From the Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India
Received for publication, July 23, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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Saporin-6 produced by the plant Saponaria
officinalis belongs to the family of single chain
ribosome-inactivating proteins. It potently inhibits protein synthesis
in eukaryotic cells, by cleaving the N-glycosidic bond of a
specific adenine in 28 S rRNA, which results in the cell death.
Saporin-6 has also been shown to be active on DNA and induces
apoptosis. In the current study, we have investigated the roles of rRNA
depurination and the activity of saporin-6 on genomic DNA in its
cytotoxic activity. The role of putative active site residues,
Tyr72, Tyr120, Glu176,
Arg179, and Trp208, and two invariant residues,
Tyr16 and Arg24, proposed to be important for
structural stability of saporin-6, has been investigated in its
catalytic and cytotoxic activity. These residues were mutated to
alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A,
R179A, and W208A. We show that for the RNA N-glycosidase
activity of saporin-6, residues Tyr16, Tyr72,
and Arg179 are absolutely critical; Tyr120 and
Glu176 can be partially dispensed with, whereas
Trp208 and Arg24 do not appear to be involved
in this activity. The residues Tyr72, Tyr120,
Glu176, Arg179, and Trp208 were
found to be essential for the genomic DNA fragmentation activity,
whereas residues Tyr16 and Arg24 do not appear
to be required for the DNA fragmentation. The study shows that
saporin-6 possesses two catalytic activities, namely RNA
N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.
Ribosome-inactivating proteins
(RIPs)1 are toxic translation
inhibitors produced by plants and bacteria. RIPs have been classified into two types; type I RIPs are composed of a single polypeptide chain,
whereas type II RIPs consist of two chains, A and B, linked by a
disulfide bond (1). Type I RIPs (e.g. saporin,
trichosanthin, and pokeweed antiviral protein) and A-chain of
type II RIPs (e.g. ricin and abrin) inhibit protein
synthesis by removing a specific adenine from 28 S rRNA of eukaryotic
ribosomes. The former also removes an equivalent adenine residue,
A2660, from 23 S rRNA of Escherichia coli
ribosomes (2). In both cases, the site of action is located in a highly
conserved, purine-rich stem and loop structure of rRNA termed the
Saporin is a family of single-chain ribosome-inactivating proteins
present in abundance in the plant Saponaria officinalis (18). Among RIPs, various peculiar features of saporin, in terms of its
remarkable stability and activity on a wide variety of substrates, make
it an interesting protein to study for structure-function relationships
(19, 20). The crystal structure of saporin-6 has been solved recently
(21). The structure superimposition of saporin-6 has shown that
corresponding to ricin A-chain active site residues, the residues
Tyr72, Tyr120, Glu176,
Arg179, and Trp208 constitute the active site
of saporin-6 (21, 22).
In the present study, analytical mutagenesis of saporin-6, the most
active isoform, was carried out to study the role of residues present
in its active site. The active site residues Tyr72,
Tyr120, Glu176, Arg179, and
Trp208 were mutated to alanine, and the activities of the
mutants were compared with the wild type protein using various
functional assays. An interesting feature found in primary sequence
alignment of RIPs was the invariance of residues, tyrosine and
arginine, corresponding to position 21 and 29, respectively, in the
ricin sequence. An analysis of ricin-A chain structure showed that a
central Construction of Saporin-6 Mutants--
Saporin-6 is a protein
consisting of 253 amino acid residues. pSap-6 is a plasmid containing
the 759-base pair saporin-6 gene cloned downstream of a T7 promoter in
bacterial expression vector pVex 11. pSap-6 was used as a template to
mutate the codons for active site residues Tyr72,
Tyr120, Glu176, Arg179, and
Trp208 to that for alanine. Similarly, the codons for the
invariant residues Tyr16 and Arg24 were also
mutated to that for alanine. All of the mutations were carried out by
oligonucleotide-mediated site-directed mutagenesis (25).
Uracil-containing DNA template was prepared by infecting the CJ236
strain of E. coli cells with the recombinant phage and growing it in the presence of uridine and chloramphenicol (25). Mutagenesis was performed using the DNA primers JKB54, JKB55, JKB56,
JKB57, JKB58, JKB59, and JKB60 containing the mutations Y16A, R24A,
Y72A, Y120A, E176A, R179A, and W208A, respectively. The sequences of
various primers used are mentioned in Table
I. The primer extension products
were transformed into E. coli strain DH5 Expression and Purification of the Recombinant
Proteins--
Saporin-6 and the mutants were expressed in BL21
( Structural Characterization by Circular Dichroism--
For CD
spectral analysis, 200 µg of protein was dissolved in 3 ml of 10 mM sodium phosphate buffer (pH 7.0), and spectra were recorded in the far-UV range (200-250 nm) at room temperature, using a
JASCO J710 spectropolarimeter. A cell with a 1-cm optical path was used
to record the spectra at a scan speed of 50 nm/min with a sensitivity
of 50 millidegrees and a response time of 1 s. The sample
compartment was purged with nitrogen, and spectra were averaged over 10 scans. The results are presented as mean residue ellipticity. Yang's
reference parameters were used to perform secondary structure analyses
from CD measurements (28).
