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THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
Vol.
255,
No.
14.
Issue
of
July
25,
pp.
6947-6953.
19w
Prtnted
m
U.S.A
Gelonin,
a
New Inhibitor
of
Protein Synthesis, Nontoxic to Intact Cells
ISOLATION, CHARACTERIZATION, AND PREPARATION
OF
CYTOTOXIC COMPLEXES WITH
CONCANAVALIN A*
(Received for publication, November 20, 1979)
Fiorenzo
Stirpe,S
Sjur Olsnes,§
and
Alexander
Rh18
From
8
Norsk
Hydro's
Institute for Cancer Research
and
the Norwegian Cancer Society, Montebello,
Oslo,
Norway and
+
Institute
of
General Pathology, University
of
Bologna, Bologna, Italy
The
protein
here
named
gelonin
was
extracted
from
the
seeds
of
Gelonium multiflorum
by
a
buffered
phos-
phate
solution
and
purified
in
a
single
step
by
chro-
matography
on
a
carboxymethyl
cellulose
column.
Polyacrylamide
gel
electrophoresis
in
the
presence
of
sodium
dodecyl
sulfate
and
gel
filtration
on
Sephacryl
300
superfine
showed
that
gelonin
consists of
one
poly-
peptide
chain
with
a
molecular
weight
of
about
28,000
to
30,000.
Gelonin
bound
to
a
column
containing
im-
mobilized concanavalin
A
and
could
be
eluted
with
a-
methylmannoside.
Pretreatment
of gelonin
with
jack-
bean
mannosidase
prevented
this
binding,
indicating
that
it
is
a
glycoprotein
containing
terminal
mannose
residues.
The
protein
proved
to
be
extremely
stable,
as
measured
by
its
biological
activity,
toward
treatment
with
sodium
dodecyl sulfate,
urea,
acid,
base,
and
heat.
Gelonin
strongly
inhibited
protein
synthesis
in
a
re-
ticulocyte
lysate.
It
inactivated
the
60
S
ribosomal
sub-
unit with
no
effect
on
the
40
S
ribosomal
subunit.
At
a
concentration
of
100
pg/ml,
gelonin
only
slightly
in-
hibited
protein
synthesis
in
intact
HeLa cells
and
it
gave
no
microscopically
visible
cytopathogenic effect.
When
the
inhibitor
was
linked
by
a
disulfide
bridge
to
concanavalin
A,
the
complex
gave
50%
inhibition
of
cellular
protein
synthesis
at a
concentration
of about
1
pg/ml,
corresponding
to
about
0.2
pg/ml of
gelonin.
The
results
show
that
gelonin
is
a
single
chain
protein
which
acts
in
a
cell-free
system
like
the
A
chains
of
abrin,
ricin, and
modeccin
and
suggest
that
it
lacks
the
ability
to
bind
to
the
cell
surface
and
to
enter intact
cells.
When
coupled
through
a
disulfide
bond to
a
pro-
tein
capable
of
binding
to
cells,
it
is
rendered
toxic
to
intact
cells.
Studies during recent years have shown that several bac-
terial and plant cytotoxins consist of two functionally different
domains. The one moiety is involved in the binding of the
toxins to receptors on the cell surface, whereas the other
somehow enters the cytoplasmic phase and acts by inhibiting
protein synthesis. Thus, the A fragments
of
diphtheria toxin
and
Pseudomonas
aeruginosa
A
toxin are enzymes capable
of inactivating elongation factor
2
by ADP ribosylation
(I),
whereas the A chains of abrin, ricin, and modeccin irreversibly
damage the
60
S
subunit
of
eukaryotic ribosomes by an
*
This work was supported by
a
European Molecular Biology
Organization short tem.fellowship (to
F.
S.)
and by Consiglio Na-
zionale delle Ricerche, Rome, within the Progetto finalizzato "Con-
troll0 della crescita neoplastica." The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "aduertisement" in accord-
ance with
18
U.S.C. Section
1734
solely to indicate this fact.
enzymatic mechanism, the nature of which has not been
elucidated
(2-4).
All these toxins are extremely potent, and
recent data indicate that a cell is killed when a single toxin
molecule (or its
A
chain) enters the cytoplasmic phase
(5,
6).
Recently, it has been found that many plants contain pro-
teins that in ceU-free systems inactivate eukaryotic ribosomes,
apparently in the same way
as
the
A
chains
of
abrin, ricin,
and modeccin, but have little or no effect on intact cells. Such
proteins have been purified
from
the roots of
Phytolacca
americana
(7,
8),
from wheat germ
(9),
from the seeds of
Momordica
charantia
(IO),
and they have been partially
purified from the seeds of
Croton
tiglium
and
Jatropha
curcas
(11, 12).
