(Received for publication, January 20, 1995; and in revised form, July 5, 1995)
From the
A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.
Cell-surface structures mediating the efficient internalization
of cell-bound molecules are frequently selected as targets of
monoclonal antibody/ligand-toxin conjugates (immunotoxins
(IT))(1) . Rapid internalization, however, is not
always synonymous with fast intoxication rates of the target cells as a
result of cell mechanisms leading to inactivation of the internalized
IT molecules (e.g. recycling, degradation, slow routing to
subcellular compartments competent for toxin translocation) (1) . The ricin toxin A chain (RTA) is a potent
ribosome-inactivating enzyme used in the synthesis of highly selective
IT. However, RTA-based IT exert their effect at relatively high
concentrations due to poor translocation of RTA to the cell cytosol
from the endocytic compartments where the IT are
internalized(1) .
Viruses utilize specialized envelope structures that allow them to enter the cytosol of the infected cells. We reasoned that it might be possible to modify a cytotoxic enzyme (i.e. RTA) by fusing it to a protein structure derived from viral envelopes, thus conferring to the cytotoxic enzyme the cytosol targeting properties of the virus. A peptide representing the primary sequence of the 25 N-terminal amino acids of protein G of the vesicular stomatitis virus envelope (KFT25) was found to have pH-dependent membrane destabilizing properties(2, 3) . In particular, at low pH, KFT25 was shown to be hemolytic, to mediate hemagglutination, to be cytotoxic for mammalian cells, and to effect gross changes in cell permeability(2, 3) . Such a virus-derived structure might be endowed with the ability to facilitate the translocation of heterologous proteins across cell membranes when they are routed to acidic intracellular compartments.
The transferrin receptor is a cell-surface structure known to deliver internalized protein-protein conjugates to acidic compartments (i.e. endosomes)(4, 5) . The physiology of the transferrin receptor and of its ligand has been well studied, and Tfn-toxin conjugates have found applications in the laboratory as well as in the clinic as antitumor reagents(6, 42) . Internalized Tfn and Tfn-toxin conjugates are directed to acidic prelysosomal compartments within the cell(4, 5) . Tfn was therefore chosen as an appropriate vehicle molecule to investigate whether RTA cell entry would be improved by fusion with KFT25.
In this preliminary report, we show that a KFT25-containing RTA (chimeric RTA (cRTA)) exhibits a greater cytotoxic activity when delivered to tumor cells by Tfn than analogous conjugates containing either native RTA (nRTA) or unmodified recombinant RTA (rRTA). These results open up the possibility of taking advantage of specialized viral structures to increase the cytosolic localization of toxins or other biologically active proteins within target cells.
For expression, pRICA or pRAK25 recombinant plasmids were
introduced into E. coli strain SURE (Stratagene) by rubidium
chloride-mediated transformation. Cultures were grown at 37 °C in 1
liter of M9 medium supplemented with 0.2% glucose, 1 mM MgSO, 0.1 mM CaCl
, and 30
µM vitamin B
in the presence of 27 µM ampicillin to A
= 0.8. The
temperature of the growth culture was then lowered to 30 °C, and 1
mM isopropyl-
-D-thiogalactopyranoside was added.
After 3 h, the cells were pelleted, incubated for 15 min in prechilled
lysis buffer (phosphate-buffered saline/EDTA (5 mM),
phenylmethylsulfonyl fluoride (8 µl/g of pellet of a 8 mg/ml
solution in isopropyl alcohol), lysozyme (80 µl/g of pellet of a 10
mg/ml solution), and sonicated on ice in four
45-s bursts using
a Labsonic-U ultrasonic disintegrator (B. Braun Bitech International).
Lysates were cleared by centrifugation at 12,000
g for
30 min at 4 °C. Cell-free lysates were dialyzed overnight against 5
mM phosphate buffer (pH 6.5) and passed through an
ion-exchange column (2.5
12 cm) of CM-Sepharose equilibrated in
dialysis buffer. Bound proteins were eluted with a 0-500 mM NaCl gradient, and fractions were then analyzed by
SDS-polyacrylamide gel electrophoresis and Western blotting. The purity
of rRTA- and cRTA-containing fractions was >90% as evaluated by
scanning densitometry with a GS-300 gel-scanning apparatus (Hoefer
Scientific Instruments). The biological activity of purified rRTA and
cRTA was determined by their ability to inhibit
[
S]Met incorporation into proteins in a rabbit
reticulocyte lysate (Boehringer Mannheim) and was compared with that of
nRTA. nRTA was kindly provided by Dr. P. Casellas (Sanofi Recherche,
Montpellier, France).
