From the
The diphtheria toxin receptor (DTR) has been identified as the
precursor of heparin-binding epidermal growth factor-like growth
factor, which may interact with other membrane proteins to form the
functional receptor. To test if mammalian DTR is able to confer toxin
sensitivity onto phylogenetically distant cells, we expressed monkey
DTR in the baculovirus system and tested infected insect cells for
toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding
epidermal growth factor-like growth factor precursor (DTR
Diphtheria toxin (DT)
While cells
of human and monkey origin are in general sensitive to DT to varying
extent, cells from rats and mice are toxin resistant(14) . The
variation in sensitivity of different cells appears to be largely due
to differences in the number of
DTRs(15, 16, 17) . Other factors have been
implicated, such as different affinity of the receptors for the toxin
and the presence of additional proteins associated with the receptor,
that could play a role in the translocation
process(18, 19) .
Transfection of DTR has been shown
to confer toxin sensitivity onto mouse cells that are normally
resistant to the toxin. It is not clear if DTR alone is required for
toxin translocation or if other proteins, such as CD9, are also
required. We therefore decided to test if monkey DTR can confer
sensitivity onto a cell of non-vertebrate origin, as the possibility of
correct interaction with any required additional membrane proteins was
considered to be much lower in these cells than in mammalian cells. We
chose to express DTR in the baculovirus system. This system utilizes
insect cells that can be infected with the naturally occurring
baculovirus AcNPV. During lytic infection, the gene product for the
baculovirus polyhedrin is expressed in high amounts and can account for
about 50% of the total cellular protein(20) . The polyhedrin
gene is not essential for production of infectious virions. Therefore,
expression vectors that use the strong AcNPV polyhedrin promoter to
drive expression of foreign genes have been developed(21) . By
using this approach, large amounts of eukaryotic proteins have been
successfully produced, including a variety of receptors. In addition,
insect cells can perform many of the post-translational modifications
carried out by higher eukaryotes (for review see Ref. 22). These
features make the baculovirus system an attractive choice for large
scale production of normal and mutant DTRs, as recently reported by Ono et al.(23) . The biological activity as DTR in the
infected cells was not tested by these authors.
In the present work,
we have studied the expression of DTR by SF9 and HF cells infected with
recombinant baculovirus. We have made a construct with an epitope (B3,
against which we have an antibody) added to the COOH-terminal
(cytoplasmic) end of the receptor to allow immunoprecipitation from
lysed cells. Expression of the DTR
To construct the recombinant baculovirus
expressing DTR (vAc-DTR
In the PCR, a band of about 800 bp is
expected for the wild type viral DNA, whereas for the recombinant virus
the expected band is about 1,300 bp. If contamination with the wild
type viral DNA occurs or if the recombinant virus originated from a
single crossover, two bands of about 800 and 1,300 bp appear upon PCR
amplification. All the recombinants tested by PCR gave a single band of
the expected size (1,300 bp), demonstrating that vAc-DTR
For experiments, SF9
cells were infected with the wild type or the recombinant
vAc-DTR
Vero cells in 12-well
plates were incubated for 30 min at room temperature with
Vero cells were incubated in binding medium with
increasing amounts of nDT for 30 min at the indicated temperature in
the absence or presence of 10 µM monensin as indicated.
Medium and unbound toxin was washed away, and low pH buffer was added
for 5 min at 37 °C; then, 3 ml of complete medium (with or without
10 µM monensin and anti-DT serum) was added, and the cells
were incubated or for 2 h at 37 °C. The incorporation of
[
SDS-PAGE-SDS-PAGE was
carried out in 12% gels. The gels were fixed in 4% (v/v) acetic acid,
27% (v/v) methanol for 30 min and then treated with 1 M sodium
salicylate, pH 5.8, in 2% (v/v) glycerol for 30 min. To visualize
proteins labeled with
To test
directly the expression of DTR, we monitored the ability of the
transfected cells to bind
To
visualize the expressed DTR by immunofluorescence, we used HF insect
cells because these cells were attached more strongly to glass
coverslips than SF9 cells. We allowed DT to bind to the cells, which
were then incubated with antibodies against DT followed by an
FITC-labeled second antibody (Fig. 1). No fluorescence was
observed in uninfected cells or in cells infected with wild type virus.
In cells infected with recombinant virus, it was possible to detect
receptor expression at 24 hpi. The fraction of cells showing
fluorescence and the intensity of the fluorescence increased with time
from 24 to 48 hpi, whereas at 72 hpi it was not possible to stain the
cells because of lysis.
