Cold stress defense in the freshwater sponge Lubomirskia baicalensis Role of okadaic acid produced by symbiotic dinoflagellates ´ Werner E G Muller1,2, Sergey I Belikov2, Oxana V Kaluzhnaya1,2, Sanja Perovic-Ottstadt1, ¨ Ernesto Fattorusso3, Hiroshi Ushijima4, Anatoli Krasko1 and Heinz C Schroder1 ă Institut fur Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitat Mainz, Germany ă ă Limnological Institute of the Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia ` Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli ‘Federico II’, Italy Department of Developmental Medical Sciences, Institute of International Health, The University of Tokyo, Japan Keywords dinoflagellates; heat shock protein; Lubomirskia baicalensis; okadaic acid; protein phosphatase Correspondence W E G Muller, Institut fur Physiologische ă ă Chemie, Abteilung Angewandte Molekularbiologie, Universitat, ă Duesbergweg 6, 55099 Mainz, Germany Fax: +49 6131 3925243 Tel: +49 6131 3925910 E-mail: wmueller@uni-mainz.de Website: http://www.biotecmarin.de/ Database The sequences from Lubomirskia baicalensis reported here have been deposited in the GenBank database under the accession numbers AM392283 (protein phosphatase LUBAIHSP70PP1) and AM392284 (heat shock protein-70 LUBAIHSP70) Note This article is dedicated to Professor Michele ` Sara, Professor of Zoology at the University of Genova, for his outstanding contributions to marine biology, 1926–2006 The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0°C, and in summer in open water at 17 °C (September) In March, specimens show high metabolic activity as reflected by the production of gametes L baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1) Only cDNA corresponding to PP1 could be identified in L baicalensis and subsequently isolated from a L baicalensis cDNA library The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer) PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 °C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 °C In the enriched extract, PP1 activity at °C is close to that measured at 17 °C Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at °C From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge (Received 16 August 2006, revised 21 October 2006, accepted 27 October 2006) doi:10.1111/j.1742-4658.2006.05559.x The taxon sponges (phylum Porifera) has been surprisingly successful during evolutionary development This metazoan phylum is the only one to have survived the severe Varanger–Marinoan ice age (605–585 million years ago) of the Neo-Proterozoic eon (1000–520 million years ago), during which the earth was almost Abbreviations hsp70, heat shock protein-70; OA, okadaic acid; PP1, protein phosphatase-1; PP2A, protein phosphatase-2A FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 23 Cold stress defense in sponges W E G Muller et al ă completely covered by ice; most organisms became extinct during this period [1] As ‘living fossils’ [2], sponges represent evolutionarily the oldest extant taxon, and thus allow insight into the genome organization of animals that lived prior to the ‘Cambrian Explosion’ At that time, sponges existed exclusively in the marine environment, whereas later some taxa also occupied freshwater biotopes (during the Cenozoic period) From cosmopolitan freshwater species, e.g Ephydatia fluviatilis, endemic species branched off in ‘old lakes’ Lake Baikal (Siberia), for example, harbors many prominent endemic sponges [3] Major reasons for the recent rapid evolution of the endemic sponge fauna in some areas, that still continues, are: (a) successful adaptation to environmental conditions, (b) dominance of sexual reproduction over asexual reproduction in sponges, and (c) differences in the habitats (littoral on rocks or on calcifying algae, e.g Chara sp.) [4] In Lake Baikal the dominant endemic sponge species Lubomirskia baicalensis lives in a cold environment; in March at an ambient temperature of )0.5 °C and in September at around 17 °C [5] These animals, which grow at depths of 1–20 m, maintain constant metabolic activities with pumping rates similar to those of species that live at 15–20 °C [6,7] Surprisingly, L baicalensis produces gametes and embryos in March when the lake is completely covered by m of ice One major source of essential organic carbon for the animals during this season is their ecological, symbiotic relationship with chlorophyllcontaining dinoflagellates [5] It has been reported previously that these symbiotic ‘Zoochlorellae’ exist in a 3-mm thick external layer of the sponge [8] Field observations revealed that, in the absence of light, the dinoflagellates die and are removed from the sponge specimens which likewise die (W E G Muller, University of Mainz, ă unpublished results) In L baicalensis these protists, which are closely related to Gymnodinium sanguineum, produce glycerol and transfer this intermediate metabolite via an aquaporin channel into the sponge cells (W E G Muller, University of Mainz, submitted) This ă raises two questions: how the sponges live in the cold environment; or, more specifically, (a) how they solve the problem of protein folding at low temperature, and (b) how they overcome the barrier of the required activation energy for the enzyme-mediated catalysis? This study mainly addresses the first question It is known that the growth of Escherichia coli at low temperature is facilitated by chaperonins [9] The question is, which sensor in these microorganisms regulates the expression of the respective heat shock proteins? Here we tested the hypothesis that secondary metabolites produced by the symbiotic ⁄ commensalic organisms in sponges contribute to the cold stress response 24 The dinoflagellates of the taxon Gymnodinium identified in L baicalensis are related to G sanguineum (Alveolata; Dinophyceae; Gymnodiniales; Gymnodiniaceae; Gymnodinium), which has been described as a component of harmful algal blooms and has been found to be hemolytic and ichthyotoxic [10] One toxin often produced in these algae [11] is okadaic acid (OA), a polyether C38 fatty acid, originally isolated from Halichondria okadaii [12] The major targets of OA in all metazoans hitherto studied are the catalytic subunits of the proteins phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) which are sensitively inhibited at