Aliquots of lipid vesicles (12 µg) prepared as described above were introduced into a quartz cuvette (optical length of 1 cm) containing 1 ml of phosphate-buffered saline/EDTA. The increase in light scattering after addition of the toxins to the SUV suspension was measured using a FluoroMax spectrofluorometer (Spex Industries) at excitation and emission wavelengths of 340 nm. Data were acquired and then processed by computer analysis with dM 3000 software (Spex Industries). Data are expressed as arbitrary units. During light scattering determinations, the temperature was regulated and maintained constant at 25 °C by a circulating heating bath, and the solution in the sample cuvette was continuously stirred magnetically. The pH of the SUV solutions containing toxins was adjusted to the desired acidic values by addition of acetic acid. To compare the activity of the toxins in kinetic assays, we considered the time required to reach 50% of the maximal change in the light scattering properties of the SUV suspension (t).
To measure the amount of active toxin present in each
conjugate, we performed cell-free assays of protein synthesis
inhibition. Equal concentrations of Tfn-nRTA, Tfn-cRTA, and Tfn-rRTA
were preincubated overnight at 4 °C with 0.14 mM dithiothreitol in order to break the S-S bond introduced
between Tfn and the toxins. This procedure was required because
conjugated RTA is enzymically inactive(11) . The full reduction
of the S-S bond was monitored by SDS-PAGE analysis. The samples
were then serially diluted and added to a rabbit reticulocyte lysate.
Incorporation of [S]Met into proteins was
measured. Dithiothreitol did not interfere with the incorporation of
the radiolabeled amino acid.
Three forms of RTA were used in our experiments: nRTA (i.e. RTA prepared from native ricin that had been purified from the seeds of Ricinus communis), unmodified rRTA, and cRTA (recombinant RTA fused to KFT25). A cRTA cloning strategy was developed to preserve in the expressed cRTA molecule the N-terminal orientation possessed by the KFT25 peptide in vesicular stomatitis virus protein G (Fig. 1A). The insertion of the KFT25 DNA sequence at the 5`-end of the RTA coding region did not affect the expression level or the post-translational localization of cRTA with respect to rRTA. With both pRICA and pRAK25 expression vectors, the amount of toxin purified from bacterial cultures ranged from 1.5 to 2 mg/liter. No cRTA was found into the periplasmic fraction, in the culture medium, or stored into inclusion bodies (data not shown).
Figure 1:
Cloning and purification of recombinant
toxins. A, the BamHI-BamHI RTA coding region
from pRICA(7) . The reported oligonucleotide sequence,
corresponding to the KFT25 peptide, was ligated between BamHI
and XbaI restriction sites present within the RTA fragment. A
partial post-translational proteolytic cleavage of KFT25 from purified
cRTA molecules takes place at Arg. B, SDS-PAGE
migration and Western blot identification of purified nRTA (lane1), rRTA (lane2), and cRTA (lane3). The two electrophoretically separated cRTA forms were
both recognized by anti-RTA antibody (arrowheads).
Fig. 1B shows the SDS-PAGE migration and Western blot identification of
rRTA and cRTA following expression in E. coli and purification
by ion-exchange chromatography. Control nRTA typically migrated in two
distinct bands because of the different glycosylation of the toxin
molecules(15) . As expected, rRTA migrated faster than
glycosylated nRTA. cRTA separated instead into two electrophoretically
distinct forms, both recognized by anti-RTA antibody. Microsequencing
of proteins recovered from SDS-PAGE revealed the presence of a
proteolytic cleavage site at Arg responsible for the
removal of the KFT25 peptide in
30% of cRTA molecules (Fig. 1), which accounted for the doublet in lane3.
To investigate whether cRTA retained the enzymic
properties of the original molecule, the protein synthesis inhibition
activity of nRTA, rRTA, and cRTA was compared in a rabbit reticulocyte
lysate. cRTA inhibited protein synthesis in a manner that was
comparable to nRTA and rRTA (IC = 10, 26.6, and 40
pM, respectively). These results demonstrated that fusion to
KFT25 had not affected the enzymic properties of RTA.
Protein G of the vesicular stomatitis virus, reconstituted in phospholipid vesicles, was shown to induce liposome fusion at pH <5.0 using PC/PS (1:1 molar ratio) SUV as target vesicles(16) . The rate of fusion dramatically increased at pH values in the range 2.0-4.0(16) .
