Tài liệu Báo cáo Y học: Induction of (2¢)5¢)oligoadenylate synthetase in the marine sponges Suberites domuncula and Geodia cydonium by the bacterial endotoxin lipopolysaccharide docx

Eur. J. Biochem. 269, 1382–1392 (2002) Ó FEBS 2002

Induction of (2¢)5¢)oligoadenylate synthetase in the marine
sponges Suberites domuncula and Geodia cydonium
by the bacterial endotoxin lipopolysaccharide
Vladislav A. Grebenjuk1, Anne Kuusksalu2, Merike Kelve2, Joachim Schutze1, Heinz C. Schroder1
ă
ă
and Werner E. G. Muller1
ă
1

Institut fuăr Physiologische Chemie, Abteilung fuăr Angewandte Molekularbiologie, Johannes Gutenberg-Universitaăt, Mainz, Germany;
Institute of Chemical Physics and Biophysics, Tallinn, Estonia

2

Recent studies have shown that the Porifera, with the
examples of the demosponges Suberites domuncula and
Geodia cydonium, comprise a series of pathways found also
in the immune system of Deuterostomia, such as vertebrates,
but are absent in Protostomia, with insects or nematodes as
examples. One pathway is the (2¢)5¢)oligoadenylate synthetase [(2–5)A synthetase] system. In the present study we
show that crude extracts from tissue of S. domuncula collected from the sea display a considerable amount of (2–5)A
synthetase activity; 16% of the ATP substrate is converted to
the (2–5)A product, while tissue from specimens which were
kept for 6 months in an aquarium shows only 1% of conversion. As aquarium animals show a lower bacterial load,
those specimens were treated for the experiments with the
bacterial endotoxin lipopolysaccharide (LPS); they respon-

ded to LPS with a stimulation of the (2–5)A synthetase

activity. To monitor if this effect can be obtained also on the
in vitro level, primmorphs which comprise proliferating and
differentiating cells, were incubated with LPS. Extracts
obtained from LPS-treated primmorphs also convert ATP
to the (2–5)A products mediated by the synthetase. In parallel to this effect on protein level, LPS causes after an
incubation period of 12 h also an increase in the steady-state
level of the transcripts encoding the putative (2–5)A synthetase. It is postulated that in sponges the (2–5)A synthetase
is involved in antimicrobial defense of the animals.

Sponges (phylum Porifera) are with the other metazoan
phyla of monophyletic origin [1]. These aquatic sessile filter
feeders existed already prior to the ÔCambrian explosionÕ [2],
which has been dated back 550 million years [3]. This
implies that they must have developed powerful mechanisms to protect themselves against unfavorable conditions,
e.g. environmental stress (ultraviolet exposure or xenobiotics) [4,5]. Because sponges have the capacity to process their
own volume of water every 5 s in order to extract edible
material [6] they are exposed to a huge amount of bacteria
and also viruses that are present in the seawater [7,8]. To cope
with these threats, sponges have developed an efficient
chemical defense system [9] as well as humoral and cellular

defense mechanisms [10], that provided also the basis for the
evolution to metazoan organisms [10].
One efficient protection against invading microorganisms
is the (2¢)5¢)oligoadenylate synthetase [(2–5)A synthetase]
system [11–13]. The (2–5)A synthetase(s) is activated by
certain classes of RNA, mainly double-stranded RNA [14].
In vertebrates the (2–5)A pathway is also induced by
interferons [15]. The major enzyme in this pathway, the
(2–5)A synthetase catalyzes the synthesis of a series of 2¢)5¢linked oligoadenylates, termed (25)A [ ẳ pppA(2Âp5ÂA)n

[pnAn], with chain lengths of 1 £ n £ 30] from ATP
[16,17]. (2–5)A acts as an allosteric activator of a latent
endoribonuclease, the RNase L, which degrades singlestranded, viral or cellular RNA [18].
Only very rarely viruses have been observed in sponges
[19], while intracellular bacteria are frequently present [20].
Some of the bacteria (both Gram positive and negative)
found in sponges might act as symbionts [21], while others
are presumably infectious [22]. In a previous contribution
we demonstrated that sponges react to bacterial infection
with suppression of cell proliferation and apoptosis [23].
In sponges the apoptotic pathway is well established
on molecular level; genes coding for both pro- (death
domains-containing proteins) and anti-apoptotic proteins
(Bcl-2 polypeptides) have been isolated from sponges
[24,25]. The (2–5)A synthetase-mediated inhibition of cell
growth [26] as well as induction of apoptosis [27,28] have
also been reported for vertebrate cells. In addition it was
demonstrated recently that besides the oligoadenylate
synthesizing activity, the murine (2–5)A synthetase isoezyme

Correspondence to W. E. G. Muller, Institut fur Physiologische
ă
ă
Chemie, Abteilung Angewandte Molekularbiologie, Johannes
Gutenberg-Universitat, Duesbergweg 6, 55099 Mainz, Germany.
ă
Fax: + 61 31 3925243, Tel.: + 61 31 3925910,
E-mail:
Abbreviations: LPS, lipopolysaccharide; CDP, disodium
2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate.

Note: The two cDNA sequences from Suberites domuncula for the
(2–5)A synthetase form 1, termed SD25A-1, and for the (2–5)A
synthetase form 2, termed SD25A-2, have been deposited in the
EMBL/GenBank database under accession numbers AJ301652 and
AJ301653, respectively.
(Received 26 September 2001, revised 7 January 2002, accepted
11 January 2002)

Keywords: Suberites domuncula; Geodia cydonium (2¢-5¢)
oligoadenylate synthetase; sponges; Porifera.