Specific RNA N-Glycosidase Activity of Saporin-6 and the
Mutants--
The RNA N-glycosidase activity of saporin and
its mutants was evaluated as their ability to specifically depurinate
28 S rRNA and produce a characteristic 390-base fragment on aniline
treatment. Rabbit reticulocyte lysate was taken as the source of
ribosomes and treated with different concentrations of proteins at
30 °C for 0.5 h as described by May et al. (29). The
reaction was stopped by adding 10 µl of 10% SDS solution and 170 µl of water and incubated at room temperature for 5 min. Total RNA
was isolated using Trizol reagent as per the manufacturer's
instructions. The RNA pellet was dissolved in 20 µl of water and
divided in two parts. To one part, 10 µl of freshly prepared 2 M aniline-acetate, pH 4.5, was added. The samples were
incubated at 60 °C for 3 min, aniline was evaporated under vacuum,
and the treated RNA was dissolved in 10 µl of water. To the
aniline-treated and -untreated samples, buffer containing 32%
formamide, 4 mM EDTA, 0.04% xylene cyanol, and 0.04%
bromphenol blue was added. The samples were heated at 65 °C for 5 min and analyzed on a 2% agarose gel. The RNA was visualized by
ethidium bromide staining.
Assay for in Vitro Protein Synthesis Inhibition--
The
capacity of saporin and its mutants to inhibit protein synthesis was
measured using a rabbit reticulocyte lysate-based in vitro
translation assay system. The rabbit reticulocyte lysate was prepared,
and the assay was carried out as described (30). The reaction mix in a
final volume of 30 µl contained 10 µl of rabbit reticulocyte
lysate, 1 mM ATP, 0.2 mM GTP, 75 mM
KCl, 2 mM magnesium acetate, 3 mM glucose, 10 mM Tris-HCl, pH 7.6, 4 µM amino acid mix
without leucine, 0.16 µCi of [3H]leucine, 1.33 mg/ml
creatine phosphokinase, 2.66 mg/ml creatine phosphate, and different
concentrations of the toxin, diluted in 0.2% RNase-free bovine serum
albumin. The endogenous globin mRNA in the reticulocyte lysate was
used for translation. The reaction was carried out at 30 °C for
1 h and stopped by adding 0.25 ml of 1 N NaOH
containing 0.2% H2O2, followed by an
incubation at 37 °C for 10 min. The proteins were precipitated with
15% trichloroacetic acid on ice for 30 min and harvested on 26-mm
glass fiber filters. The dried filters were counted using a liquid
scintillation counter. Activity was expressed as the percentage of
control where no toxin was added. ID50 represents the
concentration of toxin that inhibited in vitro protein
synthesis by 50%.
Cytotoxic Activity of Saporin-6 and the Mutants--
The
cytotoxic activity of saporin-6 and its mutants was assayed on four
different cell lines: U937 (human histiocyte lymphoma), L929 (mouse
fibroblast), J774A.1 (mouse monocyte-macrophage), and HUT102 (human
cutaneous T-cell lymphoma). Adherent cells were plated at a density of
5 × 103 cells/well in a 96-well plate in 0.2 ml of
RPMI/Dulbecco's modified Eagle's medium containing 10% FCS for
16 h. The medium was replaced with 0.2 ml of leucine-free medium
containing 2% FCS for evaluating the cytotoxicity. The suspension
cells were seeded at 104 cells/well in 0.2 ml of
leucine-free medium containing 2% FCS and used immediately. The cells
were incubated with various concentrations of toxins, diluted in 0.2%
human serum albumin in Dulbecco's PBS for 34 h followed by
labeling with 0.75 µCi of [3H]leucine/well for 2 h. The cells after freezing and thawing were harvested on filtermats
using a 96-well plate automated harvester, and the filters were counted
using an LKB Assay for Genomic DNA Fragmentation--
U937 cells were used to
evaluate genomic DNA fragmentation ability of saporin-6 and the
mutants. 5 × 105 cells were cultured in RPMI
containing 10% FCS in the presence of different concentrations of
proteins. After the indicated time of incubation, cells were harvested
and lysed with 0.2 ml of lysis buffer consisting of 10 mM
Tris-Cl, pH 8.0, 100 mM NaCl, 25 mM EDTA pH
8.0, 0.5% SDS, and 0.1 mg/ml proteinase K at 50 °C for 16 h.
The DNA was extracted with phenol/chloroform and precipitated with
isopropyl alcohol. After treating with RNase A, the DNA samples were
run in a 1.5% agarose gel and visualized by staining with ethidium bromide.
Intracellular Localization of Saporin--
Saporin-6 was
iodinated using the iodogen method as described by Harlow and Lane
(31). 10 µg of protein (0.2-1.0 mg/ml in PBS), 25 µl of iodination
buffer, and 1 mCi of Na125I were added to the
iodogen-coated tube. The tube was tapped at room temperature for 10 min
followed by the addition of 50 µl of 0.2% KI. The labeled protein
was purified using PD-10 column (Amersham Biosciences) presaturated
with bovine serum albumin.