Lipid vesicles possess the well defined property of deviating light in a way highly dependent on their dimensions and aggregation state. Hence, the amount of light scattered by the lipid suspension is a very sensitive parameter of phenomena leading to liposome aggregation and/or fusion that may be triggered by the interaction of a protein with the lipid layer(17, 18, 19) . To directly evaluate the acquired pH-dependent membrane destabilizing properties of cRTA, we have measured the changes in the light scattering shown by a PC/PS (1:1 molar ratio) SUV suspension in the presence of cRTA at different pH values. For comparison, phospholipid vesicles were also treated with nRTA.
Fig. 2shows the pH dependence of the changes in light
scattering of SUV treated with cRTA and nRTA. At pH values below 5.0,
the light scattering of SUV increased rapidly following the addition of
cRTA (t 2 s; Fig. 2A, inset).
The kinetics of interaction of nRTA with PC/PS SUV were instead slower (t
30 s; Fig. 2B, inset). Even
though at early times cRTA had a greater effect than nRTA on PC/PS SUV (Fig. 2A), at later times (stationary state), both
toxins induced a comparable increase in the light scattering properties
of the SUV suspension (Fig. 2B). These results confirm
previous observations that the ricin A chain has intrinsic properties
of membrane interaction(20, 21) . It is noteworthy
that the results shown for cRTA in Fig. 2A overlap
those obtained for the pH-dependent protein G-mediated fusion of PC/PS
vesicles reported by Eidelman et al. (16) and therefore are a
direct demonstration that at least part of the membrane destabilizing
properties of protein G have been transferred to the ricin A chain by
linkage of the KFT25 peptide with the toxin sequence.
Figure 2:
pH dependence of the effects of cRTA and
nRTA on PC/PS SUV. cRTA () and nRTA (
) at a final
concentration of 165 nM were added to a PC/PS (1:1 molar
ratio) SUV suspension, and the light scattering properties of the
liposome mixtures at different pH values were measured in kinetic
experiments. The plots represent light scattering measurements at the
time points of 20 s (A) and 125 s (stationary state; B) after addition of the toxins to the lipid vesicles. Insets, kinetics of the changes in the light scattering
properties of the liposomes induced by treatment with cRTA (A)
and nRTA (B) at the reported pH values. In both insets, the arrows show the time at which the toxins (TOX) were added to the SUV suspension. a.u.,
arbitrary units.
Fig. 3shows the dose-dependent pattern of the change in the light scattering of SUV as a function of the amount of cRTA added at pH 3.1. cRTA increased the light scattering of PC/PS SUV at low concentrations, and this increase appeared to reach a plateau at the higher concentrations tested (Fig. 3). At the end of the assays, visible precipitates of phospholipid vesicles were observed, indicating a great extent of vesicle aggregation and/or fusion. It should be noted that cRTA is active on phospholipid vesicles at acidic pH at concentrations comparable to those observed also for diphtheria toxin, whose pH-dependent membrane destabilizing properties are well documented(22) .
Figure 3: Dose dependence of the effects of cRTA on the light scattering properties of PC/PS SUV at pH 3.1. Varying amounts of cRTA were added to lipid vesicles. The total effect at pH 3.1 on PC/PS (1:1 molar ratio) SUV was measured after 125 s (stationary state). a.u., arbitrary units.
The membrane destabilizing properties of
KFT25 are activated at pH values below 6.0 in erythrocytes and
nucleated cells(2, 3) . On the other hand, Tfn is
internalized and transported within endosomes whose pH was shown to be
5.5(23) . To investigate whether cRTA had acquired cytosol
localizing properties, we synthesized Tfn-cRTA conjugates and compared
their cytotoxic effect with that of Tfn-nRTA and Tfn-rRTA conjugates.
