(Received for publication, August 22, 1994; and in revised form, October 21, 1994)
From the
The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.
Diphtheria toxin (DT) is a cytotoxic protein (M
= 58,342) that inhibits cellular protein
synthesis in eukaryotes by inactivating elongation factor 2 through
ADP-ribosylation(1, 2) . The toxin binds to a specific
receptor on the cell surface(3, 4) , then is
internalized by receptor-mediated endocytosis (5) and finally,
the enzymatically active A fragment is translocated to the cytosol (6, 7) where the A fragment inactivates elongation
factor 2.
The DT sensitivity is determined primarily by the presence or absence of specific receptors for DT(8, 9, 10) . DT receptor (DTR), originally identified in monkey Vero cells(3, 4, 11) , has been shown to be identical to the membrane-anchored form of heparin-binding EGF-like growth factor (HB-EGF)(12, 13) , first identified in the conditioned medium of macrophages and macrophage-like U937 cells(14, 15) . Analysis of the nucleotide sequence of human HB-EGF (hHB-EGF) cDNA predicts a membrane-anchored precursor protein composed of putative signal peptide, pro sequence, heparin-binding, EGF-like, transmembrane, and cytoplasmic domains(14) . The HB-EGF precursor can be cleaved to yield a mature biologically active growth factor containing 75-86 amino acids(16) . Many cell types can release a spontaneously soluble form and/or substantial amounts of the membrane-anchored form of HB-EGF which serves as DTR(13) . HB-EGF/DTR forms a complex with membrane protein DRAP27(13). DRAP27 has been shown to be identical to CD9(17) , and it up-regulates the number of functional HB-EGF/DTR and DT sensitivity in the presence of HB-EGF/DTR (13, 17, 18) .
HB-EGF is expressed in multiple tissues of human, monkey, mouse, and rat species and with a very similar tissue distribution(19) . However, while cells from human and monkey are highly sensitive to DT, those from mouse and rat are resistant(20, 21, 22, 23) . Given these observations, the question arises as to why mouse (e.g. L cells) and rat cells are resistant to DT despite expressing a considerable amount of endogenous HB-EGF(19) . Transfection of hHB-EGF cDNA into mouse L cells confers sensitivity to DT(12, 13) , suggesting that mouse L cells have the machinery needed for DT sensitivity but that for some reason mouse HB-EGF (mHB-EGF) is not a functional DTR. Since the HB-EGF precursor shows about 80% amino acid sequence identity between human and mouse (19) , one possibility is that mHB-EGF may not serve as a functional receptor for DT because of amino acid substitutions.
In this study, we have used serial human/mouse HB-EGF chimeras to study the relationship between HB-EGF primary structure and its function as a DTR. The results of these studies demonstrate that DT binds to the EGF-like domain of hHB-EGF but not of mHB-EGF due to amino acid substitutions. We also show that binding of non-toxic mutant DT, CRM197, to hHB-EGF, but not mHB-EGF, results in the inhibition of the mitogenic activity.
Figure 1:
Structures of human HB-EGF, mouse
HB-EGF, and human/mouse HB-EGF chimeras. A, alignment of amino
acid sequences of human and mouse HB-EGF. The amino acid residues are
numbered on each lane. Single and double underlines indicate the predicted signal sequence and the transmembrane
domain, respectively(14) . A basic amino acid cluster which
serves as a heparin-binding domain (HBD) is represented by a bold underline. EGF-like domains are indicated by shaded
boxes. Restriction enzyme sites used for constructing chimera
plasmids are indicated by the upper lines. B,
schematic structures of the HB-EGF chimeras and the summary for the
results of DT sensitivity and DT binding assays are shown. Shaded and open boxes indicate the regions of human HB-EGF and
mouse HB-EGF, respectively. ED were determined from the
data of Fig. 2. Specific binding of DT was determined by the
incubation with 100 ng/ml
I-DT as described under
``Materials and Methods.'' The values represent the average
of two independent experiments, and variations from the means are shown
in parentheses.
Figure 2:
DT sensitivity of LC cells transiently
expressing hHB-EGF, mHB-EGF, and the human/mouse chimeras. LC cells
were transfected with various plasmids and cultured for 2 days. Cells
were incubated with various amounts of DT for 2 h, followed by
incubation with 1 µCi/ml [H]leucine for 1 h.