In screening studies, extracts from several other seeds
strongly inhibited protein synthesis in a cell-free system, but
lacked toxic effect on intact cells
(13,
14).
One of the most
potent extracts was obtained from the seeds of
Gelonium
multiflorum
(Euphorbiaceae).
In the present study, we have
isolated from such seeds a single chain protein, gelonin, with
properties similar to those
of
the
A
chains of abrin, ricin, and
modeccin. When gelonin was linked to concanavalin A. the
complex formed was toxic to intact cells.
EXPERIMENTAL PROCEDURES
Materials-Seeds of G. multiflorum, grown in India, were pur-
chased
from
F.
G.
Celo, Zweibriicken, West Germany. Jackbean
a-
mannosidase, concanavalin
A,
trypsin, chymotrypsin, pepsin, and
protease type
VI,
from Streptomyces griseus, were obtained from
Sigma Chemical Co. Abrin
A
chain was prepared as described earlier
(15). Sepharose-concanavalin A and markers for molecular weight
determinations were obtained from Pharmacia Fine Chemicals, Upps-
ala, Sweden. Carboxymethyl cellulose (CM52) was purchased from
Whatman Ltd., Maidstone,
U.
K.
The protein iodination reagents
were from Bio-Rad Laboratories, Richmond, Ca.
~-[U-~'C]leucine (specific activity, 342
Ci/mol),
N-ethyl-P,3-["C]-
rnaleimide (specific activity, 2.1 Ci/moI), Na'"1
(13
to
17
mCi/mg of
iodine), and [14C]phenylalanyl-tRNA from Escherichia coli (specific
activity, 0.192 pCi/mg) were obtained from The Radiochemical
Centre, Amersham, England.
Extraction
of
Gelonin-Shelled seeds of G. multiflorum were
ground in a
sorvd
OmniMixer with
8
volumes of
0.14
M
NaCI,
5
mM
sodium phosphate (pH
7.4).
The homogenate was left at 2-4°C on a
magnetic stirrer overnight, then further cooled in an ice bath, and
finally centrifuged at
35,000
X
g
for
20 min at 0°C.
A
cloudy yellow
supernatant was separated from the sediment and from a floating
layer of solidified fat. The supernatant was dialyzed against 5 mM
sodium phosphate (pH 6.5). Any precipitate formed during the di-
alysis was removed by centrifugation and discarded.
Polyacrylamide
Gd
Electrophoresis-Protein fractions were made
up to contain 2% (w/v) sodium dodecyl sulfate,
60
mM Tris-HC1 (pH
6.8),
1OW
(w/v) sucrose, and, in some cases, 0.7
M
2-mercaptoethanol.
The samples were then incubated at 56°C
for
5
min and layered into
slots
of polyacrylamide slabs consisting of a
10
to
20%
exponential
gradient separating gel (0.85 rnm
X
16
cm
X
16
cm) and a
3%
concentrating gel (0.85 mm
X
2
cm
X
16
cm) with the buffers described
6947
6948
Gelanin,
a
Protein Synthesis Inhibitor
by
Laemmli (16). The gels were run at constant voltage (150
V)
until
the tracking dye (bromphenol blue) reached the lower edge of the
gel. The gels were stained with Coomassie brilliant blue and in some
cases dried and exposed to Kodak Royal X-Omat film.
Iodination-Labeling of pure gelonin with
1251
by the lactoperoxi-
dase-glucose oxidase method was carried out with the reagents sup-
plied from Bio-Rad Laboratories as described by the manufacturer.
Gelonin (100 pg in
60
pl
of buffer) was labeled with
2
p1
(1.1
mCi) of
Na’2sI. After
20
min at room temperature, the protein was separated
from free NaIz5I by filtration through a Sephadex G-25 column,
equilibrated with 0.14
M
NaCI,
5
m~
sodium phosphate (pH 7.4). The
labeled product contained 634 cpm/ng.
Cell-free Protein Synthesis-A rabbit reticulocyte lysate, prepared
as earlier described (17), was supplemented as described by Pelham
and Jackson (18) and then stored in small aliquots in liquid nitrogen.
Protein synthesis was measured in 50-pl samples containing
0.5
pCi of
[14C]leucine, with
or
without inhibitor, as described in the legends to
figures. Aliquots (10
pl)
were removed at the times indicated and
poured into tubes containing
1
ml
of 0.1
M
KOH, and then the
described
(17).
trichloroacetic acid-precipitable radioactivity was measured as earlier
Ribosomes and Ribosomal Subunits-Ribosomes were isolated
from rabbit reticulocyte lysate as described earlier (19). The ribosomal
subunits were obtained after incubation of ribosomes with puromycin
and GTP and then treated with high salt concentration and separated
on a sucrose gradient as described (19). Poly(U)-directed polyphen-
ylalanine synthesis with isolated ribosomes and ribosomal subunits
was measured as earlier described (20).