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(2¢)5¢)Oligoadenylate synthetase in sponges (Eur. J. Biochem. 269) 1383

9-2 functions as a proapoptotic protein of the Bcl-2 family
[29].
Considering the fact that in sponges the molecules
involved in immune response are closer related to deuterostomian (vertebrate) animals than to Protostomia (insects or
nematodes; reviewed in [10]), we postulated that also
elements of the (2–5)A system exist in sponges. The first
sponge species studied was Geodia cydonium (Demospongiae) which in fact showed high levels of (2–5)A oligoadenylate synthesis in comparison to vertebrate cells [30]. The
reaction products were identified by thin-layer chromatography, immunologically and by high-performance liquid
chromatography. The biological activity of (2–5)A oligomers was verified by inhibition of the protein synthesis in
rabbit reticulocyte lysate [30]. The (2–5)A synthetase
reaction products were also confirmed by MALDI-MS
and by NMR analysis [31]. We succeeded in cloning the
sponge (2–5)A synthetase from G. cydonium [32]. A calculation based on the rates of amino-acid substitutions
revealed that the sponge enzyme branched off from a

common ancestor  520 million years ago.
In view of the finding that sponges do contain the (2–5)A
synthetase system like vertebrates, while this enzyme is
lacking in Protostomia [32] it was pressing to study in
functional assays if also in sponges the (2–5)A synthetase
responds in the protection against foreign, pathogenic
microorganisms. The sponge cellular system, which proved
to be suitable for this approach are the sponge primmorphs
[33,34]. Primmorphs are formed from dissociated single cells
after usually 5 days and reach sizes of  5 mm; they contain
proliferating cells and their interior is surrounded by an
almost complete single-cellular layer of epithelial-like cells,
pinacocytes; the cells inside the primmorphs are primarily
spherulous cells, amoebocytes and archaeocytes.
In the present study, we use tissue and primmorphs from
the marine sponge Suberites domuncula (Demospongiae)
and tissue of G. cydonium (Demospongiae; as a reference
sponge) and treated them with lipopolysaccharide (LPS), an
endotoxin derived from the outer cell wall of Gram-negative
bacteria that binds to the cell surface molecule CD14 [35].
The data revealed that tissue as well as primmorphs reacted
to LPS treatment with a rapid increase in (2–5)A synthetase
activity. To determine if LPS has the same effect on the gene
expression level, two cDNAs that might encode the putative
(2–5)A synthetase have been isolated and characterized
from S. domuncula. Northern blot studies revealed that the
steady-state level of transcripts for the (2–5)A synthetase
gene strongly increased in tissue as well as in primmorphs
after LPS treatment. Because LPS is known to strongly
inhibit protein synthesis in sponge cells [22], it is concluded

that the (2–5)A synthetase system is involved in defense
against microorganisms, very likely by inhibition of cell
proliferation or induction of apoptosis.

MATERIALS AND METHODS
Materials
Restriction endonucleases and other enzymes for recombinant DNA techniques and vectors were obtained from
Stratagene (La Jolla, CA, USA), Qiagen (Hilden, Germany),
Roche (Mannheim, Germany), USB (Cleveland, OH, USA),
Amersham (Buckinghamshire, UK) and Promega (Madi-

son, WI, USA). In addition, DIG (digoxigenin) DNA
labeling kit, DIG-11-dUTP, anti-DIG AP Fab fragments,
disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2¢(5¢-chloro)-tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate
(CDP) and positively charged nylon membrane (no.
1209272) were from Roche (Mannheim, Germany)
and Na-hexafluorosilicate from Aldrich (Deisenhofen,
Germany). Shrimp alkaline phosphatase was purchased
from USB Corporation (Cleveland, OH, USA), [14C]ATP
(542 mCiỈmmol)1) from Amersham International PLC
(Buckinghamshire, England), LPS from Escherichia coli
(L2880) and adenosine 5¢-triphosphate from Sigma Chemical
Co. (St Louis, MO, USA), and polyethyleneimine cellulose
TLC plates from Schleicher & Schuell (Keene, NH, USA).
Rotiquant reagent was purchased from Roth (Germany).
Sponge
Live specimens of S. domuncula [Porifera, Demospongiae,
Hadromerida], and G. cydonium (Porifera, Demospongiae,
Geodiidae) were collected by scuba diving near Rovinj
(Croatia) from depths between 15 and 35 m (nonpolluted

site). The sponges were brought to Mainz (Germany) and
there kept in 1000-L tanks at 17 °C before use in the
experiments. In one series of experiments the animals were
kept for only 2 days in the aquarium before use (termed Ôsea
animalsÕ). In the studies to determine the effect of LPS on
the expression/activity of (2–5)A synthetase sponges
remained for 6 months in the aquarium prior to use in the
experiments (Ôaquarium animalsÕ; Fig. 1A).
Formation of primmorphs
The procedure for the formation of primmorphs from single
cells was applied as described previously [33,34]. Starting
from single cells obtained by dissociation in Ca2+ and Mg2+free artificial seawater [36] primmorphs of at least 1 mm in
diameter (average, 3–7 mm) are formed after 2 days. For the
experiments, 5-day-old primmorphs were used. They were
cultivated in natural seawater supplemented with 0.2% of
RPMI1640 medium and with silicate to the optimal concentration of 60 lM as described previously [37]. The silicate
concentration in the natural, nonsupplemented seawater was
1 lM. The incubation temperature was set to 17 °C. The
experiments were performed with six animals per assay, each.
Incubations
Sponges were cut into cubes with a side of approximately
0.5 cm. For each set of experiments (exposure to LPS and
control) samples from the same sponge specimen were used.
Incubations were performed in filtered, oxygenated seawater. The sponge cubes or primmorphs were treated for 0–
24 h in the absence or presence of 1 lgỈmL)1 or 10 lgỈmL)1
of LPS in seawater at ambient temperature. In control
experiments the samples remained untreated for the
entire incubation period. Thereafter the sponge cubes/
primmorphs were immediately frozen ()80 °C).
Cell extracts