nanomolar concentrations (the 50% inhibitory concentration for PP1 is 3–150 nm and that for PP2A is 0.03–0.2 nm) [11] Here we show that OA is present in L baicalensis, where it is synthesized by the dinoflagellates In order to perform functional studies between OA and PP1, the cDNA coding for PP1 had to be identified in L baicalensis This polypeptide shares high sequence similarity with mammalian PP1 Antibodies against PP1 allowed its assessment during temperature-dependent expression in vitro (cell culture) and in vivo (animals) At lower concentrations (< 100 nm), OA has no effect on the level and activity of the protein phosphatase(s) but induces the expression of heat shock protein-70 (hsp70) From earlier studies it is known that OA can trigger the expression of heat shock proteins in tissues [13] We applied the in vivo sponge cell culture system, the primmorphs [2], to demonstrate that in primmorphs at °C, OA upregulates both the expression of hsp70 transcripts and the amount of hsp70 protein to levels found at the ambient temperature of 17 °C Subsequent depletion experiments with antibodies against hsp70 showed that functionally active chaperon ⁄ hsp70 molecules are required for full protein phosphatase activity at low temperature From the data we conclude that at lower concentrations (< 100 nm) the secondary metabolite OA mediates ⁄ controls in L baicalensis the cold stress defense, whereas higher concentrations are required to inhibit the protein phosphatase(s) It has been established that sponges, like Suberites domuncula [14] or L baicalensis, which contain symbiotic microorganisms, display a stronger ‘resistance’ to OA; with regard to S domuncula only concentrations > 300 nm have a significant effect on protein synthesis Results L baicalensis specimens in winter and summer Animals were collected during September 2005 and March 2006 (Fig 1A,B) During these seasons, the FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS W E G Muller et al ă Fig L baicalensis specimens during late summer (September) (A) and the ice cover season (March) (B) In both seasons sponges contain associated dinoflagellates (taxon Gymnodinium) Cross-sections were prepared and examined using transmission electron microscopy (C, D) Sections through a branch from a September specimen show the abundantly present dinoflagellates (d) that are assembled at the rim of the green branches (E) Frequently the specimens during the March season contain spermatogenic cysts (sc) Identification of OA-producing dinoflagellates in L baicalensis (F–K) (F, I) Slices were prepared from tissue of L baicalensis and the cells were visualized Nomarsky interference contrast optics; dinoflagellates (d) as well as sponge cells (s) are marked (G) In one series, the slices were reacted with pAb-OA and then with Cy3-conjugated goat anti-(rabbit IgG) and finally inspected by immunofluorescence (wavelength ¼ 546 nm) (H) Autofluorescence of the chlorophyll in the dinoflagellates was detected at a wavelength of 490 nm (J) In parallel, the slices were reacted with pAb-OA, which had been pretreated with OA, coupled to the FID-33 oligopeptide and then with the labeled secondary antibodies (K) The same area was also analyzed with a wave-length of 490 nm Scale bar ¼10 lm animals have a bright green color, which is due to the high abundance of dinoflagellates (Fig 1C,D) related to the taxon G sanguineum (Alveolata; W E G Muller, ă University of Mainz, submitted) Interestingly, during winter the sponges form sexual propagation bodies, reflecting an active metabolism Figure 1E shows one spermatogenic cyst Cold stress defense in sponges A C D B E F I G J H K Presence of OA in L baicalensis The OA concentration in L baicalensis was determined using HPLC ⁄ MS analysis to be 83 ± ngỈg)1 wet weight (100 nm; March), whereas tissue from specimens collected in September had a lower OA content of 57 ± ngỈg)1 (70 nm) These values were confirmed by competitive ELISA giving concentrations of 75 ngỈg)1 (March) and 45 ngỈg)1 (September) Protein-coupled OA in L baicalensis Protein extracts were prepared from specimen tissue collected in March This preparation was subjected to SDS ⁄ PAGE (10% gel; Fig 2, lane a) After transfer, blots were incubated with anti-OA sera (pAb-OA) This revealed one prominent protein band of 14 kDa (Fig 2, lane b) The specificity of the reaction was proven using antibodies that had been adsorbed with OA bound to the FID-33 peptide; under these conditions the immunoreaction of the 14 kDa band was strongly suppressed (Fig 2, lane c) In contrast, the signal at 14 kDa was of the same strength when membranes were incubated with pAb-OA, which had been pretreated with FID-33 prior to use in the western blots (not shown) Identification dinoflagellates in tissue using anti-OA sera Slices of sponge tissue were prepared and reacted with pAb-OA The antibodies stained the dinoflagellates very brightly, whereas the sponge cells did not react FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 25 Cold stress defense in sponges W E G Muller et al ă Fig Identication of protein-coupled OA in L baicalensis Extracts were prepared and the protein size separated by SDS ⁄ PAGE (10% gel) Lane a, the gel was stained with Coomassie Brilliant Blue The proteins were blot transferred and the filters were either incubated with anti-OA sera (lane b) or with the antibody preparation, which had been preincubated with FID-33-OA which had been adsorbed with FID-33-OA (lane c) M, marker proteins (Fig 1G); using Nomarsky interference contrast optics the granule-containing dinoflagellates could be identified (Fig 1F) As further evidence for the localization of the dinoflagellates, slices were illuminated with green fluorescent light (490 nm) to identify dinoflagellates based on the autofluorescence of their chlorophyll (Fig 1H) Again, the dinoflagellates were highlighted in areas positive for pAb-OA In a parallel series, sections were treated with adsorbed antibodies against OA; this preparation showed a slight signal only very occasionally (Fig 1J) Dinoflagellates could again be visualized by their autofluorescence (Fig 1K) L baicalensis PP1 catalytic subunit Complete cDNA encoding the L baicalensis PP1 protein (LBPP1) was obtained from a cDNA library using a degenerate primer against the Ser ⁄ Thr-specific protein