Axial (Apical-Basal) Expression of Pro-apoptotic and Pro-survival Genes in the Lake Baikal Demosponge Lubomirskia baicalensis

DNA AND CELL BIOLOGY Volume 25, Number 3, 2006 © Mary Ann Liebert, Inc. Pp. 152–164

Axial (Apical-Basal) Expression of Pro-apoptotic and Pro-survival Genes in the Lake Baikal Demosponge Lubomirskia baicalensis* MATTHIAS WIENS,1 SERGEY I. BELIKOV,2 OXANA V. KALUZHNAYA,1,2 HEINZ C. SCHRÖDER,1 BOJAN HAMER,3 SANJA PEROVIC-OTTSTADT,1 ALEXANDRA BOREJKO,1 BÉRENGÈRE LUTHRINGER,1 ISABEL M. MÜLLER,1 and WERNER E.G. MÜLLER1,2

ABSTRACT Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical–basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of the branches and comparably low at the top, the pro-survival gene follows an opposite gradient. Parallel with the tuned expression of these genes, the activities of the apoptosis-executing enzymes caspase-8 (IETDase activity) and caspase-3 (DEVDase activity) are lowest at the top of the branch and highest at their base. This characteristic expression/activity pattern of the genes/enzymes, which had been determined in a few specimens, collected from an unpolluted, natural site, appears reversed in specimens collected from an anthropogenically polluted site. These findings indicate the involvement of apoptotic proteins in the axis formation (branches) in L. baicalensis.

INTRODUCTION

B

Y APPLICATION OF MOLECULAR BIOLOGICAL TECHNIQUES it be-

came overt that sponges (phylum Porifera) are provided with structural and regulatory molecules very similar to those found in other metazoans (Pfeifer et al., 1993); consequently, the monophyly of animals could be established (Müller, 1995). Especially, the class of Demospongiae, siliceous sponges, has been extensively studied, taking the marine species Suberites domuncula as the model species. The genes were functionally studied by immunohistological methods and after raising antibodies against recombinant sponge proteins it was clear that they contribute to cell–cell/cell–matrix interactions (Müller, 1997), to cell differentiation or to immune response (Müller et

al., 1999) in a homologous—or at least very related—way to the corresponding molecules of higher metazoans. Finally, in situ hybridization analysis could be successfully applied to study differentiation of cell lineages in the evolutionary oldest metazoan phylum (Porifera) (Perovic et al., 2003), for example, of sclerocytes during the formation of the skeletal elements of sponges (Schröder et al., 2004). The synthesis of these data made it possible to formulate the underlying genomic systems that regulate the formation of a Bauplan (body plan), and are common to sponges and other metazoan taxa (Müller et al., 2004; Müller, 2005). Furthermore, surprising observations indicated that during embryogenesis sponge larvae undergo gastrulation (Leys, 2004). Critical for the sculpting of an organism during development and later on to ensure tissue homeostasis, apoptosis (the mor-

1Institut

für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität Mainz, Mainz, Germany. Institute of the Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia. 3Ruder Boskovic Institute, Center for Marine Research, Rovinj, Croatia. *Note: The following sequences from Lubomirskia baicalensis have been deposited (EMBL/GenBank): cDNA for apoptosis-linked gene-2 protein (LBALG-2l) under AM040444; BAK-2 (LBBAK-2l) AM040445; MA-3 sequence (LBMA-3) AM040447; the Bcl-2 polypeptide (LBBCL2a) AM040446; sorcin from Suberites domuncula (SORC_SUBDO) under AM040448. 2Limnological

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PRO-APOPTOTIC AND PRO-SURVIVAL GENES IN LUBOMIRSKIA BAICALENSIS phological manifestation of programmed cell death) allows the elimination of redundant, damaged, or even dangerous cells (Kerr et al., 1972). It had long been postulated that apoptotic processes were conserved during evolution (Vaux and Strasser, 1996). However, only after the first identification of apoptotic genes in the demosponges Geodia cydonium and/or S. domuncula it could be demonstrated that also in sponges the core elements of the apoptotic machinery exist (Wiens et al., 2000a). Transfection studies confirmed the apoptotic function of the sponge molecules in the heterologous system of mammalian cell lines (Wiens et al., 2001). Since the initial studies by Vogt (1842), it became increasingly obvious that initiation and execution of “apoptotic” pathways are crucial for the normal development of embryos or embryonic tissue as well as for the maintenance of the tissue organization in adults (see Hsu and Hsueh, 2000). In the present study we investigated whether the expression of apoptotic molecules also contributes in sponges to the formation of the Bauplan (body plan). The Lake Baikal freshwater demosponge Lubomirskia baicalensis displays a complex Bauplan with a pronounced axis (Kaluzhnaya et al., 2005a, 2005b). Hence, this species seems to be a suitable biological model to study the role of apoptotic molecules in the maintenance of cell/tissue homeostasis. Specimens of L. baicalensis can grow higher than 1 m; the arborescently growing animals show a pronounced branching architecture with highly ordered arrangement of spicules. During growth, new, serially arranged modules are added at the tips of the branches; such architecture was earlier described as radiate accretive pattern (Kaandorp, 1994). Apoptosis is controlled by pro-apoptotic and pro-survival molecules (Liston et al., 2003). The function of the executing enzymes, the caspases, is governed by members of the Bcl-2 family (reviewed in Adams and Cory, 1998; Hsu and Hsueh, 2000). To understand their roles in different regions in the sponge body along the apical-basal axis of L. baicalensis, the cDNAs of three putative pro-apoptotic molecules, the apoptosis-linked gene-2 protein (ALG-2), the Bcl-2-antagonist/killer (BAK) polypeptide, the MA-3/TIS apoptotic protein, and one pro-survival molecule, the apoptosis regulator Bcl-2 have been isolated and their expression levels were determined. We selected these apoptotic molecules in light of earlier studies with the demosponge S. domuncula, which revealed that ALG-2 is a highly conserved molecule that is expressed in tissue grafts undergoing apoptosis (Aubry et al., 2002; Wiens et al., 2004). BAK is a major pro-apoptotic member of the Bcl-2 family and characterized by the lack of the BH4 domain (Tsujimoto, 1998; Scorrano and Korsmeyer, 2003) and MA-3/TIS is known to be expressed in vertebrate and insect tissues after induction of apoptosis (Shibahara et al., 1995) and represents a topoisomerase suppressor inhibited gene (Stalberg et al., 2001). MA-3 is also in S. domuncula, one of the dominant apoptotic molecules (Wagner et al., 1998). Finally, Bcl-2, the best characterized member of apoptosis regulators, is the founder of the Bcl-2 family (Tsujimoto, 1998). Bcl-2 homologues have been among the first poriferan molecules that have been used for transfection of mammalian cells; they conferred resistance against apoptotic stimuli in those cells (Wiens et al., 2001). In an approach to understand the expression patterns of apoptotic genes during cell death in L. baicalensis, a few specimens