To measure DT binding to infected
cells, we tested SF9 cells 48 h after the infection with
vAc-DTR
Control
experiments with uninfected cells showed that SF9 cells are resistant
to diphtheria toxin, indicating the absence of functional receptors.
Cells infected with the wild type virus were also resistant (data not
shown).
We tested the toxin sensitivity of
vAc-DTR
In the presence of heparin, more
To test to
what extent the translocation process in the
vAc-DTR
To test if DTR is able to promote toxin translocation in
non-vertebrate cells, the cDNA encoding the monkey HB-EGF precursor was
expressed in insect cells using the baculovirus system. Binding and
immunofluorescence experiments indicate that the insect cells were able
to synthesize and process the DTR so that it was inserted in the
correct transmembrane orientation and recognized by diphtheria toxin.
Also, the expression of the receptor conferred very high sensitivity to
diphtheria toxin of otherwise resistant SF9 cells.
The time course
experiments showed that 48 hpi is the best time to carry out functional
studies of the expressed DTR, as at this time a high number of
receptors is obtained and virus-induced cell lysis has not yet started.
This time course is typical for proteins expressed under the control of
the polyhedrin promoter(29) , and the time period between
48-72 hpi has been used for the study of other receptors
expressed in the baculovirus system, such as the platelet-derived
growth factor receptor(30) , the
Specific binding
of
Naglich et al. (18) and Brown et
al. (19) have shown that LM mouse cells transfected with
Vero DTR expressed a higher number of DTR than Vero cells, but the
transfected cells were not more sensitive to DT than Vero cells. The
lack of increased sensitivity was attributed to the lower affinity of
the DTR in the transfected LM cells. Also, it could be due to other
rate-limiting steps in the intoxication process such as the presence of
DRAP27(36) , a 27-kDa membrane protein corresponding to
CD9(37) . In fact, Vero cells express 7.5
The effect of heparin on
binding and toxicity of DT to vAc-DTR
Upon immunoprecipitation
with antibodies against the tag at the carboxyl-terminal end of DTR and
by precipitation with DT immobilized on Sepharose, a protein of about
25 kDa was obtained. The apparent molecular weight, which exceeds the
molecular weight calculated from the polypeptide size, indicates that
the expressed receptor is glycosylated by the insect cells. The
receptor was recognized by the anti-B3 serum, but DT-Sepharose was much
more efficient in precipitating the receptor. Recently, Iwamoto et
al.(35) found a higher amount of DTR precipitated with
antibodies against DT bound to the receptor than with antibodies
against the receptor as such.
Two additional bands were also
detected in lysate precipitated with DT-Sepharose. Additional bands
have been observed both after precipitation with anti-HB-HGF antibodies
and with anti-DT(35) . These different bands could represent
different processing in the amino terminus of the receptor, as
described by Higashiyama et al. (26) in mature HB-EGF, and in
insect cells infected with a recombinant virus encoding human
recombinant transmembrane HB-EGF, as described by Ono et
al.(23) .
The high efficiency expression of functional
DTR with the baculovirus expression system reported here represents a
convenient model to study the role of the DTR in toxin translocation to
the cytosol. The possibility of simultaneously infecting with two
recombinant viruses allows concomitant expression of other proteins
that could be involved in the translocation process.
We are grateful to Unni Spaeren for help with the
baculovirus expression system, to Ramon Ruiz for excellent technical
assistance, and to Drs. Markus Lanzrein and Harald Stenmark for
critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
was inserted into the virus genome by allelic replacement to construct
the recombinant virus vAc-DTR
. SF9 cells infected with
vAc-DTR
expressed functional DTR, which could be
precipitated from the solubilized membrane fraction of infected cells
with Sepharose-immobilized diphtheria toxin. The highest level of
expression (about 5
10
receptors/cell) was observed
48 h after infection, at which time the infected cells were highly
sensitive to diphtheria toxin. Uninfected SF9 cells and cells infected
with the wild type virus were resistant to the toxin. The presence of
heparin increased both the binding and the toxin sensitivity of
vAc-DTR
-infected SF9 cells. Translocation of toxin A
fragment was induced when cells with surface-bound toxin were exposed
to low pH, and the translocation was optimal at pH
5.5. It was
100 times more efficient at 24 °C than at 4 °C. The data
indicate that monkey DTR is fully functional when expressed in insect
cells.
(
)is a cytotoxic
protein (M
58,348) secreted by Corynebacterium
diphtheriae(1) as a single polypeptide, which is easily
nicked by trypsin-like proteases into the disulfide-linked fragments A (M
21,147) and B (M
37,195)(2) . The toxin binds to cell surface receptors by
the B fragment(3, 4) . The DTR has recently been
identified as the precursor of HB-EGF-like growth factor(5) .