phosphatase signature of mammalian protein phosphatases The ORF between nucleotides 60–62 and 1057–1059(stop) codes for a 319 amino acid polypeptide (PP1_LUBAI) with a predicted size of 36 802 Da (Fig 3A); the sequence was termed PP1_LUBAI Like the related mammalian sequences, the sponge protein comprises a characteristic Ser ⁄ Thrspecific protein phosphatase signature (amino acids 121 and 126) and the conserved matallophosphoesterase (amino acids 57 and 252) The calcineurin-like phosphoesterase (amino acids 57 and 252) overlaps with the latter region Similarity between the sponge molecule and other metazoan PP1 sequences is high; the sponge PP1 shares 288 similar and 271 identical 26 amino acids with human PP1 (length: 323 amino acids), known to bind to OA (Fig 3A) The overall similarity ⁄ homology to metazoan sequences is  80% ⁄ 70% For the alignment in Fig 3A, the human protein with the highest similarity score was used (‘Expect value [E]’ 2e-162) [15]; this phosphatase is known to function as PP1 (catalytic subunit, gamma isoforms) A phylogenetic tree was computed after alignment of the metazoan sequences with yeast and plant-related PP1 (Fig 3B) The tree was rooted with the highest similar phosphatases from Arabidopsis thaliana (protein phosphatase-type 1; NP_181514.1) and Saccharomyces cerevisiae (type Ser ⁄ Thr protein phosphatase; NP_011059.1) Among the metazoan proteins, the sponge phosphatase clusters together with that of Drosophila melanogaster (Ser ⁄ Thr protein phosphatase; CAA49594.1), while the Caenorhabditis elegans protein (even-like phosphatases family member (NP_001022616.1) forms a separate branch together with the human sequence Both branches are separated only with low significance Relative PP1 content Because of the high sequence similarity between L baicalensis PP1 and the corresponding mammalian phosphatases it was possible to use a commercial antibody for the western blots Extracts were size-separated by SDS ⁄ PAGE and either stained with Coomassie Brilliant Blue (Fig 3C, lane a) or the proteins were transferred to poly(vinylidene difluoride) membranes and reacted with antibodies against PP1, as described in Experimental procedures Extracts from animals obtained in both March (Fig 3, lane b) and September (Fig 3, lane c) show a strong signal at  37 kDa, corresponding to the size of sponge PP1 In contrast, if the blots were reacted with adsorbed antibodies, the signals were strongly reduced (lanes d and e) Phosphatase activity in the extract Extracts were prepared from sponge tissue specimens collected in March or September and subjected to the protein phosphatase assay described in Experimental procedures The specific enzyme activities in tissues from winter and summer animals were almost identical (22.6 ± 4.7 versus 23.4 ± 4.2 nmolỈmin)1Ỉmg)1) If OA was added, the reactions in the two series of experiments were inhibited dose-dependently; at 100 nm the activity differed from that seen in the controls (March, 17.3 ± 3.9 nmolỈmin)1Ỉmg)1; September, 19.2 ± 4.3 nmolỈmin)1Ỉmg)1) However, at 300 nm the activity FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS W E G Muller et al ă Cold stress defense in sponges A B C Fig PP1 from L baicalensis (A, B) The PP1 sequence from L baicalensis (A) Alignment of the sponge PP1 protein (PP1_LUBAI) with the human PP1 which binds to OA (PP1_HUMAN; accession number 1JK7_A) Amino acids, identical in both sequences, are in inverted type and those similar in both sequences are shaded The characteristic Ser ⁄ Thr-specific protein phosphatase signature (S ⁄ Tp) and the conserved metallophosphoesterase (MetPhoEsterase) regions are marked (B) The phylogenetic tree is constructed from the two abovementioned sequences as well as the PP1 from D melanogaster (PP1_DROME; CAA49594.1), from C elegans (seven-like phosphatases family member) (PP1 °CAEEL; NP_001022616.1), from S cerevisiae (PP1_YEAST; NP_011059.1) as well as from A thaliana (PP1_ARATH; NP_181514.1), which was used as outgroup to root the tree After alignment the tree was built Scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position in the sequence (C) Identification of PP1 in tissue from L baicalensis In all lanes 10 lg of protein were separated Lane a, the separated proteins were identified with Coomassie Brilliant Blue Western blot experiments: lanes b and c, the membranes were reacted with antibodies against PP1 (PcAb-PP1) and then with labelled secondary antibodies to visualize the immunocomplexes with the BM chemoluminescence substrate kit Samples from March (lane b) and September (lane c) were analyzed In parallel, membranes with the extracts were treated with adsorbed PcAb-PP1 (lanes d and e) The size markers are given decreased to  80% (March, 3.7 ± 1.9 nmolỈmin)1Ỉ mg)1; September, 5.2 ± 1.6 nmolỈmin)1Ỉmg)1) Expression level of hsp70 in animals and primmorphs The semiquantitative steady-state level of hsp70 transcripts in animals was in the same range, regardless of whether the RNA had been isolated from specimens collected in March or in September For these Northern blot studies the EST probe for hsp70 from L baicalensis (LUBAIHSP70) was used (Fig 4A) In order to assess the expression level under controlled laboratory conditions, in vivo primmorphs were incubated for 24 h at and 17 °C Primmorphs were incubated in the dark to suppress the metabolic activity of the remaining symbiotic algae Setting the expression level at °C to onefold, the amount of hsp70 transcripts in the primmorph cells incubated at 17 °C was much higher (10-fold; Fig 4A) However, if primmorphs were incubated at °C together with 100 nm OA the amount of hsp70 transcripts was the same as that measured for cultures maintained at 17 °C The toxin had no strong effect on hsp70 expression in cells at 17 °C (Fig 4A) In controls, a-tubulin expression was determined using the L baicalensis probe LUBAITUB in a parallel Northern blotting experiment Almost identical signal intensities were seen, confirming that the same amount of RNA was loaded onto the gels From these results, we conclude that OA induces hsp70 expression at the lower incubation temperature To support these studies, comparative Northern and western blot experiments were performed using hsp70 (LUBAIHSP70) or antibodies (mAb-HSP70) as the respective probes (Fig 4B) Again, the