A role for the intersubunit disulfides of seminal RNase
in the mechanism of its antitumor action
Aurora Bracale
1,
*, Francesco Castaldi
1,
*, Lucio Nitsch
2
and Giuseppe D’Alessio
1
1
Dipartimento di Chimica Biologica and
2
Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’,
Universita
`
di Napoli, Italy
The dimeric structure of seminal ribonuclease (BS-RNase)
is maintained by noncovalent interactions and by two
intersubunit disulfide bridges. Another unusual feature of
this enzyme is its antitumour action, consisting in a cyto-
toxic activity selective for malignant cells. This cytotoxic
action is exerted when the protein reaches the cytosol of
the affected cells, where it degrades ribosomal RNA, thus
blocking protein synthesis and leading cells to death. The
current model proposed for the mechanism of antitumour
action of BS-RNase is based on the ability of the protein
to resist the neutralizing action of the cytosolic RNase
inhibitor, a resistance due to the dimeric structure of the
enzyme. Monomeric RNases, and monomeric derivatives
of BS-RNase, are strongly bound by the inhibitor and

Such conclusion has been subsequently confirmed through
different experimental approaches [4], and explained [4–6]
by the resistance of the enzyme in its dimeric state to the
inhibitory action of CRI (the cytosolic RNase inhibitor).
When the structure of CRI [7] and CRI complexed to
RNases [8,9] were elucidated, it became clear how native,
dimeric BS-RNase cannot fit into the horseshoe cavity of
the inhibitor, whereas a monomeric form of the enzyme
can, and is fully inhibited by CRI. Indeed, monomeric
RNases lacking cytotoxic activity, such as bovine pancre-
atic RNase and monomeric BS-RNase, could be engine-
ered into cytotoxic agents by rendering them resistant to
CRI [6,10].
In a survey of monomeric derivatives of BS-RNase, we
found that some of them, although fully inhibited by CRI,
were active as cytotoxic agents, and selective for malignant
cells. Further investigation revealed that monomeric deri-
vatives of BS-RNase are cytotoxic only when they conserve
the intersubunit cystine residues, so that they can be
re-converted into dimers, an event primed by cell sulfhydryls.
These results indicate that the intersubunit disulfide bonds
of BS-RNase have a key role in the mechanism of
antitumour action of the enzyme.
Materials and methods
Materials
Iodoacetic acid (IAA), iodoacetamide (IAM), 2-bromo-
ethylamine hydrobromide, 5,5¢-dithio-bis(2-nitrobenzoic
Correspondence to G. D’Alessio, Dipartimento di Chimica Biologica,
Universita
`

assay [13].
Other methods
The monomeric derivative MSSAE (monomeric bis-
Cys31,Cys32-S-ethylamine-BS-RNase, 100 lg) was labelled
with 1 mCi carrier-free Na
125
I (Amersham) using IODO-
BEADS (Pierce) following the manufacturer’s instructions,
and desalted on PD10 Sephadex G-25 M columns (Phar-
macia), equilibrated with NaCl/P
i
. The specific activity of
labelled MSSAE was approximately 1 lCi per mg of protein.
Sulfhydryl content was determined as described by [14].
RNase inhibition by the cytosolic RNase inhibitor was
determined as described previously [15].
Cell cultures
SV40-transformed mouse fibroblasts and the parental
nontransformed Balb/C 3T3-line were obtained from
American Type Culture Collection (USA) and grown in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco-Life
Technology) supplemented with 10% fetal bovine serum
(Gibco-Life Technology) and Penicillin-Streptomycin-Glu-
tamine 1X (Gibco-Life Technology). Cell lines were main-
tained at 37 °C in a humidified incubator containing 10%
CO
2
mixedwithair.
Cytotoxicity assay
Cells were seeded in 24- or 96-well plates (1.2 · 10

was assayed for protein concentration and frozen at )80 °C,
or processed immediately. All steps were performed at 4 °C.
Preparation of the membrane fraction
Cells were grown to confluency in 150 mm plates, washed
twice with NaCl/P
i
and scraped with a rubber policeman in
homogenization buffer (10 m
M
Tris/HCl, pH 7.5, 0.25
M
sucrose containing the protease inhibitors cocktail). Cells
were homogenized by 25 strokes with the tight pestle of a
Dounce homogenizer. The homogenate was centrifuged at
1000 g for 10 min and the supernatant was centrifuged at
16 000 g for 30 min. The pellet, representing the plasma
membrane enriched fraction (PM), was resuspended in
NaCl/P
i
, assayed for protein concentration and frozen at
)80 °C or processed immediately. All steps were performed
at 4 °C.
Immunofluorescence studies
Immunofluorescence experiments were performed as previ-
ously described [11]. Briefly, mouse fibroblasts were incu-
bated with the RNase under test and fixed with 3.7%
formaldehyde in NaCl/P
i
for 15 min at room temperature.
RNases were detected with the BS-RNase antiserum. To

