From the Microbial Pathogenesis Section, NIAID, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, October 8, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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The interaction of anthrax toxin protective
antigen (PA) and target cells was assessed, and the importance of the
cytosolic domain of tumor endothelium marker 8 (TEM8) in its function
as a cellular receptor for PA was evaluated. PA binding and proteolytic processing on the Chinese hamster ovary cell surface occurred rapidly,
with both processes nearly reaching steady state in 5 min. Remarkably,
the resulting PA63 fragment was present on the cell surface only as an
oligomer, and furthermore, the oligomer was the only PA species
internalized, suggesting that oligomerization of PA63 triggers
receptor-mediated endocytosis. Following internalization, the PA63
oligomer was rapidly and irreversibly transformed to an
SDS/heat-resistant form, in a process requiring an acidic compartment. This conformational change was functionally correlated with membrane insertion, channel formation, and translocation of lethal factor into
the cytosol. To explore the role of the TEM8 cytosolic tail, a series
of truncated TEM8 mutants was transfected into a PA receptor-deficient Chinese hamster ovary cell line. Interestingly, all of the cytosolic tail truncated TEM8 mutants functioned as PA receptors, as determined by PA binding, processing, oligomer formation, and translocation of an
lethal factor fusion toxin into the cytosol. Moreover, cells transfected with a TEM8 construct truncated before the predicted transmembrane domain failed to bind PA, demonstrating that residues 321-343 are needed for cell surface anchoring. Further evidence that
the cytosolic domain plays no essential role in anthrax toxin action
was obtained by showing that TEM8 anchored by a
glycosylphosphatidylinositol tail also functioned as a PA receptor.
Anthrax toxin, the major virulence factor of Bacillus
anthracis, consists of three polypeptides: protective antigen
(PA,1 83 kDa), lethal factor
(LF, 90 kDa), and edema factor (EF, 89 kDa) (1, 2). These three
proteins are individually non-toxic. To intoxicate mammalian cells, PA
binds to a ubiquitously expressed, recently identified cellular
receptor, tumor endothelium marker 8 (TEM8) variant 2 (3), and is
cleaved at the sequence RKKR167 on the cell surface by
furin or furin-like proteases (4, 5). Proteolysis yields the
amino-terminal 20-kDa fragment (PA20), which is released into the
medium, and the carboxyl-terminal 63-kDa fragment (PA63), which remains
bound to the receptor and self-associates to form a ring-shaped
heptamer (6, 7). The heptamer binds up to 3 molecules of LF or EF (8,
9). The resulting oligomeric complex is then internalized into
endosomes, where the decreased pH causes the PA63 heptamer to insert
into the endosomal membrane and produce a channel through which LF and
EF translocate to the cytosol (10). Therefore, PA is the central part
of anthrax toxin, serving as the delivery vehicle for binding and
translocation of LF and EF into the cytosol of the cells. The
combination of PA plus LF kills animals (11, 12) and certain cells,
including mouse macrophages (13, 14). LF is a
zinc-dependent metalloprotease that cleaves several
mitogen-activated protein kinase kinases (MAPKK) in their
amino-terminal regions (15, 16). How this cleavage triggers the lethal
effects of the toxin and whether there are additional cellular
substrates remains unclear. EF is a calmodulin-dependent
adenylate cyclase that elevates intracellular cAMP concentrations (17),
thereby causing diverse effects in cells including the impairment of
phagocytosis (18).
Previous studies on the interaction of PA with host cells have often
used cytotoxicity assays to infer internalization mechanisms, or have
used radiolabeled or chemically labeled PA that may behave differently
due to modification. In the present studies, we directly assessed PA
binding, proteolytic processing, and internalization by target cells
using a highly sensitive and specific rabbit antiserum to PA. We found
that following binding and processing by cell surface furin, the
cleaved PA immediately forms the PA63 oligomer and that this oligomer
is the only species of PA that is internalized. In addition, following
internalization, the oligomer is quickly transformed into an
SDS/heat-resistant form, a process coincident with insertion and
channel formation in endosomal membranes.
In related studies we extended the understanding of toxin
internalization obtained in the recent breakthrough that identified TEM8 variant 2 as a PA receptor (3). Currently, there are three reported cDNAs that result from splicing variations in TEM8
(GenBankTM accession number NM_032208, NM_053034, and
NM_18153). The physiological functions of these have not been studied.
Beyond the fact that TEM8 variant 2 functions as a PA receptor, the
only information available is that implicit in the initial
identification that TEM8 expression is up-regulated in tumor
endothelium (19, 20). Thus, it remains unknown whether other TEM8
variants can also function as PA receptors, and whether TEM8 has
functions beyond binding PA in anthrax toxin action. To answer these
questions, in the present work, we constructed a series of TEM8
truncated mutants, transfected them into a PA receptor-deficient
Chinese hamster ovary (CHO) cell mutant, and found that all constructs having a membrane anchor functioned as PA receptors.
