(Received for publication, July 7, 1995; and in revised form, September 19, 1995)
From the
Diphtheria toxin receptor (DTR), which is identical to the
membrane-anchored form of heparin-binding EGF-like growth factor
(proHB-EGF), has a high affinity for heparin. We studied the effect of
heparin-like molecules on the binding of diphtheria toxin (DT) to
DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in
heparan sulfate (HS) proteoglycans were about 15 times less sensitive
to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin
was added to the culture medium, DT sensitivity of the mutant cells was
fully restored. Studies of binding of I-labeled DT to
HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA
indicated that the increased sensitivity to DT after addition of
heparin is due to increased binding of DT to cells. Vero cells display
a relatively large amount of heparan sulfate residues compared to
CHO-K1 cells or L cells. Enhancement of DT binding by the addition of
heparin was also observed with CHO-K1 cells and L cells that had been
transfected with human DTR/proHB-EGF cDNA, but the degree of
enhancement was less than that observed with the HS-deficient CHO
cells. Addition of heparin did not affect DT binding or DT sensitivity
of Vero cells. Heparin-dependent binding was observed when intact Vero
cells were treated with heparitinase or when the cell membrane was
solubilized with a neutral detergent. Scatchard plot analysis for the
binding of DT to a recombinant HB-EGF in vitro and to L cells
expressing human DTR/proHB-EGF revealed that heparin increases the
affinity of DTR/proHB-EGF for DT but does not change the number of
binding sites. Although DRAP27/CD9 is known to enhance DT binding to
DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9
increase DT binding by independent mechanisms. Thus, heparin-like
molecules, probably in the form of heparan sulfate proteoglycan on the
cell surface, are a third factor required for maximal DT binding
activity of cells.
Diphtheria toxin (DT) ()inhibits protein synthesis of
eukaryotic cells by catalyzing the ADP-ribosylation of elongation
factor-2, which results in its inactivation(1, 2) .
The process of DT cytotoxicity includes (i) binding of DT to a specific
receptor on the cell surface, (ii) internalization of the toxin by
receptor-mediated endocytosis, (iii) translocation of the enzymatically
active A fragment from endosome to the cytosol, and (iv) inactivation
of elongation factor-2. Although a number of cellular factors are
involved in this process, binding of DT to the diphtheria toxin
receptor (DTR) is a key step(3) .
DTR, the principal protein involved in binding of DT to cells, was first identified and purified from monkey Vero cells(4, 5) , one of the cell lines most sensitive to DT(6) . A cDNA-encoding DTR was cloned from this cell line(7) . DTR is identical to the precursor form of heparin-binding EGF-like growth factor (proHB-EGF) (7, 8) which was originally identified as a heparin-binding growth factor(9) . While proHB-EGF is cleaved by an unidentified protease on the cell surface to yield the soluble mature growth factor (HB-EGF)(10) , a significant amount of proHB-EGF is left uncleaved on the cell surface, where it acts as a membrane-anchored growth factor (11) and as a DTR. Although proHB-EGF is expressed in species including human, monkey, rat, and mouse with a similar tissue distribution, cells derived from mice and rats are resistant to DT(6, 12, 13) . The insensitivity of mouse and rat cells to DT is due to amino acid substitutions in the EGF-like domain of proHB-EGF that reduce binding of DT(14) .
DTR/proHB-EGF forms a complex with DRAP27/CD9(8, 15) , which belongs to a newly identified tetra membrane-spanning protein family(16) . Although direct interaction of DRAP27/CD9 with DT has not been observed, DRAP27/CD9 greatly up-regulates the binding of DT(8, 16, 17) and the juxtacrine mitogenic activity (11) of DTR/proHB-EGF. Thus, DRAP27/CD9 is a second factor determining binding of DT to cells. Although the mechanism of up-regulation by DRAP27/CD9 is still not clear, it is neither due to increased expression of the DTR/proHB-EGF gene nor increased DTR/proHB-EGF protein on the cell surface(8) . Scatchard plot analysis shows that DRAP27/CD9 increases the number of DT-binding sites but does not change their affinity for DT(8) .
