Eur J Biochem 271, 1924–1937 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04102.x A (1fi3)-b-D-glucan recognition protein from the sponge Suberites domuncula Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression ´ Sanja Perovic-Ottstadt1, Teresa Adell1, Peter Proksch2, Matthias Wiens1, Michael Korzhev1, Vera Gamulin3, Isabel M Muller1 and Werner E G Muller1 ă ă Institut fuăr Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitaăt, Mainz, Germany; 2Institut fuăr Pharmazeutische Biologie, Heinrich-Heine-Universitaăt, Duăsseldorf, Germany; 3Institute Rudjer Boskovic, Department of Molecular Biology, Zagreb, Croatia Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1fi3)-b-D-glucans, e.g curdlan or laminarin This receptor, the (1fi3)-b-D-glucan-binding protein, was identified and its cDNA analysed The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor Expression of the (1fi3)-b-D-glucan-binding protein and the fibrinogen-like Sponges (phylum Porifera) are, among all metazoan taxa, those animals which contain the widest range of specific and very effective bioactive compounds [1,2] It has been Correspondence to W E G Muller, Institut fur Physiologische ă ă Chemie, Abteilung Angewandte Molekularbiologie, Universitat, ă Duesbergweg 6, 55099 Mainz, Germany Fax: + 49 6131 39 25243, Tel.: + 49 6131 39 25910, E-mail: wmueller@uni-mainz.de Abbreviations: EGF, epidermal growth factor; LPS, lipopolysaccharide; MAPK, mitogen-actived protein kinase; PoAb, polyclonal antibody Note: This article is dedicated to Professor Zeeck (University of Gottingen) on the occasion of his 65th birthday ă Note: The cDNA sequences from Suberites domuncula have been deposited in EMBL/GenBank as follows: the (1fi3)-b-D-glucanbinding protein (GLUBPp_SUBDO) under the accession number AJ606470, the fibrinogen-like molecule (FIBl_SUBDO) under the accession number AJ606471, and the epidermal growth factor precursor (EGFl-PREC_SUBDO) under the accession number AJ606469 (Received 23 January 2004, revised March 2004, accepted 22 March 2004) protein occurred in cells on the sponge surface, in the pinacoderm By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein Their application revealed that a kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin In turn, the increased expression of the downstream genes was suppressed We conclude that sponges possess a molecular mechanism for recognizing fungi via the D-glucan carbohydrates on their surfaces Keywords: D-glucan binding protein; epidermal growth factor; fungi; sponges; symbiosis assumed that most of these secondary metabolites are produced by symbiotic microorganisms which are harbored by the sponges [3] Among these microorganisms, bacteria [4] and fungi are the most potent producers of secondary metabolites in sponges Hence, sponges must be provided with mechanisms to distinguish between harmful (perhaps infectious) and symbiotic bacteria and fungi At a molecular level, most of the work carried out towards understanding this host–microorganism symbiotic relationship has been performed with the demosponge Suberites domuncula Sponges are provided with a very efficient immune system, reminiscent of that found in higher metazoan phyla, particularly deuterostomians [5] In addition, sponges produce the same proteinaceous defense molecules (e.g tachylectin) that are known to be induced in protostomians as a defense against bacteria [6] It has also been found that S domuncula recognizes the lipopolysaccharide (LPS) molecule on the surface of bacteria and responds by activation of the mitogen-activated protein kinase (MAPK) pathway [7] Until the present study was undertaken, nothing was known, at a molecular level, about the system by which sponges recognize fungi In an approach to elucidate this mechanism, we activated sponge cells by selected model glucan polymers, including the (1fi3)-b-D-glucans, Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1925 which have been isolated from cell walls of plants, but also from bacteria and fungi [8] Prominent purified glucan molecules of this group are (a) curdlan, a linear polysaccharide from Alcaligenes faecalis [9,10] and (b) laminarin, a poly (1fi3)-b-D-glucan with some interstrand (1fi6)-b-D-glucan branch points [11], isolated from the alga Laminaria digitata (1fi3)-b-D-Glucans induce immune responses in protostomians [12,13] and deuterostomians [14] In the first series of experiments, using the model compound curdlan, we demonstrated that sponges (S domuncula) indeed react to incubation with (1fi3)-b-D-glucan First, we analysed whether curdlan influences the phosphorylation of MAPKs by S domuncula; however, no change in the phosphorylation level was seen (data not shown) Subsequently, we determined whether treatment with curdlan modulates the tyrosine kinase pathway of sponges Using an antibody specific for phosphotyrosine we showed that at least one protein species underwent phosphorylation after incubation with this glucan To determine the specificity of this reaction, the known tyrosine kinase inhibitor, aeroplysinin, isolated from the sponge Verongia (syn: Aplysina) aerophoba [15,16] was used After proving that S domuncula recognizes (1fi3)-b-Dglucan, the respective (1fi3)-b-D-glucan-binding protein had to be identified Such a molecule has previously been isolated and cloned from a number of protostomians – from crustaceans [13,17], earthworm [12] and insects [18], as well as from sea urchins [19] After successfully cloning the (1fi3)-b-D-glucan-binding protein from S domuncula, we continued our search for other potential binding proteins that might be involved in recognizing the D-glucan Promising candidates were molecules that display lectin properties, e.g the horseshoe crab acetyl group-recognizing lectin [20] or lectin molecules with fibrinogen domains, e.g the ficolins [21] This rationale led to the isolation of a fibrinogen-like molecule from S domuncula It is known that cells from deuterostomians react to fungal cell wall polysaccharides by producing cytokines [22] The epidermal growth factor (EGF) domain occurs very frequently in cytokines; for sponges this domain has already been described [23] Therefore, degenerate primers were designed to identify genes which comprise this domain and that are expressed by the stimulation of sponges with D-glucans This approach resulted in the identification of a cDNA whose deduced polypeptide, termed EGF precursor, comprises three EGF domains Our data provide, for the first time, an insight into the response of sponges to stimulation with (1fi3)-b-D-glucan We show that the polysaccharide binds to the (1fi3)-b-Dglucan-binding protein; subsequently, a gene encoding a fibrinogen-like protein, and also one for a cytokine, are strongly expressed Materials and methods Chemicals and enzymes The sources of chemicals and enzymes used were as given previously [24,25] Laminarin from L digitata, curdlan from A faecalis and LPS from Escherichia coli O55:B5, as well as monoclonal antibody (mAb) against phosphotyrosine were purchased from Sigma-Aldrich (Deisenhofen, Germany) Aeroplysinin was isolated from the sponge Aplysina aerophoba, as described previously [15,16] Curdlan was labeled with biotin according to Novotna et al [26] The glucans were dissolved as described previously [12,27] Sponges Live specimens of S domuncula (Porifera, Demospongiae, Hadromerida) were collected near Rovinj (Croatia) and maintained in aquaria in Mainz (Germany) for more than 10 months prior to use Exposure of tissue samples from S domuncula to curdlan and Western blotting Tissue samples (2 g) were maintained for 1–3 days in seawater in the presence or absence of curdlan (10 lgỈmL)1) and were then processed as described previously [28] Where indicated, the tissue was additionally treated with lgỈmL)1 of aeroplysinin Samples were homogenized in lysis buffer [1 · Tris-buffered saline (TBS), pH 7.5, mM EDTA, 1% Nonidet-P40, 10 mM NaF, protease inhibitor cocktail (one tablet per 10 mL) and mM sodium orthovanadate], centrifuged and the supernatants analysed by Western blot To determine the phosphorylation of tyrosine, the tissue samples were treated for h with polysaccharide Total tissue extracts (20 lg per lane) were subjected to electrophoresis in 8% polyacrylamide gels containing 0.1% SDS, as described by Laemmli [29] Western blotting experiments were performed as described previously [30] The membranes were incubated with mouse mAb-antiphosphotyrosine (mAb-aTyr) (1 : 2000 dilution) After washing, the blots were incubated with peroxidase-coupled goat antimouse IgG (1 : 2000 dilution) Detection of the immunocomplex was carried out using the BM Chemoluminescence Blotting Substrate kit from Roche (Mannheim, Germany) Ligand-binding blot The assay was performed as described previously [12] Extracts from tissue were incubated for day with 10 lgỈmL)1 curdlan, then treated with 0.2% SDS, but not with 2-mercaptoethanol (the samples were not boiled prior to separation) The samples were then size separated by SDS-PAGE (12% gel) After separation, the proteins were transferred to poly(vinylidene difluoride)-Immobilon After blocking with BSA (1%, w/v), the blots were incubated with biotin-labeled curdlan (5 lgỈmL)1) Visualization was performed with peroxidase-avidin, using 4-chloro-1-naphthol as the substrate In competition experiments, after transfer of the proteins, the blots were first incubated with either 10 lgỈmL)1 laminarin or lgỈmL)1 LPS The blots were then washed and incubated with biotin-labeled curdlan followed by peroxidase-avidin/4-chloro-1-naphthol Isolation of cDNA for the (1fi3)-b-D-glucan-binding protein The cDNA encoding a potential (1fi3)-b-D-glucan binding protein (GLUBPp_SUBDO) was isolated from the Ó FEBS 2004 ´ 1926 S Perovic-Ottstadt et al (Eur J Biochem 271) S domuncula cDNA library [24] by PCR The primers were designed against the highly conserved region within the (1fi3)-b-D-glucan-binding proteins; in the b-1,3-glucanbinding protein from the black tiger shrimp, Penaeus monodon (accession number AF368168-1) the stretch reads MLWPAIWM (amino acids 160–167) The degenerate primer, 5¢-TGGCTITGGCCIGCIATA/C/GTGGATG-3¢, was used in the PCR reaction, together with the vector primer The PCR was carried out as follows: initial denaturation at 95 °C for min, followed by 30 amplification cycles at 94 °C for 30 s, 62 °C for 45 s and 70 °C for 1.5 min, and a final extension at 70 °C for 10 The reaction mixture was as described previously [31] The fragments obtained were used to isolate the cDNA from the library [32] and identified one clone with a 1327 nucleotide insert [excluding the poly(A) tail] The clone was termed SDGLUBP; it was sequenced using an automatic DNA sequencer (Li-Cor4200; MWG Biotech, Ebersberg, Germany) cDNA corresponding to the fibrinogen-like protein Following the strategy described in the Introduction, a conserved fibrinogen domain was selected for the design of degenerate primers Aligning different fibrinogendomain containing proteins, fibrinogens, fibroleukins/ techylectins, angiopoietins, ficolins and tenascins, the following consensus was deduced: FSTxDNDND It is located in the human fibrinogen a/a-E chain precursor (accession number P02671) between amino acids 785 and 793 The degenerate forward primer, 5¢-TTC/TTCIACI TGGGAC/TACC/TGAC/TACC/TGAC/T-3¢, was used in the PCR reaction The PCR conditions were as described above, except that 65 °C were used during the amplification cycles Only one species of insert was obtained, with a size of 1079 nucleotides This clone was termed SDFIBI cDNA encoding the putative EGF-like precursor, EGFI-PREC_SUBDO The EGF precursor, EGFI-PREC_SUBDO, was