From the Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, United Kingdom and the
¶ Institute for Cancer Research, Norwegian Radium Hospital,
Montebello, 0310 Oslo, Norway
Received for publication, October 18, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Here we demonstrate that ricin is able to
interact with the molecular chaperone calreticulin both in
vitro and in vivo. The interaction occurred with
ricin holotoxin, but not with free ricin A chain; and it was prevented
in the presence of lactose, suggesting that it was mediated by the
lectin activity of the ricin B chain. This lectin is
galactose-specific, and metabolic labeling with [3H]galactose or treating galactose oxidase-modified
calreticulin with sodium [3H]borohydride indicated that
Vero cell calreticulin possesses a terminally galactosylated
oligosaccharide. Brefeldin A treatment indicated that the intracellular
interaction occurred initially in a post-Golgi stack compartment,
possibly the trans-Golgi network, whereas the reductive
separation of ricin subunits occurred in an earlier part of the
secretory pathway, most probably the endoplasmic reticulum (ER).
Intoxicating Vero cells with ricin whose A chain had been modified to
include either a tyrosine sulfation site or the sulfation site plus
available N-glycosylation sites, in the presence of
Na235SO4, confirmed that
calreticulin interacted with endocytosed ricin that had already
undergone retrograde transport to both the Golgi and the ER. Although
we cannot exclude the possibility that the interaction between ricin
and calreticulin is an indirect one, the data presented are consistent
with the idea that calreticulin may function as a recycling carrier for
retrograde transport of ricin from the Golgi to the ER.
Ricin is a member of a large family of protein toxins produced by
certain plants and bacteria that damage or kill mammalian cells by
catalytically modifying target substrates in the cytosol (1, 2). In the
case of ricin, the modification catalyzed is the removal of a specific
adenine residue from a highly conserved loop present in 28 S rRNA (3).
The adenine residue removed by ricin action is crucial for the binding
of elongation factors, and a consequence of the toxin-mediated
depurination is the cessation of protein synthesis, leading inevitably
to cell death.
Structurally, ricin is a disulfide-linked heterodimer in which the rRNA
N-glycosidase (termed the ricin A chain
(RTA)1) is covalently joined
to a galactose-specific lectin (the ricin B chain (RTB)) (4). For ricin
to act, RTA must enter cells to encounter ribosomes in the cytosol.
Cell entry is mediated by RTB, through which the toxin binds to
cell-surface galactosides. Some of the surface-bound ricin then enters
cells by endocytosis and is initially delivered to the endosomal system
(5). Before endocytosed ricin can act, however, RTA must traverse an
intracellular membrane to reach the ribosomes. This membrane
translocation step is now known to be achieved by a proportion of the
endocytosed toxin that undergoes intracellular transport beyond
endosomes. Initially, the toxin is transported to the
trans-Golgi network (TGN) and then, via retrograde vesicular
transport through the Golgi stack, to the lumen of the ER (6).
Translocation of free RTA into the cytosol from the ER possibly occurs
because the toxin is perceived as a candidate for ER-associated protein
degradation (7, 8). Clearly, however, a proportion of translocated RTA must escape the subsequent proteolytic degradation to then be capable
of intoxicating the cell.
Electron microscopic analysis of the cellular fate of endocytosed ricin
first showed that a proportion reaches the TGN (9). This raises the
question of how ricin achieves retrograde transport from the TGN to the
ER lumen. Several bacterial toxins also follow this trafficking route,
and structural features of some of these toxins have provided an
explanation of how this transport step is achieved. Pseudomonas
aeruginosa exotoxin A, cholera toxin, and Escherichia
coli heat-labile enterotoxin have the tetrapeptide sequence
-Lys-Asp-Glu-Leu (KDEL), or a functionally related homolog, at the C
terminus of the translocated catalytic polypeptide (10-12). It has
been shown that cholera toxin and P. aeruginosa
exotoxin A utilize the KDEL receptor, which recycles between the TGN
and the ER to retrieve escaped ER lumen resident proteins from all Golgi compartments (13, 14), to undergo retrograde Golgi-to-ER transport (15, 16).
An outstanding question concerns how ricin, which lacks a C-terminal
KDEL or related sequence, achieves Golgi-to-ER transport for its
subsequent translocation into the cytosol? Our earlier work
indicates that the lectin activity of RTB may be important in this
regard (17-20). Primary sequence analysis and x-ray crystallographic studies have shown that RTB forms two distinct globular domains with
identical folding topologies (17, 18). Each domain can bind a galactose
residue (18). We have shown that mutationally abrogating one or other
of these two galactose-binding sites does not abolish either the lectin
activity of RTB or the cytotoxicity of the ricin holotoxin (19, 20).
