Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A)
Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A)
Luigi
Barbieri*
,
Paola
Valbonesi
,
Elena
Bonora
,
Paola
Gorini
,
Andrea
Bolognesi
and
Fiorenzo
Stirpe
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna Via San Giacomo 14, I-40126
Bologna
,
Italy
Received October 21, 1996
;
Accepted December 3, 1996
ABSTRACT
Ribosome-inactivating proteins (RIP) are a family of plant enzymes for which a
unique activity was determined: rRNA
N
-glycosidase at a specific universally conserved position, A
4324
in the case of rat ribosomes. Recently we have shown that the RIP from
Saponaria officinalis
have a much wider substrate specificity: they are actually polynucleotide:adenosine glycosidases. Here we extend studies on substrate specificity to most known RIP: 52 purified
proteins, both type 1 (single-chain) and type 2 (two chain, an enzymatic chain and a lectin chain) were
examined for adenine release on various substrates including RNAs from different sources, DNA, and poly(A). All RIP depurinated extensively DNA and some released adenine from all adenine-containing polynucleotides tested. From experimental evidence the entire
class of plant proteins, up to now called ribosome-inactivating proteins, may be classified as polynucleotide:adenosine glycosidases. The
newly identified substrates may be implicated in the biological role(s) of RIP.
INTRODUCTION
The class of plant proteins called ribosome-inactivating protein (RIP) are enzymes which catalyse an irreversible
damage to ribosomes hydrolising the glycosidic bond of a unique, highly
conserved adenosine residue on the major rRNA: A
4324
in the case of rat liver rRNA (reviewed in ref.
1
). Until recently this was the only known substrate for RIP.
This homogeneity in substrate recognition by the various RIP has been challenged
by several observations: (i) some saporins, RIP from
Saponaria officinalis
, and, to a lesser extent, other type 1 RIP, release more than one molecule of
adenine from each ribosome (
2
,
3
); (ii) PAP (a RIP from the leaves of
Phytolacca americana
) is capable of depurinating artificial polynucleotidic loops which are not substrate for ricin (
4
); (iii) saporin-L1 is capable of depurinating a variety of polynucleotides (
5
) and (iv) some other RIP, namely
Hura crepitans
RIP and some isoforms of PAP, depurinate both ribosomal and non-ribosomal substrates (
6
). The substrate specificity of saporin-L1 has been studied in detail, together with the optimal conditions for
the enzymatic activity (
7
). We decided to extend the study on substrate specificity to as many RIP as
available, taking into account that optimal conditions for enzymatic activity
as determined for saporin-L1 appear to be quite different from those used so far for the
determination of enzymatic activity of RIP on ribosomes. The results presented
here show that all RIP tested do act on DNA and many on different
polynucleotidic substrates, releasing adenine from the sugar phosphate backbone
of poly- and polydeoxy- nucleotides. The plant proteins formerly called RIP may thus be
classified as polynucleotide:adenosine glycosidases.
MATERIALS AND METHODS
Materials
Sources of polynucleotide:adenosine glycosidases (i.e. RIP) and botanical
classification of RIP producing plants can be found in (
1
). Ricin,
Ricinus
agglutinin (RCA120), volkensin and saporins were purified as described in (
3
,
8
,
9
), respectively. Pokeweed antiviral protein (PAP) has been fractionated by
chromatography onto Blue-Sepharose as practised for PAP-C (
10
) into two isoforms: isoform 1 (lower affinity for the dye) and isoform 2
(higher affinity for the dye). Recombinant saporin-S6 [saporin-S6r: SAP-1 and SAP-3 (
11
), SAP-C (
12
)], trichosanthin and recombinant ricin A chain were generous gifts,
respectively from Professor M. Soria, Milan, Italy, from Dr H.W. Yeung, Hong
Kong, and from Professor J.M. Lord, Coventry, UK. Other polynucleotide:adenosine glycosidases were prepared as described in the appropriate references cited in the review by Barbieri
et al.
(
1
).