The receptor-bound toxin undergoes endocytosis via clathrin-coated pits
(6). The low pH environment in the endosomes induces a conformational
change in the B fragment, and the enzymatically active A fragment is
translocated to the
cytosol(7, 8, 9, 10, 11) .
There, the A fragment catalyzes ADP-ribosylation of elongation factor
2, resulting in inhibition of protein synthesis and cell
death(2, 12) . Translocation of diphtheria toxin can
also be induced directly from the cell surface by exposing cells with
bound DT to low pH medium, thereby mimicking the conditions inside the
endosome(8, 9, 11, 13) .
in COS cells
(
)showed that the presence of this epitope does not
impair the ability of DTR to ensure binding and translocation of the
toxin.
Materials
[S]Methionine,
1-[3,4,5,-
H]leucine, and
I were
purchased from DuPont NEN. Pronase E (from Streptomyces
griseus), trypsin (TPCK-treated), PMSF, N-ethylmaleimide,
heparin (sodium salt grade 1A from porcine intestinal mucosa),
monensin, Nonidet P-40, sodium deoxycholate, dithiothreitol, Triton
X-100, leupeptin, aprotinin, DNase, antipain, and X-gal were from
Sigma. Chymostatin and pepstatin A were from Fluka (Buchs,
Switzerland). Diphtheria toxin (crude) was purchased from Connaught
Laboratories (Willowdale, Canada) and purified as described(9) .
Protein-A Sepharose and CNBr-Sepharose were from Pharmacia LKB
(Uppsala, Sweden).
Antibodies
Rabbit immune serum against
the B3 peptide (GVNEYNEMPMPVN) was prepared by Cambridge Research
Biochemicals (Cambridge, United Kingdom).
Buffers
The following buffers were used:
PBS (140 mM NaCl, 10 mM
NaHPO
, pH 7.2); lysis buffer (50 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM MgCl
, 0.02% (w/v) sodium azide, 0.1% (w/v) SDS, 1%
(v/v) Triton X-100, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium
deoxycholate, 1 mM dithiothreitol, 20% (v/v) glycerol, 1
mM PMSF, 2 µg/ml leupeptin, 2 µg/ml chymostatin, 2
µg/ml aprotinin, 1 mg/ml DNase, 1 µg/ml pepstatin A, 2
µg/ml antipain, and 10 IU/ml heparin); Hepes medium (bicarbonate-
and serum-free Eagle's minimum essential medium buffered with 20
mM Hepes at pH 7.4); low pH buffer for SF9 cells (TNM-FH
medium plus 10 mM sodium gluconate adjusted to the indicated
pH); low pH buffer for Vero cells (Hepes medium plus 10 mM sodium gluconate adjusted to the indicated pH).
Cells
The insect cell line SF9 was
obtained from Unni Spaeren (Tromsø, Norway), HF insect cells
were purchased from InVitrogen Corp. (Oxon, UK). The cells were
propagated in suspension at 27 °C in TNM-FH Medium (Sigma)
supplemented with 10% fetal calf serum, 10,000 IU/liter penicillin, and
10 mg/liter streptomycin (Life Technologies, Inc., Uxbridge, UK).
Viral and Plasmid DNA
Wild type
linearized AcNPV DNA and plasmid pBlueBacIII were purchased from
InVitrogen. In the transfer vector, the cloning site downstream of the
polyhedrin promoter and the LacZ gene are flanked by two
recombinant sequences identical to sequences in the polyhedrin region
of the AcNPV DNA.
), the 690-bp segment of the DNA
corresponding to the monkey HB-EGF precursor with the COOH-terminal B3
epitope was cloned into the BamHI and PstI sites of
the pBlueBacIII. 3 µg of plasmid DNA was cotransfected with 1
µg of linearized wild type AcNPV total viral DNA into 2.5
10
SF9 cells using cationic liposome-mediated transfection
as indicated in the instructions from InVitrogen. A double crossover
recombinant was insolated by the plaque purification method as
described by O'Reilly et al.(22) by screening in
the presence of X-gal for blue plaques with the occlusion body negative
(polyhedrin negative) phenotype. The chosen plaques were tested by
binding of
I-labeled DT to infected cells, and those that
showed high binding were tested by PCR for the total absence of any
wild type AcNPV virus DNA.
but not wild type viral DNA (800-bp band) was obtained. After
confirmation, one recombinant was expanded to 3.10
plaque-forming units/ml by infecting SF9 cells at a multiplicity
of infection of 0.1 plaque-forming units/cell.
at a multiplicity of infection of about 20
plaque-forming units/cell or were mock infected with TNM-FH medium
(SF9) for 1 h. Then, complete medium was added, and the infection was
followed for the indicated periods of time (hpi).