Reagents--
Protein toxins produced as described previously
included PA (21), PA- Cell Lines and Culture Media--
CHO cell clone 6 (CHO CL6) is
a line recloned in this laboratory from CHO 10001, a subclone of CHO-S
(26), which was obtained from Dr. Michael Gottesman (National
Institutes of Health, Bethesda). CHO FD11, a furin-deficient derivative
of CHO CL6, was developed in our laboratory by chemical mutagenesis
(27). CHO PR230 is a spontaneous PA receptor-deficient mutant derived
from CHO WTP4, which is derived from the thioguanine- and
ouabain-resistant cell WTB111 (28), which was derived from CHO-K1. All
CHO cells were grown in PA Binding and Internalization by CHO Cells--
PA binding was
assessed at both 37 and 4 °C. Cells were grown in 24-well plates to
confluence. Cells were incubated with 1 µg/ml PA for different
lengths of time and then washed five times with Hanks' balanced salt
solution (HBSS) (Biofluids, Rockville, MD). The cells were lysed in 100 µl of modified RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl,
1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
1 µg/ml each of aprotinin, leupeptin, and pepstatin). In measurements of PA internalization, the cells were first treated with 0.5 ml of 0.5 mg/ml trypsin in HBSS per well at 37 °C for 5 min to remove proteolytically the cell surface-bound PA, then washed, and lysed. The
cell lysates were subjected to SDS-PAGE or native-PAGE using 4-20%
Tris-glycine gradient gels (NOVEX, San Diego). Prior to loading, the
cell lysates were boiled for 10 min in 1× SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
dithiothreitol, 0.01% bromphenol blue, 6% glycerol) for SDS-PAGE or
were incubated for 10 min at room temperature in 1× native buffer
(NOVEX) for native-PAGE. The proteins were then transferred to
nitrocellulose membranes, followed by Western blotting as described
(29). PA was visualized by chemiluminescence using the West Pico Kit
(Pierce). For the two-dimensional analysis, cell lysate was first
separated on native-PAGE, and the gel strip was then sequentially
equilibrated for 15 min each in Buffer I (125 mM Tris-HCl,
pH 6.8, 1% SDS, 8.7% glycerol, 5 mM dithiothreitol) and
Buffer II (125 mM Tris-HCl, pH 6.8, 1% SDS, 8.7%
glycerol, 2% iodoacetamide) and subjected to SDS-PAGE, followed by
Western blotting as described above.
LF Translocation and MAPKK1 Cleavage Assay--
In measurements
of LF translocation, CHO CL6 cells grown in 24-well plates were
incubated with 1 µg/ml LF along with 1 µg/ml PA or PA- Effect of Vacuolar pH Elevation on the Formation of the
SDS/Heat-resistant PA63 Oligomer--
To assess the
effect of vacuolar pH elevation on de novo formation of
SDS/heat-resistant PA63 oligomer, CHO CL6 cells were incubated at
4 °C with 1 µg/ml PA for 3 h and then washed five times with
ice-cold HBSS. The cells were then incubated in fresh medium in the
presence or absence of 0.2 µM bafilomycin A1 at 37 °C.
Incubations at 37 °C varied from 5 to 180 min. To assess the effect
of vacuolar pH elevation on the preformed SDS/heat-resistant PA63
oligomer, cells were incubated with 1 µg/ml PA at 37 °C for 1 h and washed five times. One set of cells was then further incubated in
fresh medium containing 0.2 µM bafilomycin A1 and the
other in medium without the drug. The incubations at 37 °C varied
from 10 to 180 min. The cells were lysed, and the lysates were
processed by SDS-PAGE and Western blotting as described above.
Construction and Transfection of Human TEM8 Variants into CHO
PR230 Cells--
Human TEM8 variant cDNA fragments were isolated
by reverse transcriptase-PCR from human fetal brain mRNA (catalog
number 11438-017) purchased from Invitrogen. First-strand cDNA was
synthesized by using the SuperScript First-strand Synthesis System
(catalog number 11904-018) purchased from Invitrogen. We used 5' primer
PR5
(AAGTGTACAATGGCCACGGCGGAGCGGAGAGCCCTCGGCATCGGCT, the start codon ATG is underlined and the BsrGI site for
cloning is in boldface) in combination with various 3' primers to
amplify different carboxyl-terminal truncated TEM8 variants, as
diagramed in Fig. 5A). Thus, primer 115 aa
(CCCACAAGGCATCGAGTTTTCCCTT, stop codon provided by the
expression vector) was used to obtain the TEM8 variant TEM8-115 aa,
having a 115-residue cytosolic tail. Similarly, primer 26 aa
(CGGGATCCTAAGCGTAATCTGGAACATCGTATGGGTAACCATCATCATCTTCTTCCTCACTCTCCTCGGCA, the antisense of stop codon is underlined, the BamHI site is
in boldface, and the sequence encoding for an influenza virus
hemagglutinin (HA) tag is in italic) was used to obtain TEM8-26 aa.
Primer 16 aa
(CGGGATCCTAAGCGTAATCTGGAACATCGTATGGGTAGGCAGGGGGTGGAGGGACCTCCTTGATAAT, underlining, etc., as above) was used to obtain TEM8-16 aa. Primer 0 aa
(CGGGATCCTACCAGAACCACCAGAGGAGAGCCAGGGCTA,
underlining, etc., as above, and having no HA tag) was used to obtain
TEM8-0 aa. Primer ED
(CGGGATCCTAACCGTCAGAACAGTGTGTGGTGGTGATGATGACA, underlining, etc., as above) was used to obtain TEM8-ed, the variant having only the extracellular domain, residues 1-320. Finally, primer
v3
(CTATTCCATGCAAGCAGCTGTTGTGGGGCCTGATGCAATTTTGTGGAGGCTACAGTGTGTGGTGGTGATGATGACAGAACTGGA, the antisense of stop codon is underlined) was used to obtain TEM8
variant 3. We found it is difficult to amplify full-length cDNA for
TEM8 variant 1 due to its exceptionally high GC content, and instead we
synthesized the 3' cDNA region of variant 1 chemically and ligated
it into TEM8-115 aa, resulting in full-length TEM8 variant 1. The TEM8
variant cDNA fragments were digested by BsrGI alone or
BsrGI and BamHI and then cloned between the
BsrGI and EcoRV or BsrGI and
BamHI sites of pIRESHgy2B (catalog number 6939-1, Clontech Laboratories, Inc., Palo Alto, CA). This
bicistronic mammalian expression vector contains an attenuated version
of the internal ribosome entry site of the encephalomyocarditis virus, which allows both the gene of interest and the hygromycin B selection marker to be translated from a single mRNA. We also constructed a
glycosylphosphatidylinositol (GPI)-anchored TEM8 by fusion of the TEM8
extracellular region to the GPI anchoring sequence of urokinase
plasminogen activator receptor (uPAR) (31). To do so, the GPI sequence
of human uPAR was amplified by using primers U5
(TATCGTACGTTGTAACCACCCAGACCTGGATGTCCAGT, the
BsiWI cloning site is in boldface) and U3
(AATTCCAGCACACTGGTTAGGTCCAGAGGAGAGTGCCT, the
antisense of the stop codon is underlined, and the BstXI
site is in boldface), with a template of uPAR plasmid phuPAR (kind gift
from Dr. Thomas H. Bugge, National Institutes of Health, Bethesda). The
PCR product was digested by BsiWI and BstXI and cloned between the BsiWI and BstXI sites of the
plasmid encoding TEM8-ed, resulting in an expression plasmid encoding
the TEM8 extracellular part (residues 1-317) and the GPI anchoring
sequence of uPAR (residues 293-335) linked by short tripeptide IVR.