Simultaneous abrogation of both sites, however, does predictably
abolish lectin activity and significantly reduces cytotoxicity (20). We
have also shown that the ricin mutant unable to bind galactose did not
kill macrophages, even though the mutant toxin (which was
N-glycosylated) could still bind to mannose receptors and
was endocytosed by these cells (20). An explanation that could account
for these findings is that wild-type ricin normally has to be bound, or
transferred, to a galactosylated component that itself undergoes
retrograde transport, e.g. an endogenous KDEL-containing
protein capable of recycling between the TGN and the ER (21). This
prompted us to examine which, if any, Golgi/ER protein(s) might bind
ricin holotoxin. Rat liver calreticulin is a potential candidate in that it contains a C-terminal KDEL motif and is galactosylated (22).
The data presented here indicate that calreticulin interacts directly
or indirectly with ricin both in vitro and in
vivo. As such, it is a candidate for a Golgi-to-ER recycling
cellular protein that might be opportunistically used by ricin to reach
the ER lumen, an essential transport step in the entry process of this protein.
Materials--
Antibodies against calreticulin, calnexin, BiP
(immunoglobulin heavy chain-binding protein),
and protein-disulfide isomerase were purchased from Stressgen Biotech
Corp. (Victoria, British Columbia, Canada), and antibody against Rab5
was from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
RTA, RTB, and fibroblast growth factor intracellular binding protein
were raised in rabbits by standard immunization. Radioisotopes were
from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom).
Native RTA was from Inland Laboratories Inc. (Austin, TX).
Production of RTA--
Plasmids encoding RTA, RTA-sulf1 (RTA
modified to include a C-terminal tyrosine sulfation site (6)), or
RTA-sulf2 (RTA modified to include a tyrosine sulfation site and
overlapping N-glycosylation sites at the C terminus (6))
were expressed in E. coli, and the recombinant proteins were
purified to homogeneity by two rounds of CM-Sepharose chromatography
and quantified as described previously (23).
Preparation of RTB--
Ricin (10 mg) in 50 mM
Tris-HCl (pH 8.5), 0.1 M lactose, and 5% (v/v)
Reassociation of RTA and RTB--
Equimolar mixtures of RTA (or
RTA-sulf1/RTA-sulf2) and RTB were incubated in
phosphate-buffered saline (PBS) containing 0.1 M lactose
and 1% (v/v) Purification of Rat Liver Microsomes--
A fresh liver from a
male Harlan Sprague-Dawley rat was homogenized by passage through a
150-µm mesh sieve (24) into ice-cold 0.5 M sucrose/KM
buffer (0.1 M potassium phosphate (pH 6.7) and 5 mM MgCl2), and the homogenate was adjusted to
25 ml with 0.5 M sucrose/KM buffer. Stepped sucrose
gradients were prepared by successively layering 2 ml of 1.3 M sucrose/KM buffer, 4.5 ml of 0.86 M
sucrose/KM buffer, 4 ml of homogenate, and 2.5 ml of 0.25 M
sucrose/KM buffer. After centrifugation at 29,000 rpm in an SW 40 swing-out rotor (Beckman Instruments) for 1 h at 4 °C, the
ER-enriched fraction was collected from the 0.86/1.3 M
sucrose interface. After dilution to 0.5 M sucrose using KM
buffer and centrifugation at 14,000 rpm for 20 min at 4 °C,
microsomes were pelleted from the resulting supernatant by
centrifugation at 50,000 rpm for 1 h. The pellet was resuspended
in 50 mM Tris-HCl (pH 7.5), 25 mM KCl, and 5 mM MgCl2 (ER resuspension buffer) and stored in
aliquots at Binding of ER Components to Immobilized Ricin--
100 µl of
rat liver microsomes (containing 4.05 mg/ml protein) was solubilized by
mixing with an equal volume of 1% Triton X-100 and standing at room
temperature for 20 min. The sample was then applied to a 1-ml column of
agarose-bound ricin (Vector Labs, Inc., Burlingame, CA) that had been
equilibrated with 50 ml of ER resuspension buffer to remove stabilizing
sugars. The column was washed with 10 ml of ER resuspension buffer, and
0.5-ml fractions were collected. Bound material was eluted with 5 ml of
0.1 M lactose in ER resuspension buffer and collected as a single fraction. An equal volume of ice-cold acetone was added to
collected fractions, which were kept on ice for 30 min. After recovery
by centrifugation at 15,000 rpm for 30 min, pellets were air-dried before being dissolved in 40 µl of ER resuspension buffer. Proteins present in each fraction were separated by SDS-PAGE, and
calreticulin was visualized by enhanced chemiluminescence Western blotting (ECL detection system, Amersham Pharmacia Biotech).