Radioactivity incorporated in protein was determined. The values
represent the average of two independent experiments, and bars indicate variations from the means. Some points omitted bars to
avoid the complication of lines, but variations from the means were
less than 6%. A, LC cells expressing hHB-EGF (
), and
mHB-EGF (
), and LC cells transfected with the vector, pRc/CMV
(
). B, LC cells expressing H(1-50) (
),
H(1-68) (
), H(1-84) (
), H(1-106)
(
), H(1-136) (
), and H(1-186) (
). C, LC cells expressing H(186-208) (
),
H(136-208) (
), H(106-208) (
), H(84-208)
(
), H(68-208) (
), and H(50-208) (
). D, LC cells expressing H(106-136) (
),
H(136-186) (
), H(106-186) (
), and
H
(63-105) (
).
To show that mHB-EGF cannot serve as the DTR, the plasmid containing mHB-EGF cDNA, cloned from a macrophage library(19) , was introduced into recipient cells. LC cells, which are the stable transfectants of mouse L cells expressing DRAP27, were used as the recipient cells through this study. Although DRAP27 itself does not confer any DT sensitivity without HB-EGF/DTR, it up-regulates the number of functional HB-EGF/DTR and DT sensitivity in the presence of HB-EGF(13, 17, 18) . LC cells were transiently transfected with mHB-EGF cDNA. Immunoprecipitation of surface-biotinylated HB-EGF with anti-HB-EGF antibody showed the expression of substantial amounts of mHB-EGF protein on the cell surface (data not shown), but cells did not show any DT sensitivity (Fig. 2A). In contrast to mHB-EGF, transfection with hHB-EGF cDNA made these cells sensitive (Fig. 2A), confirming previous reports(13) . The rate of protein synthesis of LC cells was not diminished below 50% in the range of DT concentrations studied because the transfection efficiency was about 50% throughout the experiments, and the remaining cells were left untransfected. These results indicate that mHB-EGF does not have the optimal amino acid sequence for being a functional DTR.
To define
which part of substitutions are needed to confer DT sensitivity,
plasmids encoding various human/mouse HB-EGF chimeras were constructed.
Since the amino acid substitutions between human and mouse HB-EGF are
distributed throughout the entire region of the HB-EGF open reading
frame (Fig. 1A), serial chimeras were constructed,
covering the entire structure (Fig. 1B). The various
chimera plasmids were transiently transfected into LC cells and assayed
for DT sensitivity and DT binding. Among the chimeric HB-EGF constructs
containing human sequence in the N-terminal region, only the expression
of H(1-186) conferred DT sensitivity at the same level as
hHB-EGF; others, H(1-50), H(1-68), H(1-84),
H(1-106) and H(1-136), did not (Fig. 2B).
Among the chimeric HB-EGF constructs containing mouse sequence in the
N-terminal region, the expression of H(50-208), H(68-208),
H(84-208), and H(106-208) conferred full DT sensitivity at
the same level as hHB-EGF. Cells expressing H(136-208) showed
moderate sensitivity (about 100 times less sensitive than cells
expressing hHB-EGF), and cells expressing H(186-208) did not show
any sensitivity at all (Fig. 2C). These results
suggested that the human sequence between Asp and
Tyr
was sufficient to confer full DT sensitivity to LC
cells but that the homologous region in mHB-EGF was not. The importance
of the human sequence between Asp
to Tyr
for DT sensitivity was confirmed by three other chimeric HB-EGFs,
H(106-136), H(136-186), and H(106-186). Of these,
only the expression of H(106-186) conferred full DT sensitivity (Fig. 2D). The expression of H(136-186) slightly
increased DT sensitivity, similar to H(136-208), but the
expression of H(106-136) had no effect at all. These experiments
showed directly that the human sequence from Asp
to
Tyr
is essential for the expression of complete DTR
activity. Furthermore, to define the essential region for DT
sensitivity more precisely, we constructed plasmid encoding
H
(63-105) which is hHB-EGF with a deletion from Asp
to Arg
. LC cells expressing H
(63-105)
showed full DT sensitivity (Fig. 2D), indicating that
the region between Asp
to Arg
is not
necessary for DT sensitivity. These results were not due to different
transfection efficiencies or different expression levels of chimeric
HB-EGF molecules on the cell surface as could be shown by
immunofluorescence staining or immunoprecipitation of surface
biotinylated HB-EGF with anti-HB-EGF antibody (data not shown).
