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INTRODUCTION |
The process of protein folding in the living cell is a more
complicated process than previously recognized. It is dependent on a
milieu that differs between cellular compartments. It is assisted by a
panel of molecular chaperones and folding factors and affected by a
variety of processes such as targeting, membrane translocation,
covalent modification, and degradation (1-3). Moreover, because
folding of proteins begins already at the level of the growing nascent
chain, it is likely to occur in a vectorial fashion from the N to C
terminus of the growing polypeptide chain (4, 5).
Whereas the classical in vitro refolding approach used by
Anfinsen and Scheraga (6) has provided much basic information about the
biophysical principles behind the folding of polypeptides, it does not
allow for conditions prevailing in the cell to be adequately
reproduced. To study protein folding in cells, we and others have
therefore made extensive use of a pulse-chase approach in which the
folding of a radioactively labeled cohort of proteins is followed in
tissue culture cells (7, 8). Although it reflects the folding process
inside the live cell, this technique is also not problem-free. Because
the labeled, partially folded proteins must be extracted from the cells
and biochemically analyzed, there is no guarantee that their
conformation after cell lysis faithfully reflects the conformation they
have inside the cell. Ideally, one should be able to monitor the
conformation of the folding proteins in situ.
In this study, we describe an approach that allows analysis of protein
folding inside the unperturbed endoplasmic reticulum (ER).1 It is based on the use
of conformation-dependent monoclonal antibodies. The
antigen whose folding is to be monitored and antibodies against it are
co-expressed in the same cell, and the effects of their interaction on
antigen folding are analyzed by the pulse-chase approach. As shown here
using influenza hemagglutinin (HA) as a model protein, it is possible
to determine whether specific conformations occur during the folding
process and whether antibody binding affects it.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Virus--
All hybridoma cell lines were grown in
Iscove's Dulbecco's modified Eagle's medium with 10% fetal calf
serum, 1% gentamycin, and 1% Glutamax I (Life Technologies, Inc.) at
37 °C and 5% CO2 atmosphere in a humidified incubator.
They were split at a density of 1.0 × 106 cells/ml
into 0.1 × 106 cells/ml. The P5D4 hybridoma cells
produce monoclonal antibodies against the cytosolic tail of vesicular
stomatitis virus G-protein (9). The F1 hybridoma cells produce
monoclonal antibodies against nascent chains and early folding
intermediates of HA (10), and the N2 hybridoma cells produce antibodies
that are specific for trimerized HA (11, 12).
The X31/A/Aichi/1968 strain of influenza virus was prepared as
described previously (13). The rabbit polyclonal antiserum raised
against X31 influenza virus immunoprecipitates all forms of HA, the
viral nucleoprotein, and matrix protein (7, 14).
Reagents--
The 35S-labeled cysteine and
methionine mixture (Promix) was purchased from Amersham Pharmacia
Biotech. CHAPS was from Pierce, and Endo H was from New England
Biolabs. Media and reagents for cell culture were obtained from Life
Technologies, Inc. All other reagents were purchased from Sigma.
Viral Infection and Pulse-Chase Analysis--
After hybridoma
cells were grown to 1.0 × 106 cells/ml, 1.0 × 107 cells per time point were collected by centrifugation
for 5 min at 200 × g in a Beckman GS-15 centrifuge at
4 °C, washed once with PBS, and resuspended in RPMI 1640 medium with
20 mM HEPES (pH 6.8) and 0.2% bovine serum albumin. X31
influenza virus was added at a multiplicity of infection of 10 and
bound to the cell surface for 1 h at room temperature on a rocker.
Cells were collected as before and resuspended in normal growth medium
and incubated for 14-18 h under normal growth conditions. The cells
were then washed with PBS and starved in Cys/Met-free medium (300 µl
per 1.0 × 107 cells) for 30 min. Subsequently, the
pulse was started by adding 500 µCi of 35S-labeled
cysteine and methionine to 1.0 × 107 cells. The pulse
was stopped and the chase started by adding 10 mM unlabeled
cysteine and methionine and 2 mM cycloheximide to inhibit
further translation (7). After the pulse or after additional periods of
chase the cells were immediately lysed. When looking for secreted
components in the cell supernatant, the pulse or chase was stopped by
adding 700 µl of ice-cold PBS containing 20 mM
N-ethylmaleimide (NEM), and the cells were separated from
the supernatant by centrifugation at 1,500 rpm in an Eppendorf centrifuge at 4 °C.