Northern blot FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 27 Cold stress defense in sponges W E G Muller et al ă Fig Expression of heat shock protein hsp70 transcripts in animals and primmorphs (A) Northern blot analysis RNA was extracted both from animals, collected in March or September, and from primmorphs cultivated in vitro As indicated, the primmorphs were cultivated either °C or at 17 °C for 24 h in the absence (–) or presence of 100 nM of OA (+) Then, total RNA was isolated and fractionated by electrophoresis, transferred to nylon membrane, and hybridized with the respective labeled probes; hsp70 (LUBAIHSP70) or a-tubulin (LUBAITUB) lg of total RNA were loaded into each slot The relative degree of expression was correlated with that seen for the minimal expression in primmorphs at °C (set to onefold) (B) Comparison of the level of hsp70 transcripts and hsp70 protein in primmorphs, incubated at or 17 °C in the absence (–) or presence of OA (+) As marked, the primmorphs were incubated at these two temperatures for 24 h and - ⁄ +OA Then extracts were prepared for Northern blotting (RNA) or western blotting (protein) (a) Northern blot: after size fraction and transfer the filter was hybridized with the hsp70 probe: N1, incubation at °C in the absence of OA; N2, at °C in the presence of OA; N3, incubation at 17 °C in the absence of OA; N4, incubation at 17 °C in the presence of OA In parallel, a filter was hybridized with a a-tubulin probe (b) From the same samples the proteins were extracted and subjected to western blot analysis Samples from cultures incubated at °C in the absence (lane W1) or presence of OA (W2) or at 17 °C without (lane W3) or with OA (lane W3) are loaded onto the gel, and after separation and transfer probed with the antibodies mAb-HSP70 The relative expression levels, correlated to the values assessed for cultures at °C and without OA (set to onefold), are given A B a b studies showed low expression of hsp70 in primmorphs incubated at °C or in the absence of OA, in comparison with those incubated with OA or at 17 °C (Fig 4B,a) Steady-state expression of the a-tubulin gene is shown using the same amount of RNA for analysis The data from western blot experiments showed a similar expression pattern; low levels of hsp70 in cultures incubated at °C and without OA, in comparison with those incubated with the toxin and at higher temperature (Fig 4B,b) From these data, we conclude that the level of hsp70 is controlled in primmorphs at both a transcriptional and translation level Effect of depletion of hsp70 from extracts on the activity of protein phosphatase(s) An immunodepletion study was performed as described in Experimental procedures Extracts were prepared from animals collected in September and incubated at or 17 °C Unexpectedly, enzyme activity at °C was 28 only 20% lower (18.1 ± 4.2 nmolỈmin)1Ỉmg)1) than that measured at 17 °C (22.7 ± 5.1 nmolỈmin)1Ỉmg)1) However, after incubation of the extracts for 60 with antibodies against hsp70 the activity of the protein phosphatase was reduced at °C to 5.3 ± 2.9 nmolỈ min)1Ỉmg)1, whereas the antibodies had no effect on activity at 17 °C (Fig 5) The adsorbed mAb-HSP70 Fig Effect of antibodies against hsp70 on the activity of the protein phosphatase in extracts from animals Extracts were prepared from animals, collected in summer and tested for protein phosphatase activity at or 17 °C, as described in Experimental procedures Where indicated the samples were pretreated either with mAb-HSP70 or with adsorbed mAb-HSP70 FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS W E G Muller et al ă preparation did not show a significant effect on the enzyme activities From these results we conclude that: (a) hsp70 proteins, which are supposedly functionally active, are present in sponge extracts together with the enzyme; and (b) hsp70 is required for the full enzyme activity during incubation at lower temperature Discussion OA is a secondary metabolite produced by free-living microalgae, primarily by Prorocentrum lima [16] Other dinoflagellates, e.g Gymnodinium sp., are also considered to be producers [17] Secondary metabolites are surely not without metabolic function for the producer or the host, because then they would have been eliminated during evolution They have, however, no direct role in the growth of the producing organism and are considered not to play a key role in the maintenance of cellular function but in defense [18] It remains unexplained, however, why some secondary metabolites, like OA, are synthesized not only by one taxon, but by a whole range of microorganisms These dinoflagellates are harbored in a series of hosts, e.g mussels [11] or sponges such as H okadaii [12], S domuncula [14] and Geodia cydonium (W E G Muller, University of Mainz, unpublished results), or, ă as shown here, in L baicalensis This latter finding is surprising, because L baicalensis is a freshwater sponge, in contrast to the others which are marine animals These findings suggest that OA has a crucial role in the maintenance of a symbiotic relationship between algae and host With S domuncula it has been shown that OA augments the concentration-dependent immune defense system against bacteria [14], and as described recently, kills symbiotic ⁄ parasitic annelids [19] The dinoflagellates present in L baicalensis are related to G sanguineum, a species that has been found worldwide, especially in coastal waters [20] We have shown that the dinoflagellates (G sanguineum) produce OA in L baicalensis For identification, we applied antibodies raised against OA, which have been previously qualified as specific for this secondary metabolite [14,19] In cross-sections though L baicalensis these antibodies reacted specifically with the dinoflagellates; their signals could be suppressed by adsorption with free OA Analytical measurements revealed that the concentration of OA in L baicalensis is  50 ngỈg)1 of tissue (70 nm), a level comparable with that found in other sponges [14] In addition, western blot analysis has shown that, like in extracts prepared from S domuncula and L baicalensis, OA exists in a covalent linkage with a protein of 14 kDa Cold stress defense in sponges This finding, first described in S domuncula [19], can be explained as a depot ⁄ storage form of the free OA It is