Co-immunoprecipitation from ER Extracts--
300 µl of rat
liver microsomes (containing 2 mg/ml protein) was solubilized by adding
an equal volume of 4% CHAPS in ER resuspension buffer and allowed to
stand at room temperature for 20 min. The sample was split into four
150-µl aliquots, and 10 µg of ricin was added to each in 150 µl
of ER resuspension buffer, in 150 µl of 200 mM mannose in
ER resuspension buffer, in 150 µl of 200 mM lactose in ER
resuspension buffer, or in 150 µl of ER resuspension buffer
containing 1.5 µl of 1 M CaCl2. All samples
were tumbled at room temperature for 90 min; split into three 100-µl
aliquots; and immunoprecipitated with anti-calreticulin, anti-calnexin, or anti-protein-disulfide isomerase antibodies in a total volume of 1 ml of ER resuspension buffer. After tumbling at room temperature for a
further 2 h, 90 µl of a 7% slurry of protein A-Sepharose was
added, and tumbling was continued for a further 2 h. Protein A-Sepharose beads were recovered by centrifugation; washed four times
with 50 mM HEPES-KOH (pH 7.6), 600 mM NaCl, and
1% Triton X-100; and finally resuspended in 30 µl of reducing
SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and probed
using a sheep antibody to ricin and ECL Western blotting.
Cell Culture--
Vero cells were propagated and maintained
under standard conditions (5% CO2 in Dulbecco's modified
Eagle's medium containing 5% fetal calf serum). The day prior to an
experiment, the cells were seeded onto six-well plates at 3 × 105 cells/well.
Labeling Experiments--
Cells seeded as described above were
either washed twice with methionine/serum-free Dulbecco's modified
Eagle's medium and then incubated for 4 h in the same medium
containing 25 µCi of [35S]methionine or washed twice
with sulfate/serum-free minimal essential Eagle's medium for
suspension cells (SMEM) (BioWhittaker UK, Ltd., Wokingham, United
Kingdom) and incubated for 4 h in the presence of 200 µCi of
Na235SO4. Following labeling, cells
were incubated for up to 4 h in the presence of toxin. Where
appropriate, cells were pretreated with brefeldin A (BFA; 10 µg/ml)
for 1 h prior to adding toxin.
Calreticulin Labeling--
Vero cells were labeled by incubation
for 24 h with 200 µCi of [3H]galactose.
Alternatively, Vero cells were washed once with PBS and then lysed in
lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and 200 units/ml aprotinin. The cleared lysate was incubated for 1 h with 20 units/ml galactose oxidase (Sigma). Subsequently, the lysate was incubated with 10 mCi/ml sodium
[3H]borohydride. The reaction was terminated by adding
100 mM lactose and 1 mM sodium borohydride. The
lysate was then immunoprecipitated using immobilized anti-calreticulin
antibodies. Precipitated protein was analyzed by reducing SDS-PAGE and
visualized by fluorography. A control sample was treated in the same
way, but in the absence of galactose oxidase treatment.
Preparation of Cell Extracts--
Cells were washed twice with
PBS containing 0.1 M lactose and then with PBS alone. Cells
were lysed on ice by adding 0.5 ml of PBS containing 1% Triton X-100
and 1 mM N-ethylmaleimide. After 5 min, cells
were scraped from the dishes and centrifuged to remove nuclei.
Immunoprecipitation from Cell Extracts--
Extracts were
incubated with rabbit antibodies that had been immobilized on protein
A-Sepharose beads at 4 °C overnight. The beads were then washed
twice with PBS containing 0.1% (v/v) Triton X-100 and once with PBS
alone prior to resuspension in nonreducing sample buffer and analysis
by SDS-PAGE.
Ricin Interacts with Calreticulin in Vitro--
The productive
intoxication pathway for ricin requires retrograde transport from the
TGN to the ER lumen, from where RTA translocates into the cytosol (6,
25). To achieve this retrograde trafficking, ricin possibly interacts
with a cellular component(s) normally capable of recycling between the
Golgi and the ER. To examine whether any Golgi/ER components
could interact with ricin in vitro via the RTB lectin
activity, we passed solubilized rat liver microsomal preparations down
an immobilized ricin column. Since the galactose-binding activity of
RTB seems to be essential for optimal ricin cytotoxicity (20, 26), any
lectin-interactive components were eluted from the column with lactose.
Immunoprecipitation followed by ECL Western blotting showed that the
lactose eluate contained calreticulin (Fig.
1A). The lactose eluate did
not contain calnexin, protein-disulfide isomerase, or BiP, but we
cannot exclude the possibility that these chaperones bind to ricin in a
non-lectin association. Free RTA or ricin holotoxin was next added to
the microsomal extracts; calreticulin was immunoprecipitated; and any
interaction between calreticulin and toxin was demonstrated by probing
reducing gels of the immunoprecipitates with anti-RTA antibodies. As
shown in Fig. 1B, RTA was detected only in calreticulin
complexes containing ricin rather than free RTA, suggesting that the
toxin was interacting with the chaperone via its B chain. The addition
of either mannose or Ca2+ to the microsomal extracts did
not prevent the association of toxin and calreticulin, whereas the
addition of lactose did (Fig. 1C). This suggests that the
association with RTB required a galactosylated oligosaccharide on
calreticulin (Fig. 1C).