Cells-HeLa
Sa
cells were maintained in monolayer culture in
Eagle’s minimum essential medium containing 10% calf serum as
earlier described (21). For toxicity tests, cells were seeded into Linbro
tissue culture plates with 16-mm troughs
(lo”
ceUs/well) and then
increasing amounts of gelonin were added to the wells. On the next
day, the medium was removed and replaced by serum-free medium
containing
%II
of the normal concentration of leucine and 0.1 pCi/ml
of [‘4C]leucine, and the incorporation of radioactivity for 2 h was
measured as earlier described (22). Human lymphocytes were pre-
pared and cultured
as
described by Godal et al. (23).
Coupling Experiments-Concanavalin A (25 mg) in
1
rd
of 0.1
M
sodium phosphate, pH 7.5, 0.1
M
NaCl was treated with a 10-fold
molar excess of
N-succinimidyl-3-(2-pyridyldithio)propionate,
ob-
tained from Pharmacia Fine Chemicals, Uppsala, Sweden, for 30 min
at room temperature with occasional stirring (24). Excess reagent was
removed by affinity chromatography on a 2-ml column of Sephacryl
300 supertine, equilibrated with 0.14
M
NaC1,5
m~
sodium phosphate
(pH 7.4). After washing, concanavalin
A
was eluted with 0.1
M
a-
methylmannoside in the same buffer. Finally, a-methylmannoside
was removed by dialysis.
Gelonin
(0.5
mg) was mixed with a trace
of
‘251-labeled gelonin
(los
cpm) and dialyzed against 0.1
M
sodium phosphate (pH 7.5), 0.1
M
NaCI. Then a 10-fold molar excess of SPDP’ was added and the
mixture was incubated for 30 min at room temperature. (Control
experiments showed that even treatment with
a
25-fold molar excess
of SPDP did not impair the ability of gelonin to inhibit cell-free
protein synthesis, whereas some reduction in activity was observed
after treatment with a 100-fold molar excess of SPDP.) Then, suffi-
cient acetic acid was added to reduce the pH to 4.5, dithiothreitol was
added to a final concentration of
50
m~,
and the mixture was
incubated for 20 min at room temperature. Excess reducing agent was
removed by gel filtration on Sephadex G-25 (medium), equilibrated
with 0.1
M
sodium phosphate (pH 7.5),
0.1
M
NaC1. Finally, the
gelonin thus treated was mixed with approximately equimolar
amounts of concanavalin A, pretreated with SPDP as described
above, and the mixture was incubated at room temperature overnight.
On the next day, the mixture was submitted to centrifugation at
300,000
X
g
for
18
h in
5
to
20%
(w/v) sucrose gradients containing
0.1
M
a-methylmannoside in 0.14
M
NaCI,
5
m~
sodium phosphate, pH
7.4. Fractions (0.25
ml
each) were collected by puncturing the bottom
of
the tubes, the radioactivity in each fraction was measured to
localize gelonin, and the absorbance at 280 nm was measured to
localize concanavalin
A.
Analysis of the different fractions by poly-
acrylamide gel electrophoresis in the presence
of
sodium dodecyl
sulfate and subsequent autoradiography of the gel showed that the
radioactive material sedimenting in front of the main peak of material
absorbing at 280 nm, moved more slowly than gelonin alone, indicat-
’
The abbreviation used
is:
SPDP,
N-succinimidyl-3-(2-pyridyldi-
thio)propionate.
ing that it consisted of complexes with concanavalin
A.
Other Methods-Free thiol groups were determined by measuring
the binding of N-ethyl[’4C]maleimide as earlier described (25).
Pro-
tein was determined spectrophotometrically (26). I4C radioactivity
was measured in a Beckman LS-330 liquid scintillation spectrometer
and
Iz5I
radioactivity in an Intertechnique gamma spectrometer.
RESULTS
Purification of Gelonin-Seeds of G. multiftorum were
extracted as described under “Experimental Procedures,” and
the dialyzed extract was applied to a CM52 column equili-
brated with
5
mM sodium phosphate (pH 6.5). The adsorbed
material
was
eluted with a linear
0
to
0.3
M
NaCl gradient in
the same buffer. The results are shown in Fig.
1.
The different
fractions were tested for their ability to inhibit protein
syn-
thesis in a cell-free system from rabbit reticulocytes. Most of
the inhibitory activity became adsorbed to the column and
was eluted corresponding to Peak IV.
Polyacrylamide gel electrophoresis of the material in the
different fractions showed that the material
in
Peak
IV
con-
sisted of one polypeptide chain with molecular weight of about
30,000
(Fig.
2,
A
and
B).
It
is
clear from Fig.
