(Received for publication, October 23, 1995; and in revised form, November 29, 1995)
From the
Pseudomonas exotoxin (PE) binds the heavy chain of the
-macroglobulin receptor/low density lipoprotein
receptor-related protein (LRP). To understand the significance of this
interaction, novel toxin-derived gene fusions were constructed with two
ligands that also bind this receptor. A 39-kDa cellular protein, termed
RAP, binds LRP with high affinity and often co-purifies with it. Two
RAP toxins were constructed, one with PE and one with diphtheria toxin
(DT). RAP, which replaced the toxins binding domains, was combined with
each of the corresponding translocating and ADP-ribosylating domains.
Both RAP-toxins bound LRP with an apparent higher affinity than native
PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE.
Apparently, RAP-toxin molecules bound and entered cells but used a
pathway that afforded only low efficiency of toxin transport to the
cytosol. This was evident because co-internalization with adenovirus
increased the toxicity of RAP-toxins by 10-fold. We speculate that the
high affinity of RAP binding may not allow the toxin's
translocating and ADP-ribosylating domains to reach the cytosol but
rather causes the toxin to take another pathway, possibly one that
leads to lysosomes. To test this hypothesis, additional RAP-PE fusions
were constructed. N-terminal or C-terminal fragments of RAP were joined
to PE to produce two novel fusion proteins which were likely to have
reduced affinity for LRP. Both of these shorter fusion proteins
exhibited greater toxicity than full-length RAP-PE. A second
ligand-toxin gene fusion was constructed between plasminogen activator
inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1
formed a complex with tissue-type plasminogen activator and inhibited
its proteolytic activity. However, like the RAP-toxins, this hybrid was
less toxic for cells than native PE.
The -macroglobulin receptor/low density
lipoprotein receptor-related protein (LRP) (
)is one of the
largest membrane-associated proteins characterized to date. Its primary
amino acid sequence was derived from overlapping cDNA
clones(1) . LRP is located both on surface and intracellular
membranes of many eukaryotic cell types. It is synthesized as a
4525-amino acid single chain precursor. After synthesis, possibly in
the Golgi, single chain LRP is cleaved to give a 515-kDa heavy chain
and an 85-kDa light chain(2) . The light chain contains both a
membrane-spanning region and cytoplasmic tail. The heavy chain which
remains membrane-associated via non-covalent interactions with the
light chain, contains the ligand-binding sites. LRP mediates the
binding and endocytosis of several unrelated ligands: 1) apoprotein
E-enriched
-migrating very low density
lipoprotein(3, 4) ; 2) protease- or
methylamine-activated
-macroglobulin(5) ; 3)
the complex between tissue-type plasminogen activator (tPA) or
urokinase with plasminogen activator inhibitor type 1
(PAI-1)(6, 7) . A 39-kDa receptor-associated protein,
called RAP, also binds to LRP(8, 9) . Recently, it was
shown that RAP has two regions that can independently, although less
efficiently, interact with LRP and another protein from the same
family: gp330(10, 11, 12) . RAP inhibits the
binding and or uptake of all known ligands that interact with LRP. At a
minimum, RAP binds LRP with high affinity at two sites and possibly
causes a conformational change in receptor structure. The addition of
either heparin or
-mercaptoethanol inhibits the interaction of RAP
with LRP(13) . Recently, LRP was proposed as the cell surface
receptor for Pseudomonas exotoxin
(PE)(14, 15, 16) .
Both PE and diphtheria toxin (DT) inactivate the protein synthetic apparatus of eukaryotic cells in a series of steps that include: binding to a surface receptor, endocytic uptake, cell-mediated proteolytic processing to generate an enzymatically active fragment, translocation of the fragment to the cell cytosol, and ADP-ribosylation of elongation factor 2.