SDS-PAGE analysis of the three conjugates under reducing and
nonreducing conditions followed by Western blot analysis with anti-Tfn
and anti-RTA antibodies and scanning densitometry revealed the presence
of comparable amounts of nRTA, cRTA, and rRTA conjugated to Tfn
(Tfn/RTA ratios of 1:1.27, 1:1.32, and 1:1.33 for Tfn-nRTA, Tfn-cRTA,
and Tfn-rRTA, respectively). To make sure that the conjugation
procedures had not inactivated the enzymic properties of nRTA, cRTA,
and rRTA, the protein synthesis inhibition activity of the three
conjugates was compared in a rabbit reticulocyte lysate. The measured
IC values were 7, 14, and 10 pM for Tfn-nRTA,
Tfn-cRTA, and Tfn-rRTA, respectively, further demonstrating that the
three conjugates have comparable enzymic and biochemical properties. It
should be noted that these IC
values are calculated for
molecules with M
values of 118,100, 119,600, and
119,900 for Tfn-nRTA, Tfn-cRTA, and Tfn-rRTA, respectively, and hence,
they are not directly comparable with those obtained with unconjugated
toxins (see above). Correction of IC
values obtained with
Tfn-toxin conjugates for molecular composition revealed that the toxins
were not inactivated by the conjugation procedures.
We then
investigated the cell killing potential of Tfn-nRTA, Tfn-cRTA, and
Tfn-rRTA and of free toxins against tumor cells. As shown in Fig. 4(upperpanel), Jurkat cells were equally
intoxicated by unconjugated nRTA and rRTA (IC = 1.5
10
and 2.2
10
EU,
respectively). cRTA was instead
10-fold less toxic against target
cells (IC
= 1.6
10
EU) despite
an enzymic activity that was comparable to that of nRTA and rRTA (see
above). cRTA conjugated to Tfn (Tfn-cRTA) was, however,
10-20-fold more toxic against tumor cells than Tfn-nRTA or
Tfn-rRTA (IC
= 1.6, 37, and 20 EU, respectively, in
a 24-h protein synthesis inhibition assay) (Fig. 4, center
panel). Results comparable to those obtained with Jurkat cells
were also observed with Raji, CEM, K562, and MCF7 cell lines (data not
shown). Addition of the Tfn-RTA IT enhancer monensin increased the
cytotoxicity of Tfn-cRTA, Tfn-nRTA, and Tfn-rRTA (IC
= 1.2, 0.6, and 2.0 EU, respectively, in a 6-h assay) (Fig. 4, lower panel). Monensin also abrogated the
differences in cell killing between Tfn-cRTA and the other two
conjugates. Monensin neutralizes the pH of endocytic
vesicles(24) . Thus, the higher cytotoxic activity observed for
Tfn-cRTA in the absence of monensin strongly suggests that the
pH-dependent membrane destabilizing properties of KFT25 might
facilitate the translocation of cRTA to the cell cytosol.
Figure 4:
Cell killing effects of toxins and
Tfn-toxin conjugates on Jurkat cells. Upperpanel,
protein synthesis inhibition activity of nRTA (), rRTA (
),
cRTA (
), and scRTA (
) in a representative 24-h assay; center and lower panels, cytotoxicity of Tfn-nRTA
(
), Tfn-rRTA (
), Tfn-cRTA (
), and Tfn-scRTA
(
) in 24-h assays (centerpanel) or in 6-h
assays carried out in the continuous presence of monensin (lowerpanel). The assays were repeated two to four times with
<10% variability. IT or toxin concentrations are expressed in enzyme
units (see ``Experimental
Procedures'').
The KFT25
peptide contains a potentially reactive Cys residue that could
intervene in the disulfide-based linkage of cRTA molecules to Tfn
during IT synthesis. To rule out that the greater cytotoxic effect
shown by Tfn-cRTA in the absence of monensin could be due to a spacer
effect(25) , we also created a new chimeric toxin (scRTA) by
genetically fusing to the RTA gene an oligonucleotide coding for the
the 25-amino acid unrelated peptide
Gly-Ser-(Gly)-(Ser-(Gly)
)
-Ser-(Gly)
-Cys-Pro.
The scRTA sequence was then expressed in E. coli, and the
purified toxin was conjugated to Tfn following the same experimental
procedures as described for cRTA and rRTA. As shown in Fig. 4,
unconjugated scRTA as well as Tfn-scRTA displayed cytotoxic activity
against Jurkat cells comparably to nRTA and rRTA and their Tfn-toxin
conjugates. These results demonstrate that the Cys residue present in
the KFT25 peptide is unlikely to play a role in the molecular
mechanisms leading to the higher cytotoxic activity of Tfn-cRTA.