J774A.1 cells, 6 × 106 were seeded in 6 ml of
Dulbecco's modified Eagle's medium containing 10% FCS in a T-25
tissue culture flask and incubated at 37 °C for 16 h. The
medium was replaced with 1 ml of Dulbecco's modified Eagle's medium
containing 2% FCS, and cells were incubated for various time intervals
with 100 µl (~107 cpm) of iodinated saporin-6. After
incubation, the cells were washed once with PBS and further incubated
for 1 h so that membrane-bound saporin-6 would either dissociate
or internalize. Cells were fractionated into nuclear, cytosolic, and
membrane fractions as described by Dignam et al. (32). The
cell pellet was resuspended in 500 µl of buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin and incubated on ice for 10 min. The cells were centrifuged
at 3000 × g, resuspended in 0.5 ml of the same buffer,
and homogenized using a motor-driven homogenizer (30 strokes at 1500 rpm). The homogenate was checked microscopically for cell lysis and
centrifuged for 10 min at 3000 × g at 4 °C. The
pellet constituted the nuclear fraction, and the supernatant contained
cytosol and membranes. To the supernatant, 0.11 volume of buffer
containing 0.3 M HEPES, pH 7.9, 1.4 M KCl, and
0.03 M MgCl2 was added, and the mixture was
centrifuged at 100,000 × g for 30 min at 4 °C in an
ultracentrifuge. The supernatant and the pellet constituted cytosolic
and membrane fractions, respectively. The nuclear and membrane pellets
were resuspended in 500 µl of PBS. The proteins in various fractions
were precipitated with 1 ml of 20% trichloroacetic acid. The
precipitated proteins were boiled in SDS loading dye for 3 min and
analyzed on a 12.5% SDS-polyacrylamide gel. The gel was dried, exposed
to an x-ray film, and later developed.
The present study evaluates the role of various invariant residues
present in the ribosome-inactivating protein saporin-6 in its catalytic
and cytotoxic activity. These include Tyr72,
Tyr120, Glu176, Arg179, and
Trp208, which have been proposed to be present in the
active site and play an important role in the catalysis (21). The role
of two other invariant residues, Tyr16 and
Arg24, proposed to be important for structural stability of
ricin (33), has also been studied. These residues were mutated to
alanine to generate seven single mutants, Y16A, R24A, Y72A, Y120A,
E176A, R179A, and W208A. The target residues were substituted with
alanine, since it does not impose new hydrogen bonding, sterically
bulky, or unusually hydrophobic side chains (34).
Expression, Purification, and Structural Characterization of
Saporin-6 Mutants--
The mutants of saporin-6 were expressed in BL21
(
The effect of the mutation on the overall structure of saporin-6 was
studied by CD spectral analysis of purified mutants in the far-UV
region. Saporin-6 showed the spectra characteristic of RNA N-Glycosidase Activity of Saporin-6 Mutants--
The effect of
substitution of various active site and invariant residues with alanine
on the specific RNA N-glycosidase activity of saporin-6 was
evaluated. Rabbit reticulocyte lysate was treated with various
concentrations of the mutants, and total RNA was extracted. Half of the
extracted RNA was treated with aniline, and both aniline-treated and
-untreated RNA samples were run on an agarose gel. As shown in Fig.
3, aniline treatment of saporin-6-treated RNA samples resulted in the release of the classic 390-base
Endo-fragment. Whereas saporin-6 produced the endofragment at as
low as 40 ng/ml, the mutants Y72A and Y16A failed to affect rRNA even
up to 1000 ng/ml (Fig. 3). With the mutants Y120A, E176A, and R179A, a
faint endofragment was seen at 1000 ng/ml in the aniline-treated
samples (Fig. 3). Substitution of Trp208 or
Arg24 with alanine did not have any effect on the RNA
N-glycosidase activity of saporin-6, since the mutants W208A
and R24A released the Endo-fragment from 28 S rRNA at concentrations
similar to that of the native toxin (Fig. 3). The intensity of
endofragment increased with increasing concentrations of the toxin,
indicating a dose-dependent response. The decrease in size
of 28 S rRNA upon release of the 390-base fragment was also apparent in
aniline-treated samples of the mutants W208A and R24A (Fig. 3).
The release of Endo-fragment from 28 S rRNA as a result of saporin
action results in potent inhibition of protein synthesis. The ability
of the saporin-6 mutants to inhibit protein synthesis was tested in a
rabbit reticulocyte lysate-based in vitro translation assay.