Frozen sponge cubes were ground in liquid nitrogen and an
equal amount (v/w) of the polymerase assay buffer (PAB)


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1384 V. A. Grebenjuk et al. (Eur. J. Biochem. 269)

Fig. 1. S. domuncula: animals and primmorphs. (A) The siliceous sponge S. domuncula (red) has been kept for more than
6 months together with the second demosponge D. avara (violet) in the aquarium
(·0.1). (B–D) Primmorph formation of
S. domuncula. (B) Dissociated single cells
(·200). (C) Primmorphs formed after 5 days;
(·5). (D) Cross section through a primmorph,
which has been subsequently subjected to
incubation with antiserum raised against
S. domuncula cells (·5).

[20 mM Tris/HCl, pH 7.5, containing 100 mM KCl, 5 mM
MgCl2 and 5% (v/v) glycerol] was added during homogenization. The primmorphs were suspended in polymerase
assay buffer followed by 2 cycles of freezing ()10 °C)thawing for lysis. The supernatant obtained after centrifugation (10 000 g; 10 min; 4 °C) was immediately frozen
(Ôcrude extractÕ).

column (Supelcosil LC-18, 30 cm · 4 mm, 5 lm; Supelco)
and separated in a 0.5–30% methanol gradient in 50 mM
NH4H2PO4 pH 7.0 at 40 °C [39]. The absorbance
was measured at 254 nm. The 3.05 software version
(Waters Corporation) was used to quantify the synthesis
products.
Dephosphorylation of (2–5)A oligomers

(2–5)A synthetase assay
(2–5)A synthetase activity in crude extracts was determined
after binding of the enzyme to a positively charged nylon
membrane. The assays of the same series were normalized to
the protein content. All incubations were performed in
microtiter plate wells at room temperature. Sponge extract
was added to a piece of membrane (0.16 cm2) in the well.
After incubation for 30 min with gentle shaking the
membrane was washed 4 · 5 min with PAB and subsequently dried. 10 lL of reaction buffer containing 1 mM
ATP and 2 · 104 c.p.m. [14C]ATP, 30 mM Tris/HCl
pH 7.5, 100 mM KCl and 5 mM MgCl2 was added [38].
The wells were sealed tightly and the synthesis of (2–5)A was
allowed to occur usually for 4–12 h. For HPLC analysis
50 lL of the reaction buffer without radioactive tracer was
used to produce the oligomers and the reactions were
performed in microcentrifuge tubes.
Thin-layer chromatography
The reaction products were eluted with distilled water
and separated by TLC on polyethyleneimine cellulose using
0.4 M Tris/HCl pH 8.6, 30 mM MgCl2 as the mobile phase
[30]. The TLC plates were exposed to a CS-imaging screen
and scanned with the GS-525 Molecular Imager (Bio-Rad;
Hercules, CA, USA). The amounts of ATP and (2–5)A
oligomers were quantified by the relative intensities of the
corresponding spot areas on the autoradiograms.
High-performance liquid chromatography
The 2¢-5¢ linked oligoadenylates produced in the assay in
their triphosphorylated forms were applied to the HPLC

The synthesis products of S. domuncula were verified and
quantified as the dephosphorylated (ÔcoreÕ) forms of the
oligomers. For that purpose the mixture of products was
treated with shrimp alkaline phosphatase 0.04 lL)1 for
1 h at 37 °C followed by the inactivation of the enzyme
for 15 min at 65 °C. After centrifugation at 20 000 g for
10 min, the mixture was applied to the HPLC column.
Cloning of the putative S. domuncula (2–5)A
synthetase cDNA
Two complete sponge cDNAs, termed SD25A, encoding the
putative (2–5)A synthetase (25A_SD), were cloned by
screening the cDNA library from S. domuncula [40] using
the GC2–5AS (accession number Y18497 [32]) as a probe.
Screening of the library was performed under low stringency
hybridization as described previously [40]. Positive clones
were detected with an alkaline phosphatase conjugated antiDIG Ig using 5-bromo-4-chloroindol-2-yl phosphate/nitro
blue tetrazolium as substrate [41]. All cDNAs have been
obtained from two different cDNA libraries resulting in three
independent clones each. DNA sequencing was performed
with an automatic DNA sequenator (Li-Cor 4000S). Two
different complete sequences have been obtained; they
were termed SD25A-1 and SD25A-2. The corresponding
deduced proteins were named 25A-1_SD and 25A-2_SD.
Sequence comparisons
The sequences were analyzed using computer programs
BLAST [42] and FASTA [43]. Multiple alignments were
performed with CLUSTAL W ver. 1.6 [44]. Phylogenetic trees
were constructed on the basis of amino-acid sequence