Poly(A), rRNA from
Escherichia coli
, genomic RNA from tobacco mosaic virus (TMV) and MS 2 were from Boehringer
GmbH, Mannheim, Germany. Genomic RNA (m ssRNA positive + one small satellite)
from artichoke mottled crinkle virus (AMCV), a generous gift from Dr E.
Benvenuto, Rome, Italy, was prepared by phenol extraction and ethanol
precipitation from purified virus isolates. DNA from herring sperm (hsDNA) from
Sigma (St Louis, MO, USA) was mechanically sheared and made RNA-free by treatment with DNase-free RNase A (Boehringer) for 2.5 h at 37oC. DNA was then repeatedly precipitated in ethanol to remove
the enzyme and, when indicated, was melted by heating at 90oC for 5 min, followed by rapid cooling in ice. Adenine used as standard was
from Sigma. Materials and equipment for low pressure chromatography were from
Pharmacia LKB (Uppsala, Sweden). All other reagents were of analytical or
molecular biology grade and, when possible, RNase-free. Water was Milli-Q (Waters-Millipore). Chloroacetaldehyde was prepared according to (
13
).
Determination of polynucleotide:adenosine glycosidase activity
Polynucleotide:adenosine glycosidase activity was determined by measuring
adenine released from the substrate by HPLC after derivatisation to its
fluorescent derivative ethenoadenine essentially as described in (
7
). Reaction conditions, previously determined as optimal for saporin-L1 (
7
), are reported in the legends to the pertinent Tables and Figure
1
.
RESULTS
Effect of ribosome-inactivating proteins on genomic viral ribonucleic acids
Fifty-two purified proteins, representing the vast majority of known RIP, were
tested for depurinating activity on MS 2 genomic RNA (Table
1
). Reaction conditions chosen were those optimal for the determination of RIP
activity on rat liver ribosomes [inhibition of poly(U)-directed phenylalanine polymerisation] as determined in previous
experiments (results not shown). These screening experiments were performed
with a high concentration of RIP to detect activity under conditions which
could be non-optimal, and in order to overcome the possible lack of cofactors (
14
). These enzyme concentrations are anyhow lower than those naturally occurring
in subcellular compartments where RIP are localised [e.g.
Phytolacca americana
and
Saponaria officinalis
tissues (
15
,
16
)] and lower than those necessary to show activity of ricin and other RIP on
naked ribosomal RNAs [for a review see (
1
)].
Only some saporins, RIP from
Saponaria officinalis
, released significant amounts of adenine from MS 2 RNA, in excess of one mol
per mol of RNA (Table
1
). All other RIP showed low or no detectable activity on this substrate in
present experimental conditions. A variability in the activity of different
batches of natural saporin-S6 was found, which may be due to the fact that preparations of natural
saporin-S6 are a variable mixture of different isoforms (
17
). Thus we tested three different recombinant saporin-S6 gene products, which were all inactive on viral RNAs. Saporin-L1, the RIP with the highest depurination rate on MS 2 RNA, was also
active on plant virus RNAs, from tobacco mosaic virus (TMV) and artichoke mottled crinkle virus (AMCV), although with lower rates.
.
Effect of various RIP on viral genomic RNAs of MS 2, TMV and AMCV
Substrate source
Adenine released (pmol)
MS 2
TMV
AMCV
Dianthin 32
2 (3)
a
0
PAP (isoform 2)
traces (2)
a
traces
PAP-R
2-5 (16)
a
5
traces
PD-S2
2 (10)
a
Saporin-L1
1140
744
635
Saporin-L2
607
Saporin-R1
45 (686)
a
Saporin-R2
504
Saporin-R3
39 (389)
a
Saporin-S5
61
33
Saporin-S6r
b,c
0
0
Saporin-S8
8 (18)
a
Saporin-S9
4 (46)
a
Trichosanthin
traces (5)
a
0
Ricin
0
0
traces
Reaction conditions were: 20 mM Tris/HCl, pH 7.8, 100 mM NH
4
Cl, 10 mM Mg acetate, 10 pmol of RIP, 10 [mu]g of nucleic acid (equivalent to 8.5 pmol of MS 2 RNA, 4.8 pmol of TMV RNA
and 6.7 pmol of AMCV RNA) in a final volume of 50 [mu]l for 40 min at 25oC. Type 2 RIP were reduced at 37oC for 1 h in the presence of 1% [beta]-mercaptoethanol prior to incubation with nucleic
acids. Other experimental conditions are described in Materials and Methods.