LC
50
(lgÆmL
)1
)
M–(CH
2
–S–CH
2
–COO
–
)
2
(MCM) No >200
M–(CH
2
–S–CH
2
–CONH
2
)
2
(MCA) No >200
M–(CH
2
–S–CH
2
–CH
2
–NH

–S–S–CH
2
)
2
–M (BS RNase) 25 ± 4
Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1981
intersubunit disulfides. The MSSG monomer (mono-
meric bis-Cys31,Cys32-S-glutathione-BS-RNase) was a by-
product of the preparation of recombinant BS-RNase, in
which Cys31 and Cys32 residues form mixed disulfides with
glutathione moieties.
All monomeric derivatives retained full RNase activity, in
fact they were more active than the parent dimeric enzyme,
as previously reported [12]. As for their sensitivity to the
inhibitory action of the cytosolic RNase inhibitor (CRI), it
is known that MCM is fully inhibited by CRI [16]. We
tested MCA and MAE with increasing concentrations of
CRI and found that they were inhibited by approximately
90% with a 2–4 molar excess of CRI. Monomers MSSAE
and MSSG could not be tested as such for inhibition by
CRI, because the strongly reducing conditions of the assay
produce the cleavage of their mixed disulfides. This in turn
generates, from either MSSAE or MSSG, M(SH)
2
mono-
mers, i.e. BS-RNase monomers with exposed sulfhydryls at
Cys31 and Cys32, and free thioethylamine or glutathione,
respectively. As M(SH)
2
has been shown to be fully

undergo reactions with cell thiolates, which could lead to
their transformation into dimers, as described below:
where M is a BS-RNase monomer, R is the thioethylamine
or the glutathione moiety, CELL-S
–
are cell thiolates
present in n molar excess, and M-(S-S)
2
-M is a reconsti-
tuted dimer, in fact indistinguishable from native
BS-RNase.
The presence of sulphydryls on the surface of SVT2
cells was tested with 5,5¢-dithio-bis(2-nitrobenzoic acid) a
reagent impermeable to cell membrane [17]. We found
63 nmol of reactive, surface sulphydryls per 10
6
SVT2
cells. In a typical experiment, this would give a molar
excess of cell thiol groups of approximately 50-fold over
the disulfides introduced in the cell culture upon treatment
with the RNase monomers. It should be added that the
intersubunit disulfides of BS-RNase are hyper-reactive to
reduction, even to mild reducing agents, with respect to
intrachain disulfides [18,19], and are completely cleaved by
a 10-fold molar excess of dithiothreitol [19]. This hyper-
reactivity is a feature also of the mixed disulfides formed
by Cys31 and Cys32 with glutathione [12], and of the
mixed disulfides of MSSAE (unpublished results).
To verify the hypothesis described above, SVT2 fibro-
blasts were grown at 37 °C in the presence of 20 lgÆmL

BS-RNase as a positive control (d).
2M-ðS-S-RÞ
2
þ n CELL-S
À
! M-ðS-S-CELLÞ
2
þ M-ðS
À
Þ
2
þ 4RS
À
þ n-2 CELL-S
À
ð1Þ
M-ðS-S-CELLÞ
2
þ M-ðS
À
Þ
2
þ n-2CELL-S
À
! M-ðS-SÞ
2
-M þ n CELL-S
À
ð2Þ
1982 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003

immunoblotting with an anti-BS-RNase serum. The results
of these experiments, illustrated in Fig. 3, show that inside
the cells BS-RNase, MSSG and MSSAE are all present as
dimers. These dimers are covalent, as when the electro-
phoresis run was performed under reducing conditions,
most of the dimeric proteins dissociated into monomers
(Fig. 3). Identical results were obtained when cell lysis was
carried out in the presence of 2 m
M
iodoacetamide (IAM) to
block any free sulfhydryls (Fig. 3). This indicates that dimer
formation through disulfide bonding did not occur as an
artifact during lysis.
These results led us to conclude that indeed BS-RNase
monomers linked through disulfides to thioethylamine or
glutathione moieties are reconstituted in the presence of
growing fibroblasts into the parent dimeric protein, which
is internalized as a native-like dimeric RNase. They also
indicate for the first time that when BS-RNase is internal-
ized by malignant cells, it maintains its dimeric structure.
We have previously demonstrated by immunofluores-
cence studies that BS-RNase binds to the surface of SVT2
cells and is internalized inside the cells, whilst the MCM
monomer does not bind and is not internalized [11]. We
repeated these experiments with the MSSAE monomer and
treated exponentially growing SVT2 cells with 50 lgÆmL
)1
of
MSSAE for 75 min at 37 °C. When treated cells were tested
with anti-BS-RNase serum MSSAE was found to bind