X-Gal Staining of Infected Cells
At 24,
48, and 72 hpi, the cells were washed with PBS and fixed with 1% (v/v)
formaldehyde, 0.2% (v/v) glutaraldehyde in PBS for 5 min. Subsequently,
the cells were washed three times with PBS and incubated overnight in 4
mM potassium ferrocyanide, 4 mM potassium
ferricyanide, 2 mM MgCl, and 0.4 mg/ml X-gal. The
reaction was stopped by washing twice with PBS, and the cells were
observed under the microscope, screening the number and the intensity
of blue cells.
Immunofluorescence Detection of Expressed
DTR
At 24, 48, and 72 hpi, HF cells adherent to
poly-L-lysine-treated glass coverslip were washed with
ice-cold PBS, and then 10 µg/ml DT was added and allowed to bind to
the infected cells for 30 min at room temperature as described below.
The cells were washed three times with ice-cold PBS and subsequently
incubated with anti-DT antibodies and then with FITC-labeled goat
anti-rabbit IgG, and the coverslip was mounted on a microscopy slide
with Mowiol and examined under UV light.
Radiolabeling of DT
DT or nDT were
labeled with I by the IODO-GEN method to a specific
activity of about 1
10
cpm/ng as
described(24) .
DT Binding Assay
At 48 hpi, SF9 cells
were incubated in binding medium (Grace's insect medium plus 10%
fetal calf serum and with or without 10 IU/ml heparin as indicated)
with increasing amounts of I-labeled DT in the absence or
presence of 10 µg/ml unlabeled DT for 30 min at room temperature.
The reaction was stopped by filtration through Whatman GF/A filters,
which were then washed five times with 10 ml of ice-cold PBS, and the
radioactivity associated with the filters was measured in an autogamma
spectrometer. The specific binding was calculated as the difference
between the radioactivity associated with the filters in the absence
and presence of 10 µg/ml unlabeled DT. The unspecific binding to
cells infected with the recombinant virus varied from 10 to 40% of the
total binding for different experiments, depending of the batch of
labeled DT. The specific binding was plotted as described by Scatchard
(25) to determine the number and the affinity of DT to the DTR. Binding
curves were generated by regression analysis.
I-labeled DT in Hepes medium plus 0.1 mg/ml bovine serum
albumin in the absence or presence of 10 µg/ml unlabeled DT. The
cells were then washed four times with PBS and lysed in 0.1 M KOH, and the radioactivity associated with the cells was measured.
Nicking of DT
DT at a concentration of
1.1 mg/ml was nicked with 0.5 µg/ml trypsin for 30 min at 37
°C; the reaction was stopped by adding 25 µg/ml soybean trypsin
inhibitor.
Cytotoxicity Assay
To assess the rate of
protein synthesis, the incorporation of
[H]leucine into cellular proteins was measured at
36 or 48 hpi. Although maximum expression of DTR was at 48 hpi, for
some of the toxicity experiments binding of the toxin was carried out
at 36 hpi to allow the toxin to act overnight to produce maximal effect
on protein synthesis without lysis of the cells. At the indicated hpi,
the cells were recovered by centrifugation for 5 min at 1000
g at room temperature and then washed with 10 ml of PBS. The
cells were resuspended in binding medium and incubated with increasing
amounts of nDT for 30 min at the indicated temperatures in the absence
or presence of 10 µM of monensin. The medium and unbound
toxin was removed, and the cells were recovered by centrifugation and
resuspended in buffer at the indicated pH for 5 min at 27 °C to
allow translocation of bound toxin to occur; then, 3 ml of complete
TNM-FH medium with or without 10 µM monensin and anti-DT
serum was added, and the cells were incubated for 2 h at 27 °C to
allow the translocated toxin to inhibit protein synthesis. In some
experiments, the low pH pulse was omitted. After the incubation, the
cells were recovered by centrifugation and washed with 1 ml of
leucine-free Grace medium and incubated in 1 ml of leucine-free Grace
medium with 1 µCi/ml [
H]leucine for 30 min at
room temperature. The cells were recovered by centrifugation and
treated twice with 1 ml of 5% trichloroacetic acid for 10 min; the
pellets were recovered by centrifugation for 10 min at 14,000 rpm and
dissolved in 300 µl of 0.1 M KOH, and the radioactivity
was measured.