Cell intoxication by IT is a multistep process, first involving
binding of the IT molecules at the cell surface. It is well known that
the cell killing kinetics of the IT are strictly dependent on the
affinity of the interaction between the IT and the target
receptors(26) . To rule out that the higher cytotoxic effect
shown by Tfn-cRTA was due to a more efficient binding to target cells,
we compared the binding capacity of Tfn-nRTA, Tfn-cRTA, and Tfn-rRTA on
Jurkat cells by applying the method described by Schild (14) as
modified by Ittelson and Gill(12) . This method is based on the
inhibition by a specific competitor (i.e. Tfn) of the
cytotoxic effects mediated by a cytotoxin (i.e. Tfn-RTA
conjugates). It should be mentioned that this procedure allows the K of the competitor and not of the cytotoxin to be
measured. However, if the competitor inhibits to the same extent the
cytotoxic effect of different cytotoxins directed against the same
receptor, then it can be concluded that the cytotoxins bind the common
receptor with equal affinity. This procedure was chosen because it does
not require radioisotope labeling of the molecules involved, thus
preventing inactivation of the ligands or alteration of the
ligand/receptor interactions. Moreover, the sensitivity of this method
is considerable because it is based on the biological activity of
enzymic cytotoxins (e.g. RTA). As shown in Fig. 5,
binding of Tfn-nRTA, Tfn-cRTA, and Tfn-rRTA to Jurkat cells was
comparable. Displacement of Tfn-vehicled toxins by Tfn also
demonstrated that the three conjugates bound the transferrin receptor
in a specific manner.
Figure 5:
Schild plot of the antagonism by Tfn of
the cytotoxic action of Tfn-toxin conjugates on Jurkat cells. Dose
ratios (T`/T) calculated at a response level of 0.5 from several pairs
of dose-response curves with Tfn-nRTA (), Tfn-rRTA (
), or
Tfn-cRTA (
) were plotted against the log of the concentration of
the inhibitor Tfn (I). The linear relationship between log((T`/T)
- 1) and log(I), with a slope of
= 1, is the result
expected if Tfn and Tfn-toxin conjugates compete for a common cellular
target. Linear regression through data points revealed overlapping
binding properties of the three conjugates.
Unlike other pharmacological antitumor reagents, IT are effective at very low concentrations both in vitro and in vivo(1) . However, the fraction of IT molecules reaching the target cells of a solid tumor is often despairingly low due to a number of physiologic barriers preventing diffusion of the IT within the tumor and drastically impairing their therapeutic efficacy (e.g. high interstitial pressure, low diffusion rates of macromolecules within the tumor, antigen-site barrier, inadequate pharmacokinetics, immune-mediated clearance mechanisms)(27) . To enhance RTA IT cytotoxicity in vivo, the combined use of RTA IT and of the carboxylic ionophore monensin has been proposed. However, in vivo application of monensin or of its protein-conjugated derivative human serum albumin-monensin may be problematic due to the monensin inactivating properties of the serum(28, 29, 30) . An alternative approach involves the possibility of enhancing IT cytotoxicity by directing them to intracellular compartments where translocation of the IT to the cytosol is facilitated. Retention signals have been added to Pseudomonas exotoxin and to RTA to ease their delivery to the endoplasmic reticulum lumen from where Pseudomonas exotoxin and RTA are thought to enter the cytosol(31, 32) . This approach may not have general validity, however, because internalized toxins that are preferentially routed to lysosomes would not be potentiated by addition of endoplasmic reticulum retention signals. Instead, our strategy may be more generally applicable inasmuch as most viral envelope proteins are triggered to translocate across cell membranes in prelysosomal acidic compartments. With an approach similar to ours, Wagner et al.(33) greatly increased gene transfer into target cells by linking a peptide from the N terminus of HA-2 influenza hemagglutinin to polylysine-DNA complexes using Tfn as the targeting molecule.