The decrease in the incorporation of [3H]leucine in the
nascent peptides was taken as the measure of protein synthesis
inhibition by the toxin. Saporin-6 caused a dose-dependent
inhibition of protein synthesis with an ID50 of 4.5 ng/ml
(Table III). The mutations of
Trp208 and Arg24 did not affect the protein
synthesis-inhibitory activity of saporin-6, and mutants W208A and R24A
also showed a dose-dependent inhibition of protein
synthesis with respective ID50 values of 6.0 and 5.8 ng/ml
(Table III). The substitution of invariant residue Tyr16
and the active site residue Tyr72 with alanine abolished
the protein synthesis-inhibitory activity of the protein, and even in
the presence of 1000 ng/ml Y16A or Y72A, there was no inhibition of
protein synthesis (Table III). The mutants Y120A and E176A,
however, caused protein synthesis inhibition but at a relatively
higher concentration compared with the native toxin. The respective
ID50 values of Y120A and E176A were 480 and 100 ng/ml,
showing these mutants to be 100- and 20-fold less active than the
native toxin (Table III). The mutant R179A showed an extremely poor
protein synthesis inhibitory activity; its ID50 was found
to be 200-fold less than the native toxin (Table III). The reduced
protein synthesis-inhibitory activity of the mutants is in agreement
with the inability or reduced activity of these mutants to generate the
Endo-fragment.
Thus, it appears that among the active site residues, Tyr72
and Arg179 are absolutely essential for the RNA
N-glycosidase activity of saporin-6. The residues
Tyr120 and Glu176 can be partially dispensed
with, whereas Trp208 and Arg24 are not required
for the RNA N-glycosidase activity. The invariant residue
Tyr16 also appears to be critical for the RNA
N-glycosidase activity of saporin-6.
The three-dimensional structure reveals the active site residues,
Glu176, Arg179, and Trp208, of
saporin-6 to be completely superimposable on those of other RIPs (21).
However, the residue Tyr72, shown to be responsible for the
interaction with the target adenine, assumes different side chain
conformations among all analyzed RIPs.
The role of active site residues of ricin Tyr80,
Tyr123, Glu177, and Arg180,
equivalent to Tyr72, Tyr120,
Glu176, and Arg179 in saporin, has been studied
earlier (22, 35-37). Tyr80 in ricin has been shown to make
the firmest physical contact with adenine and is more crucial to
substrate recognition than Tyr123 (22). In ricin, although
Glu177 is proposed to be essential for transition state
stabilization, its substitution with alanine facilitates the nearby
Glu208 to move into the active site and fulfill a role
similar to that of Glu177 (35, 36). The complete loss of
activity of saporin-6 mutant Y72A and a partial loss in activity of
Y120A indicate a similar mechanism of action in saporin-6 and ricin.
Compared with saporin-6, a 20-fold reduction was observed in the
activity of E176A mutant. It appears that in the mutant E176A,
Glu205 occupies the position of Glu176;
however, carboxylate of Glu205 in the saporin-6 mutant
E176A may provide less stabilization to the oxycarbonium ion transition
state than Glu176. The mutation of Arg179 to
alanine reduced the protein synthesis-inhibitory activity of saporin-6
by 200-fold. Arg180, corresponding to Arg179 of
saporin-6, has been shown in ricin to lie parallel to the conserved
Trp211 residue, Trp208, in saporin-6 and make
strong hydrogen bonds with O-78 of the protein backbone,
Glu177 and an active site water that may be involved in the
RNA N-glycosidase reaction (37). This conserved arginine may
also bind to the phosphate backbone of the 28 S rRNA substrate. A
similar mechanism seems to be operative in saporin-6 also.
The mutation of the only tryptophan residue of saporin-6 to alanine did
not affect the enzymatic activity of the protein. Studies on pokeweed
antiviral protein and abrin have suggested that Trp208 and
Trp198, respectively, in these proteins, corresponding to
Trp208 in saporin-6, are crucial for structural
integrity of these proteins (38, 39).
Tyr16 and Arg24 are two of the nine invariant
residues outside of the active site conserved among various RIPs (33).
Whereas mutation at Tyr16 to alanine resulted in complete
loss of activity, mutating Arg24 did not have any effect on
the enzymatic activity of saporin-6. Studies with trichosanthin have
suggested that the residues Tyr14 and Arg22,
corresponding to Tyr16 and Arg24 of saporin-6,
interact with the residues on the adjacent helix, which contains the
active site residues Glu160 (Glu176 of
saporin-6) and Arg163 (Arg179 of saporin-6)
(40). The mutation of Tyr14 to Phe resulted in only a
5-fold decrease in activity (40). In ricin A-chain, deletion of
residues 21-23 (thereby deleting Tyr21, equivalent to
Tyr16 in saporin-6) did not affect the functional activity
of the protein (41). These observations suggest that conserved Tyr at
this position is not absolutely essential for the activity of RIPs. However, mutation of Tyr16 in saporin-6 to alanine resulted
in complete loss of the RNA N-glycosidase activity.