H]leucine was then measured for 30 min at 37
°C in leucine-free Hepes medium supplemented with 1 µCi/ml
[
H]leucine. The cells were treated twice with 1
ml of 5% trichloroacetic acid for 10 min and dissolved in 300 µl of
0.1 M KOH; the radioactivity was then measured.
Pronase Protection Experiments
At 48 hpi,
100 ng of I-labeled nDT was bound to
vAc-DTR
-infected cells for 30 min at 4 °C in presence
of 10 µM monensin. Unbound toxin was removed by washing
the cells five times with 10 ml of PBS. Then, the cells were exposed
for 5 min to the indicated pH values at 27 °C to allow
translocation of the bound toxin to occur. The cells were incubated for
10 min at 27 °C with 6 mg/ml Pronase E in the presence of 10
µM monensin and subsequently recovered by centrifugation
for 3 min at 14,000 rpm. After washing with PBS containing 1 mMN-ethylmaleimide and 1 mM PMSF, the cells were
lysed for 10 min in lysis buffer containing 1 mMN-ethylmaleimide and 1 mM PMSF on ice, and the nuclei
were removed by centrifugation. Cellular proteins were precipitated
with 5% trichloroacetic acid for 15 min on ice and pelleted by
centrifugation. The pellets were washed with ether and subjected to
SDS-PAGE under non-reducing conditions.
DTR Precipitation
At 48 hpi, uninfected
SF9 cells, SF9 cells, and SF9
cells
were labeled with 1 µCi/ml [
S]methionine for
4 h and then lysed with 1 ml of lysis buffer for 30 min on ice; the
nuclei were removed by centrifugation for 5 min at 14,000 rpm at 4
°C, and the supernatant was used for DTR precipitation. For
precipitation of DTR, 500 µl of cell lysate was incubated for 2 h
at 4 °C with DT-Sepharose or anti-B
bound to
protein-A-Sepharose. The pellets were washed three times with lysis
buffer, once with PBS, and once with water. They were then treated with
sample buffer, and the extracted material was analyzed by SDS-PAGE
under reducing conditions.
I or with
[
S]methionine, dried gels were exposed to Kodak
XAR-5 films with intensifying screens at -80 °C for
fluorography.
Receptor Expression
Recombinant
baculovirus encoding the DTR gene was constructed by standard
techniques and checked by PCR as described under ``Experimental
Procedures.'' To check the time course of the virus infection as
well as the level of viral protein production, we first monitored the
expression of the LacZ gene visualized by staining the insect
cells with X-gal at different times post-infection. The number of blue
cells and the intensity of the blue color reflect the amount of
infected cells and the level of viral protein expression. By this
method, we found that 24 hpi almost 90% of the
vAc-DTR-infected cells were blue, whereas cells infected
with wild type virus as well as uninfected SF9 cells remained
colorless. At 48 and 72 hpi, 100% of the cells became blue, and also
the intensity of the color was increased from 48 to 72 hpi, indicating
an increase in the viral protein production (data not shown). About 72
hpi, virus-induced cell lysis starts. As the LacZ gene is
produced from an earlier promoter (pEtl promotor) than that of
polyhedrin, the X-gal staining of the cells indicates the progress in
the virus infection but not the levels of DTR production.
I-labeled DT at 24, 48, and 72
hpi. The specific binding increased to a maximum at 48 hpi, and it was
again lower at 72 hpi, probably due to beginning cell lysis.
Figure 1:
Immunofluorescence staining of
vAc-DTR-infected HF cells. 24 h after infection, the cells
were incubated for 30 min with 10 µg/ml DT at room temperature,
washed to remove unbound toxin, and subsequently incubated with rabbit
anti-DT serum for 30 min at room temperature. The bound antibodies were
visualized with FITC-labeled anti-rabbit IgG in TNM-FH medium plus 20%
aggregated
-globulin for 30 min at room temperature. Finally, the
cells were washed with PBS, mounted with Mowiol on microscopy slides,
and observed in a fluorescence microscope.
To immunoprecipitate the expressed
receptors, we used an anti-B3 serum against the COOH-terminal epitope
added to the receptor. We also precipitated the DTR with DT immobilized
on Sepharose. As it is shown in Fig. 2(lanes3 and 6), a band of about 25 kDa (arrow) was
observed in both cases. When the DTR was precipitated with
DT-Sepharose, the amount precipitated was much higher than in the case
of immunoprecipitation with anti-B3 serum, indicating that the B3
epitope is either not well exposed or partly removed in the cells.