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from normal, unpolluted, and polluted sites were taken and the expressions of the genes were determined along the axes of the branches. It had been reported earlier that marine sponges (S. domuncula) undergo apoptosis in response to environmental toxins (Batel et al., 1993; Wagner et al., 1998). One site near the paper mill at Baikalsk at Lake Baikal is highly exposed to industrial anthropogenic pollution, while the rest of the lake is unaffected (Efremova et al., 2002). The Baikalsk Pulp and Paper Plant (BPPP) has earlier been described as a potential source of pollution of the lake (see Belt, 1992; Zuev et al., 1996; Schröder et al., 2005). Examination of the influence of the waste water from the final refinement and aeration pond of BPPP revealed a concentration-dependent increase in Hsp70 expression and an induction of DNA damage in sponges (Efremova et al., 2002). Therefore, Baikalian sponges seem to be suitable bioindicators to monitor water quality and ecosystem health of the lake around the paper plant, especially in comparison with unpolluted site(s) (Schröder et al., 2005). This polluted area is loaded with microorganisms and heavy metals (Efremova et al., 2002) which cause an increase in reactive oxygen species. H2O2 is the major reactive oxygen species, which is formed as a byproduct of (ab)normal cellular function by superoxide dismutase and monoamine oxidase. Excessively produced H2O2 is normally broken down by glutathione (GSH) peroxidase and catalase. Under pathophysiological stress, excessive H2O2 provokes oxidative stress and causes apoptosis through activation of caspases (Halliwell, 1992). Therefore, the activities of caspases, mainly of caspase8 and caspase-3, as well as of glutathione (GSH) peroxidase were determined in tissue from sponge specimens collected in natural, unaffected as well as polluted areas. The results were compared to the steady-state level of Bcl-2 genes. It was found that the level of expression of the pro-apoptotic genes in sponges growing in unaffected, natural sites increases from the top to the base, while it proceeds in the opposite direction in animals from anthropogenically polluted sites. A considerable expression of the pro-survival gene Bcl-2 is seen in animals from the nonpolluted site (with a gradient from top to base), while almost no transcripts along the axis can be detected in animals from the polluted area. The gene expression studies are supported further by our data on caspase activity that showed an increased activity from the top towards the base of the branches; however, in some specimens from the polluted region caspase activity almost doubled. Likewise, the expression level of the enzyme involved in the detoxification of free radicals, the glutathione peroxidase, is elevated along the axis of branches of L. baicalensis from polluted sites.

MATERIALS AND METHODS Chemicals, materials, and enzymes The restriction enzymes, SNAP “Total RNA Isolation Kit,” and reagents for the RACE procedure were purchased from Invitrogen (Carlsbad, CA); the TriplEx2 vector from BD (Palo Alto, CA); the TRIzol Reagent from GibcoBRL (Grand Island, NY); the Hybond-N⫹ nylon membrane from Amersham (Little Chalfont, Buckinghamshire; UK); the PCR-DIG-Probe-Synthesis Kit and CDP from (Roche, Mannheim; Germany); apro-

154 tinin, sodium orthovanadate, Nonidet-P40, NaF, NADPH, GSH reductase, caspase assay kit, AC-DEVD-pNA, and Ac-IETDpNA from Sigma (St. Louis, MO).

Sponges and collection sites Live specimens of Lubomirskia baicalensis (Porifera, Demospongiae, Haplosclerida) were collected in Lake Baikal from an unaffected, natural site (near the village Listvianka [natLi]) and from the anthropogenically polluted site (paper mill at Baikalsk; Efremova et al., 2002 [antBai]; Fig. 1); the distance between Listvianka and Baikalsk is 100 km. In contrast to the natLi site, the antBai region is polluted with microorganisms, heavy metals, organochlorines and pentachlorophenol, which are released by pulp bleaching. Where indicated, protein and RNA were isolated either from tissue of the top region of the branches (the top module), the middle part, or from the base (see Efremova et al., 2002).

Tissue extract Tissue samples of 10 g were homogenized in lysis-buffer (1⫻ TBS [Tris-buffered saline], pH 7.5, 1 mM EDTA, 1% Nonidet-P40, 10 mM NaF, 0.1 ␮M aprotinin, 1 mM sodium orthovanadate). After centrifugation (10,000 ⫻ g, 5 min) the supernatants were collected and assayed for enzyme activity as well as for the determination of protein content. Five parallel experiments with five different individuals each from the two sites (natLi and antBai) were performed; the animals had been collected within an area of 4 km2 at each of the sites.

