Properties of the ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, and dianthins.
ABSTRACT The amino acid and sugar compositions of four ribosome-inactivating proteins (gelonin, Momordica charantia inhibitor, dianthin 30 and dianthin 32) were determined. The proteins are all basic glycoproteins (pI greater than 8) containing mannose (more abundant in gelonin), glucose, xylose, fucose (absent from gelonin) and glucosamine. The ribosome-inactivating properties of the proteins examined are not modified by pretreatment with N-ethylmaleimide. Precipitating and inactivating antibodies can be raised against ribosome-inactivating proteins; a weak cross-reaction was observed only between dianthin 30 and dianthin 32.
- Methods in Enzymology 02/1978; 50:323-30. · 2.00 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Gelonin, a plant protein which can powerfully reduce the protein-synthetic capacity of ribosome preparations, was covalently coupled to anti-Thy1.2 antibody. The conjugate was prepared using N-succinimidyl-3-(2-pyridyldithio)propionate which generates a disulphide linkage between the component molecules. Two conjugate fractions were obtained with Mr of 180 000 and greater than 200 000. After its linkage of the antibody, gelonin suppressed those Thy1.1-bearing T lymphocytes from AKR mice which will respond to phytohaemagglutinin and concanavalin A in tissue culture. The [3H]leucine incorporation with the T-cell mitogens was inhibited by 50% with the 180 000-Mr fraction at a concentration of 0.4 nM and with the greater than 200 000-Mr fraction of pM. Unconjugated gelonin induced comparable reductions in T-cell responsiveness but at concentrations of 30 nM. The conjugates exerted little or no effect upon B lymphocytes or T lymphocytes from CBA mice (Thy1.2 + ve). Thy1.1-expressing AKR lymphoma cell lines, AKR-A and BW5147, were found to be sensitive to the conjugates, albeit much less so than the normal T lymphocytes. The conjugates injected in vivo significantly prolonged the life of CBA mice bearing in an AKR-A lymphoma allograft. It is concluded that gelonin can, by its linkage to an antibody, be rendered cytotoxic with a potency to match or exceed those of the toxins abrin and ricin.European Journal of Biochemistry 07/1981; 116(3):447-54. · 3.58 Impact Factor
- Journal of Biological Chemistry 08/1980; 255(14):6947-53. · 4.65 Impact Factor
Biochem. J. (1982) 207, 505-509
Printed in Great Britain
Properties ofthe ribosome-inactivating proteins gelonin, Momordica
charantia inhibitor, and dianthins
Anna FALASCA,* Anna GASPERI-CAMPANI,t Ada ABBONDANZA,t Luigi BARBIERIt and
*Istituto di Chimica Biologica dell'Universitai, and tIstituto di Patologia generale dell'Universitd diBologna,
I-40126 Bologna, Italy
(Received 3 June 1982/Accepted 10August 1982)
The amino acid and sugar compositions of four ribosome-inactivating proteins (gelonin,
Momordica charantia inhibitor, dianthin 30 and dianthin 32) were determined. The
proteins are all basic glycoproteins (pI>8) containing mannose (more abundant in
of the proteins examined
pretreatment with N-ethylmaleimide. Precipitating and inactivating antibodies can be
raised against ribosome-inactivating proteins; a weak cross-reaction was observed only
between dianthin 30 and dianthin 32.
gelonin) and glucosamine. The
are not modified by
The 'inhibitor' from the seeds of Momordica
gelonin, from the seeds of Gelonium multiflorum
(Stirpe et al., 1980), and dianthin 30 and dianthin 32
from the leaves of Dianthus caryophyllus (carna-
tion) (Stirpe et al., 1981) are proteins that inhibit
protein synthesis by cell-free systems with little effect
on intact cells, and prevent local lesions by tobacco-
mosaic virus (Stevens et al., 1981; Stirpe et al.,
1981). These proteins are similar to the pokeweed
antiviral proteins purified from the leaves (PAP,
Obrig et al., 1973; PAP II, Irvin et al., 1980) and
from the seeds (PAP-S, Barbieri et al., 1982) of
Phytolacca americana (pokeweed), and to tritin,
Coleman & Roberts, 1981), and like these act in an
apparently identical manner by inactivating eukary-
otic ribosomes, on which they act in a, less than
equimolar ratio. All together, the ribosome-inactiv-
ating proteins are reminiscent of the A-chains of
ricin and abrin (reviewed by Olsnes & Pihl, 1977)
and, like the A-chains, could be rendered toxic to
cells by conjugation to suitable carriers capable of
binding to the cell surface. Cytotoxic complexes
were obtained by conjugating gelonin to concana-
valin A (Stirpe et al., 1980) and to a monoclonal
monoclonal antibody (Masuho et al., 1982).