Immunoprecipitation of Ricin from Extracts of Intoxicated Vero
Cells--
Before looking for an interaction between ricin and
calreticulin in vivo, we determined that we could visualize
endocytosed ricin and, in particular, its reduced subunits in extracts
from Vero cells treated with toxin. After incubation with toxin for the
times indicated in Fig. 2, the cells were
washed with 0.1 M lactose and lysed, and toxin was
recovered from extracts by immunoprecipitation with either rabbit
anti-RTA or anti-RTB antibodies and visualized on nonreducing gels by
Western blotting using sheep anti-RTA or anti-RTB antibodies. As
expected, the amount of total internalized ricin remained relatively
constant over the time course due to toxin recycling between the medium
and endosomes, whereas free subunits, released after intracellular
cleavage within the ER or cytosol, accumulated in a
time-dependent fashion. The relative accumulation rates for
free RTA (Fig. 2A) and RTB (Fig. 2B) were
approximately equal, suggesting that neither subunit was rapidly
degraded following release from holotoxin or that both subunits were
degraded to similar extents. The time course for the appearance of
reduced toxin was consistent with the observed time course for
cytotoxicity at the ricin concentration used, which showed that protein
synthesis was significantly inhibited within 30 min and was almost
abolished by 60 min (data not shown).
Accumulation of Free Toxin Subunits Can Be Prevented by
Preincubation of Cells with Brefeldin A--
Ricin toxicity is
abolished by preincubating cells with BFA (27, 28) presumably by
preventing passage of ricin through the Golgi stack to the ER, the site
of toxin entry into the cytosol. Preincubation with BFA also blocks the
sulfation of ricin containing an A chain with an introduced tyrosine
sulfation site (designated RCA-sulf1 (6)), a modification that is
trans-Golgi-specific (29). In the proposed model for
cellular intoxication by ricin, the lectin activity of RTB is required
for efficient retrograde transport of holotoxin to the ER (21). Hence,
it would be expected that that the reductive separation of RTA and RTB
would also be prevented by BFA treatment. Cells were intoxicated with
ricin for 2 h in the presence or absence of BFA, and extracts were
immunoprecipitated using either anti-RTA or anti-RTB antibodies. Fig.
3 shows that preincubation with BFA had
only a small effect (typically a 15% reduction) on the total amount of
ricin internalized, but almost completely abolished the appearance of
the free toxin subunits, suggesting that reduction normally occurs in
an early (pre-TGN) compartment of the secretory pathway such as the
endoplasmic reticulum. This is consistent with the earlier observation
that some of the RTA-sulf2 in ricin holotoxin (RTA-sulf2
is the ricin A chain modified to have overlapping
N-glycosylation sites at the C terminus (6)), as well as
reduced RTA-sulf2, becomes N-glycosylated during cell entry (6). This shows that endocytosed ricin can reach
oligosaccharyltransferase in the ER.
Ricin Interacts with Calreticulin in Vivo--
It has been
proposed that the retrograde transport of ricin from the Golgi to the
ER might be mediated, at least in part, by the KDEL retrieval system
(21). This model proposed that ricin might bind to a recycling
KDEL-tagged glycoprotein because of the lectin activity of RTB,
allowing ricin to "hitch a lift" to the ER as the KDEL protein is
retrieved by KDEL receptors. Candidates for such a carrier include the
abundant chaperones of the ER, several of which have putative
N-glycosylation sites (Grp94, calreticulin, calnexin,
protein-disulfide isomerase, etc.). Vero cells were intoxicated with
ricin and lysed in the presence of Triton X-100, and the lysates were
immunoprecipitated with selected anti-chaperone antibodies.
Immunoprecipitated proteins were analyzed by Western blotting using
anti-RTA antibodies (Fig. 4A).
Controls showed that ricin was immunoprecipitated by either anti-RTA or
anti-RTB antibodies, but not by irrelevant antibodies (anti-Rab5). As
expected, a small amount of free RTA was also precipitated by anti-RTA
antibodies. Anti-calreticulin antibodies also coprecipitated ricin (but
not free RTA), but no toxin was seen in complexes using antibodies
against calnexin or protein-disulfide isomerase (Fig. 4A) or
BiP (data not shown). Coprecipitation of ricin with calreticulin was
prevented in the presence of 0.1 M lactose, which had
little or no effect on the direct immunoprecipitation of ricin with
either anti-RTA or anti-RTB antibodies (Fig. 4B). This
observation is consistent with earlier findings that RTB plays an
intracellular role during the intoxication process, in addition to its
initial role in binding holotoxin to the target cell surface (20).
In keeping with this, Vero cells were treated with either free
recombinant or native (glycosylated) RTA (which enters via fluid-phase
uptake (30)) or free RTB for 4 h, washed, and then homogenized.
Anti-RTA or anti-RTB antibodies precipitated the appropriate
intracellular toxin subunit, whereas only RTB coprecipitated when
calreticulin was precipitated with anti-calreticulin antibodies (Fig.
5). Although equal concentrations of RTA
and RTB were added to cells, the relative amounts of RTA and RTB
precipitated predictably confirmed that the cellular uptake of RTB was
much more efficient. This is because uptake of RTB is
receptor-mediated, whereas free A chains enter cells in the fluid
phase.