FIG. 1. Map of Lake Baikal showing the unaffected, natural site (near the village Listvianka; [natLi]) and the anthropogenically polluted site (paper mill at Baikalsk [antBai]). The borders between Russia (Siberia; with the two regions around Lake Baikal [Irkutsk Oblast and Burjatija]) and Mongolia are indicated.

WIENS ET AL.

Determination of enzyme activity Glutathione (GSH) peroxidase activity was determined as described (Cheng et al., 1997). Proteins were extracted from homogenized tissue and subjected to the coupled NADPH oxidation enzyme reaction with hydrogen peroxide as substrate. One enzyme unit was defined as 1 nmol of reduced glutathione (GSH) oxidized per min. The quantification of the caspase-8 and caspase-3 activities was performed with the caspase assay kit, using the substrates AC-DEVD-pNA (caspase-3 colorimetric substrate) and Ac-IETD-pNA (caspase-8). Activity is given in nmoles of colorimetric substrate cleaved mg⫺1 (tissue extract) ⫻ h⫺1; the incubation period was 30 min for these experiments. Five parallel experiments from five different individuals each, collected within an area of 4 km2 of the two sites, natLi and antBai, had been analyzed; the mean values (⫾SD) and also the coefficient of variation (CV) are given.

Identification of the L. baicalensis pro-apoptotic genes ALG-2l, BAK-2 and MA-3 The cDNA library from L. baicalensis was prepared in TriplEx2 vector. The technique of polymerase chain reaction (PCR) was applied to identify the respective cDNAs. Degenerate primers were designed against the apoptosislinked gene-2 protein (ALG-2l) taking advantage of the conserved regions in ALG-2 from human to sponges (Wiens et al., 2004). The forward primer 5⬘-ATA/C/T TTC/T CAA/G AAA/G GTA/C/G/T GAC/T AAA/G GAC/T-3⬘, designed against the very conserved first calcium-binding EF hand motif present in ALG-2 polypeptides was successful. This motif is found in the human ALG-2 (accession number NP_037364) between amino acid [aa]31 to aa38 (Aubry et al., 2002). PCR was carried out with this primer and a vector primer at an initial denaturation at 95°C for 5 min, followed by 35 amplification cycles at 95°C for 30 sec, 54°C for 45 sec, 74°C for 1.5 min, and a final extension step at 74°C for 10 min. Fragments of the expected size were obtained and cloned into the TOPO TAII vector in Escherichia coli TOP10 cells. Sequencing was performed with primers directed to the SP6 promoter (5⬘-ATTTAGGTGACACTATAG-3⬘) and the T7 promoter (5⬘-TAATACGACTCACTATAGGG-3⬘). The sequence was completed with insert-specific primers in combination with 5⬘-RACE primer (5⬘-GTCTACCAGGCATTCGCTTCAT-3⬘) or with 3⬘RACE primer (5⬘-CTGTGAATGCTGGACTACGAT-3⬘) using the CapFishing Full-length cDNA Premix Kit. 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 L. baicalensis LBALG-2l is 720 nucleotides [nts] long (excluding the poly(A) tail). Similarly, the sponge cDNA for BAK-2 (LBBAK-2l) was identified by PCR using the degenerate forward primer (5⬘-GCI CTI CTI GGI TAC/T AGA/G TAC/T CGI CT-3⬘) against the BH1 motif of BAK-2 sequences; in human BAK2 (Q13014; Kiefer et al., 1995) BH1 is located between aa117 to aa136. After completion by “racing” (see above), the full-length sequence of 790 nts was obtained. The deduced protein of the MA-3 gene comprises in the second MA-3 domain a highly conserved region, which is in another poriferan S. domuncula MA-3 (accession number

PRO-APOPTOTIC AND PRO-SURVIVAL GENES IN LUBOMIRSKIA BAICALENSIS CAA75614) between aa348 and aa358. In combination with a vector specific primer the degenerate forward primer 5⬘-TTA/G GAA/G GTI CAC/T TTC/T CAC/T CAC/T GAA/G CT-3⬘ amplified a MA-3 fragment during PCR; the complete sequence was identified (1935 nts), and finally cloned, as described above.

Identification of the L. baicalensis pro-survival cDNA Bcl-2 This cDNA was also obtained from the library by PCR. The forward primer was designed against the conserved BH1 domain found in the vertebrate Bcl-2 genes; in Danio rerio (AAW21970; Langenau et al., 2005) this stretch is located between aa129 and aa136. Using the forward primer 5⬘-GAC/T GGI ATA/C/T AAC/T TGG GGI AGA/G AT-3⬘ a fragment of the cDNA was obtained. The complete sequence of LBBCL-2a was obtained after application of the RACE technique; the size of this cDNA is 717 nts.

Identification of the S. domuncula sorcin The S. domuncula sorcin cDNA sequence was cloned by PCR, during screening of the S. domuncula cDNA library for the presence of ALG-2l (Wiens et al., 2004). The sequence has a length of 822 nts and was termed SDSORC.

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RNA preparation and Northern blot analysis RNA was extracted from liquid nitrogen pulverized tissue with TRIzol Reagent as described (Grebenjuk et al., 2002), and then repurified using the SNAP Total RNA Isolation Kit. Five micrograms of total RNA each was electrophoresed and blotted onto a Hybond-N⫹ nylon membrane (Amersham). Hybridization was performed with the following probes: for the apoptosis-linked gene-2 protein (LBALG-2l; nt108 to nt485), BAK-2 (LBBAK-2l; nt 53 to nt 549), MA-3 sequence (LBMA-3; nt423 to nt873) and the Bcl-2 polypeptide (LBBCL-2a; nt161 to nt655). As a constitutively expressed control the housekeeping gene ␣-tubulin (LBTUB; AJ971711; nt 274 to nt 738) was used. The probes were labeled with the PCR-DIG-Probe-Synthesis Kit according to the “Instruction Manual.” After washing, DIGlabeled nucleic acid was detected with anti-DIG Fab fragments (conjugated to alkaline phosphatase; dilution of 1:10,000) and visualized by chemiluminescence technique using CDP according to the instructions of the manufacturer. The screens were scanned with the GS-525 Molecular Imager (Bio-Rad; Hercules, CA).