By
genetically linking KFT25 to rRTA, we expected a greater potentiation
than the 10-20-fold higher cytotoxic activity observed with
Tfn-cRTA with respect to twin conjugates made with nRTA or unmodified
rRTA. There are several possible explanations for this observation. 1)
As shown in Fig. 1, 30% of the cRTA molecules lack the
KFT25 peptide due to post-translational proteolytic cleavage. 2) KFT25
functions in a dose-dependent manner(2, 3) . The
concentration of KFT25 reached within intracellular vesicles following
Tfn-cRTA internalization may be suboptimal. 3) The effect of KFT25 is
also time-dependent(2, 3) . Cytotoxicity is observed
in the presence of the isolated KFT25 peptide after 20 min of
incubation at 37 °C(2, 3) . Tfn is recycled out of
the cell with a t of 4-5 min(34) . The
persistence of Tfn-cRTA within compartments at the appropriate pH might
not be of a sufficient length to allow cell entry of an adequate amount
of KFT-bearing Tfn-cRTA conjugate. 4) KFT25 might change its
conformation when genetically linked to rRTA and be prevented from
displaying its full membrane destabilizing potential. 5) The additional
Cys residue present in KFT25 might be involved in the disulfide-based
linkage of Tfn to cRTA; this might in turn hinder the interaction of
cRTA with the cell membrane and reduce its cell entry. To optimize the
yield of uncleaved cRTA molecules, site-directed mutagenesis of the Arg
proteolytic cleavage site is underway. A further improvement of the
Tfn-cRTA cytotoxic potential might be obtained by using targeting
molecules residing for longer times within acidified vesicles following
internalization.
By linkage to KFT25, the toxicity of RTA against intact cells has been reduced 10-fold, and therefore, its toxicity toward non-target cells has been concomitantly decreased by the same factor. This might be advantageous because cRTA-based IT would offer a larger therapeutic window with respect to nRTA- or rRTA-based IT. Considering the difference in cytotoxicity between conjugated and unconjugated toxins, the ``specificity factors'' are in fact 100,000, 405, and 1100 for Tfn-cRTA, Tfn-nRTA, and Tfn-rRTA, respectively (Table 1).
The interaction of positively charged biomolecules with negatively charged lipid membranes has been implicated in several biological processes. Some examples are membrane permeabilization or perturbation and membrane-membrane aggregation. Basic polypetides as well as clusters of positively charged residues in several proteins have been shown to have membrane activity(35, 36, 37) . Some examples include snake cardiotoxins, sea anemone cytolysins, and mellitin from bee venom. The KFT25 peptide added to the ricin A chain sequence bears five positive charges, two of which are carried by His residues that are more positively charged at acidic pH. This could account for the faster interaction of cRTA with liposomes under acidic conditions.
Although
we are at the present time unable to explain the precise molecular
mechanisms of the interaction of cRTA with membranes, we believe that
some indications and suggestions can be obtained from the analysis of
the KFT25 sequence. Computer simulations of the structure of KFT25 and
calculations of the hydropathicity (38) and of the mean
hydrophobic moment (39) of the KFT25 peptide indicated that the
peptide is composed of three distinct structural regions separated by
Pro residues: an N-terminal hydrophobic -helix (Lys-Pro region), a
central hydrophilic globular structure (His-Pro region), and a slightly
hydrophilic C-terminal
-structure (Ser-Pro). The N-terminal
-helix (
18 Å long) can potentially span the first layer
of the plasma membrane at pH 7.0 with an emission of 5.3 kcal/mol. This
calculation is in agreement with the data reported by Schlegel and Wade (3) that the first 6 amino acids of KFT25 (which correspond to
the
-helix region only) are hemolytic even at physiologic pH,
whereas the globular region would be implicated in the pH activation of
the hemolytic properties of the entire peptide. The properties of the
KFT25 peptide could explain the lower cytotoxic activity of
unconjugated cRTA with respect to unconjugated nRTA or rRTA. In fact,
cRTA might insert itself into the plasma membrane and, in the absence
of an acidic environment, might remain entrapped within the lipid
layers. As a consequence, unconjugated cRTA would be characterized by a
lower translocating potential as compared with nRTA or rRTA. On the
other hand, 1) once cRTA has been vehicled near the membrane by Tfn,
cRTA could insert itself into the lipid layer. 2) Upon acidification of
the environment (i.e. after internalization and transport of
Tfn-cRTA within endosomes), the cRTA would increase its N-terminal
positive charge due to the His residues. According to models proposed
also for other proteins, this would lead to a disorganization of the
bilayer structure as a response of the attracting forces to the
negative charges present on the cytosolic surface of the cellular
membranes(40, 41) . 3) The disorganization of the
bilayer structure would facilitate the translocation of cRTA molecules
to the cell cytosol. A role in the pH activation of the KFT25
properties could also be played by the Pro residues. However, further
information on the molecular aspects of cRTA interaction with
biological membranes needs to be gathered experimentally.
In conclusion, our results demonstrate that it is possible to exploit the strategies developed by viruses to enter eukaryotic cells in order to enhance the specific cytotoxic effect of IT. A frame is also set for further studies aimed at selecting the most appropriate viral structures to be linked to toxins of different origin.
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