To
examine whether the inability of mHB-EGF to promote DT sensitivity was
due to a deficiency in DT binding, the DT binding activity of several
LC cells expressing transiently the native and chimeric forms of HB-EGF
was examined (Fig. 3, summarized in Fig. 1B). DT
binding correlated positively with DT sensitivity in that cells with
higher sensitivity to DT showed greater DT binding. These results
suggest that the inability of mHB-EGF to promote DT sensitivity stems
from its inability to bind DT due to the amino acid substitutions.
Furthermore, as shown in the Fig. 3inset, Scatchard
plot analysis demonstrated that the binding constant for DT of LC cells
expressing H(106-186) (K 3.1
10
M
) was almost same as that
obtained with native hHB-EGF (K
3.6
10
M
), indicating that this
chimeric HB-EGF sequence possesses full binding activity. In addition,
LC cells expressing H
(63-105) also showed the similar
binding constant for DT (K
4.7
10
M
) and further confirms that region
Asp
to Arg
is not necessary for the binding
of DT.
Figure 3:
Binding of I-DT to LC cells
transiently expressing hHB-EGF, mHB-EGF, and human/mouse chimeras. LC
cells were transfected with various plasmids and cultured for 2 days.
Cells were incubated with various concentration of
I-DT
for 9 h at 4 °C, and the cell-associated radioactivity was
determined. The data are expressed as specific binding. Nonspecific
binding of
I-DT for LC cells expressing hHB-EGF or
H(106-186), H(136-186), and H(106-136) was less than
5%, 10-20%, and 30-40%, respectively. The values represent
the average of two independent experiments, and bars indicate
variations from the means. The symbols used are hHB-EGF (
),
mHB-EGF (
), H(106-136) (
), H(136-186)
(
), or H(106-186) (
). Inset, Scatchard
plots of DT binding for hHB-EGF (
) and H(106-186)
(
).
So far our studies have indicated that
Asp-Tyr
in HB-EGF is the critical region
for the binding of DT and subsequent DT sensitivity. However, the
critical region may be narrower. We have demonstrated previously that
recombinant mature hHB-EGF, which is composed of
Arg
-Ser
, binds DT with an affinity similar
to that of the transmembrane HB-EGF/DTR(13) . This would
suggest that the amino acid residues in Leu
-Tyr
are not necessary for DT binding and that the critical region of
HB-EGF that mediates DT sensitivity is HB-EGF106-147, which is
essentially the EGF-like domain of HB-EGF.
HB-EGF is mitogenic via
the binding of its EGF-like domain to the EGFR (14) . The
binding of DT to the EGF-like domain of hHB-EGF suggests that DT might
be an inhibitor of the mitogenic activity of HB-EGF. Thus, the
possibility that CRM197, a non-toxic mutant form of DT (24) with similar or higher binding affinity to
DTR(31) , inhibits HB-EGF mitogenic activity was examined.
EP170.7 cells (26) require IL-3 or EGFR ligands for growth.
EP170.7 cells cultured with the human mature form of HB-EGF
(HB-EGF73-147) in the absence of IL-3 were stimulated to
incorporate [H]thymidine into DNA. Addition of
CRM197 completely inhibited DNA synthesis in EP170.7 cells cultured
with mature hHB-EGF but not with mature mHB-EGF (Fig. 4A). In
addition, CRM197 did not inhibit DNA synthesis in EP170.7 cells
cultured with IL-3, which ruled out the possibility that CRM197 itself
is directly toxic for the growth of EP170.7 cells. Since CRM197
inhibited the mitogenic activity of hHB-EGF, but not that of mHB-EGF,
it appears that the inhibition is due to interactions with specific
amino acids in the EGF-like domain of hHB-EGF substituted for in
mHB-EGF.
Figure 4:
Effect of CRM197 on the mitogenic
activities of human and mouse HB-EGFs and other EGF family growth
factors. EP170.7 cells were incubated with either one of several EGF
family growth factors (10 ng) or IL-3 (WEHI-3 cell-conditioned medium),
and various concentration of CRM197 for 48 h, followed by incubation
with [H]thymidine for 4 h. Radioactivity
incorporated into DNA was measured. The values represent the average of
two independent experiments, and bars indicate variations from
the means. A, the symbols used are hHB-EGF (
), mHB-EGF
(
), or IL-3 (
). B, the symbols used are
EGF(
), TGF-
(
), amphiregulin (
), or
-cellulin (
).