Analytical technique Protein concentrations were determined as described (Lowry et al., 1951) using bovine serum albumin as standard.

Sequence analyses The sequences were analyzed with computer programs BLAST (2005; http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and FASTA (2005; http://www.ebi.ac.uk/fasta33/). Multiple alignments were performed with CLUSTAL W Ver. 1.6 (Thompson et al., 1994). Phylogenetic trees were constructed on the base of aa sequence alignments by neighbor-joining, as implemented in the “Neighbor” program from the PHYLIP package (Felsenstein, 1993). The distance matrices were calculated using the Dayhoff PAM matrix model as described (Dayhoff et al., 1978). The degree of support for internal branches was further assessed by bootstrapping (Felsenstein, 1993). The graphic presentations were prepared with GeneDoc (Nicholas and Nicholas, 1997).

Statistics For the statistical evaluation a Student’s t-test was applied; the means and the standard deviations (SD) are given (Sachs, 1984). The coefficient of variation (CV) [standard deviation/mean] (Lentner, 1982) has been calculated as well.

RESULTS Determination of GSH peroxidase activity The GSH peroxidase activity was determined along the apical-basal (top to base) axis of branches from L. baicalensis. In

FIG. 2. Glutathione (GSH) peroxidase activity and caspase activities in different regions of branches of L. baicalensis, top–middle–base. Tissue from animals collected in natural habitat (natLi site; open bars) and at the polluted site (antBai; closed bars) was used to study enzyme activities. Five parallel experiments from five individuals each were performed; the mean values (⫾SD) are given. (A) GSH peroxidase activity, (B, C) caspase activities. The activities were determined in assays with the caspase substrates for caspase-3 (DEVDase activity; B) and caspase-8 (IETDase activity; C), as described under Materials and Methods.

156 sponges from the natLi site the lowest activity has been determined in tissue from the top modules (approximately 4 units/mg), slightly increasing to 5 units/mg in extracts from the base (Fig. 2A). Animals from the polluted antBai site displayed a significantly higher level of enzyme activity in all three areas of the branches; in particular, the GSH peroxidase activity in the top modules of specimens collected at the antBai site is about twofold higher than in specimens from the natLi site.

Caspase activities The activities for caspase-3 (DEVDase activity) and caspase8 (IETDase activity) were determined in tissue extracts from animals collected at the natLi and antBai sites, along the apical–basal axis of their branches. Highest activities were measured for the

WIENS ET AL. enzymes in extracts obtained from the basal modules. The activities in this region in natLi animals were 5.8 nmol ⫻ mg⫺1 ⫻ h⫺1 (caspase-3) and 6.0 nmol ⫻ mg⫺1 ⫻ h⫺1 (caspase-8); compared to 4.1 nmol ⫻ mg⫺1 ⫻ h⫺1 (caspase-3) and 3.0 nmol ⫻ mg⫺1 ⫻ h⫺1 (caspase-8) at the top (Fig. 2B and C). However, both enzyme activities were drastically higher in specimens collected from antBai. Basal modules of those animals revealed a 2.9-fold increase of caspase-3 activity (caspase-8: 2.7-fold), whereas at the top the increase was determined to be 2.8-fold (caspase-3) and 3.1-fold (caspase-8). A strong increase could also be shown for the middle parts of antBai animals. These data show that in specimens from the anthropogenically polluted site (antBai) the levels of caspase activities are significantly (P ⬍ 0.001) higher than at the natural control site (natLi). As an example, the caspase-3 (DEVDase activity)

FIG. 3. The L. baicalensis apoptosis-linked gene-2 protein (ALG-2l_LUBAI). (A) This deduced protein (ALG-2l_LUBAI) was aligned with the related S. domuncula sequence (ALG-2l_SUBDO, AJ632071; Wiens et al., 2004) and the human ALG-2 (ALG2_HUMAN, NP_037364; Aubry et al., 2002). The typical five putative EF-hands are indicated. Residues conserved (identical or similar with respect to their physicochemical properties) in all sequences are shown in white on black. (B) These three proteins were compared with the related molecules from D. melanogaster, predicted CG40410-PA.3 protein (CG40410_DROME, EAA46044), and from C. elegans, calmodulin protein cal-1 (CAL1_CAEEL, AAB65364) and calmodulin protein cal-2 (CAL2_CAEEL, NP_495906). Furthermore, the sorcin proteins from S. domuncula (SORC_SUBDO, AM040448) and human ([sorcin-a, AAH11025; and sorcin-b, NP_944490]; SORCa_HUMAN and SORCb_HUMAN) together with the predicted protein SCYGR from Saccharomyces cerevisiae (SCYGR_YEAST, CAA97059) and the putative calcium binding protein from Arabidopsis thaliana (PCBP_ARATH, AAD15600) were included. The tree was constructed and rooted with the plant sequence as outgroup. Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.

