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
Trang 1A role for the intersubunit disulfides of seminal RNase
in the mechanism of its antitumor action
Aurora Bracale1,*, Francesco Castaldi1,*, Lucio Nitsch2and Giuseppe D’Alessio1
1
Dipartimento di Chimica Biologica and2Dipartimento 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 cytocyto-toxic
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
inactive as antitumor agents.Here we report on
mono-meric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells.These monomers are produced
by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides.We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhyd-ryl-disulfide interchange reactions, the native dimeric pro-tein, which is internalized as such, and displays its antitumour action
Keywords: antitumor; BS-RNase; disulfides; RNase
Seminal RNase from bovine seminal vesicles (BS-RNase)
(reviewed in [1]) is a dimeric RNase in which two identical
subunits are held together by noncovalent interactions
and by two intersubunit disulfide bonds bridging Cys31 and
Cys32 of one subunit with the corresponding Cys32¢ and
Cys31¢ of the partner subunit.BS-RNase is an antitumour
agent, as it is strongly and selectively cytotoxic for
malignant cells in vitro and in vivo, with no effects on
normal cells [2]
Since the early studies on the antitumor action of
BS-RNase, it has been recognized that the dimeric structure of
the enzyme is essential for its display of cytotoxic activity
[3].This conclusion was based on the lack of cytotoxic
activity in a monomeric derivative of the protein obtained
by selective, reductive cleavage of the intersubunit disul-fides followed by alkylation of the exposed sulfhydryls 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` di Napoli ‘Federico II’, Via Mezzocannone 16,
80134 Napoli, Italy.
Fax: + 39 081 5521217, Tel.: + 39 081 2534731,
E-mail: dalessio@unina.it
Abbreviations: BS-RNase, bovine seminal RNase; MCM, monomeric
bis-Cys31,Cys32-S-carboxymethylated-BS-RNase; MCA,
mono-meric bis-Cys31,Cys32-S-carboxyamidomethylated-BS-RNase;
MAE, monomeric bis-Cys31,Cys32-S-aminoethylated-BS-RNase;
MSSAE, monomeric bis-Cys31,Cys32-S-ethylamine-BS-RNase;
MSSG, monomeric bis-Cys31,Cys32-S-glutathione-BS-RNase;
CRI, cytosolic RNase inhibitor; PM, plasma membrane; IAM,
iodoacetamide.
*Note: These authors contributed equally to this work.
(Received 23 January 2003, revised 5 March 2003,
accepted 13 March 2003)
Trang 2acid) and alkaline phosphatase-conjugated anti-rabbit
secondary Ig were purchased from Sigma.Reagents for
Western blotting detection (SuperSignal West Dura
Chemiluminescent Substrate, and ImmobilonTM-P
mem-branes were purchased from Celbio, Milan,
Italy).Poly-clonal antibodies against BS-RNase, obtained from rabbits
as described previously [11], were used at a dilution of
1 : 1000.Fluorescein-tagged goat anti-rabbit secondary Ig
was obtained from Jackson ImmunoResearch (West Grove,
PA, USA).BS-RNase and its monomeric derivatives were
prepared as described [12].All monomers were
homogene-ous upon SDS/PAGE, and catalytically active by RNase
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 Na125I (Amersham) using
IODO-BEADS (Pierce) following the manufacturer’s instructions,
and desalted on PD10 Sephadex G-25 M columns
(Phar-macia), equilibrated with NaCl/Pi.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 37C in a humidified incubator containing 10%
CO2mixed with air
Cytotoxicity assay
Cells were seeded in 24- or 96-well plates (1.2· 104cellsÆ
cm)2) in the presence of the RNase to be tested.After a
48-h incubation, cells were trypsinized, resuspended in
growth medium, mixed with trypan blue solution (Sigma)
(1 : 1, v/v), and counted.Cell viability was determined in
triplicate as the percentage of trypan blue-excluding cells
with respect to the total cell count
Preparation of cell lysates
Cells treated with the RNase under test were washed first
with 1MHepes pH 7.5 containing 0.1MNaCl (Hepes/NaCl
buffer) for 5 min, then three times with NaCl/Pi, scraped
from plates with a rubber policeman, collected by
centrifu-gation at 1000 g, and resuspended in lysis buffer (1% NP-40
in 50 mMTris/HCl at pH 8.0) in the presence of a protease
inhibitors cocktail (CØMPLETETM, Roche).Cells were
lyzed by vortexing, incubated on ice for 30 min, and
centrifuged at 16 000 g for 30 min.The final supernatant
was assayed for protein concentration and frozen at)80 C,
or processed immediately.All steps were performed at 4C