All the expression plasmids were confirmed by DNA sequencing and were transfected into CHO PR230 cells using LipofectAMINE Plus Reagent (Invitrogen), and stably transfected cells were selected by growth in
hygromycin B (500 µg/ml) for 2 weeks. Hygromycin-resistant colonies
were either isolated individually or pooled for further analysis.
Cytotoxicity Assay with MTT--
Cells were grown in 96-well
plates to ~50% confluence. Serial dilutions of PA (0-1000 ng/ml)
combined with FP59 (100 ng/ml) were added to the cells to give a total
volume of 200 µl/well and were incubated for 48 h. Cell
viability was then assayed by adding 50 µl of 2.5 mg/ml
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in
RNA Isolation and Northern Hybridization--
Total RNA was
isolated from exponentially growing CHO cells by using TRIzol Reagent
(Invitrogen), separated on 1.0% agarose, 6.66% formaldehyde gels and
then transferred onto nylon membranes (Immobilon-N, Millipore).
Membranes were hybridized with a 32P-labeled 1.0-kb CHO
TEM8 cDNA fragment isolated by reverse transcriptase-PCR by using
5' primer TTCTGCCAGGAGGAGACACTTACATGC, and 3' primer CCCACAAGGCATCGAGTTTTCCCTT. DNA sequencing analysis showed that this
fragment was 90 and 89% identical to the corresponding mouse and human
TEM8 sequences, respectively. Hybridization was performed in QuikHyb
hybridization solution (catalog number 201220, Stratagene, La Jolla,
CA) containing denatured salmon sperm DNA (0.5 mg/ml) at 60 °C
overnight. Membranes were then washed in 2× SSC (catalog number
750020, Research Genetics, Huntsville, AL), 0.1% SDS at room
temperature for 5 min, and then twice with 0.1× SSC, 0.1% SDS at
60 °C for 25 min.
PA63 Produced from PA by Cell Surface Proteolytic Cleavage Rapidly
Forms Oligomers--
We used a high titered polyclonal anti-PA serum
(number 5308) to assess PA binding and its subsequent processing and
internalization by CHO cells. Analysis of unmodified PA eliminated
concerns that radiolabeled or chemically labeled PA might behave
differently from native PA due to the modification. The results showed
that PA bound to CHO CL6 cells and was rapidly cleaved to PA63. More than half the PA bound was cleaved to PA63 within 5 min (Fig. 1A, CL6 lanes).
Because furin is the major cell surface protease that cleaves PA (4,
5), we compared processing of PA by CHO FD11, a derivative of CHO CL6
cells that lacks furin (27). The FD11 cells bound PA efficiently, but
cleavage to PA63 was very slow (Fig. 1A, FD11
lanes). In addition to intact PA and PA63, another PA species
revealed on SDS-PAGE was an SDS/heat-resistant PA63 oligomer that
migrated very slowly (Fig. 1A). Because the formation of the
PA63 oligomer requires proteolysis, the PA63 oligomer was hardly
detected in cell lysates from FD11 (Fig. 1A). The cell
lysates were further analyzed by native-PAGE. The FD11 cell lysates
contained mainly the intact PA (Fig. 1B, FD11
lanes), as expected, whereas the lysates from CHO CL6 cells
contained intact PA as well as two higher order PA63 oligomers, but no
monomeric PA63 (Fig. 1B, CL6 lanes). When the
binding assay was performed by using trypsin-nicked PA in which the
furin site was pre-cleaved by limited trypsin digestion (22), as
expected the major PA species detected were the oligomers, and just a
negligible amount of monomer PA was shown (Fig. 1C). The
nature of these two distinct PA63 oligomers is unclear. The faster
migrating species, termed oligomer A in this study (Fig. 1,
B and C), is probably free PA63 heptamer, whereas
the more slowly migrating species, termed oligomer B (Fig. 1,
B and C), may be a complex of the PA63 heptamer
with cellular components such as the PA receptor or detergent-resistant membrane structures. When these oligomeric species (in Fig.
1C, lane 1h) were subjected to second dimension
SDS-PAGE, interestingly, oligomer A dissociated to PA63 monomer,
whereas oligomer B turned out to be the mixture of both SDS-sensitive
and -resistant oligomers (Fig. 1D). Together these results
showed not only that bound PA is rapidly cleaved by furin but also that
the resulting PA63 monomer very rapidly oligomerizes. Thus, the cell
surface-associated PA63 mimics the behavior of PA63 produced in
vitro, which forms the heptamer in neutral aqueous solutions (21).
These heptamers have been visualized previously by electron microscopy
(6), x-ray diffraction (7), and electrophoresis (22, 32). Absence of
PA63 monomer further indicated that oligomerization of PA63 is
effectively irreversible. PA63 detected by SDS-PAGE (Fig.
1A) evidently resulted from the resolution of PA63 oligomer
by boiling in SDS loading buffer.