cloned from the cDNA library using degenerate primers that were designed against the conserved domain, including the first Cys residue of the EGF domain from the human pro-epidermal growth factor precursor (P01133), DVNECAF; 5¢-GAC/TGAIAAC/TGAA/GTGC/TGCITTC/ T-3¢, was used in the PCR The PCR conditions were as described above, with the exception that a temperature of 57 °C was used during the amplification cycles Only one species of insert was obtained; it was 2446 nucleotides in size This clone was termed SDEGFI-PREC Sequence analysis The sequences were analyzed using the computer programs BLAST [33] and FASTA [34] Multiple alignments were performed using CLUSTAL W, Version 1.6 [35] Phylogenetic trees were constructed on the basis of amino acid sequence alignments by neighbour-joining, as implemented in the NEIGHBOR program from the PHYLIP package [36] The distance matrices were calculated using the DAYHOFF PAM matrix model, as described previously [37] The degree of support for internal branches was further assessed by bootstrapping [36] The graphic presentations were prepared using GENEDOC [38] Recombinant EGF precursor and production of antibodies The sponge SDEGFI-PREC sequence was isolated by PCR using the forward primer, f1 [5¢-CCATGGAGA AGATTCTAGCAACAGTCAATTCAAATGAC-3¢ (the NcoI restriction site is underlined), nucleotides 1060–1098], and the reverse primer, r1 [5¢-GCGGCCGCTG TATCTGAAGTTGGGGAATTACTGTGTTTGTTGTT-3¢ (the NotI restriction site is underlined); nucleotides 2206– 2241] The full-length PCR product (1143 bp) was expressed in E coli The cDNA was cloned into the bacterial glutathione-S-transferase/oligohistidine/S expression vector, pET41a (Novagen, Madison WI, USA) via the mentioned restriction sites After transformation with this plasmid, expression of the fusion protein was induced in E coli strain BL21 for h at 37 °C with mM isopropyl thio-b-D-galactoside [32] Bacterial pellets were obtained from 500 mL cultures The fusion protein was extracted and purified first with the His-tag purification kit (Novagen) and subsequently with the glutathione-S-transferase-tag purification kit (Pharmacia, Freiburg, Germany), as described by the manufacturer Finally, the fusion protein was cleaved with enterokinase (5 U; Novagen), as recommended The recombinant EGF precursor, r-EGF_SUBDO, was obtained tag-free through purification in a batch procedure using the glutathione-S-transferase-tag purification kit; the recombinant protein remained in the supernatant The purity of the material was verified by electrophoresis through 10% polyacrylamide gels containing 0.1% SDS, according to Laemmli [29] The protein was dialyzed against 25 mM Tris/HCl buffer (pH 7.2), supplemented with 10 mM DL-dithiothreitol Polyclonal antibodies (PoAb) were raised against the recombinant EGF protein in female rabbits (White New Zealand), as previously described [39] Animal experiments were registered and performed according to German law After three booster immunizations, the serum was collected; the PoAbs were termed PoAb-EGF protein In control experiments, 100 lL of the PoAb-EGF protein was adsorbed to 20 lg of r-EGF_SUBDO (30 min; °C) prior to use Western blotting of EGF For the identification of EGF in extracts from sponge tissue, extracts were prepared, as described above, and subjected to electrophoresis through 15% polyacrylamide gels containing 0.1% SDS, as described previously [29] The membranes were incubated with rabbit PoAb-EGF precursor (1 : 500 dilution); the immune complexes were visualized by incubation with alkaline phosphatase-conjugated antirabbit IgG, followed by staining with 4-chloro-1-naphthol To quantify a given signal on the blots, scanning with the GS-525 Molecular Imager (Bio-Rad) was performed The relative value, with respect to the signal seen in the nontreated extract, is given for the signal seen in extract from curdlan-treated tissue Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1927 RNA preparation and Northern blot analysis RNA was extracted from liquid-nitrogen pulverized tissue using TRIzol reagent (GibcoBRL, Grand Island, NY, USA), as described previously [40] Then, lg of total RNA was electrophoresed and blotted onto Hybond-N+ nylon membrane (Amersham, Little Chalfont, Bucks, UK) Hybridization was performed with a 550 nucleotide region of the SDGLUBP cDNA, a 220 nucleotide region of the SDFIBI cDNA and a 200 nucleotide region of the SDEGFL-PREC cDNA Regions spanning the open reading frames were selected The housekeeping gene (b-tubulin) of S domuncula, SDTUB (accession number AJ550806), was used as an internal standard The probes were labeled using the PCR-DIG-probe-synthesis kit (Roche) After washing, DIG-labeled nucleic acid was detected with antiDIG Fab fragments and visualized by chemiluminescence using CDP (Roche) In situ localization studies The method applied was based on the procedure described by Polak & McGee [41], with modifications described recently [42] Frozen sections of lm were obtained, fixed with paraformaldehyde, treated with Proteinase K and subsequently fixed again with paraformaldehyde To remove the sponge color, the sections were washed with increasing concentrations of ethanol and finally isopropanol After rehydration, the sections were hybridized with labeled probes, the 550 nucleotide SDGLUBP or the 200 nucleotide SDFIBL cDNA After blocking, the sections were incubated overnight, at 45 °C, with an alkaline phosphatase-conjugated antidigoxigenin immunoglobulin The dye reagent, Nitro Blue tetrazolium/X-Phosphate, was used for visualization of the signals Antisense and sense single stranded DNA digoxigenin-labeled probes were synthesized by PCR using the PCR DIG Probe synthesis Kit (Roche) Sense probes were used, in parallel, as negative controls in the experiments Results Effect of incubation with curdlan on the