Cell Lysis, Immunoprecipitation, and SDS-PAGE--
After chase
or directly after pulse, cells were lysed by adding an equal volume of
2× lysis buffer (4% CHAPS, 100 mM HEPES, 400 mM NaCl (pH 7.6)) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride and 20 µg/ml each of
chymostatin, leupeptin, antipain, and pepstatin) and 20 mM
NEM to alkylate any remaining free sulfhydryl groups. When the cells
were separated from their medium, they were lysed in 1× lysis buffer
containing protease inhibitors and 20 mM NEM. A postnuclear
supernatant was prepared by centrifuging the lysates at 16,000 × g for 5 min at 4 °C.
150 µl of the postnuclear supernatant was incubated with protein
A-Sepharose CL-4B beads (15-µl bead volume) and rotated at 4 °C
for at least 3 h in the presence or absence of 5 µl of
anti-influenza antiserum. Immune complexes were pelleted at 2,500 × g for 2 min and washed with agitation three times for 5 min each. In the case of immunoprecipitation with protein A only, the
immune complexes were washed with a wash buffer containing 0.5%
CHAPS/HEPES-buffered saline (200 mM NaCl and 50 mM HEPES (pH 7.6)) and in the case of anti-influenza
immunoprecipitation, with wash buffer containing 10 mM
Tris-Cl (pH 6.8), 0.05% Triton X-100, and 0.1% SDS (7). The washed
complexes were solubilized by the addition of nonreducing sample buffer
(100 mM Tris-Cl (pH 6.8), 4% SDS, 0.2% bromphenol blue,
and 20% glycerol) and heated for 5 min at 95 °C. For reducing conditions, 125 mM dithiothreitol was added to the samples.
The samples were analyzed by 7.5% SDS-PAGE on 10.5-cm gels followed by fluorography.
Endoglycosidase H Digestion--
Anti-influenza
immunoprecipitates from pulse-chased, influenza-infected hybridoma
cells were washed three times as above. The immune complexes were
resuspended in 0.2% SDS in 100 mM NaOAc (pH 5.5) and
heated for 5 min at 95 °C. An equal volume of 100 mM
NaOAc (pH 5.5) was added and the samples were divided in two. To one
half of the samples only buffer was added and to the other half, buffer
plus 0.5 unit of Endo H (15). The samples were incubated for 16 h
at 37 °C. Digestions were stopped by the addition of reducing sample
buffer, heated for 5 min at 95 °C, and analyzed by 7.5% SDS-PAGE on
10.5-cm gels followed by fluorography.
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RESULTS |
Influenza HA Folds and Is Transported to the Golgi Complex in
Hybridoma Cells--
Analysis of NEM-alkylated folding intermediates
extracted from cultured cells has shown that folding of HA (a type I
membrane glycoprotein, 84 kDa) starts co-translationally and continues posttranslationally in the lumen of the ER (7, 10). 10-30 min after
chain completion, when the molecules have acquired six intrachain
disulfide bonds, they assemble into homotrimers and are transported out
of the ER to the Golgi complex (12, 14, 16).
To analyze the folding process, immunoprecipitations of
detergent-extracted, pulse-labeled HA have been performed using
conformation-specific antibodies, and mobility differences between
differentially oxidized intermediates have been monitored by SDS-PAGE
(17). The panel of antibodies used included monoclonals that react
specifically with differentially oxidized early forms of HA, fully
oxidized HA forms, and HA trimers. As indicated by the
immunoprecipitation, some of the epitopes were expressed transiently
during the folding process (17) and some were detected already on
growing nascent chains (10).
To determine whether the epitopes that these antibodies react with are
actually present on the molecules during folding inside the ER, HA was
expressed in the hybridoma cells that produced the antibodies. Like HA,
the antibodies fold and assemble inside the ER lumen and can react with
their antigens within this compartment (18).