PE is synthesized as a single chain bacterial protein composed of three structural domains. The N-terminal domain mediates binding to LRP: specifically, Lys-57 and possibly other nearby residues mediate binding (17) . Toxicity for cells is reduced by at least 100-fold when Lys-57 is changed to glutamic acid or when most or all of this domain is deleted. The middle domain of PE has two functions: it contains sequences necessary for translocation to the cell cytosol and it serves as a substrate for cell-mediated cleavage(18, 19) . The C-terminal domain has the ADP-ribosylating activity and contains a putative endoplasmic reticulum (ER) retention sequence(20, 21, 22) . Functionally DT is quite similar to PE. However, the binding and ADP-ribosylating domains of DT are located in the opposite orientation to PE, i.e. the binding domain of DT is at the C terminus (23) while the ADP-ribosylating activity is at the N terminus(24) . Like PE, the middle domain of DT contains sequences that mediate translocation to the cell cytosol(25) . While the exact intracellular location for the translocation of either toxin has not been clearly defined, existing data suggests that the A chain of DT reaches the cytosol from an acidic endosomal compartment while the corresponding PE fragment needs to reach the ER to facilitate its translocation. Thus it appears that these functionally similar toxins use different intracellular pathways to reach the same cytosolic location.
By replacing the binding domains of either PE or DT with binding ligands of various specificities it has been possible to redirect toxic activity to cells bearing particular cell surface receptors(26) . For instance, DT and PE have been targeted to IL-2, IL-4, IL-6, and epidermal growth factor receptors. And although some losses in binding affinities has been noted, it is often possible to produce active hybrid toxins by placing a particular ligand either at the N terminus of PE or the C terminus of DT.
As a way to study both intracellular receptor traffic and possible differences in toxin processing, we have constructed a number of ligand-toxin hybrids which bind to LRP. Because ligands will interact with different portions of the same receptor and possibly bind with different affinities, it may be possible to determine which interactions lead to efficient delivery of active toxin fragments to the cell cytosol and which do not.
To create hybrid molecules with cytotoxic characteristics that can be compared with native PE, we have replaced the receptor-binding domains of DT and PE with either RAP or tPA-PAI-1. Results indicated that these hybrid proteins exhibited much lower cytotoxicity than native PE. We also produced RAP-PE fusions composed of RAP fragments in place of full-length RAP. These proteins were more active than toxin fusions made with whole RAP. Based on these data we speculate that RAP- and tPA-PAI-1-containing recombinant proteins possess lower cytotoxicity than PE because they are less capable of dissociating from LRP and therefore are transported to lysosomes more readily than PE.
To determine the
effect of adenovirus on cytotoxicity of RAP-PE, 96-well plates were
used. Cells at 4 10
per well (96-well plates) were
seeded 1 day prior to evaluating the cytotoxicity. Various
concentrations of toxins and RAP-PE were added to cells for 2 h with or
without non-toxic amounts of adenovirus type 2 (approximately 1
µg/ml of virus) at 37 °C. At the end of this period, the
existing medium was substituted by fresh medium containing
[
H]leucine (0.5 µCi/ml) and cells were
incubated further at 37 °C for another 1 h.
Figure 1:
Construction of
plasmids encoding RAP-PE. Boxes show the relative positions of
coding sequences and regulatory elements: P and lacZ`, promoter and LacZ
encoding sequence;
10 and
T
, fragments of T7 bacteriophage DNA which encode the
10
promoter and transcription termination signal, respectively; eta` and
10, sequences encoding a truncated version of PE
that lacks the first 103 N-terminal amino acid residues and a fragment
of
MR/LRP, respectively; spA and rap, sequences encoding the immunoglobulin-binding region of S. aureus protein A and RAP,
respectively.
Next we investigated whether or not the RAP fusions retained
their ability to interact with LRP. Results obtained with an
enzyme-linked immunosorbent assay indicated that RAP-PE bound to LRP at
both pH 5.5 and 7.2, and that the addition of heparin (9 µg/ml) as
a competitor reduced this binding to very low levels (Fig. 3).
Previously, Moestrup and Gliemann (13) reported that the
addition of heparin completely abolished the binding of RAP to LRP.
Furthermore, when we preincubated heparin with heparinase its blocking
activity was completely lost, indicating that specific sequences of
monosaccharides within heparin were responsible for the inhibition.