phosphorylation of tyrosine in sponge tissue It is known that (1fi3)-b-D-glucans are activators of gene expression in mammalian cells [22] Therefore, we investigated whether sponges react to curdlan with an increased phosphorylation of tyrosine Tissue samples were incubated in the presence or absence of 10 lgỈmL)1 curdlan Extracts were prepared and the proteins were size separated by SDS/PAGE After transfer, the blot was incubated with mAb-aTyr and then with a labeled secondary antibody The results show that in the absence of curdlan no bands were detected on the blots (Fig 1B; lane a); however, in the extracts from curdlan-treated tissue a strongly staining band of 32 kDa was observed (lane b) When the tissue was treated with curdlan and the tyrosine kinase-inhibitor, aeroplysinin (1 lgỈmL)1), no 32 kDa band was detected (lane c) In parallel, the gels were stained with Coomassie Brilliant Blue (Fig 1A) and no change in the banding pattern and their intensities occurred Fig Phosphorylation of a 32 kDa protein after incubation of sponge tissue with curdlan Tissue samples were incubated for h with or without 10 lgỈmL)1 curdlan Protein extracts were then prepared and size-separated by PAGE (8% gel) (A) The gel was stained with Coomassie Brilliant Blue (B) Proteins were blot transferred and reacted with mouse antiphosphotyrosine mAb and then with labeled goat anti-mouse IgG Detection of the immunocomplex was carried out as described in the Materials and methods Protein extract from tissue incubated in the absence (lane a, – cur), or in the presence (lane b, + cur) of curdlan In one series of experiments, the tissue was additionally treated with lgỈmL)1 aeroplysinin (lane c, + aero) M, protein size markers Detection of glucan-binding activity in extracts from S domuncula Tissue from S domuncula was incubated for day with 10 lgỈmL)1 curdlan Then, extracts were prepared and subjected to PAGE in the presence of a low concentration of SDS and in the absence of b-mercaptoethanol After size separation (Fig 2; lane a), the proteins were transferred and – after blocking – probed with labeled curdlan A 43 kDa polypeptide was observed in the extract (lane b) When the blot was first preincubated with 10 lgỈmL)1 laminarin and – after washing – probed with the labeled curdlan, no band was detected (lane c) However, when the blot was preincubated with lgỈmL)1 LPS and subsequently with labeled curdlan, the intensity of the band was only slightly reduced (lane d) From these data we conclude that a 43 kDa protein is present in the extract from curdlantreated tissue, and that this protein comprises a specificity for (1fi3)-b-D-glucans In parallel, incubation experiments with curdlan had been performed for only h Under these conditions, the binding between labeled curdlan and the 43 kDa polypeptide was very low (data not shown) Cloning of cDNA encoding the S domuncula (1fi3)b-D-glucan-binding protein Sequence The insert with SDGLUBP comprises one ORF, which ranges from nucleotides 46–48 to nucleotides 1252–1254(stop); the cDNA is of full length, as shown by Northern blot analysis (1.4 kb; see below) The deduced protein shows high sequence similarity to the Ó FEBS 2004 ´ 1928 S Perovic-Ottstadt et al (Eur J Biochem 271) cDNA encoding the fibrinogen-like protein Fig Detection of a glucan-binding protein in extracts from Suberites domuncula A protein sample was prepared from tissue that had been incubated for day with 10 lgỈmL)1 curdlan, as described in the Materials and methods Extract from curdlan-treated tissue was size separated by SDS-PAGE (lane a) After separation, the protein extracts were transferred to poly(vinylidene difluoride)-Immobilon and incubated with biotin-labeled curdlan (cur, lgỈmL)1) (lane b) Alternatively, the blots were first preincubated with 10 lgỈmL)1 laminarin (lam, lane c), or lgỈmL)1 lipopolysaccharide (LPS, lane d), for h, and then washed and probed with biotin-curdlan (cur), as described in the Materials and methods (1fi3)-b-D-glucan-binding proteins and was therefore termed GLUBPp_SUBDO The protein comprised 402 amino acid residues, with a calculated size of 45 040 Da, and possessed, between amino acid 49 and amino acid 296, one characteristic domain for Ôglycosyl hydrolases of the family 16Õ (PFAM: PF00722) with a high significance value (E-value) of 2e-05 Two transmembrane regions were identified [43], which ranged from amino acids to 23 and from amino acids 361 to 401 (Fig 3A) From these data we conclude that the 43 kDa protein identified in the ligandbinding blot probably corresponds to the 45 kDa (1fi3)-bD-glucan-binding protein deduced from SDGLUBP Phylogenetic analysis The sponge glucan-binding protein shares highest sequence similarity with the (1fi3)-b-Dglucan-binding proteins with average sizes 350–400 amino acids The highest similarity was calculated with the b-1,3glucan-binding protein from the black tiger shrimp, P monodon, having approximately 37% identical and 53% similar (with respect to the physico-chemical properties) amino acid residues to the sponge protein The similarity of the sponge glucan-binding protein to related insect and crustacean proteins (35% identity/50% similarity) was only slightly lower No considerable similarity was found to exist to the nonmetazoan and the protostomian/nematode putative proteins present in the database After alignment of all similar sequences, a radial tree was constructed which shows that the sponge glucanbinding protein forms the basis for the insect molecule on one side and the molecule from crabs on the other (Fig 3B) Sequence One species of insert was identified – the ORF, which spanned nucleotides 31–33 to nucleotides 877–879(stop) The full size cDNA (SDFIBI; 1.1 kb by Northern blot analysis; see below) encoded the predicted protein, termed FIBI_SUBDO, comprising 282 amino acid residues (giving a calculated Mr of 31 997) Domain searches revealed that within the polypeptide, one fibrinogen domain for b- and c-chains (PFAM: PF00147) exists between amino acids 81–270 One conserved disulphide bond exists connecting Cys225 to Cys239 and one eukaryotic secretory signal sequence can be predicted [44] (Fig 4A) The highest similarity exists with vertebrate fibrinogens; therefore the sequence was named fibrinogen-like protein Fibrinogens are the principal proteins of the vertebrate clotting system and form hexamers, composed of the three different chains: a, b and c [45] As outlined by Spraggon et al [46], the b- and c-chains are homologous throughout the complete sequence, while the a-chain comprises the highest similarity only in the first 200 residues Alignment studies with the sponge and three mammalian fibrinogens showed that the sponge fibrinogen-like protein, even though the full-length sequence is available, shares similarity only within the middle segment of the a-, b- and c-chains Hence, no further classification of the sponge protein to any of the three vertebrate chains can be made In the sponge sequence, besides the first disulfide bridge mentioned, the disulfide rings and the thrombin attack point (which exist in the human sequence) are lacking A potential arginine residue in the sponge fibrinogen at amino acid position 18 cannot be recognized by thrombin owing to a negatively charged glutamic acid residue at position P2 [47] The conserved central segments within the fibrinogen domain [46] are present in the sponge protein Phylogenetic analysis The analysis was performed with the fibrinogen domain of the sponge fibrinogen-like protein The highest similarity, with approximately 35% identical and 45% similar amino acid residues, was found to the fibrinogens in the databases (Fig 4B); the human fibrinogen c-chain precursor (P02679) was used for the alignment (Fig 4A) The sequences were compiled and an unrooted (slanted) tree was constructed (Fig 4B) The trichotomous tree shows that the families of the fibroleukins, with the human member (Q14314) as an example, together with the techylectins from the horseshoe crab, Tachypleus tridentatus [20] (AB024737.1 and AB024738.1), and the angiopoietins from mammals, e.g humans (O15123), form the second branch The third branch is built by the ficolins, with the mouse ficolin B as an example [48] (AF063217), and the tenascins, including also the precursors from humans (dJ1141O19.1), as members (Fig 4B) The basis again is the sponge-deduced protein cDNA of a potential EGF precursor Sequence One species of cDNA, which encodes a deduced protein containing EGF domains, and was therefore termed EGF precursor (SDEGFI-PREC) was isolated from the library The 2446 nucleotide contains an ORF, from nucleotides 100–102 to nucleotides 2242–2244(stop); the Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1929 Fig The Suberites domuncula potential beta-1,3-glucan-binding protein (GLUBPp_SUBDO) (A) The deduced sponge sequence (GLUBPp_SUBDO) is aligned with the most related sequence, the b-1,3-glucan-binding protein from the black tiger shrimp Penaeus monodon (GLUBP_PENMO, AF368168-1) Identical amino acids are shown in white on black The positions of the two potential transmembrane regions (TM) and the Ôglycosyl hydrolases-16Õ domain (glyco-hydr) are indicated The segment towards which the degenerate primers were designed is underlined by dashes (B) Phylogenetic analysis of these two sequences with the GLUBP from the blue shrimp, Litopenaeus stylirostris (GLUBP_LITSTY, AF473579-1), the putative Gram-negative bacteria-binding proteins from the Diptera Anopheles gambiae (ENSAN1_ANGA, XP_312118.1), (ENSAN5_ANGA, XP_312116.1) and (BACBP_ANGA, CAA04496.1), as well as the GLUBP from the lobster Homarus gammarus (GLUBP_HOGAM, CAE47485.1) and the crayfish Pacifastacus leniusculus (GLUBP_PACLE, CAB65353.1) After alignment, the radial tree was constructed Fig Suberites domuncula fibrinogen-like protein (A) The deduced sponge protein, FIBl_SUBDO, is aligned with the related fibrinogen c-B chain precursors from humans (FIBG_HUMAN; P02679) The conserved fibrinogen domain (FIBR) and one conserved disulfide bridge (C–C) are present in the sponge protein, while the second disulfide bridge found in the human sequence is absent; this is marked ([C]–[C]) The predicted eukaryotic secretory signal sequence terminates after amino acid 22 (SS) The thrombin attack point ({}) and the disulfide rings (underlined) in the human sequence are indicated The double underlined amino acids represent the regions towards which degenerate primers were designed at the nucleotide level The conserved central segments within the fibrinogen domain are marked (++) (B) A slanted cladogram was constructed using the conserved fibrinogen domains of the two sequences mentioned above and of the following sequences (i) Fibrinogens: fibrinogen a-2 chain precursor from the sea lamprey Petromyzon marinus (FIB2_PETMA; P33573), fibrinogen a/a-E chain precursors from chicken (FIBA_CHICK; P14448), human (FIBA_HUMAN; P02671) and rat fibrinogen (FIBA_RAT; P06399), and the fibrinogen c-B chain precursors from bovine (FIBG_BOVIN; P12799), rat (FIBG_RAT; P02680), frog (FIBG_XENLA; P17634), and sea lamprey (FIBG_PETMA; P04115) (ii) Fibroleukins and techylectins: the fibroleukin precursors from mouse (FGL2_MOUSE; P12804) and human (FGL2_HUMAN; Q14314), as well as the techylectins from the horseshoe crab Tachypleus tridentatus (TECL5A-TACTR; AB024737.1 and TECL5B_TACTR; AB024738.1) (iii) Angiopoietin: angiopoietin and precursors from mouse (AGP1_MOUSE; O08538 and AGP2_MOUSE; O35608), bovine (AGP1_BOVIN; O18920 and AGP2_BOVIN; O77802), and human (AGP1_HUMAN; Q15389 and AGP2_HUMAN; O15123) (iv) Ficolins: ficolin A and B from pig (FICOLA_PIG; L12344 and FICOLB_PIG; L12345), mouse (FICOLA_MOUSE; AB007813 and FICOLB_MOUSE; AF063217), rat (FICOLA_RAT; AB026057), and the echinoderm Parastichopus parvimensis (FIBA_PARPA; P19477) (v) Tenascins: the tenascin precursors from chicken (TENA_CHICK; P10039), human (TENA_HUMAN; P24821 – and – TENAl_HUMAN; dJ1141O19.1), fish Danio rerio (TENAC_DARE; CAA61489.1), and pig (TENA_PIG; Q29116 – and – TENAX_PIG; CAA60686.1), as well as the the microfibril-associated glycoprotein (MFA4_HUMAN; P55083) The numbers at the nodes are an indication of the level of confidence, given as a percentage, for the branches as determined by bootstrap analysis (1000 bootstrap replicates) ´ 1930 S Perovic-Ottstadt et al (Eur J Biochem 271) size of the transcript, based on Northern blotting, is 2.6 kb The deduced 714 amino acids have a calculated Mr of 77 901 (putative EGF precursor, EGFI-PREC_SUBDO) By comparison with the Isrec-Server [49] domain database, Ó FEBS 2004 three EGF domains were identified in EGFI-PREC_SUBDO; they span the regions amino acids 331–368 (EGF1), amino acids 364–410 (EGF2) and amino acids 407–455 (EGF3) Furthermore, three low-density lipoprotein Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1931 Fig The putative epidermal growth factor (EGF) precursor, EGFl-PREC_SUBDO, from Suberites domuncula (A) The 714 amino acid polypeptide comprises three potential low-density lipoprotein receptor repeats (LDL) and also three EGF-like domains (EGF) Within the EGF domains and 2, the three characteristic intramolecular disulfide bonds (C ¼¼ C) are marked In addition, a Ser-rich segment () and a transmembrane region () exists The essential amino acid residues involved in binding to a receptor (#) and necessary for the biological function of the factor (§) are marked in domain (B) Unrooted tree constructed from the three sponge EGF domains (EGF_SUBDO) and the next similarity domains present in the pro-EGF precursor from human (EGF_HOMO, P01133), the bovine fibrillin precursor (MP340) (FBN1_BOVIN, P98133; amino acids 2205–2229), the transforming growth factor-a precursor (TGF-a) from Ovis aries (sheep) (TGFA_SHEEP, P98135; amino acids 7–49), fibrillin from the Cnidaria Podocoryne carnea (FBN1_PODCA, AAA91336; amino acids 443–487), rat cubilin (CUBIL_RAT, NP_445784; amino acids 2819–2864), frog (Xenopus laevis) neurogenic locus notch protein homolog precursor (NOTC_XENLA, P21783; amino acids 1735–1780), and the hypothetical protein ZC116.3 from Caenorhabditis elegans (ZC116_CAEEL, CAA98952; amino acids 3000–3054) receptor repeats and one serine-rich segment were predicted (Fig 5A) One strong transmembrane region is present between amino acids 624 and 663 The EGF domains are characterized by three typical intramolecular disulfide bonds [50], which are found in the sponge domains and with high similarity (Fig 5A) The EGF domains from ´ 1932 S Perovic-Ottstadt et al (Eur J Biochem 271) S domuncula can be grouped to the calcium-binding EGFlike domains (NCBI:cd00054.2, EGF_CA); calcium is crucial for protein–protein interactions Comprehensive studies have been performed with mammalian EGF to elucidate the characteristic sites [51,52] All amino acid residues essential for ligand–receptor interaction and for biological activity are present in domain (Fig 5A) Phylogenetic analysis The three EGF domains from EGFI-PREC_SUBDO were aligned with the most closely related EGF domains found in the proteins exclusively from metazoans The unrooted tree shows that the two sponge EGF domains (2 and 3) share highest similarity to the domains present in the human EGF (P01133), the bovine fibrillin (P98133) and the Podocoryne carnea (Cnidaria) fibrillin (AAA91336) More closely related to the sponge EGF domain are the domains present in the transforming growth factor-a from sheep (P98135), the notch/xotch protein from frog (P21783) and a hypothetical protein (ZC116.3) from Caenorhabditis elegans Upregulation of gene expression for the described genes in response to curdlan: Northern blotting To assess the effect of curdlan and its subsequent binding to the cell surface [probably to the (1fi3)-b-D-glucan-binding protein] on the expression of the gene coding for this receptor, Northern blot experiments were performed The results revealed that the expression level of the glucanbinding protein at the beginning of the experiments is low However, after only day of incubation in the presence of 10 lgỈmL)1 curdlan, a strong upregulation of the expression is seen, which increases during the following days (Fig 6) Ó FEBS 2004 Parallel experiments were performed to determine the expression of the SDFIBI gene (fibrinogen-like protein) Again, in the absence of curdlan, no transcripts can be detected by this technique, while, in the presence of the polysaccharide, a strong increase in the expression of SDFIBI occurs Likewise, a strong expression pattern was determined for the SDEGFI-PREC gene (Fig 6) The level of expression of the housekeeping gene, tubulin, was not altered by the presence of curdlan In view of the above finding, that curdlan causes phosphorylation of Tyr residue(s) in polypeptides of S domuncula, which is prevented by aeroplysinin, it seemed necessary to determine whether this protein tyrosine kinase inhibitor also has an effect on the pronounced increase of expression of the three genes under study Therefore, the tissue was incubated with 10 lgỈmL)1 curdlan, together with lgỈmL)1 aeroplysinin In this co-incubation experiment it was evident that the inhibitor, aeroplysinin, completely prevented any upregulation of the expression of SDGLUBP [(1fi3)-b-D-glucan-binding protein], SDFIBI (fibrinogen) or SDEGFI-PREC (EGF precursor) (Fig 6) Identification of cells expressing glucan-binding protein and fibrinogen Data from the literature [12,13,22], as well as the binding studies reported here, suggest that cells expressing