Preparation of the membrane fraction Cells were grown to confluency in 150 mm plates, washed twice with NaCl/Piand scraped with a rubber policeman in homogenization buffer (10 mM Tris/HCl, pH 7.5, 0.25M 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/Pi, assayed for protein concentration and frozen at )80 C or processed immediately.All steps were performed
at 4C
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/Pifor 15 min at room temperature RNases were detected with the BS-RNase antiserum.To test the immunofluorescence of internalized proteins, cells were washed with Hepes/NaCl for 5 min and permeabilized with 0.1% Triton X-100 in NaCl/Pi for 5 min at room temperature.Fluorescein-conjugated secondary antibody was used at a dilution of 1 : 50.Cells were visualized by epifluorescence using an Axiophot microscope (Zeiss)
Results and discussion
The monomeric derivatives of BS-RNase employed in this study, illustrated in Table 1, were prepared following established procedures [12] for the derivatization of Cys31 and Cys32, the cysteine residues that form the intersub-unit disulfides of BS-RNase.Briefly, MCM (monomeric bis-Cys31,Cys32-S-carboxymethylated-BS-RNase), MCA (monomeric carboxyamidomethylated-BS-RNase), and MAE (monomeric bis-Cys31,Cys32-S-aminoethylated-BS-RNase) were obtained by selective reduction of the protein intersubunit disulfides followed
by alkylation of the exposed sulfhydryls with iodoacetate, iodoacetamide or 2-bromoethylamine hydrobromide, respectively.The MSSAE monomer was obtained by reaction with methyl aminoethanethiosulfonate of the sulfhydryls exposed by selective reduction of the
Table 1 Monomeric derivatives of BS-RNase LC 50 is the protein concentration producing 50% of cell death.
Mixed disulfide
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+3 ) 2 (MAE) No >200 M–(CH 2 –S–S–CH 2 –CH 2 –NH 3+) 2 (MSSAE) Yes 47 ± 6 M–(CH 2 –S–S–CH 2 –cGlu–) 2 (MSSG) Yes 31
| Gly M–(CH 2 –S–S–CH 2 ) 2 –M (BS RNase) 25 ± 4
Trang 3intersubunit 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
inhibited by CRI [5], all monomers investigated in the
present study can be considered as highly sensitive to the
inhibitory action of CRI
We tested the cytotoxic activity of the monomeric
derivatives described above on malignant SVT2-3T3
fibro-blasts by measuring cell survival after 48 h of growth in the
presence of increasing concentrations of each monomeric
derivative.The data illustrated in Fig.1 show that some
monomers (MCM, MCA, MAE) have no cytotoxic activity
on malignant SVT2 cells, whereas others (MSSG and
MSSAE) are surprisingly cytotoxic.This cytotoxic action
was selective for malignant cells, as when the latter, active monomers were tested on nonmalignant 3T3 fibroblasts, they were found to be as devoid of toxicity as native, dimeric BS-RNase (data not shown)
It is noteworthy that in the inactive MCM, MCA and MAE monomers Cys31 and Cys32, the cysteine residues originally involved in the intersubunit disulfide bonding
of BS-RNase, are irreversibly blocked through S-alkylation
In the active MSSAE and MSSG monomers, instead, the two Cys residues still form (mixed) disulfide bonds with thioethylamine or glutathione moieties, respectively (Table 1).This led us to hypothesize that the cytotoxic activity of the latter monomers was due to the presence in these proteins of disulfide bonds, with their potential chemical instability.It is well known that in the presence
of thiolates, disulfides can undergo sulfhydryl-disulfide interchange reactions.Thus, at difference with the mono-mers bearing stable, S-alkylated Cys residues, MSSAE and MSSG monomers could, when delivered to growing cells, 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 106 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 37C in the presence of 20 lgÆmL)1of radioactively labelled125I-labelled MSSAE in binding buffer (DMEM containing 1 mgÆmL)1BSA and 25 mMHepes at
pH 7.5) At increasing time intervals, cells were washed repeatedly with NaCl/Piand then treated with 0 6MNaCl
in NaCl/Pifor 5 min at 4C to detach labelled monomers bound to the cell surface [20].The detached labelled protein was then analyzed by SDS/PAGE followed by autoradio-graphy.The results shown in Fig.2 indicate that MSSAE monomers upon binding to the cell surface associate into a dimeric protein, with the molecular size of BS-RNase.A quantitation of the dimeric bands identified on the gel (Fig.2) shows that MSSAE undergoes dimerization into native-like BS-RNase in a time-dependent manner, and is
Fig 1 Dose–response effects on BALB/C 3T3-SVT2 cells of
mono-meric derivatives of BS-RNase Cells were treated for 48 h at 37 C
with MCA (h), MCM (j), MAE (e), MSSAE (r), MSSG (m) or
BS-RNase as a positive control (d).