2
that this
protein, which we named gelonin,
is
the most abundant one in
the extract. The yield was
4
to 5 mg
of
pure gelonin from
3.5
g of seeds. Approximately
40%
of
the inhibitory activity found
in the dialyzed extract could be recovered in pure gelonin.
Characterization
of
Gelonin-To test whether the native
molecule consists
of
one or more copies
of
this chain,
it
was
Ntered through a column
of
Sephacryl
300
superbe which
had been calibrated with proteins of known molecular weights.
It
was found (Fig.
3)
that gelonin was eluted at a position
corresponding to a molecular weight of about 28,000. The
different fractions were
also
tested in a cell-free protein syn-
thesizing system. As shown in Fig.
3,
there was good corre-
spondence between the elution pattern of the protein and the
ability to inhibit cell-free protein synthesis. The results indi-
cate that gelonin consists of only one copy of the peptide
chain.
A
small shoulder at the leading edge of the peak
probably corresponds to dimers of the protein.
P
U-
u-
n
40
SO
M
100
120
250
-
0.3
-0.2
-01
Fraction
number
FIG. 1.
Purification
of
gelonin
by chromatography
on
car-
boxymethyl cellulose.
Seed extracts
(20
d,
containing 100 mg
of
protein), dialyzed against
5
mM
sodium phosphate (pH
6.5).
was
applied to a CM52 column (20
X
1.5 cm) equilibrated with the same
buffer. The column was washed and then bound material was eluted
with a 400-ml0 to 0.3
M
linear NaCl gradient in the same buffer at a
rate of 25 ml/h at room temperature. Fractions (1.4
ml
each) were
collected and the absorbance at 280 nm was measured. From some of
the fractions,
5-pl
aliquots were taken and added to a cell-free system
as
described under “Experimental Procedures.” The incorporation of
[‘4C]leucine was measured after
10
min. The fractions indicated with
the bur were collected
as
pure gelonin.
Gelonin,
a
Protein Synthesis Inhibitor
6949
3
ABCDE
FIG.
2.
Polyacrylamide gel electrophoresis
of
crude and
pure gelonin in the presence of sodium dodecyl sulfate.
Aliquots
(50
pl)
from fractions in Fig.
1
were submitted to polyacrylamide gel
electrophoresis
as
described under "Experimental Procedures."
A,
fraction
127;
B,
fraction
129;
C,
crude gelonin;
D,
molecular weight
markers: phosphorylase
B
(94,000).
bovine serum albumin
(67,000),
ovalbumin
(43,0001,
carbonic anhydrase
(30,000).
trypsin inhibitor
(20,000).
a-lactalbumin
(14,000);
E,
abrin
A
chain.
The extinction coefficient for gelonin at 280 nm was esti-
mated from the weight of a sample of gelonin which had been
extensively dialyzed against distilled water and then lyophi-
lized in a preweighed tube. The value obtained
was
E!':.,
=
6.7.
Gelonin exhibits many properties in common with the
A
chains of abrin, ricin, and modeccin (see below). Since abrin,
ricin, and modeccin
all
are glycoproteins and bind to concan-
avalin
A,
we studied whether gelonin binds to a column of
immobilized concanavalin
A.
It
was
found (Fig.
4)
that most
of the labeled material was indeed bound to the column and
could be eluted with a-methylmannoside. When the different
fractions were tested for their ability to inhibit cell-free protein
synthesis, a good correspondence between radioactivity and
inhibitory activity was found (not shown). If gelonin had been
pretreated with jackbean a-mannosidase, very little radioac-
tivity
was
bound to the concanavalin
A
column. This indicates
that gelonin
is
a glycoprotein containing terminal mannose
residues. Treatment of gelonin with jackbean a-mannosidase
did not impair its ability to inhibit cell-free protein synthesis.
Less
than 0.1 mol of N-ethyl['4C]maleimide
was
bound per
mol of gelonin, indicating that it contains no free thiol group.
Stability
of
Gelonin-To test the stability
of
gelonin its
ability
to
inhibit cell-free protein synthesis after various treat-
ments
was
studied. The activity
was
unchanged after incuba-
tion for
1
h at 37°C and more than
12
h at room temperature.
Freezing and thawing for 10 consecutive times, as well as
freeze-drying, did not affect the inhibitory activity, and the
activity was only slightly decreased (about 15%) by treatment
overnight with 1% sodium dodecyl sulfate or 6
M
urea. Gelonin
was not affected by treatment overnight with 0.1
M
HCl, or
0.1
M
NaOH, or by incubating a sample of gelonin (0.35 mg/
ml)
overnight at room temperature with an equal amount of
trypsin, chymotrypsin, pepsin,
or
S.
griseus protease. Labeling
with
1
molecule of iodine/molecule of gelonin did not affect
the inhibitory activity. However, it was completely destroyed
by boiling for 20 min. Heat-denatured gelonin was completely
n
n
Fraction
number
FIG.