To test that calreticulin was able to interact with ricin that had
already been transported to the Golgi complex, where
tyrosylsulfotransferase is found, the coprecipitation experiment was
repeated, but using cells that had been intoxicated with ricin
containing RTA-sulf1 (RTA that had been modified to contain a tyrosine
sulfation site (6)) in the presence of
Na235SO4. Fig.
6A shows that
35S-labeled ricin holotoxin was immunoprecipitated by
anti-RTA, anti-RTB, or anti-calreticulin antibodies and that the
interaction was again abolished in the presence of lactose.
To test that calreticulin remained in contact with ricin until it
reached the ER (the location of oligosaccharyltransferase), coprecipitations were also performed using extracts from cells that had
been intoxicated with ricin containing RTA-sulf2 (a recombinant RTA modified to contain overlapping C-terminal
N-glycosylation sites (6)) in the presence of
Na235SO4. Fig. 6B shows
that immunoprecipitation with either anti-calreticulin or anti-ricin
antibodies yielded both non-glycosylated RTA and newly
glycosylated RTA (6). (To clearly see the gel mobility difference
between RTA and glycosylated RTA, reducing SDS-PAGE was used in this
case.) Significantly less RTA or glycosylated RTA was precipitated by
anti-calnexin antibodies, but the data shown in Fig. 6B
indicate that limited interaction with calnexin may also have occurred.
To ensure that the interaction between calreticulin and ricin was not
simply due to post-lysis binding events, time course experiments were
performed. The amount of ricin internalized remained reasonably
constant over a 4-h time course (see Fig. 2). In contrast, the amount
of internalized ricin that co-immunoprecipitated with calreticulin
increased steadily over the first 2 h and then remained relatively
constant for the remaining 2 h of the assay (Fig.
7A), perhaps suggesting that a
steady state had been reached. It would be expected that BFA
pretreatment would also reduce any specific interaction between ricin
and calreticulin since it would prevent recycling between the ER and
the Golgi complex. Fig. 7B shows that BFA treatment did
significantly reduce (by ~75%) the co-immunoprecipitation of ricin
with calreticulin, whereas it had only a small effect on the amount of
ricin internalized (~15% reduction) (Fig. 3).
To further demonstrate a specific interaction between calreticulin and
ricin, it was important to show that immunoprecipitates of ricin from
intoxicated cells also coprecipitated calreticulin. Vero cells were
metabolically labeled with [35S]methionine for 4 h
prior to intoxication with ricin for a further 4 h. Lysates were
immunoprecipitated with anti-RTA, anti-RTB, or anti-calreticulin
antibodies; the pellets were washed and boiled in 1% SDS; and samples
were diluted and re-immunoprecipitated with anti-calreticulin
antibodies. Fig. 8 shows that in the
absence of toxin treatment, calreticulin was precipitated only when the primary antibody was anti-calreticulin. In contrast, when cells had
been intoxicated with ricin, calreticulin could be precipitated by both
anti-RTA and anti-RTB antisera (Fig. 8).
Calreticulin from Vero Cells Is a Galactose-containing
Glycoprotein--
The interaction between ricin and calreticulin
described here is mediated by the toxin B chain and is prevented by
lactose. This requires that Vero cell calreticulin is glycosylated and that the glycosyl moiety contains terminal galactosyl residues. Vero
cells were therefore incubated with [3H]galactose for
24 h prior to immunoprecipitation of lysates with anti-calreticulin antibodies. Fig.
9A shows that
[3H]galactose was incorporated into calreticulin.
It could not be excluded, however, that the [3H]galactose
was being metabolized in the cells and that 3H was being
incorporated into calreticulin in another form. To test further if Vero
cell calreticulin is galactosylated, we took advantage of a biochemical
assay to selectively label proteins with terminal galactose (31).
Proteins present in a Vero cell homogenate were treated with the enzyme
galactose oxidase and then with sodium [3H]borohydride to
bind any oxidized terminal galactose residues. Immunoprecipitation with
anti-calreticulin antibodies showed that a band corresponding in
molecular mass to calreticulin was labeled after such treatment (Fig.
9B, lane 1). This band was not obtained in
immunoprecipitates with antibodies against fibroblast growth factor
intracellular binding protein (Fig. 9B, lane 2)
or ricin (lane 3) or in the absence of galactose oxidase
treatment (Fig. 9C). For comparison, Vero cells labeled with
[35S]methionine were lysed and subjected to
immunoprecipitation with anti-calreticulin antibodies. The
[35S]methionine-labeled calreticulin seen in lane
4 of Fig. 9B had the same migration rate as the
[3H]borohydride band in lane 1. The other
bands seen in lane 1-3 could be due to labeling of the
polyclonal rabbit antibodies used or other galactosylated proteins that
nonspecifically interact with the antibodies. Taken together, the data
indicate that at least a fraction of the calreticulin expressed in Vero
cells contains a terminal galactose residue.