DTR/proHB-EGF shows strong affinity for heparin and heparan sulfate (HS)(18, 19) . As has been shown for other heparin-binding growth factors(20, 21, 22) , HB-EGF requires heparin or cell surface heparan sulfate proteoglycan (HSPG) for binding to and activation of the EGF receptor(23, 24) . Because the binding site of DTR/proHB-EGF for DT seems to overlap with or be located in close proximity to the binding site for the EGF receptor(14) , we examined whether heparin influences binding of DT to DTR/proHB-EGF. The results indicate that DT requires cell surface heparin-like molecules for maximal binding to DTR/proHB-EGF. Heparin-like molecules on the cell surface, possibly in the form of HSPG, are a third factor influencing binding of DT to cells.
Figure 1: Flow cytometric analysis of cell surface HS-GAG with anti-HS antibody. Mutant 677 cells (677), CHO-K1 cells (K1), Vero cells (Vero), and L cells (L) treated with anti-HS antibody (bold traces) or without (dotted traces) were stained with FITC-conjugated secondary antibody.
Figure 2:
DT sensitivity on wild type CHO cells and
HS-deficient cells. CHO-K1 cells (open circles) and mutant 677
cells (closed circles) were incubated with various
concentrations of DT for 2 h at 37 °C, followed by incubation with
[H]leucine for 1 h at 37 °C. Radioactivity
incorporated into protein was determined. Data are expressed as percent
of control without DT and shown as means ± S.E. of three
independent experiments.
Binding of basic
fibroblast growth factor (bFGF) to 677 cells is restored by addition of
exogenous HS or heparin(20) . As shown in Fig. 3A, DT sensitivity of the 677 cells was increased
in a dose-dependent manner by addition of HS to the medium. At 100
µg/ml HS, the ED of 677 cells was about 10 ng/ml,
which is similar to the value for wild type cells in the absence of HS.
DT sensitivity of wild type cells was not increased at low
concentrations of HS (0.1-1.0 µg/ml), but was slightly
increased at high concentrations (10-100 µg/ml) (Fig. 3B).
Figure 3:
Potentiation of DT sensitivity of
HS-deficient cells by the addition of HS or heparin. Mutant 677 cells (A and C) and CHO-K1 cells (B and D) were incubated for 2 h at 37 °C with various
concentrations of DT, followed by incubation with
[H]leucine for 1 h at 37 °C. HS (A and B) or heparin (C and D) was added
to the medium throughout the incubation period at concentrations of 0.1 (squares), 1 (triangles), 10 (diamonds), and
100 µg/ml (crosses). Samples incubated without HS or
heparin were taken as a control (circles). The radioactivity
incorporated into protein was determined. Data are expressed as percent
of control without DT. Similar results were obtained in two separate
experiments.
The sensitivity of 677 cells to DT was
also increased by addition of heparin (Fig. 3C). The
ED for 677 cells at 0.1, 1, and 10 µg/ml heparin was
60, 28, and 24 ng/ml, respectively, while ED
in the
absence of heparin was 180 ng/ml. Heparin at high concentration
(10-100 µg/ml) increased DT sensitivity of the wild type
cells as well, but the effect of heparin was less in K1 cells than in
677 cells (Fig. 3D). Heparin was more effective than HS
for both cell lines, as was reported for
bFGF(34, 35) . The fact that the addition of HS or
heparin restores DT sensitivity in 677 cells indicates that the
decreased sensitivity of 677 cells is due to the absence of HSPG in
this mutant cells and that heparin-like molecules are involved in
determining DT sensitivity.
Other related glycosaminoglycans (GAG) were tested. N-Desulfated heparin, chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid did not increase DT sensitivity, but dermatan sulfate was as effective as heparin (data not shown), as was previously observed for FGF(35, 36) . These results suggest that both the saccharide core structure and the level of sulfation influence the enhancing effect.
Figure 4:
Heparin enhancement of binding of DT to
DTR/proHB-EGF. A, binding of DT to 677H cells in the presence
or absence of heparin. 677H cells were incubated with various
concentrations of I-DT in the presence (closed
squares) or absence (closed circles) of heparin (10
µg/ml) for 15 h at 4 °C. Data are expressed as specific
binding. Similar results were obtained in three separate experiments. B, comparison of heparin dependence on DT binding of 677H cell
to K1H cells. 677H (closed circles) and K1H cells (open
circles) were incubated with
I-DT (100 ng/ml) in the
presence of various concentrations of heparin for 15 h at 4 °C.
Then cells were washed, and the radioactivity associated with the cells
was determined. Data are expressed as relative values compared to
controls without heparin. In these experiments nonspecific binding of
I-DT was <20% of the total
binding.