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FIG. 4. The L. baicalensis BAK-2 putative Bcl-2 homologous antagonist/killer 2 (BAK-2_LUBAI). (A) Alignment of the sponge molecule with the human Bcl-2 homologous antagonist/killer 2 protein (BAK2_HUMAN, Q13014; Kiefer et al. 1995). The boundaries of the BH1 and BH3 regions as well as the predicted transmembrane region (TM) are marked. (B) These sequences were aligned with the CG1674-PA isoform A molecule from D. melanogaster (CG1674_DROME, NP_726554.1), the serine/cysteine protease inhibitor from C. elegans (SerCysINH_CAEEL, AAS13528.1), the S. cerevisiae predicted molecule YGR258C (YGR258C_YEAST, AAT92876.1) and the plant [A. thaliana] hypothetical protein AT3G4 (AT3G4_ARATH, AAU44484.1). Then the tree was constructed and rooted with the plant sequence.

level in the top module of the natLi animals is 4.1 ⫾ 1.0 nmol ⫻ mg⫺1 ⫻ h⫺1 (CV value: 0.24) and 11.8 ⫾ 0.8 nmol ⫻ mg⫺1 ⫻ h⫺1 (CV: 0.07) in antBai specimens. In the other regions of the specimens, collected from the different sites (natLi versus antBai), the differences were likewise significant. In order to support the observation that the apoptotic reaction(s) vary within the body regionally and also site dependently, subsequent gene expression studies were performed. First the genes were identified and then their expression levels were determined; see below.

The pro-apoptotic gene ALG-2l The cDNA encoding the L. baicalensis apoptosis-linked gene-2 protein (LBALG-2l) was obtained from a cDNA library using a degenerate primer designed against the EF-hand motif. The open reading frame (ORF), between nt43–45 and nt593–595(stop), codes for a 183 aa-long polypeptide (ALG2l_LUBAI) with a predicted size of 21,348 (Fig. 3A). Like the related mammalian sequences the sponge protein also comprises a penta-EF-hand organization (Satoh et al., 2002), which is involved in Ca2⫹ binding (Jung et al., 2001). These putative EF-hands motifs [motif-1: aa18 to aa46; motif-2: aa48 to aa83; motif-3: aa85 to aa113; motif-4: aa120 to aa149 and motif-5: aa151 to aa183 (Fig. 3A)] are located in the L. baicalensis ALG-2-like protein at positions corresponding to those of other members

of this protein family (Aubry et al., 2002). Furthermore, prediction of secondary structures according to Rao and Argos (1986) revealed eight helices located at the borders of the EFhands (not shown). A phylogenetic analysis (Fig. 3B) with ALG-2l_LUBAI and the hitherto known only other diploblastic sequence from S. domuncula (ALG-2l_SUBDO, AJ632071; Wiens et al., 2004) was performed including the closest related sequences from C. elegans (two calmodulins, AAB65364 and NP_495906), D. melanogaster (predicted CG40410-PA.3 protein, EAA46044), and the human ALG-2 (NP_037364). It should be stressed here, that the sponge sequences share the highest similarity to the mammalian ALG-2 molecule with an “Expect value [E]” [Coligan et al., 2000] ⬍ e⫺65. A lower similarity score exists to the protostomian sequence (E-value ⬍ e⫺55). Other sequences with a similar penta-EF-hand organization belong to the sorcin protein family (Celio, 1996). Therefore, two human sorcins and sorcin from S. domuncula were included in the alignment. The resulting tree, which had been rooted with the closest plant sequence (Arabidopsis thaliana) shows strikingly that the poriferan and the metazoan ALG-2 molecules cluster together and share a common ancestry with the sorcins (Fig. 3B). In this tree the C. elegans molecules cluster together with a Saccharomyces cerevisiae protein (SCYGR, CAA97059), very likely due to the loss of the ALG-2 related protein during the rapid evolutionary history of C. elegans (Ruvkun and Hobert, 1998).

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Pro-apoptotic gene BAK-2 The complete L. baicalensis cDNA for BAK-2 (LBBAK-2l) is 790 nts long and has its ORF between nt28–30 to nt760–762(stop). The size of the predicted 244 aa containing polypeptide is 27,373 (Fig. 4A). Mammalian BAK-2 sequences display two to three BH domains with high structural but rather low sequence similarity to each other (Muchmore et al., 1996; Adams and Cory, 1998). In the L. baicalensis BAK-2 protein, BAK-2_LUBAI, two BH motifs are located as follows: BH3 domain at aa72 to aa83 and BH1 domain at aa125 to aa140 (Fig. 4A), whereas the BH2 motif is absent (accession number PS01258; Prosite, 2005; http://ca.expasy.org/prosite/). This structure is characteristic for pro-apoptotic Bcl-2 homologous antagonist/killer 2 proteins (Adams and Cory,

WIENS ET AL. 1998). One transmembrane region could be predicted within the sponge sequence to span the segment aa148 to aa166 (Rao and Argos, 1986). The phylogenetic tree (Fig. 4B) also reflects the high sequence similarity of the L. baicalensis BAK-2 to the mammalian BAK-2 proteins, with the human sequence as an example (Fig. 4B). More distantly related are the sequences from D. melanogaster, C. elegans and yeast.

Pro-apoptotic gene MA-3 MA-3 proteins are characterized by the presence of two MA-3 domains (accession number 00544; SMART, 2004; http://smart.emb/-heidelberg.de). These stretches have been

FIG. 5. The L. baicalensis MA-3. The L. baicalensis deduced protein (MA-3_LUBAI) was aligned with the S. domuncula MA3 (MA-3_SUBDO, CAA75614), the mouse programmed cell death 4 (Pdcd4)/MA-3 protein (PDCD4_MOUSE, AAH55739), the predicted CG10990-PA protein from D. melanogaster (CG10990_DROME, NP_572918) and the MA3 domain-containing protein from Arabidopsis thaliana (MA-3_ARATH, NP_568968). The plant sequence, which is 702 aa long, has been truncated after aa 455. The borders of the two MA-3 domains (MA-3 dom) are marked.