2 M-ðS-S-RÞ2þ n CELL-S! M-ðS-S-CELLÞ2þ M-ðSÞ2þ 4 RSþ n-2 CELL-S ð1Þ M-ðS-S-CELLÞ2þ M-ðSÞ2þ n-2CELL-S! M-ðS-SÞ2-Mþ n CELL-S ð2Þ
Trang 4almost totally dimeric after a 24-h contact with growing
cells.When125I-labelled MSSAE was incubated in binding
buffer in the absence of cells no dimerization occurred (data
not shown)
These results indicate that a monomeric derivative of
BS-RNase in which disulfide bonds are conserved at Cys31 and
Cys32 residues can reconstitute into native-like BS-RNase
when administered to growing fibroblasts.Such a
transfor-mation can be explained by sulfhydryl-disulfide interchange
reactions occurring between cell sulfhydryls and the mixed
disulfide bonds present in the monomeric derivative
We further investigated whether the MSSAE and MSSG
monomers conserved the acquired dimeric structure upon
cell internalization.This was considered a necessary
condi-tion to attribute to the dimerizacondi-tion event a role in the
antitumour action of BS-RNase, as BS-RNase monomers
would be neutralized in the cytosol by the action of CRI
SVT2 cells were grown with MSSAE, MSSG, or native
BS-RNase at a concentration of 50 lgÆmL)1.After 24 h cells
were washed at 4C with NaCl/Pi, then with 0.6MNaCl to
remove proteins from the cell surface.Washed cells were then lysed and analyzed by SDS/PAGE followed by 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 mMiodoacetamide (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)1of MSSAE for 75 min at 37C.When treated cells were tested with anti-BS-RNase serum MSSAE was found to bind effectively to their surface (Fig.4A).SVT2 fibroblasts were then treated with MSSAE, then stripped of surface bound proteins with a high salt solution made up of 1M Hepes
pH 7.5 containing 0.1MNaCl [11], and permeabilized with 0.1% Triton X-100 The results of this experiment, illustra-ted in Fig.4B, show that BS-RNase immunoreactivity is localized inside the cells in endosome-like vesicles throughout the cytoplasm (Fig.4B).These results are identical to those obtained under identical conditions with native BS-RNase [11].Together with the results described above, they confirm that MSSAE monomers dimerize outside the cells, and are internalized as dimeric BS-RNase
As the dimerization event occurs outside the cells, before internalization, we investigated the role of plasma mem-branes (PM) in the transformation of MSSAE into a dimeric protein 125I-labelled MSSAE was incubated with isolated membranes from SVT2 fibroblasts (0.45 mgÆmL)1
of total protein) for 16 h at 37C in 0 2 mL NaCl/Pi.The membranes were either washed with 0.6MNaCl in NaCl/Pi,
or washed with NaCl and then, after removal of the supernatant by centrifugation for 20 min at 16 000 g, treated with 2 mM dithiothreitol in NaCl/Pi.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 NaCl and the
Fig 2 Time-course of dimerization of the labelled monomeric derivative
of BS-RNase125I-labelled MSSAE added to growing SVT2 cells.125
I-labelled MSSAE was detached by high salt from SVT2 cells at
increasing time intervals.In the insert, autoradiographic scans of
the SDS/PAGE runs.D and M mark the electrophoretic mobilities
of BS-RNase and monomeric BS-RNase, respectively.