K1H cells also showed increased DT binding
after addition of heparin, but the effect was smaller than with 677H
cells. Fig. 4B compares the effect of heparin on DT
binding by the two cell lines. Because 677H cells and K1H cells have
different levels of expression of DTR/proHB-EGF, DT binding for each
cell line was normalized to the value in the absence of heparin. In the
presence of 0.1 µg/ml heparin, specific binding of I-DT to 677H cells was increased 5.4-fold, whereas
binding to K1H cells was increased 2.3-fold. The effect of heparin on
DT binding is well correlated with the effect on DT sensitivity,
indicating that the decreased DT sensitivity of 677 cells is due to
reduced binding of DT to DTR/HB-EGF in the absence of heparin or HS
molecules.
Figure 5:
Effect of chlorate on DT binding to CHO
cells. K1H cells and 677H cells were incubated with or without 30
mM chlorate for 48 h as described under ``Experimental
Procedures.'' Then DT binding was determined by incubation with I-DT (100 ng/ml) in the presence or absence of heparin
(10 µg/ml) for 15 h at 4 °C. Data are expressed as specific
binding and shown as means ± S.E. of three independent
experiments. open bars, untreated cells; closed bars,
chlorate-treated cells; hatched bars, untreated cells with
heparin; dotted bars, chlorate-treated cells with heparin.
Nonspecific binding of
I-DT was <5% of the total
binding.
Figure 6:
Involvement of heparin-like molecules in
DT binding to Vero cells. A, effect of heparitinase on DT
binding to intact Vero cells. Vero cells were incubated with or without
heparitinase (0.02 unit/ml) for 1.5 h at 37 °C. Then DT binding to
Vero cells was measured by incubation with I-DT (100
ng/ml) for 15 h at 4 °C in the presence or absence of heparin (10
µg/ml). Data are expressed as specific binding and shown as means
± S.E. of three independent experiments. Open bar,
untreated cells; closed bar, heparitinase-treated cells; hatched bar, untreated cells with heparin; dotted
bar, heparitinase-treated cells with heparin. Nonspecific binding
was <5% of the total binding. B, precipitation of
DTR/proHB-EGF with DT and anti-DT antibody from VeroH cell lysates.
Surface-biotinylated VeroH cells were solubilized and incubated with or
without heparitinase (0.02 unit/ml) for 1.5 h at 37 °C. Then
DTR/proHB-EGF was precipitated with DT and immobilized anti-DT antibody
in the presence or absence of heparin (10 µg/ml). Precipitated
material was subjected to SDS-PAGE, followed by Western blotting with
streptavidin-horseradish peroxidase.
When cell surface HS-GAG
was diminished by treatment of Vero cells with heparitinase,
heparin-dependent binding was observed. Heparitinase treatment greatly
diminished cell surface HS contents, but did not affect the amount of
DTR/proHB-EGF (data not shown). Under these conditions, binding of I-DT to heparitinase-treated Vero cells was about 50% of
the binding to untreated cells. Binding was fully restored by addition
of exogenous heparin (Fig. 6A). Heparin-dependent
binding of DT was also observed in Vero cells lysates. VeroH cells,
Vero cells overexpressing DTR/proHB-EGF, were surface-biotinylated and
lysed. The lysate was precipitated with DT and immobilized anti-DT
antibody in the presence or absence of exogenous heparin. The
precipitated materials were analyzed by SDS-PAGE. Similar species of
HB-EGF/DTR with molecular masses of 21-28 kDa were precipitated
from the cell lysate in the presence or absence of heparin, but much
more DTR/proHB-EGF was precipitated in the presence of heparin (Fig. 6B). Furthermore, when cell lysate was first
treated with heparitinase and then precipitated with DT and anti-DT
antibody, the amount of DTR/proHB-EGF precipitated was greatly
diminished. Addition of heparin to heparitinase-treated cell lysate
restored the precipitation efficiency. These results indicate that
heparin-like molecules are involved in DT binding by Vero cells.