PRO-APOPTOTIC AND PRO-SURVIVAL GENES IN LUBOMIRSKIA BAICALENSIS used to identify the cDNA by PCR technique and resulted in the isolation of the L. baicalensis MA-3 sequence (LBMA-3), which has a length of 1935 nts. The ORF from nt379–381 to nt1765–1767(stop) codes for 462 aa, which corresponds to a calculated size of 51,622 Da. In the deduced L. baicalensis sequence (MA-3_LUBAI) the two MA-3 domains are located between aa157 to aa216 and aa320 to aa378 (Fig. 5). The highest similarity of the L. baicalensis protein exists to the sponge S. domuncula MA-3 protein with an E-value of ⬍ e⫺127. Weaker similarities are found to vertebrate sequences, for example, the mouse programmed cell death 4/MA-3 protein (AAH55739; E-value ⬍ e⫺90), a predicted CG10990-PA protein from D. melanogaster (NP_572918; E-value ⬍ e⫺78) and the MA3 domain-containing protein from Arabidopsis thaliana (NP_568968; E-value ⬍ e⫺41). Until now no MA-3-related sequence has been found in C. elegans.

Pro-survival gene Bcl-2a The proteins of the Bcl-2 superfamily comprise up to four BH domains (Adams and Cory, 1998). The pro-survival Bcl-2 molecules, with the founder of this family as a prototype, Bcl-2, possess the BH domains in the following order: BH4, BH3, BH1, and BH2 (Muchmore et al., 1996; Inohara et al., 1998). The sequence of the putative Bcl-2a homologue from L. baicalensis (LBBCL-2a) has a length of 717 nts and comprises an ORF from nt134–136 to nt687–689(stop); its encoded genes was named BCL-2a_LUBAI. Considering the conserved aa moieties, domain search and secondary structure analysis (Rao and Argos, 1986), the puta-

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tive BH domains could be narrowed down to: BH4 aa2 to aa22; BH3 aa26 to aa44; BH1 aa80 to aa95, and BH2 aa123 to aa139 (Fig. 6A). Interesting is the result of the phylogenetic analysis. While the sponge molecule shares highest sequence similarity to the vertebrate (E-value of 7e⫺13 for fish and 3e⫺11 for human sequence) and insect Bcl-2 (D. melanogaster protein E-value of 4e⫺5), the homology to the C. elegans Bcl-2 protein CED9 is significantly lower: the poriferan and the C. elegans proteins share only 9% identical and 25% similar aa (considering the physicochemical properties). Figure 6B displays the phylogenetic tree, rooted with the distantly related plant hypothetical molecule ATT22A6.

Expression studies With these cDNAs in hand it was possible to perform Northern blot studies, (1) in order to confirm that the full sequences had been obtained, and (2) to estimate the transcript abundance within the different regions of the branches (Fig. 7, left panel). By this approach, the size of the gene encoding the apoptosislinked gene-2 protein (LBALG-2l) was 0.7 kb, BAK-2 (LBBAK2l) was 0.9 kb, MA-3 (LBMA-3) was 2.0 kb, and Bcl-2 (LBBCL-2a) was 0.8 kb. The transcript sizes observed match the lengths of the cDNAs obtained. As a control for the expression studies the ␣-tubulin (LBTUB; AJ971711) probe from L. baicalensis was used; its size is 1.6 kb (Fig. 7, right panel). The expression of the genes involved in apoptotic processes was determined by Northern blotting, using RNA which had been extracted from the top, the middle part and the base of

FIG. 6. The deduced sponge Bcl-2–associated protein (BCL-2a_LUBAI) was aligned with the Bcl-2 protein from Danio rerio (BCL-2_DANIO, AAW21970; Langenau et al., 2005) (A). The conserved BH1 to BH4 domains were predicted as described under Results. (B) These two sequences were aligned with the human Bcl-x beta (BCL-xb_HUMAN, AAB17354), the Bcl-2 family member protein Drob-1 (Drosophila ortholog of the Bcl-2 family) from D. melanogaster (BCL-2_DROME, AAF26841), the apoptosis regulator ced-9 (cell death protein 9) from C. elegans (CED9_CAEEL, P41957) as well as the ATT22A6 hypothetical protein from A. thaliana (ATT22A6_ARATH, CAB45055). The tree was constructed and the plant sequence was used as outgroup.

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WIENS ET AL. survival gene LBBCL-2a is very low in all three different regions of the branches (Fig. 7; right panel). In order to rule out potential artifacts during the hybridization procedure, RNA was obtained from the different regions of a branch (top–middle–base) of one specimen each, collected at the natural, unpolluted site (natLi) and from another one from the anthropogenically polluted site (antBai) (Fig. 8A). RNA was

FIG. 7. Expression of both pro-apoptotic and pro-survival genes along the branches of L. baicalensis, collected at the unaffected, natural site (natLi) and at the anthropogenically polluted site (antBai). A schematic representation of the serial modular growth pattern along the apical-basal axis is given (left panel). In the right panel, Northern blots demonstrate the expression pattern of the five sponge genes from the top to the base of a branch. Following RNA isolation the same amounts were loaded onto gels and subsequently size separated. After blot transfer the membranes were hybridized with the following probes: pro-apoptotic apoptosis-linked gene-2 protein (LBALG-2l), BAK-2 (LBBAK-2l), and MA-3 (LBMA-3), as well as the pro-survival Bcl-2 (LBBCL-2a). The housekeeping gene, ␣-tubulin, was used as a control to show that the same amounts of RNA were loaded onto the gels. The relative degree of expression, for both the series of the natural, unpolluted site (natLi) and of the anthropogenically, polluted site (antBai) are given; they are correlated to the expression measured in the respective top module (set to onefold).