Using the same assay conditions, PE was shown to bind to LRP but with a
much lower apparent affinity (Fig. 3). PE binding was not
inhibited by heparin. In ligand blots following SDS-PAGE run under
non-reducing conditions, DT-RAP was seen to interact with the heavy
chain of LRP (data not shown). This binding was abolished when LRP was
treated with -mercaptoethanol. As a further indication that DT-RAP
retained LRP binding activity, we report that immobilized DT-RAP could
be used to affinity purify LRP from beef liver (data not presented).
Figure 3:
PE and RAP-PE binding to immobilized LRP.
2-fold dilutions of affinity-purified LRP were immobilized on Immulon
microtiter plates. Following the addition of bovine serum albumin, PE
(5 µg/ml) or RAP-PE (7.5 µg/ml) with or without heparin (9
µg/ml) were added to the receptor-coated wells in
phosphate-buffered saline, pH 7.2, 0.1% Tween 20. Wells were washed
three times for 10 min with either phosphate-buffered saline, pH 7.2,
0.1% Tween 20 or 0.3 M sodium acetate, pH 5.5, 0.1% Tween 20.
Evidence of PE and RAP-PE binding was obtained by adding
affinity-purified rabbit anti-PE that was detected using a Vectastain
kit (Vector Laboratories, Inc.). , PE + heparin, pH 7.2;
, PE, pH 7.2;
, PE, pH 5.5;
, RAP-PE + heparin,
pH 7.2;
, RAP-PE, pH 7.2;
, RAP-PE, pH
5.5.
Previously, it was shown that the addition of RAP to intact cells, blocked PE binding and PE-mediated cytotoxicity(14) . In Fig. 4we show that the addition of DT-RAP inhibited cytotoxic activity of PE for L929 cells. Significant reversal of PE toxicity was seen when DT-RAP was added in the range of 11-33 nM. (At these concentrations, DT-RAP by itself, did not inhibit protein synthesis.) Similar RAP concentrations are known to block the binding of physiologic ligands.
Figure 4:
Competition assay using DT-RAP as a
competitor for PE toxicity. Increasing concentrations of DT-RAP were
added to L929 cells together with PE (300 ng/ml) for a total of 4 h.
PE553 is an enzymatically inactive derivative of PE that was used
as a positive control for competition. Results are expressed as the
percent of control protein synthesis compared to cells receiving no
toxin.
We also found that the fusion of PE with RAP did not alter the susceptibility of PE to furin-mediated cleavage (Fig. 5, arrows, indicate the generation of proteolytic fragments with time of incubation).
Figure 5:
Cleavage of PE and RAP-PE by furin in
vitro. PE (lanes 1-7) and RAP-PE (lanes
9-15) were incubated in 200 mM sodium acetate, 4
mM CaCl, pH 5.5, without furin (lanes 1 and 9) or with furin for 4 h (lanes 2 and 10), 7 h (lanes 3 and 11), 10 h (lanes 4 and 12), 13 h (lanes 5 and 13), 18 h (lanes 6 and 14), and 20 h (lanes 7 and 15) followed by analysis on SDS-PAGE and staining with
Coomassie Blue. Numbers correspond to molecular masses of
protein standards (lane 10). Arrows indicate the
position of PE and RAP-PE fragments generated by
furin.
To determine if RAP toxin was being internalized to the endosomal compartment, cells were co-incubated for 2 h with RAP-PE and non-toxic amounts of adenovirus type 2 (approximately 1 µg/ml of virus). Previously, we showed that adenovirus disrupts endosomal membranes and releases the contents to the cytosol(35) . Results indicated that when adenovirus and RAP-PE were added to cells together, cytotoxicity was enhanced by approximately 5-10-fold (Fig. 6). From this we conclude that RAP-PE binds and enters cells but is less toxic than the native toxin because the toxin portion of the hybrid is transported to a location which does not allow efficient translocation to the cytosol.