Figure 7:
Binding of DT to a recombinant HB-EGF. A, effect of heparin on binding of I-DT to a
recombinant HB-EGF. A recombinant human HB-EGF was conjugated to
CH-activated Sepharose 4B. The gel was incubated with
I-DT (25 ng/ml) in the presence or absence of various
concentrations of heparin for 4 h at 4 °C. Then the gel was washed,
and the radioactivity associated with the gel was counted. B,
binding of
I-DT to recombinant HB-EGF in the presence or
absence of heparin. Recombinant HB-EGF conjugated to CH-activated
Sepharose 4B were incubated with various concentrations of
I-DT in the presence (closed circles) or absence (open circles) of heparin (10 µg/ml) for 4 h at 4 °C.
The gel was washed, and the radioactivity associated to the gel was
counted. The results are expressed as specific binding. Inset,
Scatchard plot of the specific binding of
I-DT to
recombinant HB-EGF in the presence or absence of heparin. Nonspecific
binding of
I-DT was <20% of the total
binding.
Figure 8:
Binding of I-DT to LH cells
and LCH cells in the presence or absence of heparin. A, LH (open symbols) and LCH cells (closed symbols) were
incubated for 15 h at 4 °C with binding medium containing various
concentrations of
I-DT in the presence (squares)
or absence (circles) of heparin (10 µg/ml).
Cell-associated radioactivity was determined. Data are expressed as
specific binding. B, Scatchard plot of the specific binding of
I-DT to LH cells in the presence (squares) or
absence (circles) of heparin. C, Scatchard plot of
the specific binding of
I-DT to LCH cells in the presence (closed squares) or absence (closed circles) of
heparin. Nonspecific binding of
I-DT was <10% of the
total binding.
Scatchard plot analysis more clearly demonstrates the difference in
mode of actions of DRAP27/CD9 and heparin-like molecules. Heparin
increased the affinities of both LH cells and LCH cells for DT but did
not change the binding site numbers. The K values
for LH cells in the presence or absence of heparin were 6.8
10
M
and 1.7
10
M
, respectively, while the K
values for LCH cells in the presence or absence
of heparin were 1.5
10
M
and 0.56
10
M
.
DRAP27/CD9 increased DT-binding site numbers rather than changing the
affinity(8, 17) . The difference in the affinities for
DT of the LH cells and LCH cells used here is probably due to clonal
variations. We conclude that DRAP27/CD9 and heparin potentiate DT
binding to DTR/proHB-EGF by independent mechanisms.
DT specifically binds to the diphtheria toxin receptor, the
DTR/proHB-EGF molecule. Binding of DT to the receptor is one of the key
steps in the intoxication process, and the sensitivity of cells to DT
is primarily determined by the number of
receptors(3, 37, 38) . Our recent studies
revealed, however, that a second factor, in addition to DTR/proHB-EGF
itself, is required for maximal DT binding activity. The membrane
protein DRAP27/CD9 up-regulates DT binding and DT sensitivity as a
co-factor of DTR/proHB-EGF(8, 15, 16) . We
showed here that heparin-like molecules, probably in the form of HSPG
on the cell surface, are a third factor required for maximal DT binding
and DT sensitivity. The requirement for heparin-like molecules for full
DT binding activity is indicated by the following evidence: (i)
HS-deficient CHO mutant 677 cells are less sensitive to DT than the
wild type CHO-K1 cells, and the decreased DT sensitivity is restored by
the addition of free HS or heparin; (ii) 677H cells bind I-DT in a heparin-dependent manner; (iii) treatment with
chlorate or heparitinase to diminish cell surface HS decreases the
binding of DT; (iv) binding of
I-DT to a recombinant
HB-EGF in vitro is heparin-dependent. We propose that the
heparin-like molecules responsible for enhancing DT binding probably
exist as HSPG rather than as proteoglycans with dermatan sulfate or
free heparin, because DT binding activity of Vero cells and Vero cell
lysates was greatly diminished by treatment with heparitinase, which
hydrolyses HS-GAG, but not dermatan sulfate or heparin.
Increased binding of DT after addition of free heparin or HS was most striking in HS-deficient 677 cells, but was also observed to a lesser extent in CHO-K1 cells and L cells expressing human HB-EGF (LH and LCH cells). No increase of DT binding was observed when heparin was added to Vero cells. The difference in heparin dependence among cell lines can be explained by differences in the amount of cell surface HS-GAG. The amount of HS on the surface of Vero cells is about 70 times higher than on CHO-K1 cells and about 35 times higher than on L cells. The amount of HS on CHO-K1 cells and L cells does not appear to be sufficient for interaction with all of the DTR/proHB-EGF, thus exogenous heparin or HS increases the binding of DT to these cells, although the effect is less than that seen with 677 cells. Since DTR/proHB-EGF molecules are overexpressed in LH and LCH cells, much more HSPG may be required for maximal DT binding by these cells, making the heparin dependence more apparent. Vero cells contain abundant HS on the cell surface, and exogenous heparin is not required for maximal binding. However, when cell surface HS is removed by the treatment with heparitinase, or when the cell membrane is solubilized, Vero cells show heparin-dependent binding of DT.