branches from L. baicalensis, collected either from the unaffected, natural site (natLi) or from the anthropogenically polluted site (antBai). The signals obtained after blotting and hybridization with the LBALG-2l, LBBAK-2l, LBMA-3, and LBBCL-2a probes were scanned to estimate (semi-)quantitatively the levels of the transcripts. In a control series with the housekeeping gene ␣-tubulin (LBTUB) (Eisenberg and Levanon, 2003), it was established that the same amounts of RNA were loaded on the gels (Fig. 7, right panel). At the natLi sites the expression of the pro-apoptotic genes, the apoptosis-linked gene-2 protein (LBALG-2l), BAK-2 (LBBAK-2l), and the MA-3 sequence (LBMA-3) was considerably lower in the top region of the branches than in the basal regions. Just the opposite gradient is seen for the expression of Bcl-2 polypeptide (LBBCL-2a); here, the highest level has been measured in the top region (Fig. 7; left panel). Very different is the expression of these genes in branches from the polluted site antBai. Here the expressions of the pro-apoptotic genes (LBALG-2l, LBBAK-2l, and LBMA-3) is considerably higher in the top region, compared to the base. The expression of the pro-

FIG. 8. (A) Photo of a L. baicalensis specimen growing in depths greater 5 m, showing the arborescent growth pattern, along the apical-basal axis (top to base). (B) Northern blot analysis of RNA, obtained from one specimen collected at the unpolluted site (natLi) and another one from the polluted site (antBai). The hybridization was performed with the same amount of RNA on one membrane, either using the probe for a pro-apoptotic (BAK-2 [LBBAK-2l]) or a pro-survival gene (Bcl-2 [LBBCL-2a]). The relative expression levels were determined by correlating the signals to that measured for the RNA which had been isolated at the top module from the specimen, collected at the natLi site (set to onefold). (C) Schematic representation of the results obtained from studies of gene expression (pro-apoptotic and pro-survival genes) and enzymatic activities (GSH peroxidase [GSH-pox] and caspases). The changes in activity/expression gradients along the apical-basal (top to base) axis are indicated by a trapezium or a triangle; the black symbols reflect the gradients in animals from natural site (natLi) and the gray those from the anthropogenically polluted site (antBai).

PRO-APOPTOTIC AND PRO-SURVIVAL GENES IN LUBOMIRSKIA BAICALENSIS size-separated, transferred onto one filter, and subjected to hybridization using the BAK-2 (LBBAK-2l) or the Bcl-2 (LBBCL2a) probe (Fig. 8B). The results show that the LBBAK-2l expression level of a natLi specimen at the top is low (set to onefold), and increases comparably little towards the basis (3.8fold). However, if the specimen from the antBai site is analyzed the top module shows the highest expression level with 6.7-fold. In contrast, if the LBBCL-2a is analyzed the top region of a branch from a natLi specimen shows by far the highest expression level. Again, the LBBCL-2a expression is very low in the branch of an animal from the antBai site, irrespectively of the region from which the tissue was taken.

DISCUSSION In Metazoa apoptosis is a physiological process essential for normal development and for homeostasis (Song and Steller, 1999). Enhancement of apoptosis results in chronic pathologies (degenerative diseases) while abnormal resistance against this process promotes tumorigenesis. Apoptosis is controlled by the delicate balance between the function of pro-apoptotic and prosurvival molecules. The expression of these molecules is the result of frequent interactions between proliferation and apoptotic pathways. Levels and functions of the pro-apoptotic molecules can be altered by radiation or toxic compounds (see Kaina, 2003) (Fig. 9). In Porifera, the evolutionary oldest Metazoa, a series of proteins has been identified, controlling tissue transplantation (Wiens et al., 2000b; 2004) or the reaction to toxic xenobiotics (Wiens et al., 2001). Until now, studies to elucidate the balance between pro-apoptotic and pro-survival molecules with regard to the formation of the sponge Bauplan (body plan) are lack-

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ing. For the present study the pro-apoptotic molecules BAK-2, the putative Bcl-2 homologous antagonist/killer 2 (BAK2_LUBAI), the apoptosis-linked gene-2 protein (ALG2l_LUBAI) and the MA-3 protein have been selected. In future studies it must be clarified if in sponges the MA-3 protein is also involved in apoptosis; however, earlier results strongly suggest such a function (Wagner et al., 1998). ALG-2 proteins are highly conserved metazoan molecules, interacting with a number of molecules in a calcium-dependent manner (Krebs et al., 2002). As a possible sensor of calcium ALG-2 can be considered as a prime candidate to be involved in the regulation of signal transduction pathways, thus connecting Ca2⫹ signaling and apoptotic pathways (Satoh et al., 2002). Furthermore, transient increase in Ca2⫹ concentration is associated with a series of pathways also involved in the response of cells to toxic molecules (Perovic et al., 1998; Jia et al., 2001). Hence, it can be postulated that ALG-2 functions as a link between Ca2⫹ sensing and apoptosis, very likely via binding to the ALG-2 interacting protein 1 (Jia et al., 2001). The L. baicalensis ALG-2 comprises the characteristic penta-EF-hand organization, whose involvement in Ca2⫹ binding has been shown (Jung et al., 2001; Satoh et al., 2002). BAK-2 is a pro-apoptotic molecule, like BAX and BID, modulating the release of apoptogenic cytochrome c and apoptosisinducing factor(s) from mitochondria into the cytoplasm (Nomura et al., 1999). The sponge BAK-2 comprises the characteristic BH1 and BH3 domains (Adams and Cory, 1998), classifying this protein to the pro-apoptotic molecules. BAK-2 acts in response to mitochondrial dysfunction, caused, for example, by ultraviolet radiation or growth factor deprivation (Wei et al., 2001), known to generate radical oxygen species (ROS). Hence, BAK-2 is a suitable biomarker for free radical-induced stress caused by adverse environmental factors.