Fig 3 Immunoblots of SVT2 cell lysates Lysates were from cells
treated for 24 h with BS-RNase (lane 1), monomeric MSSAE (lane 2),
monomeric MSSG (lane 3), MSSAE from a lysate performed in the
presence of 2 m M iodoacetamide (lane 4), BS-RNase from a lysate
performed in the presence of 2 m M iodoacetamide (lane 5), MSSAE as
in lane 2 after electrophoresis under reducing conditions (lane 6) and
BS-RNase as in lane 1 after electrophoresis under reducing conditions
(lane 7).
Trang 5membrane pellet was treated with dithiothreitol, the
mem-brane entrapped monomers could be released (lane 4), albeit
not completely, as some of them were still found to remain
entrapped by PM (lane 5)
The dimerization effect of PM on MSSAE was dependent
on PM concentration.As shown in Fig 6, at approximately
0.25 mgÆmL)1of PM protein concentration, dimerization
reached a plateau
These data indicate that the cell sulfhydryls responsible
for the exchange with the protein disulfides are located in
the plasma membrane.Furthermore, they show that, as
proposed in the hypothesis above, the RNase monomers are
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 with125I-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)
125I-labelled MSSAE (20 lgÆmL)1) was added to cell membranes in the presence or the absence of 10 or 50 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.
Trang 6IAM and incubated for 16 h at 37C.The labelled protein
extracted from PM by 0.6M NaCl in NaCl/Pi was
gel-filtered on a Superdex-75 column.As shown in Fig.7A,
after 16 h of incubation with PM, MSSAE was found to be
totally converted into dimers.When the incubation was
carried out in the presence of 10 mMIAM, the product of
Fig 5 Autoradiography of SDS/PAGE runs of the labelled monomeric
derivative of BS-RNase125I-labelled MSSAE incubated with plasma
membranes (PM) from SVT2 cells Lane 1, plasma membranes treated
for 16 h with125I-labelled MSSAE; lane 2, labelled proteins extracted
from PM with high salt; lane 3, labelled proteins still bound to
extracted PM; lane 4, labelled proteins extracted from the PM pellet
with 2 m M dithiothreitol; lane 5, proteins from the PM pellet after
treatment with dithiothreitol.
Fig 6 Dimerization effect of PM isolated from SVT2 cells on the
labelled monomeric derivative of BS-RNase125I-labelled MSSAE
trea-ted with increasing concentrations of PM Inset, autoradiographic scans
of the SDS/PAGE runs of 125 I-labelled MSSAE detached by high salt
from PM.D and M mark the electrophoretic mobilities of BS-RNase
and monomeric BS-RNase, respectively.
Fig 7 Gel-filtration analysis of the labelled monomeric derivative of
BS-RNase125I-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.
Trang 7dimerization decreased to 60% (Fig.7B); at the higher IAM
concentration (50 mM), 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
proposed above
It has been reported [21–23] that protein-disulfide
iso-merase (PDI) is present and active at the plasma membrane
surface of many types of cells.We thus considered the
possibility that PDI had a role in the dimerization reaction
of BS-RNase M(SSR)2 monomers.However, we did not
detect any effects of 1–10 mMconcentrations of bacitracin
(Sigma), a known inhibitor of PDI [21], on the dimerization
reaction.Likewise, an anti-PDI serum (Stressgen) had no
inhibitory effects on the reaction.These data suggest that
PDI has no role in the reconstitution of dimeric BS-RNase
from M(SSR)2monomers
Conclusion
The results reported here reveal a new, significant event in
the mechanism of cytotoxic action of BS-RNase on
malignant cells.The event consists in the interactions,
through sulfhydryl–disulfide interchange reactions, between
surface cell sulfhydryls and the intersubunit disulfides that
link the two subunits of BS-RNase.Monomeric derivatives
of the protein are inactive as cytotoxic agents when they are
prepared by reductive cleavage of the intersubunit disulfides
and the resulting free sulfhydryls are blocked through
alkylation.Monomers of BS-RNase are instead active when
obtained by linking to small thiol compounds the
sulfhy-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).
References
1 D’Alessio, G., Di Donato, A., Mazzarella, L & Piccoli, R (1997) Seminal Ribonuclease: The Importance of Diversity.In Ribonucleases: Structures and Functions (Riordan, J.F & D’Alessio, G , eds), pp.383–423.Academic Press, New York, USA.