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(2¢)5¢)Oligoadenylate synthetase in sponges (Eur. J. Biochem. 269) 1385

alignments by neighbour-joining, as implemented in the
NEIGHBOR program from the PHYLIP package [45]. The
distance matrices were calculated using the Dayhoff PAM
matrix model as described previously [46]. The degree of
support for internal branches was further assessed by
bootstrapping [45]. The graphic presentations were prepared with GENEDOC [47]. Hydropathicity analysis, based on
the method of Kyte & Doolittle [48], was performed using
the PC/GENE Soap [49].
Exposure of primmorphs to Escherichia coli
Primmorphs (5 days old), obtained from cells of aquarium
animals, were exposed to heat-killed Escherichia coli as
described earlier [23]; the concentration of bacteria was
adjusted to 10 lg of nitrogen per mL [23]. Twelve and
twenty-four hours later primmorphs were taken and RNA
was extracted which then was subjected to Northern
blotting, using SD25A-1 as a probe.
Northern blot
RNA was extracted from liquid-nitrogen pulverized sponge
tissue with TRIzol Reagent (GibcoBRL, Grand Island,
NY, USA). Then an amount of 5 lg of total RNA was
electrophoresed through 1% formaldehyde/agarose gel and
blotted onto Hybond N+ membrane following the manufacturer’s instructions (Amersham; Little Chalfont, Buckinghamshire, UK) [5]. Hybridization was performed with a
0.7-kb part of SD25A-1. The probe was labeled with the
PCR-DIG-Probe-Synthesis Kit according to the manufacturer’s instructions (Roche). In one series of experiments
poly(A)+-RNA was purified from sponge tissue with
Oligotex mRNA kit (Qiagen) and analysed. For the

quantification of the Northern blot signals the chemiluminescence procedure was applied [50]; CDP-Star was used as
substrate. The screen was scanned with the GS-525
Molecular Imager (Bio-Rad).
Immunohistological analysis of primmorphs
Fresh tissue was fixed in paraformaldehyde, embedded in
Technovit 8100 and sectioned, essentially as described
previously [23]. The 2-lm thick slices reacted with antiserum, raised against S. domuncula cells.
The polyclonal antiserum against cells from S. domuncula
was raised in female rabbits (White New Zealand). An
amount of 3 · 106 cells [34] was injected at 4-week intervals;
after three boosts, serum was prepared [51]. The antiserum
obtained was termed anti-S. domuncula.
After fixation of the slices from S. domuncula primmorphs, the cells were made permeable with 0.1% saponin,
washed in NaCl/Pi and incubated with anti-S. domuncula
for 30 min and finally with fluorescein isothiocyanateconjugated goat anti-(rabbit IgG) Ig for 2 h [52]. The sections
were inspected by immunofluorescence with an Olympus
AHBT3 microscope. Control experiments with preimmune
serum did not show any auto- immunofluorescence.
Protein quantification
Protein concentration was estimated by the Rotiquant
reagent, using bovine serum albumin as a standard.

RESULTS
Identification of (2–5)A synthetase activity
in S. domuncula ‘crude extract’
It is established that (2–5)A synthetase is present in
G. cydonium in high amounts. The product length formed
by the G. cydonium enzyme from ATP is 2–8 adenylate
residues and the 2¢)5¢ linkage was verified by NMR analysis
[31]. The p3A4 proved to be biologically active [30]. It also

appears that G. cydonium is not the only species with (2–5)A
synthesizing activity within the phylum Porifera. We have
identified (2–5)A synthesizing activity in different marine
sponges, including S. domuncula (this study and A.
Kuusksalu, A. Lopp, T. Reintamm and H. Kelve, unpublished data). The product pattern of the enzyme from
S. domuncula differs under the same reaction conditions
from that of G. cydonium. The synthesis level is significantly
lower and the main synthesis product is p3A2 as verified by
the comigration with p3A2 standard (TLC and HPLC) and
A2 standard (HPLC) after dephosphorylation with shrimp
alkaline phosphatase (not shown). Recently we have shown
in G. cydonium as well as in S. domuncula crude extracts
that the enzyme, catalyzing the formation of (2–5)A, does
not require dsRNA for activity (submitted). In the present
study we took advantage of this phenomenon and performed the assays for (2–5)A synthesis with crude cell
extracts using positively charged membranes for partial
purification of the enzymes.
(2–5)A synthetase activity from field/aquarium animals
Interestingly, samples from S. domuncula kept in the
aquarium for 6 months had lower (2–5)A synthesizing
activities compared to those which were cut into pieces and
frozen after only 2 days maintenance in an aquarium. The
extract from aquarium animals converted, under otherwise
identical conditions, only 1% of the substrate to the (2–5)A
product; in contrast extracts from the sea animals could
utilize more than 16% of the substrate during the same
synthesis period (Fig. 2; Table 1).
In the case of G. cydonium the synthetase activity was
initially high in all animals tested (total product formation
at the same reaction conditions was 82.8% in sea animals

and 33.6% in aquarium animals), still revealing significant
product decrease during the long-term incubation (for
6 months) in the aquarium (Table 2).
This result suggested that the animals kept in the
aquarium are lacking a factor that causes either the
expression of the gene encoding the (2–5)A synthetase or
the activation of the enzyme. One potential factor could be
the differential load of microbes. In a recent study it could
be established that specimens from S. domuncula, analyzed
immediately after being taken from the sea, harbor a series
of bacterial strains (> five strains; very likely commensalic
ones), while those which were kept for 6 months in an
aquarium contained only one bacterial strain (likely to be
the symbiotic one) which showed high rRNA sequence
similarity to a Pseudomonas species. The latter bacterial
species was operationally termed S. domuncula symbiont
(GenBank accession number AF324886 [22]). These symbionts were found to be encapsulated inside special cells, the
bacteriocytes, present in the vicinity of the canals. This result