Cytotoxic Activity of Saporin-6 Mutants--
A variety of cancer
cell lines were treated with different concentrations of saporin-6 or
the mutants, and protein synthesis inhibition in the cells was taken as
the measure of cytotoxicity. Saporin-6 caused a
dose-dependent toxicity to all of the cell lines. J774A.1
was the most sensitive cell line, followed by L929, HUT 102, and U937
(Table IV). On J774A.1 cells, the mutant
R24A had toxicity similar to that of saporin-6, and the mutant E176A had about 15-fold reduced activity (Table IV). Although mutants W208A and Y16A were toxic to J774A.1 cells, compared with saporin-6 they had about 200-fold lower activity (Table IV). Mutants Y120A and
R179A manifested an extremely poor cytotoxicity on J774A.1 cells (Table
IV). The mutant Y72A was totally nontoxic to J774A.1 cells. A similar
pattern of cytotoxicity was observed with all of the mutants on
the other three cell lines also, however, the extent of toxicity and
the -fold difference between saporin and various mutants varied with
different cell lines (Table IV). The effect of various mutants on
J774A.1 cells closely matched with that on U937 cells (Table IV).
The protein synthesis-inhibitory activity of all the mutants correlated
well with their ability to release the Endo-fragment. A comparison of
the cytotoxicity and enzymatic activities, in vitro
translation inhibition, and production of the endofragment revealed the
two activities for mutants R24A, Y72A, Y120A, E176A, and R179A to be in
complete correlation (i.e. the mutants having in
vitro protein synthesis inhibitory activity and the Endo-fragment release activity demonstrated cytotoxicity). The extent of cytotoxicity corresponded quantitatively to the enzymatic activities of the mutants.
Saporin-6 and the mutant R24A showed similar enzymatic activities and
similar cytotoxicities on all of the cell lines. Y72A did not have any
enzymatic activity and lacked cytotoxicity as well. E176A showed
1-25% cytotoxic activity of the native toxin (Table IV), consistent
with its partial enzymatic activity. Y120A also showed partial
cytotoxicity, agreeing with its reduced enzymatic activity. R179A was
found to be partially active in the Endo-fragment assay and had
significantly less cytotoxicity. Interestingly, for the mutants Y16A
and W208A, there was no correlation between their cytotoxic activity
and enzymatic activities. The activity of the mutant W208A was found to
be similar to that of saporin-6 in both in vitro protein
synthesis inhibitory assay and Endo-fragment release assay; however,
its cytotoxicity was found to be significantly lower than the native
toxin. On the other hand, the mutant Y16A was found to be inactive in
both protein synthesis inhibition assay and Endo-fragment release
assay, yet it showed partial cytotoxicity on all of the cell lines
tested. Thus, it appeared that cytotoxicity of saporin-6 is not solely
the consequence of its RNA N-glycosidase activity, and other
activities or factors may also be involved.
Effect of Saporin-6 and Its Mutants on Genomic DNA--
For almost
two decades it was largely assumed that RIPs act only on rRNA within
ribosomes (3, 42). Recently, however, all plant RIPs and Shiga toxin
have been shown to remove several adenine residues from naked RNAs and
from DNA in vitro (9, 43, 44). Some RIPs have been shown to
possess direct DNase activity also (24, 45). There has been a proposal
to replace the term "ribosome-inactivating protein" with
"polynucleotide:adenosine glycosidase" (9). This has raised great
interest in the study of the mechanism of action of RIPs on intact
cells, to investigate the potential contribution of various activities
of RIPs to their cytotoxicity.
The effect of saporin-6 was monitored on the genomic DNA of U937 cells
in an attempt to ascertain the contribution of this activity to the
cytotoxicity. U937 cells were incubated with 0.1 and 1 µM
of saporin-6 for various time intervals (Fig.
4A). In the presence of 1 µM saporin-6, significant DNA fragmentation started
within 24 h of incubation and became more pronounced at 36 and
48 h. The control, where no toxin was added, showed a high molecular weight genomic DNA band throughout the course of study (Fig.
4A). With 0.1 µM toxin there was no laddering
after 24 h; however, in 36 h faint small molecular weight
bands could be seen, and a further 12-h incubation resulted in a
laddered appearance of the genomic DNA (Fig. 4A). In order
to investigate the effect of mutations of active site residues and
invariant residues in saporin-6 on its ability to fragment genomic DNA,
U937 cells were incubated in the presence of 1 µM mutant
proteins for 48 h. The genomic DNA was isolated and analyzed on
agarose gels. As shown in Fig. 4B, the active site mutants
Y72A, Y120A, E176A, R179A, and W208A failed to affect the genomic DNA.
The cells treated with these proteins showed a high molecular weight
intact DNA band as seen in the control. However, with mutants Y16A and
R24A, fragmented DNA was obtained similar to that with the native
saporin-6 (Fig. 4B).