The data presented here show that ricin can interact with
calreticulin in vitro and in vivo. Interaction
in vivo did not appear to be an artifact caused by
homogenizing the cells since (i) the level of interaction was shown to
increase with time over a period when the total intracellular ricin
pool did not alter significantly and (ii) BFA treatment of cells
significantly reduced the extent of this interaction without
significantly affecting ricin uptake.
The interaction could take one of several forms, although a direct
protein-protein interaction is unlikely because of its lactose-dependent nature. An interaction of the
sugar-binding site of calreticulin with glucose residues present on RTA
and/or RTB is also unlikely since the glycans present on native RTA are complex and endoglycosidase H-resistant (32). Also, since both toxin subunits have undergone ER-to-vacuole transport with associated Golgi processing of their glycans in the producing plant (33), the
toxin subunits are not monoglucosylated, a requirement for any
interaction with the sugar-binding site of calreticulin. Furthermore, the interaction we observed involves native ricin, whereas calreticulin is known to interact with partially folded proteins. This, together with the data presented here, supports an interaction between the
galactose-specific binding sites of RTB and galactosyl groups present
on calreticulin. This contention is further supported by the facts that
calreticulin interacts with ricin, but not free RTA, indicating that
the association occurs via RTB, and that the interaction is abolished
in the presence of lactose, which is known to block the biological
activity of the RTB lectin.
An interaction mediated by the sugar-binding sites of RTB would require
that calreticulin is glycosylated and that the oligosaccharide contains
a terminal galactose residue. Until recently, the glycosylation status
of calreticulin has been unclear. Although the primary sequences of
calreticulin isolated from several different species all contain a
single putative N-glycosylation site, the search for an
attached glycan has been inconclusive. Calreticulins from rat liver
(34) and bovine liver (35) are known to be glycosylated; but in most
species, including human, calreticulin was thought to be
non-glycosylated (36). More recently, however, calreticulin from human
myeloid cells has been shown to be glycosylated, despite the fact that
neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin had any effect on the protein's mobility on
SDS-polyacrylamide gels (37). Since human glycosylated calreticulin has
the apparent properties of a non-glycosylated protein, it is possible
that other forms are also glycosylated, despite reports to the
contrary. Here we were able to metabolically label calreticulin from
Vero cells with [3H]galactose and confirm that Vero cell
calreticulin contains an oligosaccharide with a terminal galactose
residue by treatment with galactose oxidase followed by sodium
[3H]borohydride, providing an explanation for its
apparent ability to interact with RTB.
Calreticulin contains an N-terminal signal sequence and a C-terminal ER
retrieval sequence (KDEL); and as expected, its major intracellular
location is the lumen of the ER (36, 38). Calreticulin has been
reported to be present in other cellular locations, however, including
later compartments of the secretory pathway (39) and at the cell
surface (40). It is tempting to suggest that the interaction between
ricin and calreticulin that occurs in vivo is
physiologically significant in that calreticulin functions as a
retrograde carrier that transports ricin from the TGN to the ER.
Although the present data do not provide unequivocal evidence for such
a function, several features of the interaction are consistent with
this idea. To act as a retrograde carrier, calreticulin would normally
need to recycle between the ER and the trans-Golgi. This certainly seems to be the case for rat liver calreticulin since it is
terminally galactosylated (22), a modification that occurs in the
trans-Golgi cisternae (41). Likewise, we have shown here that Vero cell calreticulin is galactosylated, a feature that would
allow its interaction with the RTB subunit of incoming ricin. The
finding that BFA pretreatment of cells (to disrupt the Golgi stack, but
not the TGN), significantly reduced, but did not abolish, the
calreticulin-ricin interaction is also consistent with the TGN or a
later secretory pathway compartment being the site where the
interaction initially occurs. This supports the idea that a
ricin-calreticulin interaction can occur before ricin reaches the ER
rather than after it reaches this compartment and suggests that its
primary purpose is the delivery of toxin to the ER rather than
any subsequent events. The interaction in vivo is
stable, and the fact that it does not involve free RTA implies that
what we are seeing does not reflect events within the ER leading to membrane translocation where association with chaperones may be very
transient and involve the translocating RTA subunit. By virtue of its
C-terminal ER retrieval sequence, any calreticulin present in the TGN
and interacting there with incoming ricin could predictably be recycled
to the ER lumen via KDEL receptors. These receptors are known to
recycle between the ER and the Golgi (13) and can retrieve proteins to
the ER from the trans-most cisterna of the Golgi complex
(14). In keeping with this, we have shown that overexpressing
lysozyme-KDEL, which causes a relocation of the KDEL receptor
from the Golgi complex to the ER (42), reduced the sensitivity of cells
to ricin intoxication.2 This
implies that ricin, at least in part, might indirectly utilize the KDEL
retrieval system to undergo Golgi-to-ER retrograde transport. Because
ricin is a galactose-specific lectin, it could presumably bind any
appropriate galactosylated glycoprotein or glycolipid it might
encounter intracellularly; and if any of these were to recycle between
the TGN and the ER, they could potentially be parasitized by ricin for
retrograde transport. However, it must be stressed that much of the
data presented here could be explained by an indirect interaction
between ricin and calreticulin, and we concede that the
physiological significance of the interaction during toxin entry
remains speculative. In any event, calreticulin is unlikely to be the
exclusive carrier for ricin. Indeed, calreticulin-deficient cell lines
remain sensitive to ricin (data not shown). We suggest that
calreticulin is one of a number of as yet unidentified cellular components that can act in this way and that retrieval systems other
than that involving the KDEL receptor may likewise be utilized. Calreticulin may be the preferred carrier, however, since if it enters
the ER lumen in association with ricin, it would be well placed to
guide toxin unfolding and/or some other event required for the
subsequent RTA translocation across the ER membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol was stirred overnight at 4 °C. The reduced toxin
was applied to a 100-ml DEAE-Sepharose column pre-equilibrated in the
same buffer but containing 1%
-mercaptoethanol and washed to remove
RTA and nonreduced ricin. RTB was eluted with a linear 0-0.5
M NaCl gradient and stored at 4 °C.