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1386 V. A. Grebenjuk et al. (Eur. J. Biochem. 269)

Despite the initially high (2–5)A synthesizing activity in
tissue of G. cydonium (aquarium animals), the incubation
with LPS caused significant increase of synthesis level
(Table 2). The time course of the induction showed
similarity to the effect we had seen in the case of
S. domuncula. The increment of the products was highest

on the third hour of incubation (1 lgỈmL)1 LPS). The most
drastic increase (3 h; 1 lgỈmL)1 LPS) could lead almost to
the synthesis level of the sea animal. Longer incubation
caused again a decrease of the synthesis level. Incubation at
10 lgỈmL)1 of LPS gave a lower effect on synthetase activity
than at 1 lgỈmL)1.
Effect of LPS on (2–5)A synthetase activity
in primmorphs from S. domuncula

Fig. 2. Autoradiogram of the thin layer chromatography of the [14C]
labelled 2¢-5¢ oligoadenylates synthesized as described under Materials
and methods. Lane a: S. domuncula (aquarium animal); lane b:
S. domuncula (sea animal); lane c: G. cydonium (sea animal). Lane a
and b: 6 mg of total protein per assay, synthesis time 12 h; lane c: 2 mg
of protein, synthesis time 3.5 h. Reaction products were separated by
polyethyleneimine cellulose TLC followed by visualization with
GS-525 Molecular Imager System. The position of the authentic
compounds (AMP, ADP, ATP as well as p3A2 and p3A3) which were
run in parallel is shown.

was taken as the rationale to study if lipopolysaccharide
(LPS), a known endotoxin derived from the outer cell wall
of gram-negative bacteria, may influence the activity of the
(2–5)A synthetase.
Effect of LPS on (2–5)A synthetase activity in tissues
from S. domuncula and G. cydonium
Tissue samples from S. domuncula specimens, kept for
6 months in the aquarium are almost devoid of (2–5)A
synthetase activity, under the conditions used. Approximately 1% of the substrate was converted to (2–5)A
oligomers during 12 h synthesis. Tissue from these animals

was used to analyze if the endotoxin LPS has the capacity to
induce the enzyme. The data revealed that in the presence of
1 lgỈmL)1 of LPS the (2–5)A synthetase activity started to
increase; after 3–12 h incubation period 4% of the ATP
substrate was converted to p3A2 (Table 1). This increase
was transient and during longer incubation periods (24 h)
the product level dropped again. Higher concentrations of
LPS (10 lgỈmL)1) caused a lower effect on the (2–5)A
synthesizing activity in S. domuncula.

Primmorphs were prepared from single cells (Fig. 1B) of
aquarium animals and used 5 days later for the experiment
(Fig. 1C). In order to make certain that the cells which had
been reorganized into primmorphs indeed originated from
the S. domuncula species, cross sections through the cells
were reacted with anti-(S. domuncula) serum. The immunofluorescence analysis shows that all (> 95% of the
total) cells included in the primmorphs were stained
brightly with the antiserum (Fig. 1D). Control sections,
incubated with preimmune serum did not show any
reaction.
The experiments show again that after incubation of the
primmorphs with 1 lgỈmL)1 of LPS for 3 h an increase of
(2–5)A synthetase activity can be measured (from 1.5%
(controls) to 3.3% of the ATP substrate was converted to
p3An after this period), Table 1. This amount does not
change significantly during a prolonged incubation for up to
24 h. The identity of the p3A2 product synthesized by the
(2–5)A synthetase both in tissue and in primmorphs of
S. domuncula was verified by TLC and HPLC analysis as
triphosphorylated and/or core oligomers.

Two CDNAs encoding the putative S. domuncula (2–5)A
synthetase
Two cDNAs, named SD25A-1 (accession number
AJ301652) and SD25A-2 (AJ301653), have been isolated
which comprise 1175 and 1205 nucleotides. The longest
ORFs translate to 324 amino acids (for the predicted
polypeptide 25A-1_SD) and to 322 amino acids (25A2_SD), respectively; Fig. 3A. The start ATG for 25A-1_SD
is located at nucleotides 62–64 (stop codon, nucleotides
1034–1036) and for 25A-2_SD at nucleotides 62–64 (nucleotides 1028–1030). Northern blot analyses showed that the
transcript length for SD25A-1 is 1.4 kb (see below) and for
SD25A-2 1.3 kb (not shown), indicating that the full length
clones have been isolated. The calculated relative molecular
masses for these new synthetases are 37 846 and 37 494,
respectively. The two proteins were predicted to be unstable
with instability indices of 40.1 (25A-1_SD) and 46.0 (25A2_SD), respectively [49].
The two sponge sequences show the characteristic
domains, found in other (2–5)A synthetases, from the
sponge G. cydonium and in mammals (mouse) and chicken:
The (2–5)A synthetase signature-1 [14], is found between
amino acid 195 and amino acid 206 and signature-2 between
amino acid 258 and amino acid 268; the positions refer to


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(2¢)5¢)Oligoadenylate synthetase in sponges (Eur. J. Biochem. 269) 1387