In view of this and of other possible applications,
the main properties of the M. charantia inhibitor, of
gelonin and of dianthins were studied. Their iso-
electric points, amino acid compositions and sugar
contents were determined in the present experi-
(bitter gourd) (Barbieri
1981) or PAP to a
ments. It was ascertained also that proteins purified
from different plants are immunologically distinct
from each other, whereas a cross-reaction was
observed between dianthin 30 and dianthin 32,
which both are purified from carnation leaves.
Proteins and antisera
M. charantia inhibitor (Barbieri et al., 1980),
gelonin (Stirpe et al., 1980) and dianthins (Stirpe
For the production of antisera, New Zealand
White rabbits were immunized by subcutaneous
injections of the proteins (0.5 mg/rabbit), emulsified
with complete Freund's adjuvant for the first dose
and with incomplete adjuvant for the subsequent in-
jections, given 3 and 6 weeks after the first one.
Rabbits were bled 2 weeks after the last injection.
Sera were dialysed overnighf against 0.14M-NaCl
containing 5mM-sodium phosphate buffer, pH7.2,
and were stored frozen at -25 'C.
al., 1981) were purified as described in the
The amino acid and amino sugar compositions of
the proteins were determined in samples extensively
dialysed against distilled water. Protein samples were
hydrolysed in sealed tubes under N2 at 110C with
6M-HCI for 24, 48 and 72h, and were analysed with
acid analyser (Multichrom B 4235;
Beckman, Munich, West Germany). The assay for
cysteine was performed
after oxidation of the
0306-3275/82/120505-05$01.50/1 (C 1982 The Biochemical Society
A. Falasca and others
proteins with performic acid (Moore,
separate hydrolysis with toluene-p-sulphonic acid
was performed for determination of tryptophan and
hexosamine (Allen & Neuberger, 1975).
The neutral sugar composition was analysed by
g.l.c. as described by Dunstan et al. (1974), with
D-mannitol as a standard.
Isoelectric point was determined by isoelectric
focusing on Ampholine polyacrylamide-gel plates
(LKB, Stockholm, Sweden)
3.5-9.5, according to the instructions supplied by
at a pH range of
Treatment with N-ethylmaleimide
Ribosome-inactivating proteins (17pM) were incu-
bated at 370C for 30min in 20mM-sodium phos-
phate buffer, pH 7.4, with or without a
100-fold molar excess of N-ethylmaleimide (Sigma
Chemical Co., St. Louis, MO, U.S.A.). The proteins
were then dialysed overnight at 40C against 500vol.
of 20mM-sodium phosphate, pH 7.4.
Protein synthesis was determined with a lysate of
rabbit reticulocytes (Allen & Schweet, 1962) with
the techniques and chemicals described by Barbieri
agarose gel [1% (w/v) in 0.14M-NaCl containing
20mM-sodium phosphate buffer, pH7.21, cast to a
depth of 4mm on glass
diameter were cut in the gel for antigen and wells of
11 mm for antiserum. Diffusion was allowed to take
in humidity chambers
temperature (approx. 200C).
slides. Wells of 8mm
Results and discussion
activating proteins examined with present experi-
ments can be summarized as follows.
All proteins examined by isoelectric focusing
gave a single band with a basic isoelectric point
behaviour (the proteins bind to CM-cellulose at
A comparison of the amino acid compositions
characteristics of the ribosome-in-
1), consistent with their chromatographic
Table 1. Isoelectric points and amino acid compositions ofribosome-inactivatingproteins
Experimental conditions are described in the text.