i
,
or washed with NaCl and then, after removal of the
supernatant by centrifugation for 20 min at 16 000 g,
treatedwith2m
M
dithiothreitol in NaCl/P
i
. Labelled
proteins extracted from plasma membranes and membrane
pellets were then analyzed by SDS/PAGE and autoradio-
graphy. Figure 5 shows that after incubation with labelled
MSSAE, membranes contained radioactive protein both
monomeric and dimeric (lane 1). This indicates that under
the conditions employed a substantial fraction of MSSAE
was dimerized. In the fraction extracted from PM by the salt
treatment (lane 2), most (approximately 80%) of the protein
was dimeric. Clearly, monomers remained entrapped in the
PM pellet, which upon electrophoresis in SDS was found to
contain almost all monomeric protein (lane 3). When
membranes were extracted with 0.6
M
NaCl and the
Fig. 2. Time-course of dimerization of the labelled monomeric derivative
of BS-RNase
125
I-labelled MSSAE added to growing SVT2 cells.
125
I-
labelled MSSAE was detached by high salt from SVT2 cells at

linked through disulfides to the cell membrane, and are
released only when additional sulfhydryl–disulfide exchange
reactions occur, which eventually lead to their association
into dimers.
These results were confirmed when the separation of
monomeric and dimeric RNase species produced by treating
membranes with
125
I-labelled MSSAE was performed by
gel filtration. In these experiments the role of membrane
sulfhydryls in MSSAE dimerization was further verified by
testing the effect on dimerization of iodoacetamide (IAM).
125
I-labelled MSSAE (20 lgÆmL
)1
)wasaddedtocell
membranes in the presence or the absence of 10 or 50 m
M
Fig. 4. Fluorescence studies of SVT2 fibroblasts treated with the MSSAE monomeric derivative of BS-RNase. Cells were treated with 50 lgÆmL
)1
MSSAE for 75 min at 37 °C and fixed without permeabilization (A) or after a high-salt washing and permeabilization with Triton X-100 (B). The
RNase was detected with anti-BS-RNase serum followed by incubation with fluorescein-tagged anti-rabbit secondary Ig. The bar represents 10 lm.
1984 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003
IAM and incubated for 16 h at 37 °C. The labelled protein
extracted from PM by 0.6
M
NaCl in NaCl/P
i
was
gel-filtered on a Superdex-75 column. As shown in Fig. 7A,

BS-RNase
125
I-labelled MSSAE after a 16-h incubation with isolated
PM from SVT2 cells. The incubation was performed (A) in the absence
of iodoacetamide (IAM), (B) in the presence of 10 m
M
IAM, or (C) of
50 m
M
IAM. D and M mark the elution volumes of BS-RNase and
monomeric BS-RNase, respectively.
Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1985
dimerization decreased to 60% (Fig. 7B); at the higher IAM
concentration (50 m
M
), only 30% of dimer was produced
(Fig. 7C).
The data from the experiments on plasma membranes
indicate that the cell sulfhydryls responsible for the inter-
changes with disulfides, the reactions that reconstitute
native-like BS-RNase, belong to the plasma membranes.
They also show that BS-RNase monomers derived from
MSSAE bind covalently through disulfide bonds to the
membranes, as they can be released from the membranes as
monomers only through the action of a reducing agent, such
as dithiothreitol. The labelled RNase monomer, when
added to PM, is released from the membranes as a dimeric
protein, apparently produced by a sulfhydryl–disulfide
interchange occurring on the membranes. These are exactly
the events described in Eqns (1 and 2) of the hypothesis

dryls exposed after reductive cleavage. The latter monomers
are found to reconstitute into disulfide linked dimers when
they interact with malignant cells, or with isolated cell
membranes, and are recovered as covalent dimers in treated
cell lysates. Also native BS-RNase is found to be a covalent
dimer inside the cells. These data lead us to conclude
that the same interchange reactions occur when the native
BS-RNase dimer binds and penetrate cells, with the protein
undergoing a double transition from dimer to monomers
linked to cell sulfhydryls, to covalent dimer again. Thus, the
reported results provide a first clue to the mechanism by
which BS-RNase is endocytosed by cells.
Acknowledgement
This work was financed by grants from the Associazione Italiana per la
Ricerca sul Cancro (AIRC), Ministero dell’Universita
`
e della Ricerca
(Progetti di Rilevante Interesse Nazionale 2001) and Consorzio
Interuniversitario Biotecnologie. Aurora Bracale was supported by a
fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC).
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