Table 1. Determination of (2–5)A synthetase activity in tissue (both from sea animals and aquarium animals) and primmorphs from S. domuncula
(obtained from aquarium animals). The samples were incubated with 1 or 10 lgỈmL)1 of LPS for a period of 0–24 h. Subsequently crude extracts
were prepared and reacted in the enzyme assay with ATP (12 h synthesis time for the tissue extracts; 22 h for primmorph extracts) as described

under Materials and methods. The products were dephosphorylated after synthesis with shrimp alkaline phosphatase and analysed by HPLC. The
reaction products were also analyzed by TLC, followed by autoradiography to determine the product. Based on these data the conversion of
[14C]ATP to (2–5)A was calculated and is given in percent to the sum of ATP, ADP and AMP (The SD is less than 15%; n ¼ 5).
LPS
(lgỈmL)1)

Animals
S. domuncula tissue
Sea
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
S. domuncula primmorphs
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium

Incubation
period (h)

ATP + ADP + AMP
(%)

Product
(%)

–
–
1
1
1
10
10
10

–
–

83.84
98.88
95.34
96.61
97.78
98.78
97.80
98.90

16.26
1.12
4.66
3.39
2.22

1.22
2.20
1.10

0
1
1
1
10
10
10

–

98.48
96.71
96.44
96.28
98.41
97.93
98.38

1.52
3.29
3.56
3.72
1.59
2.07
1.62

3
12
24
3
12
24

3
12
24
3
12
24

Table 2. Determination of (2–5)A synthetase activity in tissue (from sea animals and aquarium animals) of G. cydonium. Where indicated, incubation
with 1 or 10 lgỈmL)1 of LPS was performed for 0–24 h. Extracts were prepared, reacted in the enzyme assay with ATP for 3.5 h, the products were
analysed by HPLC as described in Materials and methods. The amount of 2¢)5¢ linked dimers (p3A2) as well as trimers (p3A3) and longer were
calculated based on corresponding peak areas (The SD is less than 15%; n ẳ 5).

Animals

LPS
(lgặmL)1)

Incubation
period (h)

ATP + ADP + AMP
(%)

p3A2
(%)

p3A3 and
longer (%)

Product
(sum %)

Sea
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium
Aquarium

–
–
1
1
1
10
10
10

–
–
3

12
24
3
12
24

17.19
66.25
25.77
28.07
56.80
60.70
57.19
67.11

37.10
27.21
38.60
39.47
32.86
30.81
32.21
26.77

45.71
6.54
35.60
32.46
10.50
8.49

10.60
6.12

82.81
33.57
74.23
71.93
43.20
39.30
42.81
32.89

the 25A-1_SD sequence (Fig. 3A). The ATP-binding site
essential for enzyme activity [53,54] resides between amino
acid 273 and amino acid 284. The dsRNA binding region of
(2–5)A synthetase has been narrowed down to the segment
within amino acid 104 and amino acid 158 of the murine
enzyme [53]; in S. domuncula a related stretch has been
found between amino acid 76 and amino acid 125. The
polyA-related domain, found in enzymes such as poly(A)
polymerase (2–5)A synthetase and topoisomerase 1 (accession number IPR001201 [55]), spans from amino acid 148
and amino acid 212.
Phylogenetic analysis of sponge (2–5)A synthetases
Based on sequence similarity no sequence related to (2–5)A
synthetases from sponges or from vertebrates, is present in

the Protostomia Caenorhabditis elegans or Drosophila
melanogaster (Advanced BLAST available from http://
www.ncbi.nlm.nih.gov/blast/blast.cgi); a related enzyme is
also lacking in yeasts (e.g. Saccharomyces cerevisiae) or

plants (Arabidopsis thaliana). The two S. domuncula
sequences share with each other 95% identity and 97%
similarity with respect to amino acids and with the
G. cydonium enzyme 28% identity and 48% similarity.
The percent identity (similarity) to the mammalian (mouse)
(2–5)A synthetase is 19% (36%) and to the chicken
sequence 19% (35%); Fig. 3A.
A phylogenetic tree was constructed on the basis of
amino-acid sequence alignments (Fig. 3B) by neighbourjoining of the vertebrate and sponge (2–5)A synthetases.
The distantly related sequence of anthocyanidin synthase
from the plant Dianthus caryophyllus (U82432) was used as


1388 V. A. Grebenjuk et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 3. The two putative sponge (2–5)A synthetases from S. domuncula. (A). Alignment of the amino-acid sequence of the two sponge sequences,
25A-1_SD and 25A-2_SD, deduced from the cDNAs SD25A-1 and SD25A-2, with the related proteins from the sponge G. cydonium
(25A_GEOCY, accession number Y18497), as well as from mouse (25A_MOUSE, P11928) and from chicken (25A_CHICK, AB002586). The
alignment was performed using the CLUSTAL W program. Residues of amino acids, similar among all sequences, are in inverted type and residues
conserved in at least three sequences are shaded. The characteristic signatures of the (2–5)A synthetase are indicated: the two conserved signatures
(| Sig-1 and | Sig-2), the potential ATP-binding region (|+ ATP), the dsRNA binding segment (|– Bdg: dsRNA) and the polyA-related domain
(|::: polyA-related domain). (B) The phylogenetic relationship of the five (2–5)A synthetase sequences. The tree was routed with the distantly related
sequence of anthocyanidin synthase from the plant Dianthus caryophyllus (ANTO_DC, U82432). The numbers at the nodes are an indication of the
level of confidence for the branches as determined by bootstrap analysis (1000 bootstrap replicates). The scale bar indicates an evolutionary distance
of 0.1 amino-acid substitutions per position in the sequence.

outgroup to root the tree. The anthocyanidin synthase is
known to be involved in the catalysis of the colorless

leucoanthocyanidins to the colored anthocyanidins [56].