not known whether OA is released from the sponge into the surrounding aqueous habitat Previous studies with S domuncula have shown that in this sponge OA accumulates in the epithelial layers of the aquiferous system within the animals, suggesting that OA is involved in defense against microbial invaders [21] Because L baicalensis ingests ⁄ feeds on microorganisms and plankton [7] it is very likely that OA is accumulated in the aquiferous canal system and acts as a protecting metabolite Based on existing data, it is increasingly evident that OA in the symbiotic bacteria also affects the primary cell metabolism of the host Previously, the main focus of research has been on the effect of OA on attacking or commensalic organisms, via inhibition of enzymes (protein phosphatases) [11] In view of the data, the corresponding cDNA for one of these enzymes (PP1) needed to be identified first cDNA coding for PP1 was completely isolated and the corresponding protein deduced This polypeptide contains all the characteristic domains of other enzymes in this group, e.g the characteristic Ser ⁄ Thr-specific protein phosphatase signature and the conserved metallophosphoesterase region Based on the high sequence similarity between the sponge protein phosphatase and mammalian enzymes an antibody against the latter could be used here Signals obtained by western blot analysis, 37 kDa, matched the expected size Enzyme activity in the prepared extract was  20 nmol inorganic phosphate released per and mg of protein in tissue extracts Inhibition studies with OA were performed which revealed that the toxin blocks the enzyme(s) significantly at OA concentrations > 300 nm; at 100 nm no inhibition was seen The sensitivity of the enzyme to OA was in the range published previously [11,14] In the plant Medicago sativa it could be shown that at low temperature hyperphosphorylation of proteins occurs and this is the result of inhibition of protein phosphatase(s) [22] It has been established that OA is a more sensitive inhibitor of PP2 than of PP1 [11] Therefore, we screened our EST database from L baicalensis, which comprises over 4000 sequences, and also performed extensive screening studies using degenerate primers, designed against the conserved regions within the PP2 nucleotide sequence, to identify PP2 transcripts in the cDNA library from L baicalensis However, these attempts were without success Therefore, we focused our studies at the protein and cellular level on PP1 only Nevertheless, the data presented here not exclude that PP2 is involved in thermoregulation in L baicalensis FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 29 Cold stress defense in sponges W E G Muller et al ă As outlined earlier, L baicalensis lives in a biotope with ambient temperatures between )0.5 °C (March) and 17 °C (August–September) To date, no data have been available that could help in understanding which protection system allows these animals to maintain high metabolic activity during these extreme situations The first series of experiments now demonstrates that the relative level of enzyme(s) (protein phosphatase) in the animals is the same in March or in September Also, the sensitivities of the enzymes towards OA are very similar Based on these results, we conclude that OA has an inhibitory effect in the animals during both seasons at concentrations > 300 nm In multicellular organisms one major protection system against temperature stress is provided by the heat shock proteins [23], with hsp70 being the most thoroughly studied example In contrast to the related constitutively expressed cognate hsc70, which changes its level only slightly upon differing stresses, hsp70 is strongly upregulated upon exposure to stress [24] For the marine sponges S domuncula or G cydonium we were able to show that at both the gene-expression level [25] and the protein level [26] expression of hsp70 increases strongly after temperature change and also after exposure to xenobiotics [27] The induction of the gene with respect to the stressors proceeds with the same kinetics in the sponge and in fish [28] Focusing on Lake Baikal sponges, hsp70 proved to be a suitable biomarker for xenobiotics and temperature stress [29] Few publications are available that describe the effect of OA on the expression of heat shock proteins [13,30] These authors demonstrated that injection of 300 ng of OA into 250 g rats resulted in notable expression of hsp70 ⁄ 72 after an incubation of 72 h More importantly, Joyeux et al [30] showed that OA treatment results in a potentiation of hsp72 mRNA expression In this study, we tested whether OA changes the level of hsp70 expression at both the gene expression level and the protein level For these studies, the primmorph system was used, which allowed the study of this toxin under controlled laboratory conditions In earlier studies, primmorphs have proved to be suitable for measuring these effects [31] A hsp70 cDNA probe was used to measure semiquantitatively the steady-state expression hsp70 in animals collected in March and September; there were no significant changes As outlined, the sponges contain OA in March and September However, if primmorphs, kept in the dark to suppress the photosynthetic activity of the algae, were cultivated at °C the level of hsp70 transcripts was very low compared with primmorphs cultivated at 17 °C, or animals in the biotope, irrespective of whether they were collected at )0.5 °C or at 30 12 °C If primmorphs were cultivated at °C together with 100 nm OA the hsp70 level reached values seen in animals or primmorphs incubated at 17 °C Interestingly, the level of hsp70 protein also followed this pattern From these results we conclude that OA causes, at both the gene- and the protein level, increased expression of this heat shock protein in primmorphs at °C Interestingly, expression of a-tubulin in primmorphs is low during incubation at °C, and is upregulated in the presence of OA or at higher temperatures This suggests that OA plays an inducer role for hsp70 and tubulin in primmorphs under cold stress conditions A set of immunodepletion experiments was performed Extracts from animals collected during the summer were prepared that, according to the abovementioned data, contained greater amounts of hsp70 protein They were subjected to protein