Table V summarizes the RNA
N-glycosidase activity, cytotoxicity, and genomic DNA
fragmentation activity of saporin-6 mutants. The activity of all the
mutants on genomic DNA did not correlate with their RNA
N-glycosidase activity. The wild type saporin-6 and the
mutant R24A showed similar RNA N-glycosidase activity, cytotoxicity, and genomic DNA fragmentation activity. The mutant Y72A
did not show any RNA N-glycosidase activity and
cytotoxicity, and did not cause any genomic DNA fragmentation. The
mutants Y120A, E176A, and R179A had no DNA fragmentation activity;
however, they showed partial RNA N-glycosidase activity and
poor cytotoxic activity. The mutant W208A possessed full RNA
N-glycosidase activity and had reduced cytotoxicity but
failed to affect the genomic DNA. The mutant Y16A did not show any RNA
N-glycosidase activity and possessed reduced
cytotoxicity similar to that of W208A; however, it caused the genomic
DNA fragmentation comparable with native saporin-6. These results
clearly demonstrate that saporin-6 possesses two independent activities
(namely RNA N-glycosidase activity and genomic DNA
fragmentation activity), and both are required for the cytotoxicity of
the toxin.
Role of RNA N-Glycosidase and Genomic DNA Fragmentation Activity in
Saporin-6 Cytotoxicity--
The cytotoxicity data of various mutants
indicated that for complete cytotoxic activity of saporin-6 both RNA
N-glycosidase and genomic DNA fragmentation activities are
required (Table V). Loss of either one of these activities resulted in
a reduction or loss of the cytotoxic activity. The mutants Y16A and
W208A each possessed one of the two activities, DNA fragmentation
activity and rRNA N-glycosidase activity, respectively,
and showed similar but much reduced cytotoxicities (Table V).
In order to further confirm the contribution of the two enzymatic
activities of saporin-6 to its cytotoxic activity, U937 and J774A.1
cells were treated with various concentrations of Y16A, W208A, and an
equimolar mixture of the two proteins. As mentioned before, Y16A and
W208A showed a comparable, about 6-fold lower, cytotoxic activity than
saporin-6 on U937 cells, and on J774A.1 cells they had 230- and
125-fold less activity (Table VI). An
equimolar mixture of Y16A and W208A resulted in the cytotoxicity very
similar to that of saporin-6 on U937 cells, whereas the mixture had a
5- and 3-fold improved cytotoxicity compared with that of the
individual mutant proteins on J774A.1 cells (Table VI). The cytotoxicity of saporin-6, therefore, appears to be a combined effect
of its RNA N-glycosidase and DNA fragmentation
activities.
Intracellular Localization of Saporin-6--
To investigate
whether saporin translocates to nucleus to degrade DNA, J774A.1 cells
were treated with iodinated saporin-6 for 3, 6, 9, and 16 h, and
the presence of radiolabeled protein was checked in the nuclear,
cytosolic, and membrane fractions. As shown in Fig.
5, the concentration of saporin-6
increased in cytosol up to 6 h, decreased gradually, and became
negligible by 16 h. The membrane fraction did not show any
significant amount of protein at any time point. The concentration of
the protein in nuclear fraction was found to be comparable with that in
the cytosolic fraction up to 9 h; however, by 16 h,
concomitant with the decrease in cytosol, most of the labeled saporin-6
localized in the nucleus (Fig. 5). Apart from the intact ~30-kDa
saporin-6 band, the nuclear fraction also showed some low molecular
mass bands of ~17 and 25 kDa, seen earlier in the membrane
fraction, indicating onset of degradation of saporin-6 at longer
incubation periods (Fig. 5). The study shows that after internalization
initially the protein stays in the cytosol and later migrates to the
nucleus. Studies with Shiga toxin have shown that internalized Shiga
toxin-I reaches the nuclear envelope, and the cells treated with Shiga toxin show the toxin predominantly in the nuclear fraction (46, 47).
Saporin-6 does not possess any apparent nuclear localization signal. It appears that the primary target inside the cell is rRNA and
that activity on genomic DNA is a late event in cytotoxicity.
Recent studies on ricin and Shiga toxin suggest that these RIPs can
damage nuclear DNA in whole cells by means that are not secondary to
ribosome inactivation (48). Shiga toxin has been shown to release
adenine from DNA by its RNA N-glycosidase activity that
leads to spontaneous break of sugar-phosphate backbone, whereas saporin-6, dianthin-30, and gelonin have been reported to manifest direct DNase-like activity on plasmid DNA (24, 49). The key residues
involved in the catalytic activity of saporin appear to be functionally
similar to homologous residues in ricin A and other RIPs. The current
study demonstrates that the DNA fragmentation observed is not entirely
dependent on the RNA N-glycosidase activity. The comparison
of activities of saporin-6 mutants also suggests that there is a
considerable overlap between the residues required for RNA
N-glycosidase activity and genomic DNA fragmentation
activity. Tyr72, Tyr120, Glu176,
and Arg179 are important for the activity on both DNA and
rRNA. Trp208 appears to be required only for the genomic
DNA fragmentation activity. Substitution of Tyr16 abolished
the activity of protein on rRNA; however, it did not affect the genomic
DNA fragmentation activity. The residue Arg24 can be
dispensed with for both of the activities of saporin-6. The crystal
structure of saporin-6 and superimposition of the structure with other
RIPs has suggested that the loop between its
In conclusion, we have shown that saporin-6 possesses two catalytic
activities, namely RNA N-glycosidase and genomic DNA
fragmentation activity. The cytotoxic activity of saporin-6 is governed
by both of these activities, which share considerable overlap in terms of amino acid residue requirement. Tyr72 and
Arg179 are absolutely indispensable for RNA
N-glycosidase as well as genomic DNA fragmentation activity,
whereas Arg24 does not seem to be playing any role in any
of these activities of saporin-6. Tyr120,
Glu176, and Trp208 play an important role in
DNA fragmentation activity. However, Trp208 could be
dispensed with completely, and Tyr120 and
Glu176 partially, for the RNA N-glycosidase
activity. Tyr16 appears to be required for the maintenance
of a conformation essential for RNA N-glycosidase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sarcin/ricin loop (3). The catalytic depurination disrupts the
binding of elongation factors to the ribosomes, thus arresting protein
synthesis at the translocation step (4). Although the catalytic action
carried out by all the RIPs is identical, their activity on ribosomes from sources other than eukaryotes are markedly different (1, 5).