The phylogenetic relationship reveals that the three sponge
sequences form the basis of the tree from which the
vertebrate sequences branch off.


Ó FEBS 2002

(2¢)5¢)Oligoadenylate synthetase in sponges (Eur. J. Biochem. 269) 1389

Increase in the steady-state level of the (2–5)A
synthetase transcripts by LPS
The effect of LPS on the steady-state level of (2–5)A
synthetase transcripts, SD25A-1, was monitored by Northern blotting in a semiquantitative way both in tissue from
aquarium animals as well as in primmorphs obtained from
them. The data show that in tissue from those animals no
expression could be visualized after blotting with the
SD25A-1 probe (Fig. 4A, lane a). However, after an
incubation period for 12 h in the presence of 1 lgỈmL)1 of
LPS, a clear 1.4-kb band became visible which reflects the
size of the (2–5)A synthetase gene (Fig. 4A, lane b). A
likewise strong expression is also seen if the poly(A)+-RNA
fraction from tissue, exposed to LPS for the same period,
was subjected for Northern blotting (Fig. 4B, lane a); in
contrast poly(A)+-RNA from nontreated tissue did not
show any signal (not shown).
If single cells, kept for 3 days in Ca2+- and Mg2+-free
artificial seawater (Fig. 4B, lane b), or primmorphs, not
treated with LPS (Fig. 4B, lane c) were analyzed for

transcripts of (2–5)A synthetase, no signal in the 1.4-kb size
range could be seen. However, if the primmorphs were
treated with 1 lgỈmL)1 LPS a strong expression of the
(2–5)A synthetase gene is seen after 12 h (Fig. 4B, lane d);
the 1.4-kb signal even increased if RNA was analyzed from
primmorphs, incubated with LPS for 24 h (Fig. 4B, lane e).
Increase in the steady-state level of the (2–5)A
synthetase transcripts during incubation with E. coli
In view of our earlier finding that S. domuncula cells
respond to exposure to heat-killed E. coli with a reduced cell
proliferation and cell viability [23], primmorphs were
exposed to dead bacteria under the conditions described.
The bacteria were added at a concentration of 10 lg of
nitrogen per mL to the primmorphs; 12 and 24 h later RNA
was extracted and then probed with the SD25A-1 cDNA in
the Northern blotting experiment.
The results show that in the absence of the heat-killed
bacteria no transcripts, corresponding to 1.4 kb (2–5)A
synthetase mRNA, can be identified in the Northern blotting
approach (Fig. 4C, lanes a to c). In contrast, the steady-state
level of the transcripts increased strongly, even after the short
incubation period of 12 h (Fig. 4D, lane b vs. lane a; at time
0). A prolonged incubation for 24 h resulted in an even higher
level of the (2–5)A synthetase transcripts (Fig. 4C, lane c).

DISCUSSION
Inhibition of cell growth, apoptosis and inhibition of
protein synthesis are ways of protection of metazoan
organisms against death caused by microbes. The bacterial
endotoxin LPS causes cell growth inhibition [57] as well as

induction of apoptosis [27] in vertebrates very likely via a
(2–5)A synthetase-mediated pathway. Also in sponges LPS
inhibits cell proliferation and apoptosis [22,23]. Furthermore, LPS strongly inhibits protein synthesis in S. domuncula [22]. Therefore, in the present study we tried to answer
the question of whether also this effect is mediated or
paralleled by a stimulation/induction of (2–5)A synthetase
in sponges, using two different sponge species, S. domuncula
and G. cydonium as examples.

Fig. 4. Effect of LPS and heat-killed E. coli on the steady-state level of
(2–5)A synthetase transcripts in S. domuncula tissue from aquarium
animals (A) and primmorphs/cells (B). Sponge tissue or primmorphs
were incubated with 1 lgỈmL)1 of LPS for 0–12 h. Thereafter RNA
was isolated and Northern blotting was performed with SD25A-1 to
determine the expression of the (2–5)A synthetase gene. Five micrograms of total RNA each were loaded on the slot. In one series of
experiments single cells which remained in Ca2+- and Mg2+-free
artificial seawater for 3 days were used to isolate RNA which was
subjected to Northern blotting (B; lane b). The incubation period for
the tissue was 0 (A; lane a) and 12 h (A; lane b); the primmorphs were
incubated for 0 h (B; lane c), 12 h (lane d) or 24 h (lane e). M, marker
RNAs, which were run in parallel. In (B; lane a) poly(A)+-RNA,
isolated from tissue of an aquarium animals, which was treated with
1 lgỈmL)1 of LPS for 12 h, was analysed. Determination of the effect
of heat-killed E. coli on the steady-state level of (2–5)A synthetase
mRNA in primmorphs. Primmorphs were incubated in the absence
(C) or presence of of the heat-killed bacteria (D) for 0–24 h. At the
indicated times primmorphs were taken, RNA was extracted and
subjected at the same concentrations (5 lg) to Northern blotting
experiments using the SD25A-1 cDNA as a probe.

