, colchicine 0.057 ± 0.010, n =
2).
Figure 4:
Turnover rates for PDI in primary rat
hepatocytes. A, primary rat hepatocytes were radiolabeled with
[S]cysteine and methionine for 4 h. PDI was
recovered by immunoprecipitation from cell lysates at 0 h (lanes 1 and 5), 24 h (lanes 2 and 6), 48 h (lanes 3 and 7), and 72 h (lanes 4 and 8) after chase periods. The immunoprecipitates were analyzed
by 10% SDS-PAGE and fluorography as described under ``Experimental
Procedures.'' PDI is indicated by an arrow. Molecular
mass markers shown are kDa
10
. B,
graphic representation of the amount of recoverable PDI from cell
lysates shown in A. The relative amounts of PDI, determined by
scanning laser densitometry, are shown as arbitrary units for each time
point. The mean and standard deviation of the results of the two
experiments and the first-order linear regression are
shown.
Figure 5:
Peptide mapping of cellular and secreted
PDI. The radiolabeled secreted PDI and purified rat liver PDI were
subjected to 7.5% SDS-PAGE, and the bands corresponding to PDI were cut
from the gel. These purified bands were subjected to partial digestion
with S. aureus protease, and the resulting peptides resolved
by 15% SDS-PAGE as described under ``Experimental
Procedures.'' The peptides from purified rat liver PDI, visualized
by silver staining (lane 1), and the visualized peptides from
the secreted PDI, detected by fluorography, were similar (lanes 2 and 3). Shown are the same fluorogram exposed for 7 days (lane 2) and 17 days (lane 3). Molecular mass markers
shown are kDa
10
.
Since peptide mapping would not be sufficient to reveal the presence or absence of the KDEL sequence within PDI, sequential immunoprecipitation of secreted PDI using anti-PDI antibody and then an anti-KDEL antibody was performed. The secreted PDI was recovered with the anti-KDEL antibody following the immunoprecipitation with anti-PDI antibody, indicating the presence of the KDEL sequence in the secreted protein (Fig. 6, lane 2). The failure to recover albumin using the anti-KDEL antibody is consistent with the lack of the retention signal in this protein (Fig. 6, lane 4).
Figure 6:
Immunodetection of KDEL sequence in
secreted PDI. Primary rat hepatocytes (4 10
/dish)
were metabolically labeled for 4 h and the medium replaced for the 4-h
chase period. The medium from each dish was subjected to
immunoprecipitation, SDS-PAGE, and subsequent fluorographic analysis as
described under ``Experimental Procedures.'' Shown are
fluorographs of immunoprecipitates recovered with either anti-rat PDI
IgG (lane 1) followed by the affinity purified anti-KDEL
antibody (lane 2), or anti-albumin antisera (lane 3)
followed by the affinity purified anti-KDEL antibody (lane 4).
The M
of PDI and albumin standards are indicated.
Molecular mass markers shown are kDa
10
.
Figure 7: Effect of tunicamycin and heat shock treatment on the distribution of PDI. Shown are the graphic representation of densitometric scans (in arbitrary units, means of duplicate experiments) of fluorographs of PDI recovered by immunoprecipitation from cell lysates (lanes 1-3) and from media (lanes 4-6). Lanes 2 and 5 show results of PDI recovered from cells subjected to tunicamycin treatment; lanes 3 and 6 PDI recovered from cells subject to heat shock as described under ``Experimental Procedures.'' The inset shows the fluorographic results from one experiment.
Figure 8:
Biochemical identification of cell surface
PDI. Primary rat hepatocytes were surface labeled with iodinated
sulfo-SHPP following treatment with 0.1 M sodium carbonate, pH
9 (lanes 1 and 4) or with PBS alone (lanes 2 and 5). Cell lysates were either analyzed directly by
SDS-PAGE and autoradiography (lanes 1 and 2) or
subjected to immunoprecipitation with anti-rat PDI IgG followed by
autoradiography as described under ``Experimental
Procedures'' (lanes 4 and 5). Purified
radioiodinated rat PDI (lane 3) is shown for comparison. PDI
is indicated by the arrow. Molecular mass markers shown are
kDa 10
.
Both anti-PDI and transferrin Texas Red conjugates were visualized as present on the cell surface by immunofluorescent microscopy (Fig. 9, D and F), indicating the presence of PDI on the cell surface. The addition of unlabeled PDI to the suspension markedly reduced the detection of bound antibody (Fig. 9H), indicating the specificity of the binding of the Texas Red antibody conjugate for PDI. The failure to completely abolish the cell surface fluorescence by the addition of unlabeled PDI could be due to some association of the added PDI with the cell surface as seen in Fig. 3A, lane 7. No background autofluorescence was observed at this wavelength (Fig. 9B).
Figure 9:
Immunofluorescent detection of cell
surface PDI. Isolated primary rat hepatocytes were incubated at 4
°C without additional antibody (A and B), with
Texas Red-labeled diferric transferrin (C and D),
with Texas Red-labeled anti-PDI IgG (E and F), and
with purified rat PDI and Texas Red-labeled anti-PDI IgG (G and H). Cells were visualized at 200 by light
microscopy (A, C, E, and G) or by
fluorescence microscopy at 590 nm (B, D, F,
and H). All fluorescent photomicrographs were obtained with
constant exposure times of 2 min.
In this study we provide evidence for the hepatocellular secretion of newly synthesized PDI, its surface membrane localization on rat hepatocytes, and information regarding PDI turnover in primary cultures of rat hepatocytes.