Differences in toxicity of RIPs toward various cell lines, their
different requirements for cofactors, and variations in the minimal
structure of the adenine-containing loop that they can attack point to
their substantial diversity (6, 7). The study of molecules that bind
and inactivate RIPs has also suggested that local sequence/structure
variabilities exist among RIPs (8). The interest in RIPs has gained a
new momentum recently with the growing evidence of their action on
nonribosomal substrates (9-11). Most of the novel enzymatic activities
are related to a presumed RNase or DNase activity. Other enzymatic
activities reported for individual RIPs include phosphatase activity on
lipids, chitinase activity, and superoxide dismutase activity (12-14). It has been shown that pokeweed antiviral protein cleaves the double-stranded supercoiled DNA using the same active site required to
depurinate rRNA and that momordin has intrinsic RNase activity (15,
16). A better understanding of the catalytic mechanism of RIPs will be
extremely useful in the exploitation of their unique properties for
diverse applications like development of RIP-based immunotoxins,
abortifacients, and anti-HIV agents (17).
-helix is bent near its C terminus, and this bending allows
the catalytic residues, Glu177 and Arg180, to
reach the solvent of the active site cleft (23). This important helix
bending disrupts the normal
-helical bonding pattern. However, the resulting structure is stabilized by new hydrogen bonds to the side
chains of Tyr21 and Arg29. Alterations of these
residues could therefore affect the folding rate or thermodynamic
stability of the protein. In order to ascertain the role of
corresponding residues, Tyr16 and Arg24, in
saporin-6, they were mutated to alanine, and the mutants were analyzed
for various functional activities. Saporin-6 has been reported to
contain DNA nuclease activity, and recently saporin-L1 has been shown
to act on various forms of mammalian DNA (10, 24). DNA could be a
probable alternate polynucleotide substrate for RIPs within a cell. In
the present study, the effect of saporin-6 and the mutants has been
studied on genomic DNA of U937 cells. An attempt has been made to
correlate the cytotoxicity of saporin-6 with the effect of toxin action
on DNA and RNA. Our study shows that the cytotoxic activity of saporin
is a cumulative effect of its RNA N-glycosidase and DNA
fragmentation activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
by standard
methods. All mutations were confirmed by DNA sequencing using the
dideoxy chain termination method (26).
Sequence of primers used for mutagenesis of residues in saporin-6
DE3) strain of E. coli. Bacterial cells were transformed
with the desired construct and grown in Super broth containing 100 µg/ml ampicillin at 37 °C with shaking. Saporin-6 and all of the
mutants were found to accumulate in the form of inclusion bodies, and
they were purified using the procedure described by Buchner et
al. (27). Briefly, the resuspended cells were lysed with lysozyme,
and the membrane pellet was washed with Triton X-100 followed by
several washings without Triton X-100. The inclusion body pellet thus
obtained was dissolved in guanidine hydrochloride and reduced by adding dithioerythritol. Renaturation was carried out by 100-fold dilution of
the protein in a refolding buffer containing L-arginine and oxidized glutathione. After incubating at 10 °C for 48 h, the renatured material was dialyzed against 20 mM acetate
buffer, pH 4.5, containing 100 mM urea. The dialyzed
solution was loaded on an S-Sepharose column and eluted using a 0-1.5
M gradient of NaCl in 20 mM acetate buffer, pH
4.5. Relevant fractions were pooled, concentrated, and purified to
homogeneity by gel filtration chromatography on a TSK 3000 column in
PBS, pH 7.4.
-plate counter. Activity was plotted as percentage of
control where no toxin was added to the cells, and the results were
expressed in the form of ID50 values. The ID50
values represent the concentration of the toxin that inhibited the
cellular protein synthesis by 50%.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
DE3) strain of E. coli cells, and the overexpressed
mutant proteins were found to localize in the inclusion bodies
like the wild type protein. The recombinant proteins from
the inclusion bodies were denatured and refolded in vitro
and purified by a two-step purification scheme comprising a
cation-exchange and gel filtration chromatography. By SDS-PAGE
analysis, the mutants gave a single band at the same position as
saporin-6, indicating the preparations to be homogeneous (Fig.