How is the binding of DT to the cells increased by
heparin-like molecules? DTR/proHB-EGF, like mature HB-EGF, binds
heparin tightly, while DT itself is not bound to heparin-Sepharose (Table 1). Therefore, the effect of heparin on the binding of DT
must result from the interaction of heparin with DTR/proHB-EGF
molecules. Highly cationic amino acids clusters located within and
upstream of the EGF-like domain of HB-EGF form the putative
heparin-binding domain(19) . We have previously shown that DT
binds to the EGF-like domain of DTR/proHB-EGF molecule. Because cells
expressing a deletion mutant of DTR/proHB-EGF, H (63-105)
that lacks most of the heparin-binding domain still bind
DT(14) , it is likely that the heparin-binding domain modulates
the binding activity for DT rather than forming the binding site
itself. Therefore, we propose that binding of heparin, or HSPG, to the
heparin-binding domain brings about a conformational change in the
DTR/proHB-EGF molecule, resulting in increased affinity for DT by an
allosteric effect, similar to the induced-fit model shown originally in
the interaction of bFGF with heparin(20) .
Heparin and
DRAP27/CD9 potentiate the binding of DT by different mechanisms.
Heparin increases the affinity of DTR/proHB-EGF for DT but does not
change the number of binding sites, while DRAP27/CD9 increases the
number of DT-binding sites but does not changes the K value(8, 17) . The K
for DT
of L cells expressing both DTR/proHB-EGF and DRAP27/CD9 for DT is still
lower than that of Vero cells(8, 17) . We showed here
that K
values of LH cells and LCH cells for DT in
the presence of heparin are similar to that of Vero cells. Therefore,
the lower abundance of heparin-like molecules on the cell surface is
likely to be responsible for the lower affinity of L cells.
We propose a model for the role of HSPG and DRAP27/CD9 in the binding of DTR/proHB-EGF molecule with DT (Fig. 9). Although the precise mechanism for the enhancing effect of DRAP27/CD9 is not clear, we speculate that DTR/proHB-EGF alone may not be stably retained on the cell surface. Association of DRAP27/CD9 with DTR/proHB-EGF may stabilize DTR/proHB-EGF, and thus DRAP27/CD9 would up-regulate DT binding and mitogenic activity. Cell surface HSPG, or exogenous heparin, binds to the heparin-binding domain of DTR/proHB-EGF, and changes the conformation of the EGF-like domain, resulting in increased of affinity for DT.
Figure 9:
Proposed model for the roles of DRAP27/CD9
and heparin-like molecules in binding of DT to DTR/proHB-EGF. a, DTR/proHB-EGF alone in the plasma membrane is not available
for DT-binding. b, DRAP27/CD9 binds to orients DTR/proHB-EGF,
making it accessible to DT. c, cell surface HSPGs or free
heparin bind to DTR/proHB-EGF at the heparin-binding domain, and induce
a conformational change that results in increased affinity of
DTR/proHB-EGF molecule for DT. d, the
DTR/proHB-EGFDRAP27/CD9
HSPG
DT
complex.
The present studies demonstrate that full DT
binding activity is achieved in the presence of heparin-like molecules.
DT sensitivity is higher at low cell densities than under confluent
conditions(39) , indicating that close contacts between cells
are not required for the maximal DT sensitivity. This implies that
DTR/proHB-EGF on the cell surface can interact with its HSPG on the
same cell. We showed recently that the complex comprising DTR/proHB-EGF
and DRAP27/CD9 associates with integrin 3
1 as
well(40) . DTR/proHB-EGF may form a large functional complex
including DRAP27/CD9, integrin
3
1 and HSPG. Although present
study does not define whether a specific class of HSPG is involved in
the interaction with DTR/proHB-EGF, studies of specificity of HSPG
interacting with DTR/proHB-EGF may provide further understanding of the
physiological role of DTR/proHB-EGF as a membrane-anchored growth
factor, as well as the DT receptor.