Also, two additional bands of about 20 kDa (asterisks) were
precipitated with DT-Sepharose (lane6). These bands
were not observed in uninfected SF9 cells (lanes1 and 4) or in cells infected with wild type virus (lanes2 and 5).
Figure 2:
Expression of DTR in SF9 insect cells.
Uninfected SF-9 cells (lanes1 and 4) and
SF9 cells infected with wild type virus (lanes2 and 5) or with vAc-DTR (lanes3 and 6) for 48 h were labeled for 4 h with
[
S]methionine and lysed in 1 ml of lysis buffer
for 30 min. The nuclei were removed by centrifugation, and the
supernatant was incubated for 2 h at 4 °C with DT-Sepharose or
anti-B3 bound to protein-A-Sepharose as described under
``Experimental Procedures.'' For precipitation, immobilized
rabbit anti-B3 serum bound to protein-A-Sepharose (lanes1-3) and DT-Sepharose (lanes4-6) was used. For fluorography, the dried gel from
anti-B3 protein-A-Sepharose immunoprecipitation was exposed, as
described under ``Experimental Procedures,'' for 8 days. The
dried gel from DT-Sepharose precipitation was exposed
overnight.
Binding of Radiolabeled DT to Cell Surface
Receptors
We have in the following defined specific binding
of DT as the difference in binding of I-labeled DT in the
presence and absence of excess unlabeled toxin. In uninfected SF9 cells
and cells infected with the wild type virus, only marginal binding was
detected, indicating that the cells do not express appreciable amounts
of receptors for diphtheria toxin.
. HB-EGF constitutes part of DTR(26) . We
therefore tested if the presence of heparin in the binding medium
affected the binding of DT to the receptor. Uninfected SF9 cells and
vAc-DTR
-infected cells were incubated with 100 ng/ml
I-labeled DT and increasing amounts of heparin in the
absence and presence of 10 µg/ml unlabeled DT. The results (Fig. 3) showed that heparin induced a considerable increase in
the amount of total binding in the vAc-DTR
-infected cells
but not in uninfected cells, indicating that the binding to the
infected cells was of a different kind. The binding increased up to
10-20 IU/ml heparin and then decreased again with higher heparin
concentrations. Compared to the increase seen in
vAc-DTR
-infected cells (Fig. 4A), there was
only a small increase in the binding of
I-labeled DT in
presence of heparin in Vero cells (Fig. 4B).
Figure 3:
Effect of increasing amounts of heparin on
binding of I-labeled DT to uninfected and
vAc-DTR
-infected SF9 cells. The cells were incubated with
100 ng/ml
I-labeled DT in the absence (top of
the bars) or presence (bottom of the bars)
of 10 µg/ml unlabeled DT and increasing amounts of heparin for 30
min at room temperature. Then, the cells were washed with ice-cold PBS
on Whatman GF/A filters, and the radioactivity associated with the
filters was measured.
Figure 4:
Effect of heparin on the specific binding
of I-labeled DT to vAc-DTR
-infected SF9
cells (A) and Vero cells (B). Cells were incubated
with increasing amounts of
I-labeled DT in the absence
and presence of 10 µg/ml unlabeled DT and 10 IU/ml heparin for 30
min at room temperature. Then, the insect cells were washed on Whatman
GF/A filters with ice-cold PBS, and the radioactivity associated with
the filters was measured. The Vero cells (growing on plastic) were
washed with PBS and lysed in 0.1 M KOH, and the radioactivity
associated with the cells was measured. The specific binding indicated
on the ordinate was calculated as the difference between the
radioactivity bound in absence and presence of unlabeled DT.
, no
heparin;
, 10 IU/ml heparin.
The
vAc-DTR-infected cells were found to bind
I-labeled DT in a highly specific and saturable manner (Fig. 5A). A single binding affinity (K
of 3.7
10
M) and 5
10
binding sites/cell were
calculated. In comparison, Vero cells were found to express 22,000
receptors/cell with an affinity of 2.1
10
M (Fig. 5B).
Figure 5:
Saturation binding of I-labeled DT to vAc-DTR
-infected SF9 cells (A) and to Vero cells (B). Binding was carried out as
described in Fig. 4 in the presence of 10 IU/ml heparin. The specific
binding (
) was calculated as the difference between the total
binding (
) and the unspecific binding in presence of 10 µg/ml
DT (
). The insets represent Scatchard analyses of the
data presented in each panel. The data were fitted by
regression analysis of the specifically bound
I-labeled
DT on abscissa and bound/free toxin (B/F) on the ordinate.