The following RIP were inactive on MS 2 RNA: abrin a, asparin 2, barley RIP 1, bryodin-L, bryodin-R, colocin 1, colocin 2, crotin 1, crotin 2, crotin 3, curcin 1, curcin
2, curcin 3, dianthin 30, gelonin,
Hura
crepitans
RIP, luffin a, luffin b, lychnin, manutin 1, manutin 2, mapalmin, momorcochin-S, momordin I, momordin II (contaminated with RNase), PAP-C, PAP (isoform 1), PAP-II, PAP-S, petroglaucin 1, petroglaucin 2, ricin, trichokirin,
viscumin, volkensin. The following RIP (10 pmol) were inactive on TMV RNA:
barley RIP, bryodin-R, gelonin, momordin I, PAP-S,
Saponaria ocymoides
RIP,
Vaccaria pyramidata
RIP, volkensin.
a
Values in brackets have been obtained with 30 pmol of RIP added.
b
Three slightly different clone products have been tested with identical results:
SAP-1, SAP-3 and SAP-C.
c
Different batches of natural non-recombinant saporin-S6 released the following amounts of adenine in separate
experiments: from traces to 90 pmol from MS 2, from 23 to 100 pmol from TMV
RNA, and traces of adenine from AMCV RNA.
.
Depurination of herring sperm DNA,
Escherichia coli
rRNA and poly(A) by RIP
RIP added
Adenine released (pmol)
Herring sperm DNA
rRNA
Poly(A)
Type 1
barley RIP 1
184
14
0
bryodin-L
183
7
3
bryodin-R
336
21
16
dianthin 30
4 297
316
7
gelonin
3 858
195
0
Hura crepitans
RIP
4 271
832
69
luffin a
271
5
0
mapalmin
161
10
0
momordin I
81-118
traces
0
PAP
4 527
2 379
traces
PAP (isoform 2)
4 737
2 177
0
PAP II
4 046
418
traces
PAP-R
5 288
2 796
0
PAP-S
3 461
1 070
0
PD-S1
a
7 567
487
2 339
PD-S2
a
6 931
1 453
2 447
saporin-L1
5 578
8 331
>15 000
saporin-L2
6 060
9 570
14 400
saporin-R1
4 215
1 247
952
saporin-R2
5 850
6 898
12 700
saporin-R3
4 939
1 337
931
saporin-S5
5 599
1 360
208
saporin-S6r
b,c
2 245
396
4
saporin-S8
4 211
1 877
37
saporin-S9
6 257
2 554
156
trichosanthin
138
9
0
trichokirin
602
13
0
Type 2
abrin
native
330
13
0
reduced
d
475
traces
0
ricin
native
689
6
0
A chain r
185
10
0
RCA 120
native
220
5
0
reduced
d
121
4
0
viscumin
native
844
12
0
reduced
d
853
11
traces
volkensin
native
68
0
0
reduced
d
48
traces
0
Reaction conditions. hsDNA and rRNA: 10 [mu]g of nucleic acid, 100 mM KCl, 50 mM Na acetate, pH 4.0, 30 pmol of RIP (900
ng for type 1 or 1800 ng in the case of type 2) in a final volume of 50 [mu]l. Incubation was for 40 min at 30oC. Poly(A): 10 [mu]g of substrate, 10 mM Mg acetate, 100 mM NH
4
Cl, 20 mM Na acetate, pH 6.0, 10 pmol (300 ng) of RIP in a final volume of 50 [mu]l. Incubation was for 40 min at 30oC. Released adenine was measured by HPLC as described in Materials and
Methods.
a
20 [mu]g of substrate and reaction conditions for poly(A) identical to those of
DNA.
b
SAP-3 clone product gave similar results.
c
Various batches of natural non-recombinant saporin-S6 released variable amounts of adenine from poly(A) (from 3 to 277
pmol).
d
Reduced by treatment with either 2% [beta]-mercaptoethanol at 37oC for 1 h or 50 mM dithiotrithol, in 50 mM Tris/HCl buffer, pH
8.5 for 1.5 h at 37oC.