PA63 Oligomer Is the Only PA Species That Is Internalized--
The
data above indicated that cells exposed to PA contain intact PA and
PA63 oligomers on their surface (Fig. 1B). To explore whether these PA species are equally internalized, we performed a PA
trypsin protection assay. After incubation with PA at 4 or 37 °C,
cells were treated with trypsin to remove the cell surface-bound PA,
allowing identification of those materials internalized by endocytosis.
Remarkably, PA63 oligomer constituted the major protected PA species at
37 °C (Fig. 1, A and B, CL6 lanes),
indicating that the PA63 oligomer was the only form of PA to be
internalized. Also present were small amounts of a PA fragment,
probably the carboxyl-terminal 47-kDa receptor-binding portion
remaining bound to receptor after incomplete cleavage by trypsin (33).
Endocytosis is temperature-dependent (10), and therefore all
surface-bound PA should be removed by trypsin from cells incubated at
4 °C. Thus, in a control for the previous experiment, we showed that trypsin removed all the cell-associated PA (Fig. 1E), with
the exception of the 47-kDa fragment mentioned above. Further evidence that intact, monomeric PA is not internalized into cells was obtained using PA-U7, an uncleavable variant of PA that can bind but cannot be
proteolytically activated by cellular furin (24). This PA mutant was
not internalized to a trypsin-resistant site even when incubated with
cells at 37 °C for 4 h (Fig. 1F). These results demonstrated that the proteolytic cleavage of receptor-bound PA is an
absolute prerequisite not only for the biochemical property of
self-assembly but also for its subsequent biological activity of
undergoing endocytosis.
Oligomeric PA63 Endocytosed to Acidic Compartments Is Rapidly
Transformed to an SDS/Heat-resistant Form--
Previous
studies showed that in solution, purified PA63 can form two types of
oligomers, an SDS-sensitive type, which forms at neutral pH and can be
resolved into PA63 monomer by SDS, and the SDS-resistant type, which
forms at acidic pH and persists in the presence of SDS (34). This
suggested that the SDS/heat-resistant oligomer shown above (Fig.
1A) may be the counterpart of this SDS-resistant oligomer
formed in acidic solution, and therefore may be produced following
endocytosis and delivery to acidic compartments. In fact, when CHO CL6
cells were incubated with PA, the SDS/heat-resistant oligomer was
formed at 37 °C (Fig. 1A, CL6 lanes) but not
at 4 °C, a temperature at which endocytosis does not occur (Fig.
1E, 1h lane). Moreover, native-PAGE analysis
revealed that the PA63 oligomer A formed at both 4 and 37 °C (Fig.
2A). Based on these observations we hypothesized that the PA63 oligomer formed on the cell
surface encounters a progressively more acidic environment along the
endocytic pathway and undergoes conformational changes and membrane
insertion at acidic pH that renders it resistant to SDS/heat. To verify
this hypothesis, we incubated CHO CL6 cells with PA at 4 °C, washed,
and then shifted to 37 °C for various lengths of time in the absence
or presence of bafilomycin A1, a potent and specific inhibitor of the
vacuolar (H+)-ATPase proton pumps that maintain the pH
gradients of acidic compartments (35, 36). We showed that following
3 h of incubation at 4 °C, little, if any, SDS/heat-resistant
oligomer formed (Fig. 2B,
The chymotrypsin-sensitive loop in PA domain 2 is involved in membrane
insertion (7, 22, 37). We used the PA protein altered in this loop to
show that formation of the SDS/heat-resistant oligomer requires
membrane insertion. The mutated PA protein, PA-
In the experiment using bafilomycin, we noted that the small amount of
resistant oligomer that appeared late in the presence of bafilomycin A1
persisted for at least 120 min (Fig. 2B, + Bafilomycin lanes), suggesting both its formation and degradation were
arrested by bafilomycin A1. We therefore assessed the oligomer
degradation more systematically. The acidic pH of early endosomes is
important for sorting endocytosed material, influencing whether
materials are recycled to the plasma membrane, or transported to
lysosomes for degradation. The SDS/heat-resistant oligomer is likely
transported to lysosomes for degradation. To examine oligomer
stability, cells were incubated with PA for 1 h at 37 °C and
then washed to remove PA. At this point, all the compartments in the
endocytic pathway, including cell surface, endosomes, and lysosomes,
should be loaded with the corresponding PA species. During a subsequent
incubation at 37 °C in the absence of bafilomycin A1, it was found
that the SDS/heat-resistant PA63 oligomer remained at the same level
for 30 min and then decreased with time, completely disappearing after 180 min (Fig. 4, TEM8-mediated PA Binding, Proteolytic Processing, and Endocytosis
Are Independent of Its Cytoplasmic Sequence--
An important advance
in understanding anthrax toxin action was the recent demonstration that
TEM8 variant 2 (368 residues, designated ATR) acts as a PA receptor
(3). However, no information is available regarding the ability of
other TEM8 gene products to serve as PA receptors. TEM8-related
proteins produced through alternative splicing also include TEM8
variant 1 (564 residues) and variant 3 (333 residues) (Fig.
5A). TEM8 variants 1 and 2 contain a putative 23-residue transmembrane region (residues 321-343), whereas TEM8 variant 3 lacks this sequence and instead has a unique 15-residue carboxyl-terminal sequence whose properties suggest that the
protein will be released as a soluble form that would not function as a
receptor. TEM8 variant 2 (ATR) has a very short putative cytoplasmic
tail (25 residues) with a 360EESEE364 acidic
cluster that was suggested to cause co-localization of ATR with the
furin protease whose action is required for PA activation (3). The TEM8
variant 1 cytoplasmic region (221 residues) includes a more extended
acidic cluster 360EESEEEDDD368, a proline-rich
region (34 prolines within the last 57 residues), and 14 potential
phosphorylation sites.
To explore the roles of the various TEM8 variants as PA receptors and
experimentally evaluate the roles of the motifs in the cytoplasmic
region of TEM8 in PA binding, proteolytic processing, and
internalization, we constructed eight variants of human TEM8 (Fig.