Composition (mol/mol ofprotein)
*Redetermined by L. Barbieri (unpublished work). The value given previously (23 000; Barbieri et al., 1980) was found
to be incorrect.
t Values obtained from the hydrolysis at 24, 48 and 72h were extrapolated to zero time. Error was less than 1%.
t Determined ascysteicacid.
§ Determined as described by Allen & Neuberger (1975).
(Table 1) shows some difference in the contents of
lysine, which is lower in the M. charantia inhibitor,
and of cystine and methionine, which are more
abundant in dianthins. As could be expected, the
amino acid composition of dianthins 30 and 32 is
very similar. The amino acid composition of gelonin
is similar to that of PAP-S (Barbieri et al., 1982).
From the amino acid analysis it appears that the
groups. These can be used conveniently for linking
proteins together, and, since one of the possible uses
of these proteins is the preparation of cytotoxic
conjugates with antibodies or other proteins, it is
useful to know whether reactive thiol groups are
involved in the ribosome-damaging activity. When
ribosome-inactivating proteins were incubated in the
presence of a molar excess of N-ethylmaleimide to
block free thiol groups, no significant variation in
observations with gelonin (Stirpe et al., 1980).
The sugar contents are shown in Table 2. All
proteins contain mannose,
especially rich, glucose, xylose, fucose (absent in
gelonin) and glucosamine, the content of which is
lower in dianthin 30 than in other proteins. It should
be pointed out that dianthin 30 contains twice as
much xylose as does dianthin 32. This rules out the
1981), that dianthin 30 could be derived from
Immune sera against the ribosome-inactivating
gelonin and dianthin 30 gave a single band of
precipitation with the respective antiserum, whereas
M. charantia inhibitor and dianthin 32 gave two
bands. The precipitation was specific, except for the
two dianthins, which showed some cross-reactivity.
The antisera also abolished the inhibitory effect on
activating proteins (Table 3), again without sig-
nificant cross-reactivity between the proteins. This
strongly suggests that the functionally active sites of
the proteins are immunologically different from one
protein to another. Alternatively, they may not
coincide with an antibody-binding site, and in this
case the loss of inhibitory activity in the presence of
the antiserum could be the consequence of con-
formational changes following the binding of the
proteins to the respective antibody.
As a whole, these results show that the ribo-
some-inactivating proteins purified from different
plants are all basic proteins, have a different amino
acid composition, and are immunologically distinct
from each other. The last observation may be of
administered to animals either as such or in the form
cell-free protein synthesis was
synthesis of the respective ribosome-in-
IRibosome-inactivating proteinI (,ug/ml)
1. Activity of ribosome-inactivating proteins after
treatment with N-ethylmaleimide
Protein synthesis was measured with a lysate of
rabbit reticulocytes as described in the text. M.
charantia inhibitor (a) or gelonin (b) was added
after prior incubation, as described in the text and in
Table 3, with buffer (0) or with a 10-fold (0) or
100-fold (A) molar excess of N-ethylmaleimide.
Control values were 1692 and 1750 d.p.m. res-
pectively for (a) and (b). Results are the means of
of conjugates selectively toxic to a given type of cells
(Thorpe etal., 1981; Masuho et al., 1982).
The four proteins examined
proteins. Their different carbohydrate compositions
suggest that a specific sugar component
required for their action on ribosomes, consistent
(Barbieri et al., 1982) and in the A-chain of abrin
(Olsnes, 1978), and (ii) the inhibitory activity of
demannosylated gelonin (Stirpe et al., 1980). On the
other hand, it cannot be excluded that sugars may
(i) the lack of detectable sugars in PAP-S
A. Falasca and others
Table 2. Sugar content ofribosome-inactivatingproteins
Experimental conditions are described in the text. Results are means of at least two determinations on two separate
samples ofeach protein; the S.E.M. was <0.09.