Control experiments were first performed using a hybridoma cell line
that produces antibodies that do not interact with HA. The cell line
was P5D4, a hybridoma line that produces antibodies against the
cytosolic tail of vesicular stomatitis virus G-protein (9). The cells
were infected with the X31 strain of influenza virus using a procedure
previously described for Chinese hamster ovary cells (7). In the case
of Chinese hamster ovary cells, optimal HA expression is achieved
5 h postinfection. However, in hybridoma cells the cytopathic
effects started 24 h postinfection, and optimal expression of HA
was reached at 14-18 h after infection. As detected by indirect
immunofluorescence, 90% of the cells were infected.
At 18 h postinfection, the P5D4 cells were pulse-labeled for 5 min
with 35S-labeled methionine and cysteine and chased for
0-40 min. As a control, infected and mock-infected cells were
pulse-labeled for 15 min and chased for 40 min. After the pulse or
after the chase, the cells were lysed with detergent in the presence of 20 mM NEM to alkylate the free sulfhydryl groups and
prevent further disulfide bond formation (7). Postnuclear supernatants
were immunoprecipitated using anti-influenza antibodies and analyzed by
nonreducing and reducing SDS-PAGE.
Folding of HA proceeds via two easily recognized intermediates, IT1 and
IT2, that differ in the number of intrachain disulfide bonds. IT1
contains one or more of the small disulfide loops but lacks both of the
major loop-forming disulfides, 14-466 and 52-277. IT2 has disulfide
52-277, but lacks disulfide loop
14-466.2 Soon after reaching
the fully oxidized form (NT), HA trimerizes and is transported to the
Golgi complex where the N-linked glycans undergo terminal glycosylation.
The results showed that after a 5-min pulse all three forms, IT1, IT2,
and NT, were present (Fig. 1, lane
3). The asterisk marks IT2, which has a slightly lower
mobility than the Golgi form (lane 2 or 5). The
mobility of NT, the fastest moving of the HA bands, increased during
the chase. This is an effect caused by the trimming of glucose and
mannose residues from the six core oligosaccharide chains (19). The
Golgi form only became visible at later chase times (Fig. 1,
lanes 3-5). In nonreduced gels, it had a mobility almost
similar to IT2 but was easily distinguished after reduction by a lower
mobility, due to the extra sugars added during terminal
glycosylation.

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Fig. 1.
Folding of influenza HA in P5D4 hybridoma
cells. P5D4 hybridoma cells (their monoclonal antibodies
(mAb) are specific for the cytosolic tail of vesicular
stomatitis virus G-protein) were infected with X31 influenza virus.
18 h postinfection, the cells were pulse-labeled for 15 or 5 min
with 500 µCi of 35S-labeled cysteine and methionine and
chased for 0-40 min in the presence of cycloheximide. Reduced and
nonreduced anti-influenza immunoprecipitates of the lysates were
analyzed by nonreducing and reducing 7.5% SDS-PAGE followed by
fluorography. Lanes 1 and 6 show mock-infected
cells. DTT, dithiothreitol; IT1, first folding
intermediate of HA; G, Golgi form of HA; NT,
oxidized native form of HA; NP, nuclear protein of the
virus. The asterisk in lane 3 marks the second
folding intermediate (IT2) of HA.
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The changes in gel pattern showed that HA folded normally in hybridoma
cells and that about half of it reached the Golgi compartment within 40 min, which corresponds to the rate of maturation seen in Chinese
hamster ovary cells (7). Labeled antibody bands were also present in
both the reduced (indicated by mAb in Fig. 1) and nonreduced
gels (not shown). Thus, the production of antibodies did not interfere
with the process of normal HA folding in the ER.
Antibodies against HA Interfere with Folding and Transport--
To
test whether synthesis of antibodies specifically targeted against HA
would affect HA folding and transport, a hybridoma cell line, F1, was
chosen. F1 antibodies react with an epitope in the stem domain of HA
(7). Immunoprecipitation indicates that the F1 epitope is transient; it
is expressed on full-length and nascent HA molecules that are present
in the IT1 and IT2 forms but not at later stages of maturation (7, 10).
Fig. 2A shows that, in
contrast to the P5D4 cell control (lane marked
P5D4), the HA produced in F1 cells failed to fold properly. In nonreduced gels, the HA ran as a smear with a mobility slower or
equal to that of IT2 (lanes 2-5). No NT was formed.
Apparently, formation of the correct intrachain disulfide bonds was
disturbed. Furthermore, in the reduced gel (Fig. 2A), no
Golgi form appeared, indicating that the glycans did not undergo
terminal glycosylation (cf. Fig. 2A, lanes
7 and 10 with Fig. 1, lanes 7 and
10). This implied that the HA was trapped in the ER as a
result of interaction with the antibody.