3.
Gel
filtration of gelonin on
a
Sephacryl300 superfine
column.
Gelonin
(2
mg) was mixed with a trace of dextran blue in
0.25
ml
of buffer
(0.14
M
NaCI,
5
rn~
sodium phosphate, pH
7.4),
applied at
20°C
to a Sephacryl column
(80
X
1.5
cm), and eluted with
the same buffer. Fractions
(35
drops each) were collected, and the
absorbance at
280
and
620
nm was measured. Aliquots of the fractions
were tested for their ability to inhibit cell-free protein synthesis
as
in
Fig.
1.
The proteins used
to
calibrate the Sephacryl column were:
bovine serum albumin
(BSA)
(M,
=
67,0001,
ovalbumin
(M,
=
43,000),
carbonic anhydrase
(M,
=
30,000),
trypsin
(M,
=
24,000).
and lysozyme
(M,
=
13,000).
The aliquots tested in the cell-free system were:
A,
1
pl;
A,
0.3
pl;
X,
0.01
pl.
41
Fraction number
FIG.
4.
Binding of gelonin
to
a
Sepharose-concanavalin
A
column.
A sample of '2511-labeled gelonin
(0.2
pg,
120,000
cpm) in
0.14
M
NaCI,
5
mM sodium phosphate (pH
7.5)
was treated at
37OC
for
30
min with
50
pg/ml
of a-mannosidase. A parallel sample was incubated
without enzyme. The samples were then applied at room temperature
to columns of Sepharose-concanavalin
A
(2
X
1.5
cm). After washing
with buffer, the columns were eluted with
0.1
M
a-methylmannoside
in the same buffer. The radioactivity in each fraction was measured.
0,
gelonin incubated without enzyme;
0,
gelonin pretreated with
a-
mannosidase. The
arrow
indicates the
start
of the elution with
a-
methylmannoside.
6950
Gelonin,
a
Protein Synthesis Inhibitor
degraded by chymotrypsin and
S.
griseus protease and also
became somewhat sensitive
to
trypsin and pepsin.
Effect on Protein Synthesis in Cell-free Systems-Gelonin
is a very potent inhibitor of protein synthesis in a cell-free
system from
a
rabbit reticulocyte lysate. Thus, as shown in
Fig. 5, as little
as
0.4
ng/assay exhibited a definite effect.
It
is
clear that gelonin inhibited protein synthesis even more effi-
ciently than did abrin
A
chain. It can be seen from Fig.
6
that
the concentration giving 50% inhibition after
5
min of incu-
bation, when the rate of protein synthesis was still linear, was
about 12.5 ng/ml.
We have earlier found that the inhibitory effect of abrin,
ricin, and modeccin
is
strongly increased by pretreatment of
the toxins with 2-mercaptoethanol.
No
similar effect was
obtained with gelonin (Fig.
6).
The
A
chains of abrin, ricin, and modeccin, as well
as
curcin,
crotin, and the pokeweed inhibitor,
all
exert their effect by
inactivating the
60
S
ribosomal subunit (2-4,
7,
12). Since
washed ribosomes from gelonin-treated rabbit reticulocyte
lysate had a strongly reduced activity in a cell-free system
(not shown), we tested which of the ribosomal subunits is
inactivated by gelonin. Ribosomes were isolated from a rabbit
reticulocyte lysate, part of the ribosomes were treated with
gelonin, whereas another part was untreated. Ribosomal sub-
units were prepared
as
described under "Experimental Pro-
cedures" and a reconstituted cell-free system was prepared.
With polyuridylic acid as synthetic messenger it was found
(Table
1)
that when the
60
S
ribosomal subunit had been
exposed to gelonin, the incorporation of ['4C]phenylalanine
was very low, whereas with 40
S
subunits derived from ge-
lonin-treated ribosomes essentially the same incorporation
was obtained
as
when both subunits were untreated. It
is
therefore clear that also gelonin exerts its effect on the
60
S
ribosomal subunit.
Experiments with Intact Cells-When gelonin was added
I
I
J
Time (min)
FIG.
5.
Ability of gelonin and abrin
A
chain
to
inhibit protein
synthesis
in
a cell-free system from rabbit reticulocytes.
The
indicated amounts of gelonin or abrin A chain were added
to
a cell-
free system prepared from a rabbit reticulocyte lysate. The final
volume was 80
pl.
Aliquots
(10
pl)
were taken, as indicated, and the
alkali-stable, acid-precipitable radioactivity was measured.
X,
control;
0,
0.4
ng
of
gelonin;
0,
0.4
ng
of
abrin
A
chain;
W,
4
ng of gelonin;
0,
4
ng
of
abrin A chain;
A,
40
ng
of
gelonin;
A,
40
ng of abrin A chain.