Total neutral sugar (%)
Fig. 2. Immunodiffusionofribosome-inactivating proteins against antisera
The following rabbit antisera (lOO,l) were in the centre well: (a) anti-gelonin; (b) anti-M. charantia inhibitor; (c)
anti-dianthin 30; (d) anti-dianthin 32. Antigens (50,ug) were: gelonin in well G, M. charantia inhibitor in well M,
dianthin 30 in well D30 and dianthin 32 in well D32.
have a role in the uptake of ribosome-inactivating
proteins by some cells. It should be recalled, in this
respect, that gelonin conjugated to pentamannose
6-phosphate acquired toxicity for human normal
lymphoid (F-265) and leukaemic (K-562)
(Forbes et al., 1981).
Table 3. Effect ofantisera on theprotein-synthetic inhibitory activity ofribosome-inactivatingproteins
The reaction mixture contained, in a final volume of 125,p1: l0mM-Tris/HCI buffer, pH7.4, 100mM-ammonium
acetate, 1mM-ATP, 0.2mM-GTP,
(Boehringer, Mannheim, West Germany), 0.05mM-amino acids (minus leucine), 0.l9,uCi of L-['4Clleucine, SO,u of
rabbit reticulocyte lysate and, when appropriate, the indicated amount of ribosome-inactivating proteins and 2.5,u1 of
antiserum. Incubation was at 27°C for 5 min. The radioactivity incorporated into protein was measured in 25pl
samples. These results are accurate within + 10%.
15mM-phosphocreatine, 6,ug of creatine kinase
Protein synthesis (% of control)
M. charantia inhibitor (lOng/ml)
Dianthin 32 (25 ng/ml)
Dianthin 30 (50ng/ml)
*1006 d.p.m. incorporated.
Dianthin 32Dianthin 30
This research was supported by a contract from the
Consiglio Nazionale delle Ricerche, Rome, within the
Progetto finalizzato 'Controllo della crescita neoplastica'
and by the Pallotti's Legacy for Cancer Research.
Allen, A. K. & Neuberger, A. (1975) FEBS Lett. 80,
Allen, E. H. & Schweet, R. S. (1962) J. Biol. Chem. 237,
Barbieri, L., Zamboni, M., Lorenzoni, E., Montanaro, L.,
F. (1980) Biochem. J.
Barbieri, L., Aron, G. M., Irvin, J. D. & Stirpe, F. (1982)
Biochem. J. 203, 55-59
Coleman, W. H. & Roberts, W. K. (1981) Biochim.
Biophys. Acta 654, 57-66
Dunstan, D. R., Grant, A. M. S., Marshall, R. D. &
Neuberger, A. (1974) Proc. R. Soc. London Ser. B
Forbes, J. T., Bretthauer, R. K. & Oeltmann, T. N. (1981)
Proc. Natl. Acad. Sci. U.S.A. 78, 5797-5801
Irvin, J. D., Kelly, T. & Robertus, J. D. (1980) Arch.
Biochem. Biophys. 200,418-425
Masuho, Y., Kishida, K. & Hara, T. (1982) Biochem.
Biophys. Res. Commun. 105,462-469
Moore, S. (1963)J. Biol. Chem. 238, 235-237
Obrig, T. G., Irvin, J. D. & Hardesty, B. (1973) Arch.
Biochem. Biophys. 155, 278-289
Olsnes, S. (1978) Methods Enzymol. 50, 323-330
Olsnes, S. & Pihl, A. (1977) in Receptors and Recog-
nition, Series B (Cuatrecasas, P., ed.), vol.
129-173, Chapman and Hall, London
Roberts, W. K. & Stewart, T. S. (1979) Biochemistry 18,
Stevens, W. A., Spurdon, C., Onyon, L. J. & Stirpe, F.
(1981) Experientia 37, 257-259
Stirpe, F., Olsnes, S. & Pihl, A. (1980) J. Biol. Chem.
Stirpe, F., Williams, D. G., Onyon, L. J., Legg, R. F. &
Stevens, W. A. (1981) Biochem. J. 195, 399-405
Thorpe, P., Brown, A. N. F., Ross, W. C. J., Cumber,
A. J., Detre, S. Edwards, D. C., Davies, A. J. S. &
Stirpe, F. (1981) Eur. J. Biochem. 116,447-454