5A). The corresponding cDNA fragments were cloned into
the bicistronic mammalian expression vector pIRESHyg2b. The resulting expression plasmids were transfected into PR230, a spontaneous CHO cell
PA receptor-deficient mutant. In contrast to its parental CHO cell line
WTP4, PR230 is specifically defective in PA binding and thus is
resistant to PA plus FP59 but sensitive to diphtheria toxin and
Pseudomonas exotoxin A (data not shown). Stably transfected cells were established by hygromycin B selection for 2 weeks, and the
hygromycin-resistant colonies were either isolated individually or
pooled. We found that >80% of the hygromycin-resistant clones expressed the transfected genes (data not shown). Therefore, in addition to analyzing representative isolated clones from selected TEM8
variant constructs (Fig. 5, B and C), we fully
analyzed pools of transfectants to obtain results characteristic of
average expression levels (Fig. 5, D and E).
Remarkably, all the TEM8 variants or truncated mutants containing the
putative transmembrane domain and extracellular region, including TEM8
variant 1, TEM8-115 aa, TEM8-26 aa, TEM8-16 aa, and TEM8-0 aa, were
functional PA receptors (Fig. 5). These transfected PR230 cells
regained the ability to bind and proteolytically process PA, leading to
the internalization and conversion of the PA63 oligomer into the
SDS/heat-resistant form to an extent matching that of the parental WTP4
cells (Fig. 5, B and D). Moreover, these
transfected cells became sensitive to killing by PA plus FP59 (Fig. 5,
C and E). In contrast, cells transfected with the
TEM8-ed construct that is truncated before residues 321-343, the
putative transmembrane domain, failed to bind PA and were resistant to
PA plus FP59 (Fig. 5, D and E). Similarly, cells
transfected with TEM8 variant 3 or mouse vacuolar protein sorting
protein 11 (Vps11) (39), an irrelevant gene, also were unable to bind
PA and remained resistant to PA plus FP59 (Fig. 5, D and
E). These data support the prediction that residues 321-343
constitute a transmembrane anchor and imply that TEM8 variant 3 is a
naturally occurring soluble form of TEM8. Taken together, the results
demonstrate that the TEM8 extracellular region and transmembrane domain
together constitute the minimum PA receptor structure. As an
alternative to being anchored by transmembrane domains, some cell
surface proteins are retained at the surface by post-translational
linkage to GPI. To explore further what properties are required in a
TEM8 protein for it to act as a PA receptor, we constructed an
expression vector that expresses a fusion of the TEM8 extracellular
domain (residues 1-317) with the GPI-anchoring sequence of uPAR
(residues 294-335). Interestingly, this GPI-anchored TEM8, designated
TEM8-GPI, could also function as a PA receptor, supporting the binding
and processing of PA into PA63, formation of the SDS/heat-resistant
oligomer, and making cells sensitive to PA plus FP59 (Fig. 5). The
GPI-anchored nature of TEM8-GPI was confirmed by showing that PI-PLC
treatment to release GPI-anchored proteins (40) significantly decreased PA binding to TEM8-GPI-transfected cells without affecting binding to
parental WTP4 cells or cells transfected with TEM8-0 aa (Fig. 6). These results clearly demonstrate
that anthrax toxin receptor-mediated internalization can be mediated by
TEM8 regardless of how it is anchored to the cell surface.
Like CHO CL6 cells (Figs. 1-4), WTP4 cells can only internalize the
oligomeric form of PA63 (Fig. 7), as
demonstrated by trypsin treatment method. We found that constructs with
no cytosolic tail (TEM8-0 aa, Fig. 7, middle
panel) or with a GPI anchor (TEM8-GPI, Fig. 7,
right panel) had this same ability to internalize the oligomeric PA but not the monomer PA (Fig. 7).
Northern blot analysis revealed that CHO WTP4 cells express one major
TEM8 transcript, one having a size slightly larger than 4.4 kb (Fig.
8). This is likely to be TEM8 variant 1, which has a full-length cDNA of 5.2 kb in mouse and 5.4 kb in
human. This transcript was also expressed by PR230 cells but at a
significantly decreased level. This suggests that one allele in PR230
does not produce a stable TEM8 transcript. This first defective allele could either pre-exist in the CHO WTP4 parent or result from the mutation in PR230, whereas the second defective allele in PR230 must
consist of a more subtle change that still allows production of the
transcript observed in Fig. 8.
The CHO PR230 mutant cell line is one of a number of PA
receptor-deficient CHO mutants obtained in this laboratory by either spontaneous mutation, chemical mutagenesis, or retrovirus insertional mutagenesis.2 All the PA
receptor-deficient CHO mutants analyzed so far by cell fusion belong to
a single genetic complementation group, and their phenotype is restored
to that of parental cells by transfection with TEM8-26 aa (data not
shown). These data strongly suggest that the naturally occurring TEM8
variants, all produced from a single gene, are the only cell surface
molecules that act as functional PA receptors.
PA is a central part of anthrax toxin, serving as a cellular
binding moiety and a delivery vehicle for translocation of LF and EF
into the cytosol of cells. Therefore, the interaction between PA and
the target cell has been a central issue in studies of anthrax toxin.
Here, we took advantage of a highly sensitive and specific anti-PA
antiserum, instead of using radiolabeled or biotin-labeled PA that may
behave differently due to modification, to assess directly the binding,
processing, and internalization of PA by host cells. These analyses
revealed that PA not only rapidly binds but also is rapidly cleaved by
furin protease on the CHO cell surface, with both processes nearing
completion in 5 min. Remarkably, the resulting PA63 was very rapidly
converted to the oligomer, so that only monomeric PA (PA83) but not
monomeric PA63 could be detected. Surprisingly, we did not find any
monomeric form of PA inside the cells, and the PA63 oligomer was the
only form of PA internalized. These results are consistent with the
previous report (41) that the proteolytic activation of PA on
macrophages promotes its internalization. Taken together, these results
indicate that oligomerization of PA63 on the cell surface triggers
receptor-mediated endocytosis, explaining that only PA63 oligomer but
not the monomeric form of PA was internalized. Therefore, proteolytic
cleavage of receptor bound PA is a fundamental prerequisite not only
for PA self-assembly and LF and EF binding but also for the subsequent step of endocytic internalization.