Figure 6:
Effect of adenovirus on the sensitivity of
COS cells to RAP-PE. RAP-PE alone or together with adenovirus was added
to COS cells for 2 h. At the end of the incubation period, the level of
protein synthesis was determined by measuring the incorporation of
[H]leucine into cellular protein.
, PE;
, RAP-PE;
, RAP-PE +
adenovirus.
To examine possible reasons for the relatively low toxicity of the RAP-toxins, additional experiments were performed on three cell lines all having the same genetic background. Wild type (WT) Chinese hamster ovary cells (clone CL6) and two lines characterized as PE-resistant (13-5-1) and PE-supersensitive(221-1) were used. The 13-5-1 cells have no detectable LRP (16) while the 221-1 line appears to express slightly higher levels of LRP than wild type. Compared to WT, the LRP-negative line was 100-fold resistant to PE, while the 221-1 was 3-fold more sensitive (Fig. 7). On WT and 221-1 cells RAP-PE was 5- and 10-fold less active than native PE, respectively (Fig. 7). While 13-5-1 cells were 10-fold resistant to RAP-PE compared to WT, the absolute activity of RAP-PE and PE for this cell line was quite similar. DT-RAP had about the same activity as RAP-PE on WT and 221-1 cells (Fig. 7). However, the 13-5-1 cells exhibited only slight resistance to DT-RAP. Thus, in the context of a RAP fusion, PE toxicity was much more dependent on delivery by LRP than was DT. Since DT can translocate from the endosomal compartment and PE must reach the endoplasmic reticulum, it is possible that internalization on any receptor will allow efficient delivery of DT to the cytosol but not PE (see ``Discussion'').
Figure 7:
Toxicity of RAP-toxins for wild-type and
mutant lines of Chinese hamster ovary cells. Purified proteins were
added to wild-type Chinese hamster ovary cells as well as to lines
221-1 and 13-5-1 for an overnight incubation at 37 °C. At the end
of the incubation period, the level of protein synthesis was determined
by measuring the incorporation of [H]leucine into
cellular protein.
Figure 8:
Cytotoxicity of PE, RAP-PE, and RAP
fragments PE for COS cells. Proteins were added to COS cells for an
overnight incubation at 37 °C. At the end of the incubation period
the level of protein synthesis was determined by measuring the
incorporation of [H]leucine into cellular
protein.
, RAP(1-323)-PE;
, RAP(1-175)-PE;
, RAP(172-323)-PE; +, PE. The numbers in parentheses indicate the residues from RAP that were present
in each construction.
Figure 2: Construction of plasmids encoding DT-RAP and DT-PAI-1. toxA, toxB`, and toxB", sequences encoding fragment A and portions of DT B-fragment; pai-1, sequence encoding PAI-1. The remaining symbols are the same as in Fig. 1.
Figure 9: Interaction of DT-PAI-1 with tPA. The ability of DT-PAI-1 to interact with tPA was assessed by co-incubation of the two proteins followed by analysis on SDS-PAGE and staining with Coomassie Blue. Lanes: 1, DT-PAI-I incubated with tPA; 2, DT-PAI-I; 3, tPA; and 4, molecular weight markers.
Figure 10:
Cytotoxicity of DT-PAI-1-tPA and DT-PAI-1
for COS cells. Proteins were added to COS cells for an overnight
incubation at 37 °C. At the end of the incubation period the level
of protein synthesis was determined by measuring the incorporation of
[H]leucine into cellular protein.
, PE;
, DT;
, DTPAI-1-tPA;
,
DTPAI-1.
PE enters cells by receptor-mediated endocytosis. Endocytosis is the beginning of the toxin pathway that results in the generation of an enzymatically active fragment, the translocation of this fragment to the cell cytosol and, ultimately, the inhibition of protein synthesis. Previously, we reported that PE binds to the heavy chain of LRP and most likely uses this receptor for endocytic uptake(14) . LRP is a multiligand receptor whose function is the clearance and degradation of proteases and ligands related to lipid metabolism. Therefore, most of LRP ligands end up in the lysosome where they are degraded. For PE, some percentage of entering molecules somehow avoid this fate. Existence of an endoplasmic retention-like sequence at the C terminus of PE and results of mutational analysis of this sequence suggest that PE or its enzymatically active fragment is transported to the ER where it translocates to the cytosol.