phosphatase activity determination in the presence or absence of antibodies raised against hsp70 Hsp70 is known to bind to target protein(s) in the presence of ATP [23,32,33], which was therefore added If the extracts were assayed for protein phosphatase(s) activity it was found that at lower temperatures (4 °C) enzyme activity was close to that seen after incubation at 17 °C However, if antibodies were added to the mixture and incubation was performed at °C the activity was almost completely abolished At 17 °C the antibodies had no effect on the high expression level of hsp70 These results strongly suggest that native hsp70 binds at lower temperature to the enzyme(s) and restored the activity to values seen at higher temperature OA is a toxin present in dinoflagellates that coexist with marine animals ⁄ sponges and, as described here, in freshwater sponges Quantitative determinations showed that the level of this toxin in the freshwater sponge L baicalensis was as high as in the marine sponge S domuncula As reported here, in these freshwater animals OA is involved in processes that result in a high steady-state expression of the chaperon– hsp70 system In addition, the data suggest that the hsp system contributes to the cold thermotolerance seen in L baicalensis, because the secondary metabolite OA functions as an inducer for hsp70 Therefore, it can be postulated that OA contributes markedly to the survival strategies of these animals during unfavorable environmental conditions This view is outlined in Fig In animals, collected during winter and summer the level of hsp70 is high If extracts from these specimens were used for immunodepletion experiments the ‘activating’ effect of hsp70 on protein phosphatase activity could be demonstrated in extracts prepared from animals collected during the cold season This suggests that at low temperature ATP- FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS W E G Muller et al ă Cold stress defense in sponges Fig Proposed function of OA in L baicalensis The toxin OA is produced by the dinoflagellates from the taxon Gymnodinium In both winter and summer the dinoflagellates produce primary metabolites via their photosynthetic activity; the metabolite is taken up by the sponge cells Using the primmorph system it could be shown that the level of hsp70 is low at the lower temperature (in primmorphs at °C) However, the abundance of hsp70 increases in response to low concentrations of OA (100 nM) to levels which are seen in primmorphs incubated at a higher temperature (17 °C) or in animals collected during March and September It is postulated that a sufficiently high level of hsp70 is one prerequisite for the promotion of folding, transport and ⁄ or assembly of target proteins participating in the primary metabolism or in the composition of the fluid membranes during unfavorable environmental conditions (in nature, at )0.5 °C) Only at higher concentrations (> 300 nM) does OA inhibit protein phosphatase(s) dependent hsp70 molecules are required for the promotion of folding, transport and ⁄ or assembly of target proteins participating in the primary metabolism or, perhaps also, in the composition of the fluid membranes Energetically, the dinoflagellates supply their host with primary metabolites via their photosynthetic activity, glycerol being the major compound We recently identified that the dinoflagellates synthesize glycerol which is then taken up by cells of L baicalensis through the aquaporin channel (W E G Muller, ă manuscript submitted) In conclusion, our data suggest that OA causes induction of hsp70 at low ⁄ cold temperature stress; in turn, hsp70 contributes to the proper activity of the protein phosphatase at low temperature Experimental procedures Chemicals and enzymes Restriction enzymes, SNAP ‘Total RNA Isolation Kit’, Superscript II and reagents for RACE procedure were purchased from Invitrogen (Carlsbad, CA), TriplEx2 vector from BD (Palo Alto, CA), TRIzol Reagent from GibcoBRL (Grand Island, NY), Hybond-N+ nylon membrane from Amersham (Little Chalfont, UK), mAb against hsp70 (bovine; H 5147) was obtained from Sigma (St Louis, MO), polyclonal antibodies raised against PP1 were from Santa Cruz Biotechnology (Santa Cruz, CA), CDP from Roche (Mannheim; Germany), Technovit 8100 from Heraeus Kulzer (Wehrheim, Germany), Sephadex G-20 from Pharmacia (Uppsala, Sweden), Lake Baikal water was obtained from ‘Lake’ Comp (Irkutsk; Russia), and the protein phosphatase assay kit from Upstate Biotechnology (Lake Placid, NY) Okadaic acid was purchased from Alexis Biochemicals (Grunberg; Germany), the toxin was dissolved in ă dimethylsulfoxide Sponges, cells/primmorphs and cDNA libraries Specimens of L baicalensis (Porifera, Demospongiae, Haplosclerida) were collected in Lake Baikal (Russia) near the village Bolshiye Koty (51°58 N, 105°21¢E) from depths between and 12 m during September 2005 and March 2006 Primmorphs were prepared by immerging sponge tissue into natural Lake Baikal water, supplemented with 50 mm EDTA Lake Baikal water was used to guarantee a suitable mineral composition [34] After gentle squeezing and subsequent shaking for 30 at 16 °C on a rotatory shaker, the solution was discarded and new Baikal water ⁄ EDTA was added After 40 the supernatant was collected and filtered through a 40-lm mesh nylon net; shaking in Baikal water ⁄ EDTA and filtration were repeated once Single cells FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 31 Cold stress defense in sponges W E G Muller et al ă were harvested by centrifugation (500 g for min, Eppendorf centrifuge 5702 with rotor A-8-17) and washed once The cells of this pellet were resuspended in Baikal water, supplemented with lgỈmL)1 penicillin and 100 lgỈmL)1 streptomycin A cell suspension of 107 cells was added to mL (final volume) of medium in 10 mL flasks (Nuclon surface; #136196; Nunc Wiesbaden, Germany) Primmorphs were obtained from these single cells; they reached sizes of 3–7 mm after two days in the dark The primmorphs were incubated at or 17 °C; during the summer season the ambient water environment of L baicalensis can reach a temperature of up 22 °C [35] The cDNA library from L baicalensis was prepared in TriplEx2 vector Preparation of antibodies against OA Polyclonal antibodies against OA (pAb-OA) were raised in female rabbits (White New Zealand) as described previously [14] The antigen (OA) was covalently coupled via its C-terminus to the FID-33 oligopeptide (sequence: NH2-FIDAVWKCVTPFIDAVWKTKFICVTPFIDAVWK-COOH), using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Sigma) as described previously [14] This OA conjugate was injected at 4-week intervals into the animals; after three boosts serum was collected and the antibodies were prepared [36] The titer of the antibodies was determined to be : 2000 Where indicated the antibodies (pAb-OA; 0.3 mL of undiluted serum) were adsorbed with FID-33 coupled to OA (1 mg) for 30 at room temperature The studies with animals (antibody production) have been approved by the respective state authorities Histological analysis Tissue was fixed in paraformaldehyde, embedded in Technovit 8100 and sectioned [37] The 4-lm thick slices were incubated with pAb-OA (1 : 250 dilution) overnight Then the slides were treated with Cy3-conjugated goat anti-(rabbit IgG) for h Subsequently, the sections were inspected by immunofluorescence with an Olympus AHBT3 microscope, using an excitation light wavelength of 546 nm In addition, the slices were illuminated with light of a wavelength of 490 nm, which detects the autofluorescence of chlorophyll in the dinoflagellates In parallel, the slices were inspected directly using Nomarsky interference contrast optics In controls, the pAb-OA (10 lg) were preincubated with OA, coupled to the FID-33 oligopeptide (10 lg) Competitive ELISA The ELISA was performed similar to the procedure described earlier [14] OA, coupled to the FID-33 oligopeptide was linked to 96-well plates (Covalink-primary amine; 32 Nunc) [14] After three washing steps with NaCl ⁄ Pi (containing 0.05% Tween-20) the plates were blocked prior to use with 3% of fatty acids-free bovine serum albumin in NaCl ⁄ Pi Then 100 lL of pAb-OA were added at different dilutions to each well for 90 Subsequently, the immunocomplexes were visualized using secondary antibodies, coupled to horseradish peroxidase (1 : 1000; Sigma, Deisenhofen, Germany) under application of o-phenylenediamine as substrate The plates were read at 492 nm In the competitive ELISA procedure, OA was added at different concentrations (1 ngỈmL)1 to lgỈmL)1 NaCl ⁄ Pi) from a stock solution of mgỈmL)1, dissolved in methanol Within the range of 10 ngỈmL)1 to lgỈmL)1 the change of the absorbance was linear (logarithmic plot) For the determination in the tissue of the sponge, extracts were prepared from the tissue with 80% methanol The values for the absorbance were extrapolated using the calibration curve obtained with the free toxin Identification of OA-bound protein Extracts were prepared from tissue samples; they were homogenized in lysis buffer (1· Tris-buffered saline, pH 7.5, mm EDTA, 10 mm NaF, 0.1 lm aprotinin, mm sodium orthovanadate) Total cell extracts (10 lgỈlane)1) were subjected to electrophoresis in 10% polyacrylamide gels containing 0.1% SDS ⁄ PAGE as described previously [37] The gels were stained with Coomassie Brilliant Blue Subsequently, western-blotting experiments were performed after transfer of the proteins onto poly(vinylidene difluoride) membranes (Millipore-Roth) using pAb-OA (1 : 300 dilution) After incubation for h, the blots were incubated with goat anti-(rabbit IgG), peroxidase-coupled (1 : 5000 dilution; New England Biolabs) Detection of the immunocomplex was carried out using the BM Chemoluminescence Blotting Substrate kit Where indicated the pAb-OA had been adsorbed with OA coupled to the FID-33 oligopeptide Relative PP1 content (western blotting) The relative content of PP1 in the extracts was determined by western blotting First, the tissue extracts (see previous section) were size separated by electrophoresis (SDS ⁄ PAGE ⁄ 12% polyacrylamide); samples of 10 lg protein extract were loaded onto the gels In addition, the proteins were blot transferred and reacted with polyclonal antibodies raised against PP1 (PcAb-PP1; : 2000 dilution) After incubation for h, blots were incubated with peroxidase-coupled goat anti-(rabbit IgG); and the immunocomplexes were visualized using the BM Chemoluminescence Blotting Substrate kit In control experiments 10 lL of undiluted PcAb-PP1 was adsorbed with 10 lL of cell extract (1 mgỈmL)1; 30 min; °C) prior to the use onto the blots FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS W E G Muller et al ă Heat shock protein content (western blotting) The effect of OA on hsp70 expression was determined in primmorphs Extracts were prepared (see above) and subjected to western blot analysis using 10% polyacrylamide gels (SDS ⁄ PAGE) Ten micrograms of protein extract per lane were applied and after blot transfer reacted with antiHSP70 mAb (mAb-HSP70; : 5000 dilution) in Trisbuffered saline containing 0.1% milk powder protein and 0.05% Tween-20 for 1.5 h at room temperature, followed by alkaline phosphatase-conjugated anti-mouse IgG for h at room temperature The immunoblots were stained using 5-bromo-4-chloro-3-indolyl phosphate ⁄ nitroblue tetrazolium Phosphatase activity in the extract The relative protein in the extract phosphatase content was determined in crude extract following the described procedure [38] Cell extracts were prepared by homogenization using a 20 mm Hepes buffer (pH 7.5; containing 150 mm NaCl, 1.5 mm MgCl2, mm EDTA, mm phenylmethylsulfonyl fluoride, mgỈmL)1 aprotinin, mgỈmL)1 leupeptin, 10% v ⁄ v glycerol and mm Na-orthovanadate) After standing for 10 at °C the suspension was centrifuged for at 22 000 g (20 min, Eppendorf centrifuge 5702 with rotor A-8-17) to remove the spicules and the tissue particles The supernatant was collected and passed though a Sephadex G-20; the loading volume of the extract was 5–10% of the total volume of the column The column was eluted with the Hepes buffer Fractions were collected and protein phosphatase activity was determined The protein phosphatase activities (both PP1 and PP2A) were determined by applying the protein phosphatase assay kit and the synthetic phosphorylated substrate KRpTIRR [38] The cell extract was added and the reaction (incubation temperature at 17 °C) terminated after 20 by addition of Malachite Green solution Absorbance was determined at 660 nm to determine the inorganic phosphate release and correlated to the amount of protein (1 