-mercaptoethanol and dialyzed against the same buffer
(without
-mercaptoethanol) overnight and then against PBS for 2 more
nights. The extent of reassociation was determined by nonreducing
SDS-polyacrylamide gel electrophoresis (PAGE).
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Rat liver calreticulin binds to immobilized
ricin. A, ER extracts were passed over an immobilized
ricin-agarose column, which was then washed with ER resuspension buffer
until no further protein was present in the wash fractions. Bound
material was eluted from the column using 100 mM lactose in
ER resuspension buffer. The presence of calreticulin was detected by
SDS-PAGE and Western blotting. Lane 1, ER extract;
lane 2, first collected wash fraction; lane 3,
final wash (fraction 20); lane 4, lactose
eluate. B, ricin (5 µg) or RTA (10 µg) was added to ER
extracts, and calreticulin was recovered by immunoprecipitation. The
immunoprecipitates were subjected to reducing SDS-PAGE, and
coprecipitated RTA was visualized by Western blotting. Lane
1, extract supplemented with RTA; lane 2, extract
supplemented with ricin. C, ricin-treated ER extracts were
immunoprecipitated using anti-calreticulin antibodies, and
coprecipitated RTA was visualized by Western blotting using anti-RTA
antibodies as described for B, either in the absence of
additives (lane 1) or in the presence of mannose (lane
2), lactose (lane 3), or calcium (lane
4).
View larger version (68K):
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Fig. 2.
Appearance of free ricin subunits in cells
endocytosing the toxin. Vero cells were incubated with ricin (10 µg/ml) for various times before being washed twice with PBS
containing 100 mM lactose and once with PBS alone. After
lysing with Triton X-100, cleared lysates were treated with antibodies
to RTA (A) or RTB (B). Precipitated proteins were
separated by nonreducing SDS-PAGE. Cells were harvested immediately
after the addition of ricin or after the times indicated.
View larger version (36K):
[in a new window]
Fig. 3.
Brefeldin A treatment prevents the release of
free ricin subunits. Vero cells were preincubated in the presence
(+) or absence ( ) of 10 µg/ml BFA for 1 h prior to being
incubated with ricin for a further 2 h and subsequently treated
exactly as described in the legend to Fig. 2.
A and
B indicate
immunoprecipitation with anti-RTA and anti-RTB antibodies,
respectively.
View larger version (55K):
[in a new window]
Fig. 4.
Ricin specifically coprecipitates with
calreticulin. A and B, Vero cells were
incubated with ricin for 4 h, washed, and lysed as described in
the legend to Fig. 2 and treated with a range of immobilized antibodies
in the absence (A) or presence (B) of 100 mM lactose. Precipitated proteins were separated by
nonreducing SDS-PAGE and visualized by Western blotting using sheep
anti-RTA antibodies. A, anti-RTA antibody;
B, anti-RTB antibody;
CNX, anti-calnexin antibody;
CRT, anti-calreticulin antibody;
Rab, anti-Rab5 antibody;
PDI, anti-protein-disulfide isomerase
antibody. In A and B, the upper arrows
indicate ricin, and the lower arrows indicate RTA.
View larger version (33K):
[in a new window]
Fig. 5.
RTB coprecipitates with calreticulin.
Vero cells were incubated with 5 µg of glycosylated RTA
(gRTA; lanes 1 and 2), recombinant RTA
(rRTA; lanes 3 and 4), or RTB
(lanes 5 and 6) for 4 h prior to washing and
lysis in Triton X-100. Cleared lysates were treated with immobilized
antibodies, and the precipitated proteins were analyzed by SDS-PAGE.
Lanes 1 and 3, anti-RTA antibody
( A); lane 5, anti-RTB antibody
(
B); lanes 2, 4, and
6, anti-calreticulin antibody
(
CRT).
View larger version (40K):
[in a new window]
Fig. 6.