We confirmed that the majority of the detectable PDI in primary isolated rat hepatocytes was clearly localized to microsomes (Fig. 2), as previously reported. Quantitation of PDI by slot-blot and Western analyses indicated that PDI was present in primary hepatocytes at a concentration of 0.7% of cell protein (7 µg/mg), almost twice the 0.4% of the total liver protein reported by Freedman et al.(10) . This increased content of PDI in isolated hepatocytes compared to whole liver preparations is likely due to the exclusion of non-parenchymal cells which have significantly less PDI contents (Fig. 1).
Secretion of PDI from primary rat hepatocytes was detected by immunoprecipitation of the protein from the medium with PDI-specific antibody after metabolic labeling of cultured cells to steady state. The effect of colchicine treatment to reduce the recovery of PDI in the medium while increasing its recovery in cell lysates indicates microtubular directed secretion from cells. The identity of the secreted protein was similar to that of the cellular protein as determined by peptide mapping (Fig. 5). The presence of the carboxyl terminus ER retention signal was verified by immunoprecipitation with KDEL-specific antibody (Fig. 6), indicating that the recovery of secreted PDI was not due to the loss the peptide retention sequence. The amount of recoverable secreted PDI increased over time, reaching a steady state at approximately 4-6 h (Fig. 3B). The estimated t for secretion was between 1-2 h (Fig. 3B).
The recovery of the
newly synthesized PDI from cell lysates by immunoprecipitation was
relatively poor compared to that recovered from media. This raised the
possibility that either the majority of newly synthesized PDI was
secreted, or that there was competition for recovery of the labeled
protein by the abundant quantities of unlabeled hepatocellular PDI. The
latter possibility was suggested by experiments in which unlabeled cell
lysates added to radiolabeled secreted proteins prior to
immunoprecipitation with PDI-specific antibody significantly reduced
the amount of PDI recovered (Fig. 3C) and is consistent
with the calculated long half-life (t 96 h) in this
study and the previously reported half-life of greater than 60 h
reported by Berg et al.(21) for PDI from chicken
tendon fibroblasts.
Although the vast majority of hepatocellular PDI remains cell associated and is concentrated in microsomes, the secretion of PDI by hepatocytes raises the question of how the ER retention signal is dealt with by cells. Two possibilities have been suggested: first, the presence of ER receptors specific for -KDEL-containing proteins, and second, a retrieval mechanism which may recognize -KDEL and possibly the neighboring peptide region as well. In the first an overflow of -KDEL-containing proteins should theoretically saturate their putative receptors. This possibility is not supported by a study in which the overexpression of PDI or ERp72, both -KDEL-containing proteins, led to their secretion but not to the secretion of other -KDEL proteins(22) . Alternatively, there may be sorting of the luminal proteins for recycling to the ER from a salvage compartment, with some escape from this pathway being a small but normal infidelity to the system. This is supported by the report that several -KDEL-containing proteins were secreted under normal conditions by ARIP cells (16) and in this study by primary rat hepatocytes (Fig. 6).
Earlier studies by Dorner et al.(22) indicated that steady state PDI mRNA levels were slightly increased in cells subjected to tunicamycin treatment, while the levels of mRNA for other ER proteins, glucose-regulated proteins 78 and 94 and ERp72, were markedly increased, suggesting a cellular stress response. We subjected primary rat hepatocytes to tunicamycin treatment and found a reduction in newly synthesized PDI that was secreted, and recovery from cell lysates was increased (Fig. 7). To determine whether this was indeed just a response to a generalized cellular stress as suggested by the studies of Dorner et al.(22) , we also subjected primary rat hepatocytes to heat shock treatment. Similar results were observed for heat shock treated cells (Fig. 7), suggesting that cellular stress responses result in an increased cellular retention of PDI. Some possible explanations for this outcome include the increased biosynthesis of retention molecules, an increased affinity of the retention mechanism for PDI, or increased association of PDI with other cellular proteins in the ER. Further experimentation is needed to resolve these or other possible explanations for these findings.
Surface localization of PDI was detected by both immunofluorescent labeling (Fig. 9) and by immunoprecipitation following cell surface labeling with radioiodine (Fig. 8). PDI on the cell surface appears to interact with the surface membrane electrostatically since the majority was removed by carbonate washing (Fig. 8) in contrast to the transmembrane localization proposed by Honscha et al.(14) . This loose association of PDI with membranes has been previously described for rat liver microsomes(12) . Though the source of the surface membrane PDI in rat hepatocytes was not directly determined, data from this study indicating recovery of newly synthesized PDI from unlabeled cells incubated with the radiolabeled secreted protein (Fig. 3A, lane 7) suggests that at least a fraction of the secreted protein is likely to reassociate with the surface membrane. We did not examine the disulfide isomerase activity of the surface protein; however the study by Mandel et al.(15) indicating disulfide isomerase on the surface of Chinese hamster ovary cells suggests the rat surface PDI should also be biologically active.
At present we can only speculate the presence of PDI on the cell surface of hepatocytes that may be important for certain immune mediated pathologic processes affecting the liver. Recent studies of two distinctly different modes of hepatic injury, halothane anesthesia-induced hepatitis in man (23) and hepatic copper toxicosis in the LEC rat(24) , demonstrate the presence of autoantibodies to PDI. Cell surface localization of this protein would be required for these autoantibodies to play a significant role in the pathogenesis of any autoimmune injury. Though the cell surface localization had been suggested for PDI in rat liver by previous immunolocalization studies (11, 14) , this methodology did not exclude the possibility of reaction of PDI antisera to common epitopes on other proteins or peptides. The data presented in this study confirm the cell surface localization of intact PDI, suggesting the hypothesis that its presence there plays a role in immune mediated liver injury.