the (1fi3)-b-D-glucan-binding protein are located in areas where the polysaccharide contacts the tissue, the pinacoderm A similar localization, expression of the (1fi3)-b-Dglucan-binding protein in the cells of the pinacoderm, can be expected for fibrinogen or the related molecules, e.g ficolins, which are involved in host defense [48] Therefore, in situ hybridization studies were performed Our studies revealed that the cryosections from tissue which had not been incubated with curdlan showed no cells which hybridized with the antisense probes, either for the sponge (1fi3)-b-D-glucan-binding protein (Fig 7A, a) or for fibrinogen (Fig 7B, a) However, after only day of incubation with curdlan, the cells that are primarily located around the canals react with the antisense probes (Fig 7A, b; Fig 7B, b) After a longer incubation (for days) the density of the hybridizing cells increased considerably (Fig 7A, c; Fig 7B, c) In contrast, no reaction was observed if the cells were treated with both sense probes (data not shown) Level of low-molecular-weight EGF in tissue after treatment with curdlan Fig Steady-state expression of the SDGLUBP gene [(1fi3)-b-Dglucan-binding protein], the SDFIBI gene (fibrinogen-like protein) and the SDEGFI-PREC gene (epidermal growth factor EGF precursor) in tissue from Suberites domuncula after exposure to 10 lgỈmL)1 curdlan The housekeeping gene, b-tubulin, SDTUB (accession number AJ550806), of S domuncula was used as an internal standard In one series of experiments the tissue samples were co-incubated with lgỈmL)1 of aeroplysinin (aero) RNA extraction was performed 0, or days after incubation with curdlan Equal amounts were loaded onto the gel The RNA was size separated, blot transferred and then hybridized with the labeled probes, as described in the Materials and methods As a first step, antibodies were required that could identify the (mature) EGF product and the approximate level in tissue by Western blotting The recombinant protein was prepared in E coli using the cDNA (clone SDEGFI-PREC) spanning the three EGF domains (corresponding to amino acids 321–713) After induction with isopropyl thio-b-Dgalactoside, the bacterial extract was isolated and purified by affinity chromatography (Fig 8A; lanes a and b) The 68 kDa recombinant fusion protein (r-EGF_SUBDO) was used to raise PoAbs, as described in the Materials and methods After cleavage with enterokinase, the sponge Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1933 Fig Spatial expression pattern of (1fi3)-b-D-glucan-binding protein and fibrinogen in sections of Suberites domuncula Cryosections were performed of S domuncula tissues, which were then hybridized with DIG-labeled SDGLUBP antisense DNA (A), or SDFIBl antisense DNA (B) Subsequently, the samples were incubated with antidigoxigenin/alkaline phosphatase and the signals detected with Nitro Blue tetrazolium/XPhosphate, as described in the Materials and methods Tissue samples, which were untreated (a), or treated with 10 lgỈmL)1 of curdlan for day (b), or days (c), were analyzed The canals (ca) of the aquiferous system are lined by an epithelial layer formed from pinacocytes Magnifications: A-a and B-a, · 25; A-b and B-b, · 50; A-c and B-c, · 100 PoAb-EGF precursor, which corresponded to a molecular weight of kDa This molecular weight predicts a polypeptide of approximately 45–50 amino acids, which matches exactly a processed form of the EGF from the 78 kDa EGF precursor (Fig 8B; lanes b and c) No further major band, including the 78 kDa EGF precursor, was detected In the control experiment, using PoAb-EGF precursor which had been adsorbed with recombinant r-EGF_SUBDO, no band was seen (Fig 8B; lanes d), indicating that the immune reaction with the kDa protein is specific The relative expression value for the band corresponding to the kDa polypeptide was assessed This approach revealed that in curdlan-exposed tissue, a threefold higher level of the EGF exists Fig Level of epidermal growth factor (EGF) in tissue after treatment with curdlan (A) Antibodies against the EGF precursor were prepared from the recombinant protein expressed in Escherichia coli (A, a; stained with Coomassie Brilliant Blue) The purified recombinant 41 kDa polypeptide (A, b; stained with Coomassie Blue) was used for immunization (B) The polyclonal antibody (PoAb)-EGF precursors were used to identify the protein in extracts from sponge tissue The extracts were size separated and the gels stained with Coomassie Brilliant Blue (B, a) Then, the proteins were blot transferred and reacted with PoAb-EGF precursor The level of cross-reacting proteins was assessed by molecular imaging, as described in the Materials and methods (B, b and c) In one series (B, d) the PoAb-EGF precursor was adsorbed with recombinant r-EGF_SUBDO prior to the application The immunocomplex has been visualized by a labeled secondary antibody For further data see the Materials and methods recombinant protein, r-EGF_SUBDO, showed an expected molecular weight of 41 kDa (data not shown) This PoAb, PoAb-EGF precursor, was used for the Western blot experiments Tissue extract from S domuncula was prepared (Fig 8B; lane a) and used for the blotting studies In nontreated, as well as in curdlan-treated extracts from tissue, a strong band was detected using the Discussion Based on the known symbiotic relationship between sponges and microorganisms, such as bacteria and fungi, it can be deduced that these animals must have an efficient recognition system which is able to discriminate between self and symbiont On the next level of specificity, the sponges must be provided with pattern recognition molecules serving as biosensors for the detection of invading pathogens (parasitic), of commensalic (of benefit only for one partner) or of mutualistic organisms (benefit for both partners) It