Calreticulin binds to ricin that has reached
the Golgi complex and the ER. A, Vero cells were
labeled with Na235SO4 for
4 h prior to the addition of ricin containing RTA-sulf1. After
incubation for a further 4 h, cells were washed, lysed, and
immunoprecipitated with anti-RTA ( A), anti-RTB
(
B), or anti-calreticulin
(
CRT) antibodies in the absence or presence of
100 mM lactose. Precipitated proteins were analyzed by
nonreducing SDS-PAGE and visualized by fluorography. The upper
arrow indicates 35S-sulfated ricin, and the
lower arrow indicates 35S-sulfated RTA.
B, the same experiment was repeated, but using reconstituted
ricin containing RTA-sulf2. In this case, precipitated proteins
were analyzed by reducing SDS-PAGE.
CNX,
anti-calnexin antibody; gA and A, glycosylated
RTA and non-glycosylated RTA, respectively.
View larger version (26K):
[in a new window]
Fig. 7.
Brefeldin A reduces the coprecipitation of
ricin with calreticulin. A, Vero cells were intoxicated
with ricin for the indicated times, washed, lysed, and
immunoprecipitated with immobilized anti-calreticulin antibodies.
Immunoprecipitates were analyzed by nonreducing SDS-PAGE and visualized
by Western blotting using anti-RTA antibodies. B, Vero cells
were incubated in the presence (+) or absence ( ) of BFA (10 µg/ml)
for 1 h prior to the addition of ricin and incubation for a
further 2 h. Cells were washed, lysed, immunoprecipitated, and
analyzed as described for A.
View larger version (32K):
[in a new window]
Fig. 8.
Calreticulin coprecipitates with ricin.
Vero cells were labeled with [35S]methionine (25 µCi)
for 4 h and then incubated for a further 4 h in the absence
( ) or presence (+) of ricin. Following washing and lysis, cleared
extracts were immunoprecipitated with the appropriate primary antibody
as indicated. Immunoprecipitates were washed twice with PBS and 0.1%
Triton X-100 and once with PBS alone prior to being resuspended in 100 µl of PBS and 1% SDS and boiled for 2 min. The denatured extracts
were diluted to 1 ml with PBS and 0.5% Triton X-100, and the protein
A-Sepharose beads were removed by centrifugation. Extracts were all
subsequently immunoprecipitated using anti-calreticulin antibodies, and
precipitated protein was analyzed by SDS-PAGE and visualized by
fluorography.
CRT, anti-calreticulin antibody;
A, anti-RTA antibody;
B, anti-RTB antibody.
View larger version (36K):
[in a new window]
Fig. 9.
Vero cell calreticulin is
galactosylated. A, Vero cells were labeled for
24 h with [3H]galactose (200 µCi) or for 8 h
with [35S]methionine (25 µCi) and lysed with Triton
X-100, and cleared extracts were immunoprecipitated with immobilized
anti-calreticulin antibodies. Precipitated proteins were analyzed by
SDS-PAGE and fluorography. Lane 1,
[35S]methionine-labeled cells; lane 2,
molecular weight markers; lane 3,
[3H]galactose-labeled cells. The arrow
indicates the calreticulin (CRT) band. B, Vero
cell lysates were first treated with galactose oxidase (20 units/ml)
and then with sodium [3H]borohydride (10 mCi/ml). The
reactions were terminated by the addition of 100 mM lactose
and 1 mM sodium borohydride. Immobilized antibodies were
subsequently added to the lysates, and precipitated proteins were
analyzed by SDS-PAGE and fluorography. Lane 1,
anti-calreticulin antibody ( CRT); lane
2, anti-fibroblast growth factor intracellular binding protein
antibody (
FIBP); lane 3,
anti-RTA antibody (
RTA); lane
4, calreticulin immunoprecipitated from
[35S]methionine-labeled cells. C, the same
experiment as described for B was carried out, but with
(lane 2) or without (lane 3) galactose oxidase
treatment. Lane 1, calreticulin immunoprecipitated from
[35S]methionine-labeled cells.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work carried out at the University of Warwick was supported by Grant BO8000 from the United Kingdom Bio/Technology and Biological Sciences Research Council and the Wellcome Trust, and that at the Norwegian Radium Hospital was supported by the Norwegian Cancer Society, the Novo Nordisk Foundation, the Norwegian Research Council, the Blix Fund for the Promotion of Medical Research, Rachel and Otto Kr. Brunn's Legat, and by the Jahre Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: European Molecular Biology Laboratory, Heidelberg 69117, Germany.
Fellow of the Norwegian Cancer Society.
** To whom correspondence should be addressed. Tel.: 44-24-7652-3598; Fax: 44-24-7652-3701; E-mail: Mike.Lord@warwick.ac.uk.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009499200
2 M. E. Jackson, L. M. Roberts, and J. M. Lord, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: RTA, ricin A chain; RTB, ricin B chain; TGN, trans-Golgi network; ER, endoplasmic reticulum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BFA, brefeldin A.
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