Tissue samples from S. domuncula and G. cydonium

displayed different (2–5)A synthetase activities depending
on the time of cultivation in the aquarium. If crude extracts
from animals were taken (almost) immediately out of the
sea (sea animals) which were analyzed for (2–5)A synthetase
activity, product was detectable. In the present study this
effect was documented for S. domuncula and G. cydonium
(Tables 1 and 2). If these animals (S. domuncula) were kept


Ó FEBS 2002

1390 V. A. Grebenjuk et al. (Eur. J. Biochem. 269)

for a longer period, more than 6 months, in the aquarium
(aquarium animals) almost no enzyme activity was observed
(Fig. 2; Tables 1 and 2). One reason for this effect is the fact
that the bacterial load, with respect to the number as well as
the species diversity of bacteria, is reduced under the
controlled aquarium conditions (closed circuit). The reduction of the bacterial flora in specimens kept in the aquarium
has been recently documented [22].
To test the assumption that bacterial load of sponge
tissue is causatively connected with (2–5)A synthetase
activity, the endotoxin LPS from the outer bacterial cell
wall was used as a substitution/model component. Incubation studies with tissue from aquarium animals (S. domuncula, G. cydonium) revealed that LPS causes a significant
and rapid stimulation of the synthetase activity. The extent
of products formed in S. domuncula amounts to 1–2% of
conversion of ATP to (2–5)A, in comparison to 16%
measured in field sea animals while the corresponding
values for G. cydonium had the same tendency. It should be
mentioned that the (2–5)A synthesizing activity in G. cydonium is per se markedly higher than that in S. domuncula.

This stimulatory effect of LPS on the (2–5)A synthetase
activity was confirmed using the primmorph system from
S. domuncula. The primmorphs that contain proliferating
and differentiating cells [33,34] have been demonstrated here
to consist almost exclusively of sponge, S. domuncula, cells.
These experiments were included in order to rule out the
possibility that nonsponge cells form the aggregates. Previously it had been argued that contaminating unicellular
eukaryotic organisms could have formed the aggregates that
might have been erroneously contributed to sponge cells
[58]. The antiserum raised against S. domuncula cells was
found to stain the cells of the primmorphs brightly. Using
this primmorph system, it was demonstrated that again after
the incubation with LPS a significant amount of (2–5)A is
synthesized;  3.5% of the ATP present in the assays was
converted to dimers which comigrate with p3A2 if analyzed
by TLC or coelute with the reference compound in HPLC
runs.
Based on the incubation studies with tissue samples or
primmorphs it could be deduced that LPS causes a
stimulation of (2–5)A synthetase activity by a hitherto
unknown signal transduction pathway. In a previous study
it had been shown that the mitogen-activated protein kinase
pathway is involved in the cell response to LPS [22]. Until
now a potential involvement of this pathway in the (2–5)A
synthetase system has not been reported. Nonetheless,
the fast response of the cells to LPS argues in favor of a
post-translational/allosterical activation of the (2–5)A
synthetase.
The effect of LPS on the steady-state level of the
S. domuncula (2–5)A synthetase transcripts was analyzed

in tissue and primmorphs, incubated with LPS and heatkilled bacteria. The results revealed that the steady-state
level of the transcripts is strongly up-regulated after an at
least 12-h incubation period. This finding supports the
view that LPS causes not only a post-translational/
allosteric activation of the (2–5)A synthetase activity in
cells and tissue but also an increased transcript level. The
potency of LPS to modulate gene expression in vertebrate
cells is well established [58]; nevertheless, the involvement
of the toxin in the (2–5)A synthetase pathway in these
systems has not yet been described. However, the partici-

pation of LPS in apoptosis has been documented as
reviewed recently [59].
Even though the documentation of virus infection/
presence in sponges is very poor in contrast to that of
bacterial association/infection, which is very abundant in
Demospongiae, the data presented show that the activity of
the enzyme as well as the steady-state level of the transcripts
of the respective gene increases in cells after LPS/bacteria
treatment. Therefore, we currently subscribe to the view that
LPS affects two pathways, one which causes a posttranslational/allosteric activation of the enzyme resulting
in the formation of the p3An products and a second, that
increases the steady-state level of the transcripts of the
corresponding (2–5)A synthetase gene. In vertebrate cells it
has been demonstrated that the expression of the (2–5)A
synthetase is mediated by the jak/STAT pathway and
initiated by cytokines [28]. At present, studies on the
elucidation of this pathway in S. domuncula are in progress
in our group. As a consequence of the activation/induction
of (2–5)A synthetase the sponge specimens might protect

themselves against microbial infection or inhibition of cell
proliferation and finally may undergo apoptosis. The
existence of cytokines in sponges has been documented,
e.g. the macrophage-derived cytokine-like molecule (the
allograft inflammatory factor or glutathione peroxidase) or
the polypeptide related to the mammalian endothelialmonocyte-activating polypeptide (reviewed in [10]).
In conclusion, the data reported here suggest that the
products of the (2–5)A synthetase in sponges, p3An, could
be involved in the antimicrobial defense of the animals.
Furthermore, sequence data show that genes encoding a
putative (2–5)A synthetase are present in different sponge
species. This adds further support for the view that the
immune system in sponges is closer related to the deuterostomian, vertebrate, taxa than to the protostomian systems
[60], which are lacking not only a series of characteristic
cytokines [61] but also the (2–5)A synthetase system. Future
transfection studies must show if the genes encoding the
putative (2–5)A synthetases from S. domuncula are indeed
responsible for the (2–5)A synthetase activity measured in
cells from S. domuncula.

ACKNOWLEDGEMENTS
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Mu 348/14-1), the European Commission (project:
ă
SPONGE), the Bundesministerium fur Bildung und Forschung
ă
(project: Center of Competence BIOTEC-MARIN), the International
Human Frontier Science Program (RG-333/96-M) and the Estonian
Science Foundation.

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