Activity of ribosome-inactivating proteins on herring sperm DNA, rRNA and poly(A)
Thirty-two type 1 and type 2 RIP were tested on hsDNA, rRNA and poly(A) (Table
2
). Reaction conditions were those determined as optimal for saporin-L1 (
7
).
All RIP of either type tested released adenine from hsDNA, although with different activity: RIP from Cucurbitaceae (bryodins, luffin a, momordin I, trichosanthin and trichokirin), barley RIP, mapalmin and
all type 2 RIP were substantially less active than other RIP. Surprisingly,
ricin and related two-chain RIP, which must be reduced to act on ribosomes (
18
), were active on DNA even before reduction. Denaturation of DNA prior to assay
did not increase significantly depurination rates, except in the case of
momordin I (results not shown).
Activity on purified
E.coli
rRNA in present conditions was highly variable. To be noted however that all
type 2 RIP, either reduced or not, depurinated rRNA only marginally.
Only saporins and RIP from
Phytolacca dioica
(PD-S) depurinated extensively poly(A) at pH 6.0; saporin-L1, -L2 and -R2 being the most active (Table
2
); marginal depurination of poly(A) was observed with
Hura crepitans
RIP and bryodin-R, whilst no activity on this substrate was detected with the other RIP
tested, including recombinant saporin-S6r and those (PAP-R and trichosanthin) which were marginally active on viral genomic RNA.
The dependance of the reaction on enzyme concentration was determined for PD-S2 (a RIP from the seeds of
Phytolacca dioica
) (Fig.
1
). From linear regression analysis a release of 856 (from hsDNA) and 657 [from
poly(A)] mol of adenine per mol of enzyme was calculated.
DISCUSSION
The experiments described above show that all tested RIP remove more than one
adenine residue from DNA, and some also from rRNA and other
polyribonucleotides. The possibility of a contamination of RIP preparations by
other enzymes seems to be excluded by the most careful purification procedures
(
3
), by the low amount of enzyme necessary for activity (Fig.
1
) and by the fact that RIP from all sources, including recombinant ones, were
active on hsDNA. To our knowledge there are no enzymes with the activity
described in this work. The nearest are probably DNA glycosylases [reviewed in
(
19
)] whose similarities with RIP have been discussed in (
7
).
Within the limit of the small number of observations, there is consistency
between results obtained using bacteriophage RNA (from MS 2) and plant viral
RNAs, possible natural substrates.
Type 2 RIP do not require reduction for activity on DNA, whereas they must be
reduced to depurinate ribosomes. The different steric hindrance of ribosomes
and hsDNA may account for this difference. The recent report that a mutant non-reducible form of ricin is still cytotoxic (
20
) suggests that depurination of non ribosomal substrate(s) may contribute to the
toxicity of ricin and related toxins.
Depurination of poly(A), RNA and DNA proceeds in the absence of any cofactor,
although an influence of cofactors, as those characterised by Carnicelli
et al.
(
14
) cannot be excluded. DNA, RNA, and poly(A) all undergo multiple deadenylation
by some RIP indicating that a specific nucleotide sequence in the substrate is
not required for these enzymes to act.
Several molecular structures of RIP have been elucidated in detail and only one
possible enzymatic site has been identified indicating that the activity on
ribosomes and on other substrates is due to the same active site.
Interactions between RIP and DNA have been reported previously. Relevant to
present work are the observations that mice poisoned with ricin presented
single-strand breaks in DNA (
21
) and some type 1 RIP and ricin (
22
-
26
) introduced nicks into supercoiled double-stranded DNA. Interestingly, both ricin and camphorin, another type 2 RIP, acted unreduced (
23
), consistently with what observed in present work with hsDNA (Table
2
). It was found also that gelonin (referred to as GAP31) transforms supercoiled
DNA into relaxed and linear forms, and that gelonin-derived peptides bind to DNA and RNA (
27
). It is possible that these RIP actually removed adenine from DNA, which became
more fragile and then broke spontaneously or under the action of contaminating
nucleases.