Among the ligands that bind LRP, are RAP and the complex between tPA and PAI-1. To compare the fate of these ligands with PE, we constructed PE and DT hybrid proteins that have RAP or PAI-1 in place of the toxins' receptor-binding domains.
We found that DT-PAI-1 was able to inhibit the catalytic
activity of tPA and form an SDS-resistant complex with this protease.
This data suggest that the N terminus of PAI-1 is not essential for its
interaction with tPA. Toxicity was clearly specific for the
DT-PAI-1tPA complex since the addition of DT-PAI-1 alone was at
least 10-fold less active. However, compared with DT and PE, the
DT-PAI-1 complexed with tPA exhibited a much lower level of toxicity
for COS cells.
RAP-PE and DT-RAP were also less toxic than PE for mammalian and insect cells. We showed that these proteins retained their binding activity for the heavy chain of LRP. Since the RAP cDNA is fused at the 5` end of the PE gene and 3` end of the DT gene, it appears that neither the N or C terminus of RAP are required for interaction with LRP.
Among various cell lines we did not find a
correlation between PE and RAP-toxin sensitivities. Nevertheless, we
were able to see such a correlation when the toxins were tested on
isogenic cell lines. In particular, a PE-resistant line, 13-5-1, that
lacked detectable LRP protein was less sensitive to RAP-toxins. A
second mutant, 221-1, that expressed higher amounts of LRP than wild
type cells and was supersensitive to PE by 3-5-fold, was also
more sensitive to RAP-toxins. The lack of a good correlation between PE
and RAP-toxin toxicities may be explained by results of several authors
who have demonstrated the existence of additional receptors for RAP
besides LRP. Indeed, a recent report has indicated that Chinese hamster
ovary cells express the very low lipoprotein receptor, which in
ligand blots, interacts with RAP but not PE(39) . Thus in
13-5-1 cells, the toxicity of RAP-toxins, that is likely to be mediated
by the
very low density lipoprotein receptor, could be assessed.
From the upper and middle panel of Fig. 7it
was clear that the toxicity of both PE and RAP-PE apparently relied
heavily on the presence of LRP. However, this was not true for DT-RAP,
because the 13-5-1 line was not greatly resistant to this hybrid toxin.
Since PE translocates from the ER and DT from an acidic endosome, the
intracellular transport requirements for these two toxins is likely to
be quite different. One interpretation would suggest that 13-5-1 cells
were not very resistant to DT-RAP because interactions with the
very low density lipoprotein receptor were sufficient to mediate DT
transport to an acidic endosome. RAP-PE was less active in 13-5-1 cells
presumably because
very low density lipoprotein receptor-mediated
internalization did not facilitate transport to the ER.
To understand the reason for the lower toxicities of RAP-toxin chimeras, we checked the ability of RAP-PE to interact with LRP and to be cleaved by furin. RAP-PE appeared to interact with LRP with much higher affinity than the native toxin. At the same time substitution of the N-terminal portion of the receptor binding domain of PE by RAP did not dramatically change the ability of furin to cleave PE. It was therefore reasonable to suggest that either RAP-PE was not internalized by the cell or that after internalization, it followed a different pathway than that taken by native PE. To determine if RAP-PE was internalized to the endosomal compartment, we performed a co-incubation with adenovirus. We reasoned that if RAP-PE could reach the endosome, then adenovirus would enhance toxicity by releasing more toxin than was possible in the absence of virus. Toxicity was enhanced by 5-10-fold.
In conclusion, we speculate that a relatively low binding affinity allows native PE to dissociate from LRP and thus to avoid the fate of other LRP ligands, which is to be degraded in the lysosome. Our data showing increased cytotoxicity when RAP fragments were fused to PE instead of whole RAP seems to support this hypothesis. This finding may have wide reaching implications for the design of recombinant immunotoxins.
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