mg) Where indicated, assays were supplemented with 100 and 300 nm of OA Immunodepletion studies Extracts were prepared from animals collected during the summer season (September), using the buffer system described above Extracts (100–150 lL) containing 120 lg of protein and supplemented with 100 lm ATP were incubated in the absence or presence of 10 lL of hsp70 antibodies (mAb-HSP70) for 60 at room temperature Subsequently, the activity of the protein phosphatase(s) was determined at and 17 °C, respectively, applying the described procedure and the synthetic phosphorylated substrate In controls, 10 lL of undiluted mAb-HSP70 were Cold stress defense in sponges adsorbed with 10 lL of cell extract (1 mgỈmL)1; 30 min; °C) prior to the use onto the blots Transmission microscopy analysis For transmission microscopy analysis samples were cut into pieces (2 mm3), incubated in 0.1 m phosphate buffer (supplemented with 2.5% glutaraldehyde, 0.82% NaCl, pH 7.4) and washed in 0.1 m phosphate buffer (1.75% NaCl) at room temperature After treating the samples with 1.25% NaHCO3, 2% OsO4 and 1% NaCl, they were dehydrated with ethanol Dried samples were incubated with propylene oxide, fixed in propylene oxide ⁄ araldite (2 : 1), covered with pure araldite and hardened at 60 °C for two days prior to cutting to 60 nm ultrathin slices (Ultracut S; Leica, Wetzlar; Germany) The samples were transferred onto coated copper grids and analyzed with a Tecnai 12 device (FEI Electron Optics, Eindhoven; Netherlands) Isolation of cDNA encoding PP1 PCR was applied to identify the complete cDNA encoding for L baicalensis PP1 Degenerate primers were designed against the conserved Ser ⁄ Thr-specific protein phosphatase signature of mammalian PP1s, e.g the human PP1 which binds to OA (accession number 1JK7_A) [39] This segment is present in the human protein sequence between amino acids 123 and 130 PCR was carried out with the forward primer, 5¢-GGIAAC ⁄ TCAC ⁄ TGAA ⁄ GTGT ⁄ CGCIAGC ⁄ TAT-3¢ and the vector primer at an initial denaturation at 94 °C for min, followed by 30 amplification cycles at 94 °C for 30 s, 52 °C for 45 s, 75 °C for 1.5 min, and a final extension step at 75 °C for 10 Fragments were isolated and cloned into the pCRII-TOPO vector in E coli TOP10 cells (Invitrogen) Sequencing was performed with primers directed to the SP6 promoter and the T7 promoter The sequence was completed with insert-specific primers in combination with 5¢-RACE primer or with 3¢-RACE primer using the CapFishing Full-length cDNA Premix Kit (Seegene Inc., Rockville, MD, USA) The final sequence was confirmed by an additional PCR using primers directed against the nontranslated region of the cDNA, followed by sequencing The clone encoding the PP1 molecule from L baicalensis LBPP1 is 1359 nucleotides long, excluding the poly(A) tail EST sequence: heat shock protein In the cDNA ⁄ EST (expressed sequence tag) database from L baicalensis, which comprises 4000 sequences, more than 70 tags encoding heat shock proteins exist The dominant sequences code for hsp70 One sequence was selected which has a length of 416 nucleotides and was termed LUBAIHSP70 FEBS Journal 274 (2007) 23–36 ª 2006 The Authors Journal compilation ª 2006 FEBS 33 Cold stress defense in sponges W E G Muller et al ă Sequence analysis The deduced protein sequence of L baicalensis PP1 (PP1_LUBAI) was compared with those of the most closely related proteins, especially those from human, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana using the neighbor-joining method [40] The degree of support for internal branches was further assessed by bootstrapping [41] Accurate multiple protein sequence alignments were made using the software clustal w [42] Northern blotting RNA was isolated from primmorphs incubated for 24 h in the absence or presence of OA Samples were frozen, pulverized in liquid nitrogen and RNA was extracted using the TRIzol Reagent Total RNA (2 lg) was fractionated by electrophoresis, transferred to a Hybond-N+ nylon membrane, and hybridized overnight at 50 °C The hsp70 probe (LUBAIHSP70), labeled using the PCR-DIG-Probe-Synthesis Kit, was used As a control to assure that the same amount of RNA was loaded onto the gels, the housekeeping gene a-tubulin (LUBAITUB; AJ971711) from L baicalensis was used After washing, DIG-labeled nucleic acid was detected with anti-DIG Fab fragments conjugated to alkaline phosphatase, and visualized by chemiluminescence technique using CDP-star The screens were scanned with the GS-525 Molecular Imager (Bio-Rad; Hercules, CA) Analytical determinations Protein concentrations were determined as described previously [43] using BSA as standard The free level of OA was quantified in tissue from animals collected in March and September by coupled HPLC ⁄ MS as described [14,44] The concentrations were correlated with the weight of fresh tissue used for the analysis (n ¼ 5; mean values ± SD are given] Statistics For the statistical evaluation Student’s t-test was applied; the means and the standard errors (± SE) are given [45] Acknowledgements We thank Academician Dr Michael A Grachev (Limnological Institute, Irkutsk; Russia) very much for very important discussions This work was supported by grants from the Bundesministerium fur Bildung und ă Forschung Germany [projects: Joint German-Russian Laboratory for Biology of Sponges at the Limnological Institute RAS in Irkutsk; and Center of Excellence 34 BIOTECmarin], the European Commission, the Deutsche Forschungsgemeinschaft and the International Human Frontier Science Program; as well as by a grant from the Presidium of the Russian Academy of Science (no 25.5) and 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compilation ª 2006 FEBS ... marine animals ⁄ sponges and, as described here, in freshwater sponges Quantitative determinations showed that the level of this toxin in the freshwater sponge L baicalensis was as high as in the. .. of OA in L baicalensis The toxin OA is produced by the dinoflagellates from the taxon Gymnodinium In both winter and summer the dinoflagellates produce primary metabolites via their photosynthetic... domuncula and L baicalensis, OA exists in a covalent linkage with a protein of 14 kDa Cold stress defense in sponges This finding, first described in S domuncula [19], can be explained as a depot ⁄ storage