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Fig. 2.
Folding of influenza HA in F1 hybridoma cells
is disturbed. F1 hybridoma cells (their monoclonal antibodies
(mAb) are specific for nascent chains, IT1 and
IT2) were infected and pulse-chased as described in the
legend to Fig. 1. Abbreviations are defined in the legend to Fig. 1.
A, lysates were immunoprecipitated with anti-influenza
antiserum and analyzed with nonreducing and reducing 7.5% SDS-PAGE.
The vertical line marks the smear of folding intermediates
under nonreducing conditions. An X31-infected P5D4 cell lysate (pulsed
for 5 min) was immunoprecipitated with anti-influenza antiserum as a
marker for the folding intermediates (P5D4). B,
lysates were immunoprecipitated with protein A (Prot. A)
beads only and analyzed by nonreducing and reducing 7.5% SDS-PAGE
followed by fluorography. Again, the vertical line marks the
smear of folding intermediates of HA. The relatively high amount of
background (A and B) is due to the fact that the
lysates could not be precleared with protein A beads.
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To confirm that the HA was retained in the ER in the F1 cells, its Endo
H sensitivity was determined. Although the HA signal was weak in the F1
cells, it was apparent that it remained Endo H-sensitive throughout the
chase, in contrast to the HA in P5D4 cells (Fig.
3), half of which reached an Endo
H-resistant form within 40 min. The uninhibited secretion of antibodies
into the medium proved that the inhibition of intracellular transport
of HA was not caused by a general defect in the secretory pathway (Fig.
4).

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Fig. 3.
Endoglycosidase H digestion of HA from
X31-infected P5D4 and F1 cells. Anti-influenza immunoprecipitates
from P5D4 and F1 hybridoma cells were obtained as described in the
legends to Figs. 1 and 2. The precipitated proteins were divided in
two; one half was digested with Endo H at 37 °C for 16 h and
the other half was incubated without Endo H at 37 °C for 16 h.
They were then analyzed by reducing 7.5% SDS-PAGE followed by
fluorography. G, reduced Golgi form of HA; R,
reduced non-Golgi form of HA. Low mobility HA is Endo H-resistant, and
high mobility HA is still sensitive to Endo H.
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Fig. 4.
Antibody secretion by X31-infected F1
cells. F1 cells were infected and pulse-chased as described in the
previous figure legends. However, after the chase, the cells were
resuspended in PBS containing 20 mM NEM. The cells were
analyzed for their ability to secrete soluble antibodies by
immunoprecipitating the cell supernatant with protein A beads. The
immunoprecipitates were analyzed for radiolabeled antibodies by
reducing 7.5% SDS-PAGE followed by fluorography. mAb,
monoclonal antibody.
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To show more directly that the F1 antibodies produced in the ER were
the cause of the disturbed folding, we examined whether they were bound
to the pulse-labeled HA. To bring down cellular immune complexes, we
precipitated cell lysates with protein A-coated beads without added
antibodies. The labeled HA was precipitated, revealing the same smeary
pattern in nonreduced gels as seen with immunoprecipitation using the
anti-influenza antiserum (Fig. 2B). During the chase, the
amount of radiolabeled HA complexed to antibodies decreased (cf.
lane 5 or 10 with 4 or 9,
respectively). Although we do not know the reason for this decrease (it
might be that the interaction between antibody and HA is transient),
these results indicated that at least a fraction of the endogenous
anti-HA antibodies were, indeed, bound to incompletely or incorrectly
folded HA molecules.
We concluded that F1 antibodies were able to bind to the HA molecules
inside the lumen of the ER and that the binding effectively interfered
with normal folding and transport. Instead of trapping HA intermediates
in the familiar IT1 and IT2 forms, the antibodies caused the formation
of a more heterogeneous set of intermediates than normally seen in cell
lysates. The antibodies were clearly able to interfere with proper
maturation and transport of the newly synthesized HA.