FIG. 9. Schematic model of metazoan apoptotic pathways. Major checkpoints are the target of pro-apoptotic (BAK-2, ALG-2, and MA-3) and pro-survival (Bcl-2) proteins. Focusing on environmental stress, which results in an increase of free radical oxygen species, frequently generated within mitochondria, the BAK-2, ALG-2, and MA-3 genes are increasingly transcribed, while the Bcl-2 expression undergoes downregulation. Homeostasis between cell death and cell survival (physiological cell functions) is controlled by a tuned interaction/communication between proliferation and apoptotic pathways. Dysregulated apoptotic processes can lead to cell loss or cancer.

162 MA-3 was the first apoptotic molecule identified in sponges (S. domuncula; Wagner et al., 1998). The homologous human protein is characterized by two MA-3 domains (Shibahara et al., 1995; Stalberg et al., 2001) that are also found in the poriferan protein. The expression of MA-3 appears elevated during tumorigenesis (Stalberg et al., 2001) and during apoptosis in response to xenobiotics (Shibahara et al., 1995). The pro-survival protein Bcl-2 is a member of the core apoptotic machinery and regulates the developmentally programmed somatic deaths (Willis et al., 2003). Bcl-2 comprises four BH domains, characteristic of the Bcl-2 family. The function of Bcl-2 is largely dependent on BH1, BH2, and BH4 forming a hydrophobic groove, thus allowing the interaction with proapoptotic Bcl-2 proteins (Muchmore et al., 1996). As a consequence, interaction with pro-apoptotic BH3 proteins results in the inactivation of pro-survival Bcl-2 (Willis et al., 2003). Obviously, the ratio between pro-survival and pro-apoptotic proteins ultimately determines the life or death of a cell. Expression studies were performed with these four apoptosis-regulating molecular markers to determine their transcript levels. Studying the branches of L. baicalensis, collected from an unaffected, natural site (natLi) it is evident that the expression of the pro-apoptotic genes LBALG-2l, LBBAK-2l, and LBMA-3 is low at the tip and high at the base. This gradient of pro-survival on the top of the branches towards pro-apoptotic at the base is strengthened by the expression of the pro-survival gene LBBCL-2a. The expression level of this gene is highest at the top of the branches. Based on these molecular biological studies it becomes obvious that at the proliferation zone of the branches, the tip, the propensity to apoptotic processes is low. This gradient is reversed if specimens from an anthropogenically polluted site (antBai) are studied. In those specimens the expression of pro-apoptotic genes is high at the tip (growing zone), clearly indicating an entry into the decision phase of the apoptotic cascade (McCarthy, 2002). Only comparably few transcripts of the pro-survival gene LBBCL-2a can be detected. This pilot study implies that anthropogenic factors may impair the homeostasis of cell growth and cell death in this species; the cells in the top region are especially prone to apoptotic stimuli. A schematic summary is given in Figure 8C. The response of the animals to environmental load was determined by measuring the activity of the GSH peroxidase. GSH is a metabolite that prevents ROS-induced apoptotic cell death (Ratan and Baraban, 1995). One major enzyme that converts hydrogen peroxide to water through oxidation of GSH is the GSH peroxidase. The data presented show that the activity of this enzyme follows the gradient of apoptotic propensity. For the enzymatic studies five parallel experiments, performed with five different individuals each, had been performed; the animals had been collected within an area of 4 km2 at the two sites (natLi and antBai). The standard deviations and the coefficient of variance indicate a significant effect even though only a limited number of specimens had been included in this pilot study. Therefore, a comprehensive, more detailed analysis including more specimens has to follow. In unpolluted sites the activity is highest at the base and lower in the tip region. However, a strong GSH peroxidase activity is found throughout the branch of antBai sponges, suggesting that the load with free radicals is higher in these specimens, compared to those collected from the unpolluted area. Since GSH peroxidase is an enzyme that

WIENS ET AL. is regulated by the level of gene expression (see Baek et al., 2005), it can be deduced that environmental stress causes an upregulation of the GSH peroxidase gene. The expression studies and the determination of the enzyme activity of GSH peroxidase suggest that in specimens collected from unpolluted sites a gradient of propensity to apoptosis exists from the top to the base of the branches; the final support of this view can be taken from the determinations of caspase activities, the proteolytic key enzymes of the execution phase of apoptotic cell death. The caspase pathway has been recently identified in demosponges (Müller, 2003). Therefore, in the present study we determined the activity of the upstream and downstream caspases: caspase-8 and caspase-3. These studies also underscore the suggestion that in the growing zone of the branches the activity of apoptotic enzymes is lower compared to the base. Dramatic, however, are the increased activities in specimens collected from the polluted site. Based on the studies presented here a gradient of the expression pattern of apoptotic genes from the top to the base of the L. baicalensis specimens can be determined. This finding is supported by enzymatic data with the GSH peroxidase and the caspase-8 and caspase-3. Using these molecular and biochemical markers it is also demonstrated that in anthropogenically polluted sites the balance between pro-apoptotic and antiapoptotic molecules is disturbed, resulting in an upregulation of the pro-apoptotic pathways. With respect to biomonitoring, this pilot study might not be considered as representative to date, and should be followed by a more detailed and comprehensive analysis of the effect of environmental stressors on (1) the expression of the apoptotic genes in L. baicalensis along the axis of their branches, and (2) the level of peroxides and caspases.

ACKNOWLEDGMENTS This work was supported by grants from the Center for Environmental Research (University of Mainz), WTZ Germany–Russia (German–Russian cooperation through the BMBF), Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, Germany (project: Center of Excellence BIOTECmarin), the European Society for Marine Biotechnology, and the International Human Frontier Science Program.

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