We further assessed the role that the acidic pH of intracellular
compartments plays in the formation and degradation of the PA
oligomers. PA could be processed to form an SDS/heat-sensitive PA63
oligomer on the cell surface at 4 °C, a temperature at which endocytosis does not occur. However, when endocytosis was allowed by
shifting the temperature from 4 to 37 °C, the PA63 oligomer began to
transform to the SDS/heat-resistant form, in a process that was
efficiently blocked by bafilomycin A1, a specific inhibitor of the
vacuolar ATPases that generates the pH gradient of acidic compartments.
These results clearly demonstrated that transformation of PA63 oligomer
to the SDS/heat-resistant form was induced by acidic pH in endocytic
compartments. Furthermore, this conformational change was functionally
correlated with membrane insertion, channel formation, and
translocation of LF into cytosol, because a mutated PA protein
defective in membrane insertion, PA- Because the PA63 band formed by dissociation of the SDS/heat-sensitive
oligomer was barely detectable inside cells (Fig. 1A, trypsin + lanes), we concluded that the majority of the
internalized PA63 oligomer rapidly transformed to the
SDS/heat-resistant form in the acidic environment in endosomes
following the endocytosis. Therefore, the PA63 SDS/heat-sensitive
oligomer, the source of the PA63 band in the SDS gels, mainly exists on
the cell surface. Thus, the progressive decrease of PA63 on the SDS gel
(Fig. 4, Taken together, the data show that the anthrax toxin system has evolved
to deliver efficiently LF and EF to the cytosol. PA is retained on cell
surface receptors until it is cleaved and converted to the oligomer,
the first species able to bind LF and EF (9). Endocytosis, initiated
after oligomerization, is relatively slow compared with furin cleavage,
so that the PA oligomer remains on the surface for a sufficient time to
allow LF (or FP59) and EF to bind. This mechanism makes it probable
that the internalized PA oligomers are loaded with at least one
molecule of the enzymatic moieties before entry. This may explain why
CHO cells are very sensitive to PA, having an EC50 only
1-2 ng/ml of PA in the presence of FP59.
The other objectives of this work were to determine whether TEM8
variants 1 and 3 can also function as PA receptors, to explore the
roles of the TEM8 cytosolic tail in PA binding, proteolytic processing,
oligomerization, and internalization, and to experimentally define the
transmembrane region. The three reported TEM8 variants share the same
amino-terminal extracellular part but differ in length and sequence in
their putative cytosolic regions. TEM8 variant 1 is the longest and
possesses a more complicated cytoplasmic region. Surprisingly, all the
TEM8 mutants we constructed that retained a membrane anchor functioned
as PA receptors, as determined by PA binding, processing, oligomer
formation, and translocation of FP59 into cells. The shortest mutant of
TEM8 that acted as a PA receptor is TEM8-0 aa, which was truncated at
the carboxyl terminus of the putative transmembrane domain. When the
predicted transmembrane domain (residues 321-343) was further deleted,
the resulting mutated TEM8-ed transfected cells failed to bind PA, demonstrating that residues 321-343 are responsible for TEM8 cell surface anchoring. Cells transfected with either the naturally occurring TEM8 variant 3 or the TEM8-ed construct could not bind PA,
suggesting that the resulting proteins are soluble forms of TEM8.
Secretion of a soluble receptor by some cells may have an impact on the
normal function of TEM8 and any endogenous ligands that normally bind
to it.
The evidence that a simple transmembrane anchor was sufficient for TEM8
to function as a PA receptor suggested that other forms of membrane
anchor might also be sufficient. Surprisingly, when we linked the TEM8
extracellular domain to the GPI-anchoring sequence of uPAR, the
chimeric protein still functioned as a PA receptor, clearly
demonstrating that the ability of TEM8 to act as a receptor is
independent of the type of membrane anchor. The physiological function
of TEM8 is still unknown. A portion of the extracellular part of TEM8
shares high sequence homology with the von Willebrand factor type A
domain. Because von Willebrand factor type A domains are often found in
extracellular domains of integrins where they constitute ligand-binding
sites, TEM8 may be involved in the interaction of cells with the
surrounding extracellular matrix. Although the complex TEM8 cytosolic
domain is not required for PA receptor function, it may play a crucial role in its unidentified normal physiological function. Therefore, it
will be important to identify any natural TEM8 ligands and to determine
the consequences of ligand binding, in part because of the role TEM8
plays in anthrax toxin action. In this respect, a mutated PA defective
in TEM8 binding may be considered in the development of new anthrax
vaccines so as to avoid any potential effects of PA that follow from
its binding to TEM8.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FF (PA with 313FF314
deleted) (22), diphtheria toxin (23), PA-U7 (a non-cleavable variant of
PA with the furin site RKKR replaced by PAA) (24), and FP59, a
recombinant fusion toxin consisting of anthrax toxin LF amino acids
1-254 (LFn) fused to the ADP-ribosylation domain of
Pseudomonas exotoxin A (25). Rabbit anti-PA polyclonal
antiserum (number 5308) and LF polyclonal antiserum (number
5309) were made in our laboratory by immunization with recombinant PA
and LF. Polyclonal antibody against the amino-terminal sequence of
MAPKK1 (MEK1-NT) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat anti-rabbit IgG-HRP (sc2054) and goat anti-mouse IgG-HRP (sc2005) were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Bafilomycin A1, saponin, and
phosphatidylinositol-specific phospholipase C (PI-PLC) were
purchased from Sigma.