-
4"
50
100
250
500
Inhibitor
added
(ng/ml)
-.
FIG.
6.
Inability of 2-mercaptoethanol to alter the inhibitory
effect of gelonin in
a
cell-free system from rabbit reticulocytes.
Gelonin was incubated for 2 h at 37°C with and without
1%
2-
mercaptoethanol. Then, increasing amounts of the protein were added
to cell-free systems
as
in Fig.
5
(except that the final volume was 65
pl)
and, after incubation at 28°C for
5
min,
10-p1
aliquots were taken,
and the amount of acid-precipitable radioactivity was measured.
0,
untreated gelonin;
0,
gelonin pretreated with
1%'
2-mercaptoethanol.
TABLE
I
Ability
of
untreated and gelonin-treated ribosomal subunits to
polymerize phenylalanine
Ribosomes were isolated from
10
ml of rabbit reticulocyte lysate
and resuspended in
1
ml
of 50 mM Tris-HC1 (pH
7.4).
60
m~
KCI,
2
mM MgC12, and
9
mM
2-mercaptoethanol. The suspension was divided
into two equal
parts
to one
of
which was added
40
pg
of
gelonin. The
samples were incubated at 37°C for
10
min, then treated with puro-
mycin and GTP, and ribosomal subunits were isolated
(19).
Aliquots
(0.5
pmol)
of
each subunit as indicated were incubated
as
described
(20), and the ability to polymerize ['4C]phenylalanine was measured.
IJntreated subunit
Ge'onin-treated
Radioactivity
unit
cpm
40,
60
264
40,60 117
40 60
85
60
40
35
1
to cultures of HeLa
S:,
cells, very little effect was observed.
Thus, only very high concentrations
(100
pg/ml) of gelonin
induced some reduction (about 20%) of protein synthesis in
the cells after incubation with gelonin overnight.
To test whether gelonin is able to bind to cells, we incubated
'L'I-labeled gelonin with HeLa
S:,
cells for
60
min at
0°C.
The
cells were sedimented and washed three times with cell culture
medidm.
No
binding above background was found.
A
protein synthesis inhibitor from
M.
charantia was found
to be selectively toxic to lymphocytes.2
To
test whether ge-
F.
Licastro,
C.
Franceschi,
L.
Barbieri, and Stirpe,
F.
(1980)
Virchows Arch.
B.
Cell. Pathol.,
in press.
Gelonin,
a
Protein Synthesis Inhibitor
6951
lonin has
a
similar effect, different amounts
of
gelonin were
added to lymphocytes stimulated with
three
different mito-
gens (Table
11).
In cultures stimulated with phytohemagglu-
tinin from
Phaseolus vulgaris
and with pokeweed mitogen,
very little inhibition was observed. However, in cultures stim-
ulated with concanavalin
A,
an inhibitory effect was seen at
the highest concentration of gelonin. Since gelonin binds
to
TABLE
I1
Effect of gelonin on thymidine incorporation by mitogen-stimulated
lymphocytes
Human lymphocytes were isolated and 2.5
X
IO5
cells/well were
incubated with
or
without mitogens
as
described (23). The indicated
amounts of gelonin were added to the cultures and
after
incubation
at 37°C for 48 h, 1.25 pCi of [“Hlthymidine was added. The incorpo-
ration of radioactivity into acid-precipitable material during the fol-
lowing
18
h was measured.
Mitogen used Gelonin added
(pg/ml)
None
0.4 4.0
40
~ ~~~ ~
cPm
None (controls) 1,538 1,985 1,317 1,320
Phytohemagglutinin 87,262 64,140 51,642 62,393
Concanavalin A 32,117 27,471 23,098 7,834
Pokeweed mitogen 16,807 13,449 11,715 12,584
94
K=
67
K-
43
K-
FIG. 7.
Polyacrylamide gel electrophoresis
of
complexes
of
gelonin and concanavalin A.
‘”I-labeled gelonin and unlabeled
concanavalin A were joined by disulfide bridges
as
described under
“Experimental Procedures.” Aliquots
(50
pl)
were incubated with
sodium dodecyl sulfate with and without 2-mercaptoethanol and
analyzed by polyacrylamide gel electrophoresis in the presence
of
sodium dodecyl sulfate. The dried gel was submitted to autoradiog-
raphy.
A,
nonreduced sample;
B,
sample pretreated with 2-mercap-
toethanol. The horizontal bars indicate the migration of the same
molecular weight markers as in Fig. 2.
Protein
added
(Pg/ml)
FIG.
8.
Ability
of
complexes
of
gelonin and concanavalin A
to
inhibit protein synthesis in HeLa
Sa
cells.
Gelonin, concana-
valin A, and complexes of both were added to HeLa
S:,
cells growing
in Linbro plates (FB 16-24 TC) in serum-free medium containing
1
g/
liter
of
galactose instead
of
glucose. After
8
h of incubation at 37°C.