2 Youle, R.J.& D’Alessio, G.(1997) Antitumor RNases.In Ribo-nucleases: Structures and Functions (Riordan, J.F & D’Alessio, G., eds), pp 491–509 Academic Press, New York, USA 3.Vescia, S , Tramontano, D , Augusti Tocco, G.& D’Alessio, G (1980) I n vitro studies on selective inhibition of tumor cell growth
by seminal ribonuclease Cancer Res 40, 3740–3744.
4 Kim, J.S., Soucek, J., Matousek, J & Raines, R.T (1995) Struc-tural basis for the biological activities of bovine seminal ribo-nuclease J Biol Chem 270, 10525–10530.
5 Murthy, B.S., De Lorenzo, C., Piccoli, R., D’Alessio, G & Sirdeshmukh, R.(1996) Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase Biochemistry 35, 3880–3885.
6 Leland, P A , Schultz, L W , Kim, B -M & Raines, R T (1998) Ribonuclease A variants with potent cytotoxic activity Proc Natl Acad Sci USA 95, 10407–10412.
7.Kobe, B.& Deisenhofer, J.(1993) Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats Nature
366, 751–756.
8 Papageorgiou, A., Shapiro, R.& Acharya, K.(1997) Molecular recognition of human angiogenin by placental ribonuclease inhibitor – an X-ray crystallographic study at 20 A˚ resolution EMBO J 16, 5162–5177.
9.Kobe, B.& Deisenhofer, J.(1996) Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A J Mol Biol 264, 1028–1043.
10 Antignani, A., Naddeo, M., Cubellis, M., Russo, A & D’Alessio, G.(2001) Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor Biochemistry 40, 3492–3496.
11 Bracale, A., Spalletti-Cernia, D., Mastronicola, M., Castaldi, F., Mannucci, R., Nitsch, L & D’Alessio, G (2002) Essential stations
in the intracellular pathway of cytotoxic bovine seminal ribonuc-lease Biochem J 362, 553–560.
12 D’Alessio, G., Di Donato, A., Piccoli, R & Russo, N (2001) Seminal ribonuclease: preparation of natural and recombinant enzyme, quaternary isoforms, isoenzymes, monomeric forms; assay for selective cytotoxicity of the enzyme Methods Enzymol.
341, 248–263.
13.Kunitz, M.(1946) A spectrophotometric method for the meas-urement of ribonuclease activity J Biol Chem 164, 563–568 14.Ellman, G.& Lysko, H.(1979) A precise method for the determination of whole blood and plasma sulfhydryl groups Anal Biochem 93, 98–102.
15.Blackburn, P , Wilson, G.& Moore, S.(1977) Ribonuclease inhibitor from human placenta.Purification and properties.
J Biol Chem 252, 5904–5410.
16.Murthy, B S.& Sirdeshmukh, R.(1992) Sensitivity of monomeric and dimeric forms of bovine seminal ribonuclease to human placental ribonuclease inhibitor Biochem J 281, 343–348 17.Gitler, C.& Londner, M.(1995) Use of p-nitrophenyl disulfide to measure reductive capacity of intact cells Methods Enzymol 251, 279–286.
18 Parente, A., Merrifield, B., Geraci, G & D’Alessio, G (1985) Molecular basis of superreactivity of cysteine residues 31 and 32
of seminal ribonuclease Biochemistry 24, 1098–1104.
19 D’Alessio, G., Malorni, M.C.& Parente, A.(1975) Dissociation of bovine seminal ribonuclease into catalytically active monomers by
Trang 8selective reduction and alkylation of the intersubunit disulfide
bridges Biochemistry 14, 1116–1122.
20.Mastronicola, M R , Piccoli, R.& D’Alessio, G.(1995) Key
extracellular and intracellular steps in the antitumor action of
seminal ribonuclease Eur J Biochem 230, 242–249.
21 Couet, J , de Bernard, S , Loosfelt, H , Saunier, B , Milgrom, E &
Misrahi, M.(1996) Cell surface protein disulfide-isomerase is
involved in the shedding of human thyrotropin receptor
ecto-domain Biochemistry 35, 14800–14805.
22 Zai, A., Rudd, M.A., Scribner, A.W & Loscalzo, J (1999) Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide J Clin Invest 103, 393–399.
23 Shin, B K , Wang, H , Yim, A M , Le Naour, F , Brichory, F , Jang, J H , Zhao, R , Puravs, E , Tra, J , Michael, C W , Misek, D.E & Hanash, S.M (2003) Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function J Biol Chem 278, 7607–7616.