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 ¨ and Werner E G Muller1 ¨ Institut fu¨r Physiologische Chemie, Abteilung fuăr Angewandte Molekularbiologie, Johannes Gutenberg-Universitaăt, Mainz, Germany; Institute of Chemical Physics and Biophysics, Tallinn, Estonia 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 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 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 2Â)5Âlinked oligoadenylates, termed (25)A [ ẳ pppA(2Âp5ÂA)n [pnAn], with chain lengths of £ 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 ¨ ¨ Chemie, Abteilung Angewandte Molekularbiologie, Johannes Gutenberg-Universitat, Duesbergweg 6, 55099 Mainz, Germany ă Fax: + 61 31 3925243, Tel.: + 61 31 3925910, E-mail: wmueller@mail.uni-mainz.de 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 January 2002, accepted 11 January 2002) Keywords: Suberites domuncula; Geodia cydonium (2¢-5¢) oligoadenylate synthetase; sponges; Porifera Ĩ FEBS 2002 (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 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 days and reach sizes of  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 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 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 mm in diameter (average, 3–7 mm) are formed after 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 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 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) Ó FEBS 2002 1384 V A Grebenjuk et al (Eur J Biochem 269) Fig S domuncula: animals and primmorphs (A) The siliceous sponge S domuncula (red) has been kept for more than 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 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, mM MgCl2 and 5% (v/v) glycerol] was added during homogenization The primmorphs were suspended in polymerase assay buffer followed by cycles of freezing ()10 °C)thawing for lysis The supernatant obtained after centrifugation (10 000 g; 10 min; °C) was immediately frozen (Ôcrude extractÕ) column (Supelcosil LC-18, 30 cm · mm, 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 with gentle shaking the membrane was washed · with PAB and subsequently dried 10 lL of reaction buffer containing mM ATP and · 104 c.p.m [14C]ATP, 30 mM Tris/HCl pH 7.5, 100 mM KCl and 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 h at 37 °C followed by the inactivation of the enzyme for 15 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 Ĩ FEBS 2002 (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 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 · 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 and finally with fluorescein isothiocyanateconjugated goat anti-(rabbit IgG) Ig for 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 months had lower (2–5)A synthesizing activities compared to those which were cut into pieces and frozen after only 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 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 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 Ó FEBS 2002 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; 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 lgỈmL)1 Effect of LPS on (2–5)A synthetase activity in primmorphs from S domuncula Fig 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: mg of total protein per assay, synthesis time 12 h; lane c: 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 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 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 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 lgỈmL)1 of LPS for 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 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 Ó FEBS 2002 (2¢)5¢)Oligoadenylate synthetase in sponges (Eur J Biochem 269) 1387 Table 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 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 ẳ 5) LPS (lgặmL)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 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 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 12 24 12 24 12 24 12 24 Table Determination of (2–5)A synthetase activity in tissue (from sea animals and aquarium animals) of G cydonium Where indicated, incubation with 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 ¼ 5) Animals LPS (lgỈmL)1) Incubation period (h) ATP + ADP + AMP (%) p3A2 (%) p3A3 and longer (%) Product (sum %) Sea Aquarium Aquarium Aquarium Aquarium Aquarium Aquarium Aquarium – – 1 10 10 10 – – 12 24 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 (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 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 Ĩ 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 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 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 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 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 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 days were used to isolate RNA which was subjected to Northern blotting (B; lane b) The incubation period for the tissue was (A; lane a) and 12 h (A; lane b); the primmorphs were incubated for 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 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 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 months, in the aquarium (aquarium animals) almost no enzyme activity was observed (Fig 2; Tables 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 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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. .. 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