-minimal essential medium supplemented with
5% fetal calf serum, 2 mM glutamine, 50 µg/ml
gentamycin, and 25 mM HEPES.
FF for
1 h at 37 °C, washed once with HBSS, and treated with 0.5 ml
0.5 mg/ml trypsin in HBSS per well at 37 °C for 5 min to remove
proteolytically the cell surface-bound toxin. The cells were then
washed and permeabilized by saponin to allow efflux of cytosol as
described (30). Briefly, the cells were resuspended and incubated in
100 µl of phosphate-buffered saline containing 50 µg/ml saponin, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin for 30 min at
4 °C. The soluble fraction was separated from the particulate fraction by centrifugation at 15,000 × g for 5 min at
4 °C. The pellet was washed in HBSS and solubilized in RIPA lysis
buffer. The samples from the soluble and pellet fractions were analyzed by native-PAGE followed by Western blotting using LF antiserum (number
5309). In measurements of MAPKK1 cleavage by LF, CHO CL6 cells grown in
24-well plate were incubated with 1 µg/ml LF along with 1 µg/ml PA
or PA-
FF for 1 h at 37 °C, washed, lysed, and analyzed by
SDS-PAGE followed by Western blotting using an antibody against the
amino-terminal sequence of MAPKK1 (MEK1-NT).
-minimal essential medium. The cells were incubated with MTT for 45 min at 37 °C; the medium was removed, and the blue pigment produced
by viable cells was solubilized with 100 µl/well of 0.5% (w/v) SDS,
25 mM HCl, in 90% (v/v) isopropyl alcohol. The
plates were vortexed, and the oxidized MTT was measured as A570 using a microplate reader.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
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Fig. 1.
Binding, proteolytic processing, and
internalization of PA by CHO cells. A and B,
PA63 oligomer is the only PA species internalized. CHO CL6 and FD11
cells were incubated with PA (1 µg/ml) for different lengths of time
at 37 °C, washed to remove unbound PA, and lysed. Cell lysates
prepared directly or after the cells were incubated with trypsin to
remove cell surface-bound PA species were separated by SDS-PAGE
(A) or native-PAGE (B) followed by Western
blotting using PA antiserum (number 5308). C and
D, two-dimensional electrophoretic analysis of PA oligomers.
CHO CL6 cells were incubated with the trypsin-nicked PA
(nPA, 1 µg/ml) for 0 or 1 h, and cell lysates were
separated by native-PAGE (C). A gel slice like the 1-h
sample containing oligomers A and B was subjected to a second dimension
SDS-PAGE analysis (D). E, cell surface-associated
PA can be efficiently removed by trypsin. CHO CL6 cells were incubated
with PA at 4 °C and treated as in A. F,
uncleavable PA cannot be internalized. CHO CL6 cells were incubated
with PA-U7 at 37 °C and treated as in A.
Bafilomycin, 0 lane), although a significant amount of oligomer was present when
lysates were analyzed by native gels (data not shown). However, the
resistant oligomer formed efficiently in a time-dependent
manner when the cells were shifted to 37 °C following binding at
4 °C (Fig. 2B,
Bafilomycin lanes).
Moreover, the amount of resistant oligomer quickly declined after
peaking at 30-40 min of incubation at 37 °C, and less than half
remained after 60 min (Fig. 2B,
Bafilomycin
lanes). In contrast, formation of the SDS/heat-resistant PA63
oligomer was potently inhibited when bafilomycin A1 was included at the
time when the cells were shifted to 37 °C (Fig. 2B,
+ Bafilomycin lanes), indicating that the SDS/heat-resistant
PA63 oligomer is produced within acidic compartments. Because the
resistant oligomer began to appear as early as 5 min after shifting to
37 °C, it likely formed in early endosomes, the first compartments
along the endocytic pathway that have a significantly acidic pH.
Because only trace amounts of monomeric PA63 were detected in the
trypsinized samples shown above (Fig. 1A, trypsin + lanes), we conclude that the majority of internalized PA63
oligomer is rapidly transformed to the SDS/heat-resistant form in
endosomes following internalization.
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Fig. 2.
SDS/heat-resistant PA63 oligomer forms in
acidic endocytic compartments. A, PA63 oligomer forms
at both 37 and 4 °C. CHO CL6 cells were incubated with 1 µg/ml PA
at 4 or 37 °C for 1 h, washed, and lysed. The cell lysates were
subjected to native-PAGE, followed by Western blotting using PA
antiserum (number 5308). B, SDS/heat-resistant
PA63 oligomer formation requires internalization and endosome
acidification. CHO CL6 cells were incubated with PA (1 µg/ml) at
4 °C for 3 h, washed, and changed to fresh medium. The cells
were shifted to 37 °C in the absence or presence of bafilomycin A1
(0.2 µM) for various lengths of time and lysed. The cell
lysates were analyzed as in Fig. 1A.
FF, with
313FF314 at the tip of the
chymotrypsin-sensitive loop deleted (22), was bound, processed,
and internalized normally (Fig.
3A), but its transformation
into the SDS/heat-resistant form in acidic compartments was greatly
decreased (Fig. 3B). This in turn made the mutated PA unable
to translocate LF into the cytosol of CHO CL6 cells, observed both
biochemically (Fig. 3C) and through the failure to cause
MAPKK1 cleavage (Fig. 3D). These data show that membrane
insertion and channel formation by the chymotrypsin-sensitive loop is
the basis of the conformational changes that render the oligomer
resistant to boiling in SDS sample buffer.
View larger version (49K):
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Fig. 3.