DNA and ribosomes, that are depurinated consistently by all RIP, possibly are
the best candidates for the natural substrate of these enzymes. Action on non-ribosomal RNA and poly(A) may be either accidental or expression of a
possibly additional biological role. Thus the subdivision of RIP based on
substrate specificity may be correlated to their natural activity.
RIP with high activity on DNA come from plant species belonging to the order of
the Caryophyllales, whilst RIP from Cucurbitaceae have a lower activity, thus
suggesting that there may be a evolutionary-related difference either in the substrate fine specificity or in the
requirements for maximal activity. Moreover, it appears that the different
forms of RIP produced by the same plant (
1
) often are functionally different, all being active on mammalian ribosomes but
having very different activity on nucleic acids. This suggests that RIP with
distinct substrate specificity produced by a single plant species may have a
different functional role. No correlation between the tissue origin (seed,
leaf, etc.) and the spectrum of activity on nucleic acids was observed.
Present results may help to understand the function of RIP in Nature. Several
roles have been proposed for RIP, each one with an associated substrate for the
enzymatic action [reviewed in (
1
)]. Some of these roles may be readdressed on the basis of the newly identified
substrate specificities of RIP, if the latter apply also
in vivo
:
(i) a defensive role against plant viruses (
1
,
28
,
29
). In the widely accepted mechanism of antiviral action the target of RIP are
the autologous ribosomes: viral infection alters the cell structure, RIP gain
access to ribosomes and inactivate them, with arrest of protein synthesis,
death of the cells, and prevention of viral replication and spread. Although a
direct action on the entire virus has been excluded since first experiments,
present results suggest that the RIP deadenylating different forms of RNA could
act directly on viral RNA. Localisation of PAP in high concentrations between
cell wall and plasma membrane in pokeweed leaves (
15
) should allow for RIP-viral nucleic acid contact. Since a DNA template is present at some stage
of all viral infections, depurination of virus-derived DNA may also give protection against all viruses. Present work
indicates that retroviral DNA could be damaged as suggested for HIV (
24
), and, although definitely not physiological, anti-HIV activity of RIP is suggestive of mechanisms which may work also in plant systems;
(ii) a role in the regulation of cell metabolism. In this hypothesis the targets
are autologous ribosomes, which are inactivated to stop protein synthesis by
senescent and/or altered ribosomes, or in senescent tissues. RIP activity
appears, or increases when seeds mature (
3
) and in senescent or stressed leaves (
6
,
29
,
30
), in correlation to events leading to arrest of the metabolism of plant cells.
The activity on DNA may well contribute to this regulation, since the removal
of few or even one adenine residue may be sufficient to disrupt transcription.
An effect on poly(A) tails of mRNAs and on other RNAs may also be of some
importance in the regulation of cell metabolism. Depurination of autologous DNA
may be a cause of programmed cell death in the senescence of leaves. Also, DNA
damage may have a role in the pathogenesis of the lesions induced by RIP, and
particularly in the DNA fragmentation typical of apoptosis observed in cultured
cells exposed to RIP of either type (
37
-
37
) and in tissues of rats poisoned with ricin and abrin (
38
). It should be recalled, however, that inhibition of protein synthesis is
per se
sufficient to induce apoptosis both in rat liver (
39
) and in cultured cells (
31
,
32
,
40
), and thus the apoptosis induced by RIP could be accounted for by the ribosomal
inactivation brought about by these proteins.
The identification of new
in vitro
substrates may allow for the identification of yet undiscovered plant and/or
animal polynucleotide:adenosine glycosidases, which might not be active on
mammalian ribosomes, the substrate now used to detect RIP.
ACKNOWLEDGEMENTS
This study was supported by the Università di Bologna,
Funds for selected Topics
, by grants from the Ministero dell'Università e della Ricerca Scientifica, from the Consiglio Nazionale delle
Ricerche, special projects
Biotecnologie e Biostrumentazione
and
ACRO
, from the Associazione Nazionale per la Ricerca sul Cancro, from the Regione
Emilia Romagna, and by the Pallotti's Legacy for Cancer Research.