1
g/liter of glucose and
10%
calf serum were added and the plates
were incubated further overnight. Then, the medium was changed to
low leucine medium containing 0.05 pCi
of
[“C]leucine/ml. and the
incorporation of radioactivity into acid-precipitable material during
1
h was measured.
0,
complex of gelonin and concanavalin A, the
same as shown in Fig. 7A;
0,
the same complex,
50
m~
a-methylman-
noside added to the medium;
A,
mixture of SPDP-treated concana-
valin A and gelonin with only about
10%
of the gelonin present in
covalent complexes;
A,
the same mixture,
50
mM a-methylmannoside
added
to
the medium;
X,
SPDP-treated concanavalin A alqne.
concanavalin
A
and may inhibit the binding
of
the lectin to
its receptors on the lymphocytes, it
is
possible that the inhi-
bition seen is not due to
a
direct effect of gelonin on the cells.
Complexes
of
Gelonin and Concanavalin
A-The above
experiments indicated that gelonin
fails
to intoxicate cells
because it is unable to bind
to
the cell surface. We therefore
attempted to bind gelonin through
a
disulfide bridge to
a
molecule capable of binding to cell surface receptors. For this
purpose, we chose concanavalin
A.
Concanavalin
A
and ge-
lonin were
fit
reacted with
N-succinimidyl-3-(2-pyrimidyl-
thio)propionate, and free
SH
groups were then introduced in
gelonin by treatment with dithiothreitol as described under
“Experimental Procedures.” The proteins were then mixed
and after reaction overnight, the complexes formed were
separated from unreacted proteins by sucrose gradient cen-
trifugation. Since ‘”“I-labeled gelonin was used in this experi-
ment, complexes of gelonin with concanavalin
A
should be
revealed by autoradiography after polyacrylamide gel electro-
phoresis in the presence of sodium dodecyl sulfate under
nonreducing conditions. Since the monomer of concanavalin
A
has a molecular weight of
27,000,
complexes containing one
monomer of each protein should move in the gel correspond-
ing to
a
molecular weight
of
about
57,000.
As
shown in Fig.
7A,
a major labeled band was indeed found at this position.
Bands with higher molecular weights were also observed,
probably corresponding to complexes containing more than
one copy of each protein. When the complexes were treated
with 2mercaptoethanol in the presence of sodium dodecyl
6952
Gelonin,
a
Protein Synthesis Inhibitor
sulfate (Fig.
7B),
all
the radioactivity moved corresponding to
a
molecular weight of 30,000,
i.e.
like free gelonin. When the
treatment with 2-mercaptoethanol was carried out under non-
denaturing conditions, dissociation was observed only in part
of the complexes (not shown).
When tested in a cell-free system under conditions as in Fig.
5,
gelonin in complex with concanavalin
A
was considerably
less efficient than free gelonin in inhibiting protein synthesis.
A
considerable increase in activity was found after pretreat-
ment of the complex with 2-mercaptoethanol (not
shown).
It
has earlier been observed that treatment of diphtheria toxin,
abrin, ricin, and modeccin with 2-mercaptoethanol strongly
increased their ability to inhibit protein synthesis in cell-free
systems
(1-4).
To test the toxicity of the complex, it was added to ceils in
culture and the inhibition of protein synthesis was measured.
Since glucose and serum proteins present in the medium
compete with the cell surface receptors for binding of concan-
avalin
A,
the complex was added to cells in serum-free medium
containing galactose instead of glucose, and serum and glucose
were added after
8
h
of
incubation. It was found (Fig.
8)
that
50%
inhibition
of
protein synthesis occurred when about
1
pg
of complex, containing about
0.2
pg
of
gelonin, was added per
ml
of medium.
This
inhibition
was
prevented when a-meth-
ylmannoside was added to the medium. Since about
30
p,g
of
SPDP-treated concanavalin
A
alone was required to give the
same inhibition
of
protein synthesis and since as much as
100
pg/ml of free gelonin inhibited protein synthesis only slightly,
the data indicate that concanavalin
A,
attached to gelonin
through a disulfide bridge, sewed as a haptomer that mediated
the binding and uptake of the inhibitor by the cells.
DISCUSSION
The present results indicate that gelonin
is
a single chain
glycoprotein with biological properties
similar
to those of the
A
chains of abrin, ricin, and modeccin. Like these, gelonin
inactivates the
60
S
ribosomal subunits. From the activity of
gelonin in a cell-free system from rabbit reticulocytes, it ‘can
be estimated that
1
molecule of the toxin inactivates about
200 ribosomes/min, indicating that it acts catalytically. In a
cell-free system from rabbit reticulocytes gelonin
is
even more
active than abrin
A
chain.