PA- FF has decreased
ability to form SDS/heat-resistant oligomer and to translocate LF into
the cell cytosol. A and B, PA-
FF is
impaired in SDS/heat-resistant oligomer formation. CHO CL6 cells were
incubated with 1 µg/ml PA or PA-
FF for 1 h at 37 °C and
treated as in Fig. 1, and cell lysates were separated by SDS-PAGE
(A) or native-PAGE (B) for Western blotting using
PA antiserum (number 5308). C and D, PA-
FF
fails to translocate LF into the cytosol (C) or to produce
MAPKK1 cleavage (D). CHO CL6 cells were incubated with 1 µg/ml PA or PA-
FF plus 1 µg/ml LF for 1 h at 37 °C; the
cells were then trypsinized, permeabilized by saponin, and separated
into cell pellet and supernatant cytosol fractions as described under
"Experimental Procedures." The cell pellet lysates (p)
and the cytosol (c) were separated by native-PAGE followed
by Western blotting using LF antiserum (number 5309) (C).
CHO CL6 cells were incubated with 1 µg/ml PA or PA-
FF plus 1 µg/ml LF for 1 h at 37 °C; the cell lysates were then
separated by SDS-PAGE followed by Western blotting using a antibody
that detects the amino-terminal sequence of intact but not cleaved
MAPKK1 (MEK1-NT) (D).
Bafilomycin
lanes). In contrast, oligomer degradation was completely inhibited
in the presence of bafilomycin A1 (Fig. 4, + Bafilomycin
lanes). Bafilomycin A1 probably arrested the degradation of the
SDS/heat-resistant PA63 oligomer by inhibiting its transport to
lysosomes and also by decreasing the activity of lysosomal proteases
that prefer an acidic environment (38). The stability of the
SDS/heat-resistant PA63 oligomer in cells treated with bafilomycin A1
also suggests that the acid-induced conformational change is
effectively irreversible, because elevating the pH of endocytic
compartments does not produce monomeric PA63.
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Fig. 4.
Bafilomycin A1 inhibits PA63 oligomer
degradation. CHO CL6 cells were incubated with 1 µg/ml PA for
1 h at 37 °C, washed, and changed to fresh medium without
( ) or with (+) bafilomycin A1 (0.2 µM) for different lengths of time. The cells were then
washed and lysates analyzed as in Fig. 1A.
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Fig. 5.
TEM8-mediated PA binding, proteolytic
processing, and endocytosis are independent of its cytoplasmic sequence
and the type of cell surface anchoring. A,
schematic representation and the sequences alignment of various TEM8s
constructed and transfected into CHO PR230 cells. TM,
putative transmembrane region shown in green; AC,
acidic cluster shown in red; PC, proline cluster
shown in blue. Residues 1-300 are not shown in the sequence
alignment; HA tag is not capitalized when present; GPI sequence is
underlined; the additional five residues DRTLT at the
carboxyl terminus of TEM8-115 aa was produced due to DNA manipulation.
Interactions of PA with the various TEM8-transfected PR230 cells either
isolated (B) or pooled (D). The stably
transfected cells were incubated with 1 µg/ml of PA for 1 h at
37 °C; the cells were then analyzed by SDS-PAGE and Western blotting
as in Fig. 1A. Cytotoxicity of PA plus FP59 to the various
TEM8 transfected PR230 cells either isolated (C) or pooled
(E). Cells were incubated with various concentrations of
PA plus 100 ng/ml of FP59 for 48 h, and the cell viability was
assessed as described under "Experimental Procedures."
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Fig. 6.
PI-PLC treatment reduces PA binding to
TEM8-GPI-transfected PR230 cells. Cells were treated with PI-PLC
for 1 h at 37 °C, washed, and immediately incubated with 1 µg/ml PA-U7 for 1 h at 37 °C. Cell lysates were analyzed by
SDS-PAGE and Western blotting using PA antiserum as in Fig.
1A.
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Fig. 7.
Different membrane-anchored forms of TEM8
internalize only the PA63 oligomer and not intact PA. Transfected
CHO cells were incubated with 1 µg/ml PA at 37 °C. Cell lysates
were analyzed by Western blotting as in Fig. 1A.
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Fig. 8.
Northern blot analysis of TEM8 expression by
CHO cells. 30 µg of total RNA from each cell line was used for
analysis. RNA integrity, equality of loading, and evenness of transfer
to a nylon membrane were assessed by hybridization with mouse Vps11
cDNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FF, underwent significantly
decreased SDS/heat-resistant oligomer formation and failed to
translocate LF into cytosol as assessed by MAPKK1 cleavage. It was also
noted that the SDS/heat-resistant PA63 oligomer formation was
irreversible, because bafilomycin A1 could not affect the resistant
oligomer already formed inside the cells. Another finding in this work
is that bafilomycin A1 could completely inhibit the PA63 oligomer
degradation which otherwise is complete within 3 h, suggesting
that the degradation takes place in a low pH compartment, probably the lysosome.
Bafilomycin lanes) reflects the endocytic
process. The clearance of PA63 (Fig. 4,
Bafilomycin lanes)
required about 2 h, indicating that the endocytic process is a
slow and rate-limiting step in toxin internalization.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dana Hsu for assistance with toxin purification and to Drs. F. Gisou Van der Goot, Laurence Abrami, M. J. Rosovitz, Rebecca Schubert, and Ghulam J. Chaudry for critically reading the manuscript and for helpful comments.
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FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Microbial Pathogenesis
Section, NIAID, National Institutes of Health, Bldg. 30, Rm. 303, MSC
4350, Bethesda, MD 20892-4350. Tel.: 301-594-2865; Fax: 301-480-0326;
E-mail: Leppla@nih.gov.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210321200
2 S. Liu and S. H. Leppla, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: PA, protective antigen; TEM8, tumor endothelium marker 8; CHO, Chinese hamster ovary; LF, lethal factor; HA, hemagglutinin; aa, amino acid; MAPKK, several mitogen-activated protein kinase kinases; PI-PLC, phosphatidylinositol-specific phospholipase C; HBSS, Hanks' balanced salt solution; GPI, glycosylphosphatidylinositol; uPAR, urokinase plasminogen activator receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
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