Antibodies against HA Trimers Do Not Interfere with Transport of HA
to the Golgi Complex--
We then asked whether retention of HA in the
ER could also be observed when antibodies were complexed to fully
folded HA molecules. For this we used the N2 hybridoma cells that
produce antibodies specific for fully folded HA trimers (14). As shown
in Fig. 5A, HA folded normally
to NT in N2 cells,and a large fraction was transported to the Golgi
complex where it was terminally glycosylated. Precipitation with
protein A-coated beads without added antibodies only brought down the
terminally glycosylated Golgi intermediates, indicating that only late
forms of HA bound to the antibodies (Fig. 5B). It has
previously been demonstrated that the majority of the HA subunits
trimerize already in the ER (12). This suggests that HA trimers
associated with N2 antibodies in the ER were able to move to the Golgi
complex. The retention of HA in F1 hybridoma cells is therefore most
likely not caused by the antibody-antigen complex formation but by
misfolding of HA. Thus, association with an antibody as such may not be
the cause of HA retention in the ER but rather the effect it has on the
antigen's folding and maturation.

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Fig. 5.
Folding and transport of HA in N2 hybridoma
cells is not disturbed. N2 hybridoma cells (their monoclonal
antibodies (mAb) are specific HA trimers) were infected and
pulse-chased as described in the previous figure legends. Cell lysates
were immunoprecipitated with anti-influenza antiserum (A) or
with protein A (Prot. A) beads only (B). The
immunoprecipitates were analyzed by nonreducing and reducing 7.5%
SDS-PAGE followed by fluorography. Abbreviations are defined in the
legend to Fig. 1.
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DISCUSSION |
The approach taken was to use the ER lumen of the hybridoma cell
as a "reaction vessel" in which antigens and antibodies were mixed
under unperturbed, physiological conditions. The antigens in this case
were nascent and newly synthesized HA glycoproteins. Because HA was
continuously being translated and translocated into the ER, it was
presented to the antibodies in all the different conformations that
folding chains normally display within the authentic, lumenal ER environment.
On the basis of prior immunoprecipitation data, the hybridoma cells
were chosen so that the antibodies were expected to react with nascent
chains and early folding intermediates or with fully folded molecules.
In both cases, immune complexes were formed. In the case of antibodies
against nascent chains and early folding intermediates, it was clear
that the immune complexes formed in the ER because no Golgi
modifications could be observed in the HA. The antibodies that reacted
specifically with early intermediates of HA were found to disturb
folding and intracellular transport. The antibodies to the mature
trimeric HA had no effect on folding or intracellular transport.
The effects of the antibodies were likely to be caused by
antibody-induced misfolding. Attachment of a bulky IgG molecule to the
F1 epitope on a growing nascent chain or newly synthesized full-length
HA molecule would limit the freedom of the folding chain and prevent
interactions with molecular chaperones and folding enzymes. This would
result in the formation of incorrectly oxidized, non-native conformers
retained in the ER by the quality control system. Alternatively, the
presence of HA bands that have not been seen previously could mean that
folding of HA does not have as distinct a set of intermediates as
suggested by previous data.
Another conclusion that can be drawn from these results is that the
conformational epitopes identified by immunoprecipitation using
solubilized folding intermediates of HA were also present in the
protein during in situ folding. Among the three cell lines tested, we did not find a single case in which a specific epitope would
not cause antibody binding. This suggested that, at least as far as HA
is concerned, the emerging picture of in vivo folding based
on the pulse-chase approach is on the right track.
Expression of specific antibodies has been used in the past to block
the formation of functional nuclear pores and to identify the location
of antigens in the Golgi complex (18, 20). In these studies the
hybridoma cells were not used directly, but the mRNA for the heavy
and light chains of an antibody were microinjected into tissue culture
cells. More recently, studies have been performed in which single-chain
antibodies were expressed intracellularly and targeted to specific
compartments using specifically designed vectors (21, 22). In one
study, the ER-targeted single-chain antibodies were shown to interfere
with the post-ER cleavage of human immunodeficiency virus, type I
gp160, indicating that the antigen was retained in the ER (23).
However, it is very likely that this retention was caused by the ER
retention signal on the antibody and not by misfolding of the antigen.
From the results described here, it is clear that antibodies can be
successfully used to analyze protein folding in the ER of living cells
and for targeted interference with the maturation of specific antigens. In the future, one might be able to screen single-chain antibody libraries (24) expressed intracellularly for the identification of new
factors (antigens) that affect folding of proteins in the ER.