The EGF homologous growth factors, TGF-, EGF,
-cellulin, and amphiregulin also are mitogenic via the EGFR. As
shown in Fig. 4B, CRM197 did not inhibit any of these
EGF-like growth factors, demonstrating that CRM197 specifically
inhibits the mitogenic activity of HB-EGF.
DTR has been shown to be identical to the membraneanchored form of HB-EGF(12, 13) . HB-EGF/DTR is expressed in multiple tissues of many species including primate and murine(19) . Yet it has been known for a long time that murine cells are much less sensitive to DT than are primate cells. For example mouse L cells are about 100,000 times less sensitive to DT than are monkey Vero cells(9) . Several lines of evidence indicate that DT sensitivity is determined primarily by the number of DT-specific receptors, i.e. the amount of HB-EGF on the cell surface(4, 8, 9, 10) . Nevertheless, expressing cell surface HB-EGF is insufficient to produce DT sensitivity in mouse cells. We have demonstrated this directly in transfection experiments in which expression of hHB-EGF confers DT sensitivity to mouse cells, but expression of mHB-EGF does not. In exploring the mechanism of the differential sensitivity of human and mouse HB-EGF/DTR to DT, we have found that the ability to bind DT and the consequent toxicity is inherent in the primary structure of HB-EGF/DTR, particularly in the EGF-like domain. Non-conserved amino acid substitutions in mHB-EGF results in loss of DT sensitivity.
We
have concluded that the critical area for DT binding resides in the
residues Asp to Ser
as found in the human
sequence, but not in the mouse sequence. This is the EGF-like domain of
HB-EGF. The strongest evidence is that (i) a chimeric HB-EGF
encompassing human, but not mouse, amino acids Asp
to
Tyr
is fully active in mediating DT toxicity, (ii) that a
deletion mutant encompassing human Asp
to Arg
is also fully active, and (iii) that as previously shown,
secreted HB-EGF, encompassing Arg
to Ser
is
sufficient for full DT binding activity(13) .
The critical
amino acids within Asp to Ser
needed for DT
sensitivity have not been refined any further. Altogether there are 10
substitutions between human and mouse HB-EGF, 8 non-conserved residues
in the region Asp
to Pro
, and, in addition,
human Glu
mouse His
and human
Ser
mouse Thr
. That the chimera
H(106-136), which contains human residues 106-136, and the
chimera H(1-136), which contains human residues 1-136, are
mostly inactive in DT binding and sensitivity implicates the importance
of the downstream human Glu
and human Ser
residues in DTR activity. The relative inactivity of the chimera
H(136-186), which contains human residues 136-186, and the
chimera H(136-208), which contains human residues 136-208,
suggests that the human residues upstream of Pro
are
important for DTR activity. Further studies involving single amino acid
substitutions might provide additional insight into exactly which amino
acid residues in the EGF-like domain are the critical ones for DT
sensitivity.
We have also demonstrated that the non-toxic DT mutant
CRM197 inhibits specifically the mitogenic activity of hHB-EGF but not
the mitogenic activity of mHB-EGF. Studies of the structure-function
relationships for EGF-EGFR interactions indicate that several amino
acid residues located in anti-parallel -sheets of EGF as well as
those highly conserved amino acid residues in the other EGFR ligands
contribute to the EGF-EGFR interaction without drastic alteration of
the overall structure(32, 33, 34) . This
suggests that EGF binds to EGFR at the entire region of the molecule.
Present studies suggest that the interaction of hHB-EGF with DT may
also occur at the entire region of the EGF-like domain. Thus, it is
reasonable to speculate that inhibition of mitogenic activity of
hHB-EGF by CRM197 is due to the masking of or competition for binding
sites for EGFR on the hHB-EGF molecule.
Moreover, CRM197 does not inhibit other EGFR-binding growth factors. Therefore, although these EGF family members are also produced as membrane-anchored precursors and bind to EGFR in a manner similar to HB-EGF, HB-EGF is probably the only EGF family member that can function as an efficient receptor for DT among these growth factors. Thus, CRM197 may be useful as a specific inhibitor for hHB-EGF. Alteration of CRM197 structure, based on knowledge of the three-dimensional structure of DT, may be useful for producing highly potent inhibitors of HB-EGF, stronger than CRM197, or compounds of altered specificity that would inhibit other EGF family members.