1). On Western blots, all of the mutants
reacted equally well with a polyclonal antibody raised against
saporin-6 (Fig. 1).
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Fig. 1.
SDS-PAGE and Western blot analysis of
saporin-6 and the mutants. The proteins were expressed in BL21
( DE3) cells of E. coli and purified from inclusion
bodies by cation exchange and gel filtration chromatography. The
recombinant proteins were analyzed by 12.5% SDS-PAGE under reducing
conditions, followed by Coomassie Blue staining (A). Western
blot analysis of the mutants was done using a polyclonal antibody
raised against saporin-6 (B). The different lanes
in B correspond to the same proteins as in
A.
+
structure (Fig. 2). The substitution of
active site residues Tyr72, Glu176, and
Trp208 with alanine resulted in a modest shift of the CD
spectrum, whereas the mutants Y120A and R179A appeared to have similar
spectra as that of saporin-6 (Fig. 2, A and B).
The replacement of the invariant residue Tyr16 with alanine
resulted in a significant shift in the CD spectra (Fig. 2C).
The
-helical content of the mutants Y16A, Y72A, Y120A, E176A, R179A,
and W208A was found to be similar to that of saporin-6; however, there
was a decrease in
-sheet content of these mutants compared with the
native toxin (Table II). The mutant R24A
appeared to be similarly folded as the native toxin (Fig.
2C). The quantitative values of various secondary structure
elements of R24A were found to be similar to saporin-6 (Table II).
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Fig. 2.
CD spectral analysis of saporin-6 and the
mutants. CD spectra were recorded in the far-UV region (200-250
nm) at 25 °C, and the spectra are presented as the mean residue
ellipticity. A, saporin-6 (solid
line), Y72A (dotted and
dashed line), and Y120A (dotted
line). B, saporin-6 (solid
line), E176A (dotted and dashed lines), R179A
(dotted line), and W208A (dashes
and double dots). C,
saporin-6 (solid line), Y16A (dotted and
dashed line), and R24A (dotted line).
Secondary structure analysis of saporin-6 and the mutants
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Fig. 3.
Specific RNA N-glycosidase
activity of saporin-6 and the mutants on 28 S rRNA. Rabbit
reticulocyte lysate was treated with various proteins at the indicated
concentrations. The reaction was carried at 30 °C for 30 min
followed by termination of the reaction using 0.4% SDS. Total RNA was
extracted, and half of it was treated with aniline acetate at 60 °C
for 3 min. Following vacuum drying, the aniline-treated (+) and
-untreated ( ) RNA was resolved by electrophoresis on 2% agarose
gel.
In Vitro protein synthesis inhibitory activity of saporin-6 and the
mutants
Cytotoxic activity of saporin-6 and the mutants
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Fig. 4.
Genomic DNA laddering by saporin-6 and the
mutants. DNA was isolated from equal number of U937 cells treated
with saporin-6 or the mutant. The samples were run on 1.5% agarose gel
and visualized by staining with ethidium bromide. A, genomic
DNA profile after treatment of U937 cells with saporin-6 for indicated
time and concentration; B, genomic DNA isolated from U937
cells after treatment with 1 µM mutant protein for
48 h.
Comparison of various activities of saporin-6 and the mutants
Cytotoxic activity of Y16A, W208A, and saporin-6
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Fig. 5.
Intracellular localization of radiolabeled
saporin-6. J774A.1 cells were incubated with radiolabeled
saporin-6 for the indicated time points, and cells were washed and
incubated further for 1 h. The cells were homogenized and
separated into the nuclear, cytosolic, and membrane fractions by
ultracentrifugation. Proteins were precipitated from the cytosolic and
membrane fractions using 20% trichloroacetic acid, the samples were
analyzed by 12.5% SDS-PAGE followed by autoradiography. H,
total homogenate; N, nuclear fraction; C,
cytosol; M, membrane fraction.
7 and
8 strands is particularly short, and it makes
active site more accessible to various different adenine-containing
substrates (21). Therefore, it appears that saporin-6 binds to DNA and rRNA through the same active site; however, the local positioning of
the two substrates could be different, enabling different residues to
interact. The cytotoxic activity appears to require both the activities, and loss or reduction of one results in a loss or reduction
in the cytotoxic activity of saporin.
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FOOTNOTES |
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* This work was supported by grants from the Department of Biotechnology, Government of India (to the National Institute of Immunology).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Senior Research Fellow of the Council of Scientific and Industrial
Research, India.
§ To whom correspondence should be addressed. Tel.: 91-11-6183009/6162281; Fax: 91-11-6162125/6109433; E-mail: janendra@nii.res.in.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M207389200
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ABBREVIATIONS |
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The abbreviations used are: RIP, ribosome-inactivating protein; FCS, fetal calf serum.
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