Sensitivity of Infected SF9 Cells to Diphtheria
Toxin
After binding to DTR on toxin-sensitive cells, DT is
normally endocytosed, and, upon acidification, the enzymatically active
A fragment is translocated across the endosomal
membrane(7, 8, 9, 10, 11) . This
normal route can be mimicked at the level of the cell surface membrane
by acidification of the culture
medium(8, 9, 11, 13) .
-infected cells and Vero cells under conditions
where the toxin was bound to the cells for 30 min at 4 °C, and then
the cells were exposed to either neutral or low pH medium and incubated
at neutral pH in the presence of monensin to inhibit toxin entry by the
normal route. The rate of protein synthesis was measured 2 h later.
When the low pH pulse was omitted, protein synthesis inhibition was not
observed in either cell type (Fig. 6). In contrast, when cells
with bound toxin were exposed to pH 4.8, protein synthesis in the
vAc-DTR
-infected SF9 cells was inhibited at very low toxin
concentrations. Both Vero cells and vAc-DTR
-infected
insect cells were more sensitive to DT when incubated with the toxin
over night to let the toxin be endocytosed and enter by the normal
route than when the toxin entry was induced by acidification of the
medium (Fig. 7, A and B).
Figure 6:
Effect of pH on the sensitivity to DT of
vAc-DTR-infected SF9 cells (A) and Vero cells (B). Increasing amounts of nicked DT were added to cells for
30 min at 4 °C in binding medium with 10 µM monensin.
The medium with unbound toxin was removed, and medium adjusted to pH
4.8 (
) or to pH 6.5 (
) was added. The incubation lasted
for 5 min at 27 °C for SF9 cells and 37 °C for Vero cells.
Subsequently, 3 ml of complete medium with 10 µM monensin
and 5 µl of anti-DT serum were added, and the cells were incubated
for 2 h at 27 or 37 °C for SF9 and Vero cells, respectively. Then,
the ability of the cells to incorporate
[
H]leucine was measured. The results are
expressed as percent of the control values (no toxin
added).
Figure 7:
Comparison of sensitivity of
vAc-DTR-infected SF9 cells (
) and Vero cells (
)
to DT translocated from the plasma membrane at low pH (A) and
from endosomes (B). In A, increasing amounts of
nicked DT were added to vAc-DTR
-infected SF9 and Vero
cells for 30 min at 4 °C in the presence of 10 µM monensin. The medium with unbound toxin was removed, and the cells
were incubated for 5 min at 27 °C (SF9) or 37 °C (Vero) with
medium adjusted to pH 4.8. Then, 3 ml of complete medium with 10
µM monensin were added, and the cells were incubated for 2
h at 27 or 37 °C for SF9 and Vero cells, respectively. In B, the cells were incubated with DT overnight at 27 or 37
°C for SF9 and Vero cells, respectively. Then, the ability to
incorporate [
H]leucine was measured. The results
are expressed as percent of the control values (no toxin
added).
The sensitivity to
DT of the infected insect cells was 200 times higher than that of
Vero cells (Fig. 7), both when the toxin was translocated
directly from the plasma membrane and when it was endocytosed by the
normal route. This is in accordance with the
250-fold higher
number of DTR on the insect cells with somewhat lower affinity as
compared to Vero cells.
I-labeled DT was bound to the
vAc-DTR
-infected cells than in the absence of heparin, and
we tested if this was accompanied by an increase in the sensitivity to
DT. This was indeed the case. In contrast, in Vero cells, where toxin
binding was not much increased by heparin, the sensitivity was the same
in absence and presence of heparin (data not shown).
Pronase Protection of the Translocated
Toxin
Translocation of DT from the plasma membrane can be
assayed directly by treating the cells with Pronase E after the low pH
pulse to remove surface-bound toxin. Successful translocation yields
two protected polypeptides of 21 and 25 kDa. The 21-kDa band represents
the toxin A fragment translocated to the cytosol, whereas 25-kDa
polypeptide is part of the B fragment inserted into the
membrane(11) . In Vero cells, translocation of nicked DT from
the plasma membrane occurs only at pH <5.3(9) .