Inhibitors similar to gelonin have been found in a variety of
plants. The first one described
is
an antiviral protein (PAP or
Phytolacca americana protein) present in the roots of poke-
weed
(8).
This protein
(Mr
-
27,000)
which
is
non-toxic to
cells, may prevent the multiplication of
viruses
in the cells
(27).
Like gelonin, it inhibits cell-free protein synthesis by
catalytically inactivating the
60
S
ribosomal subunit
(7).
This
is
also
the case
with
the less well characterized inhibitors
crotin and curcin, present in the seeds of
C.
liglium
and
J.
curcas
(11,
12).
It
is
not clear whether the wheat germ and
the
M.
charantia inhibitors act on the
60
S
subunit, although
in
both cases it has been found that the target
is
on the
ribosomes (9,
10).
Several other seed extracts contain a heat-labile inhibitor
of cell-free protein synthesis (13,14). Since such inhibitors are
found in a variety of unrelated plant species, they may be
present in all plants and have an important function. Possibly,
by the methods
now
available, such proteins
are
only found in
those cases where, for one reason or another, they are synthe-
sued
in
exceptionally high amounts.
Probably, the most interesting finding in this paper is that
geIonin which is nontoxic to intact cells, acquired toxic activity
when linked by a disulfide bridge to concanavalin
A.
Presum-
ably, the lectin serves as a “haptomer” that binds the complex
to cell surface receptors, permitting uptake of gelonin to take
place.
In several laboratories, attempts have been made to alter
the specificity of diphtheria toxin, abrin, and ricin by replacing
the binding part (the
B
chains) with a protein having a
different binding specificity. The goal of these experiments
has been to bind the “effectomers,” the
A
chains, to molecules
that may direct the
A
chains to particular targets in the
organism. Thus, diphtheria toxin
A
fragment has been bound
by a disulfide bridge to human placental lactogen
(28,
29),
concanavalin
A
(30), Wistaria floribunda lectin
(31),
Fab
fragments of antibodies against L1210 ceUs (32), and, by the
aid of a derivative of chlorambucil, to antilymphocyte globu-
lins
(33).
Similarly, ricin
A
chain has been linked through a
disulfide bridge to the
/3
chain of human chorionic gonadotro-
pin (34,
35)
and to concanavalin
A
(36).
In most cases, the
hybrid molecules have proved to be toxic to cells having the
appropriate receptors, albeit to a much lesser extent than the
native toxins. Apart from the fact that the isolated
A
chains
may be diffcult to prepare in sufficient quantities, an inherent
difficulty in such experiments is that the purified
A
chains
may contain traces of intact toxins. Since gelonin is an
A
chain
like protein, it appears to be ideal for such studies. It is present
in the seeds of
G.
multiflorum in high concentrations, it
is
easily isolated, it is exceptionally stable
to
chemical and
physical treatments, and it
is
nontoxic to cells, unless linked
to a haptomer. In mice, no toxic effect was observed after
intravenous injection of 1 mg of gelonin/100 g body weight.
The complex of gelonin and concanavalin
A
here prepared
was much less toxic to cells than the toxins abrin, ricin, and
modeccin. There may be several explanations for the low
toxicity of artificial complexes. First, it is possible that the
natural
B
chains of the toxins have functions other than that
of merely binding to cell surface receptors. Probably, the
B
chains somehow facilitate the entry of the
A
chain through
the plasma membrane. Second, the artificial binding moieties
may not bind to those receptors that are most efficient in
internalizing the toxins. Possibly, the artificial complexes enter
the cells by mechanisms entirely different from those used by
the natural toxins. Third, reduction of the interchain disulfide
bridge has been shown to be essential for inhibitory effect of
the natural toxins on ceU-free protein synthesis. Possibly, the
disuKde bridge in the artificial complexes may be less easily
split than those in the natural toxins. These questions may be
elucidated by binding gelonin to several carrier (haptomer)
molecules by different coupling agents and comparing the
toxic effect of the complexes on cells in culture. Possibly,
similar
complexes can be formed with other
A
chain like
inhibitors present in plant material.
An intriguing question is how toxins like abrin, ricin, and
modeccin have evolved. These toxins contain in addition to
the effectomer
a
binding or haptomer part which in effect is
a lectin. Nontoxic lectins are widely distributed in the plant
kingdom. Possibly, abrin, ricin, and modeccin have been as-
sembled by the plants from inhibitor molecules and lectin
molecules already present for other purposes. The fact that
these toxins, which are closely related in structure and func-
tion, are found in plants belonging to three different orders is
consistent with this possibility.
Acknowledgments-We
are
indebted
to
Dr.
T.
Godal for help with
lymphocyte cultures and to
Miss
Jannikke Ludt
for
her
skiuful
technical assistance.
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