-infected insect cells resembles that in Vero
cells, we analyzed the amount of protected 21- and 25-kDa polypeptides
obtained at different pH values. As shown in Fig. 8, also in
insect cells the translocation was a function of the pH during the
exposure to low pH medium. The toxin entry was maximal at pH
4-4.8, as in Vero cells, but whereas pH <5.3 is required for
translocation in Vero cells(27) , some protected material was
observed even at pH 6.5 in the insect cells (Fig. 8, lanes3 and 4). It should be noted that some whole
toxin bound to the cells was protected against Pronase, the amount of
which increased with pH. This effect, which is not seen in Vero cells,
could be due to reduced accessibility of the toxin on the insect cells,
possibly by efficient pinching off of DTR-containing coated pits, a
process that could be inhibited by low pH(28) .
Figure 8:
Effect
of pH on Pronase protection of cell-bound DT in
vAc-DTR-infected SF9 cells. vAc-DTR
-infected
cells at 48 hpi were incubated with 100 ng
I-labeled
nicked DT for 30 min at 4 °C in presence of 10 µM monensin. Unbound toxin was removed by washing with PBS, and the
cells were exposed for 5 min to pH 4 (lane1), pH 4.8 (lane2), pH 5.5 (lane3), and pH
6.5 (lane4) at 27 °C to allow translocation of
the bound toxin to take place. The cells were then incubated for 10 min
with 6 mg/ml Pronase E in the presence of 10 µM monensin.
The medium with enzyme was removed, and the cells were washed with
medium and lysed in lysis buffer. Proteins in the lysate were
precipitated with 5% trichloroacetic acid and analyzed by SDS-PAGE
under non-reducing conditions.
-adrenergic
receptor(31, 32, 33) , and the human
follicle-stimulating hormone receptor(34) .
I-labeled DT can be demonstrated readily in highly
sensitive cells such as Vero, but not in cells with intermediate DT
sensitivity such as HeLa cells, or in resistant mouse
cells(15) . Naglich et al. (18) and Brown et
al. (19) showed specific and saturable binding in mouse LM cells
transfected with DTR from Vero cells. In these cells, the number of DTR
was about 50,000 receptors/cell, which is twice that in Vero cells, but
the affinity was lower in the transfected cells (K
of 5.8-15
10
M) than in Vero cells (5.9-12
10
M). Iwamoto et al. (35) found that the K
of DT to
recombinant HB-EGF immobilized on heparin-Sepharose was 7.7
10
M. The same authors found 8.1
10
receptors/cell with a K
of
10
M in mouse L cells transfected with a
DTR cDNA. In our expression system, we have obtained the highest
density of DTR per cell so far reported, viz. about 5
10
receptors per cell with an affinity (K
3.7
10
M) similar to that reported by Brown et al. (19) in transfected mouse cells. The lower affinity of the
expressed receptor as compared to Vero cells has been attributed to the
absence of proteins associated with the receptor in the transfected
cells(18) . In the insect cells, it could also be due to
different post-translational processing of the receptor as compared to
Vero cells.
10
copies/cell of DRAP27, while LM cells display only 1,900
molecules/cell(36) . Transfection of DRAP27/CD9 was found to
increase the DT binding and sensitivity in a human-mouse hybrid cell
line that expresses DTR(37) . Also, Brown et al. (19) and Iwamoto et al. (35) have demonstrated
an increase in the number of DT binding sites and DT sensitivity in
mouse cells expressing DTR and CD9. In both cases an increase in the
receptor number, but not in the receptor affinity, was observed. In our
system, the vAc-DTR
-infected cells express about 250 times
more receptors than Vero cells, and the cells were about 200 times more
sensitive to DT than Vero cells despite the observation that the
affinity of the expressed receptor was lower than in Vero cells. The
very high concentration of receptors under our test conditions (2
10
cells with 5
10
receptors
each) reduces the importance of the high affinity of the receptors. It
can not be excluded that proteins present in insect cells can increase
the sensitivity of the infected cells to the toxin. Conversely,
inhibitory factors of the translocation mechanism could exist in Vero
cells and be absent in insect cells.
-infected cells was
unexpected, since heparin did not increase much the binding and toxin
sensitivity in Vero cells. It is not clear if this is due to
DTR-associated proteins that may differ between Vero cells and SF9
cells. Also, it could be due to differences in the amount and types of
proteoglycans expressed at the surface. On Vero cells, most of the DTR
are associated with surface heparans,
(
)and this
may not be the case in the insect cells.
,
recombinant baculovirus expressing DTR; SF9, uninfected Spodoptera
frugiperda cells; HF, high five uninfected cells; PMSF,
phenylmethylsulfonyl fluoride; X-gal,
5-bromo-4-chloro-3-indolyl-
-D-galactoside; hpi, hours
post-infection; PBS, phosphate-buffered saline; bp, base pair(s); PCR,
polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein isothiocyanate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.