has been shown, in sponges, that some microbial secondary metabolites, e.g okadaic acid [4], are beneficial for the host However, the origin of most secondary metabolites identified in sponges is not clear The understanding of the pathways which result in the synthesis of these compounds in sponges is crucial for their sustainable production/ exploitation for human benefit [53,54] It is now established that sponges can recognize bacteria and react to them with an increased phosphorylation of key kinases of the MAPK pathway [7,28] Furthermore, the first genes encoding antibacterial proteins, such as perforin [55] or the lectin tachylectin [6], have been identified in the ´ 1934 S Perovic-Ottstadt et al (Eur J Biochem 271) sponge S domuncula In addition, evidence has been presented that the antibacterial alkyl-lipids, lyso-plateletactivating factors, are produced in S domuncula in response to bacterial infection [56] The molecular basis for the interaction between fungi and sponges has not yet been studied It is well established that fungi present in sponges, e.g Penicillium sp in Ircinia fasciculata, produce bioactive products such as sorbicillactone [57] In this study, we present the first molecular data to demonstrate that sponges, with S domuncula as the model, have recognition receptors for fungi on their cell surface The fungal coat carbohydrate – the (1fi3)-b-D-glucan, curdlan – served as a model compound of choice Fungi are known to bind, via this carbohydrate, to the surface of insects [58], crustaceans [59], and also to human cells [22] The (1fi3)-b-D-glucan-binding protein was first identified in crustacean blood [60] and then cloned from the earthworm Eisenia foetida [12] In plants, the (1fi3)-b-D-glucan binds to a b-glucan elicitor-binding protein of different structure [61] Following a previously described approach [12], the (1fi3)-b-D-glucan-binding protein was identified biochemically The binding of the linear carbohydrate, curdlan, was abolished by the branched molecule, laminarin, indicating that in S domuncula a binding protein with specificity to (1fi3)-b-D-glucan exists Subsequent cloning studies revealed a cDNA coding for a putative protein which shares the characteristic feature of other metazoan (1fi3)-b-Dglucan-binding proteins The expression of the gene was induced after exposure to (1fi3)-b-D-glucan The sponge polypeptide shares a high similarity to the related insect/ crustacean molecules, but shows no significant relationship to protostomian/nematode, deuterostomian or non-metazoan molecules in the database In order to further support the assumption that the sponge (1fi3)-b-D-glucan-binding protein is involved in the recognition of potential fungi, in situ hybridization studies were performed They demonstrated that the expression of this gene is especially high in those regions of the sponge – the aquiferous canal system (pinacoderm) – which are exposed to the carbohydrate during the incubation period Next, it was attempted to identify downstream molecules, potentially involved in the response of the sponge to (1fi3)b-D-glucan A search for sequences in the S domuncula EST database, coding for potential clotting enzymes or coagulin, known to be involved in the coagulation cascade in the horseshoe crab [62], was unsuccessful Therefore, the S domuncula cDNA library was screened for an alternative scavenging system against carbohydrates, with lectins as the first candidates A C-type lectin has been isolated and cloned from the hexactinellidian sponge, Aphrocallistes vastus [63], which has been implicated in cell–cell interactions Furthermore, a galectin has been identified in S domuncula, which was also found to be an adhesion molecule causing morphogenetic effects [64] Hence, the next promising lectins are those which are known, from higher metazoan phyla, to be involved in innate immunity and comprise the fibrinogen-like domain Molecules with this domain exist from the crown taxa to echinoderms [65] or ascidians [21] in the deuterostomian branch, and to the horseshoe crabs [20] in the protostomian line; this domain is thought to play a role in carbohydrate binding [66] PCR-based identification and subsequent cloning of a full-length cDNA coding for a Ó FEBS 2004 fibrinogen-like protein was successful The putative protein comprises only one domain, which, however, has high similarity to the fibrinogen domain; unexpectedly, no further domain, e.g a collagen domain as in the ficolins [21], or lectin as in the horseshoe crab [20], or a coiled-coil domain in angiopoietin [67], could be detected in the sponge protein The sponge fibrinogen-like protein identified shares the highest sequence similarity with the deuterostomian fibrinogens, with the sea lamprey (Petromyzon marinus) sequence as the most closely related The lamprey fibrinogens [68] and, to a smaller extent, also the echinoderm related molecule [65], are known to be the ancestors for the three homologous polypeptide chains (a2b2c2) in the vertebrate blood clotting system The a-, b- and c-subunits of the fibrinogens are evolutionarily closely related; they differ especially in the N- and/or C-terminal regions [45,46] Only one characteristic disulfide bridge exists in the predicted fibrinogen-like protein from S domuncula; in addition, the thrombin cleavage site is lacking However, the centrally located conserved segments within the fibrinogen domain [46] are present in the sponge protein It is proposed that this sponge protein represents the ancestor of all fibrinogen domain-containing proteins found in Metazoa In consequence, the other vertebrate fibrinogens emerged from the sponge protein, probably by gene duplication [65] and domain shuffling [69] The phylogenetic relationship (the slanted cladogram) presented here, demonstrates that the related molecules – the fibroleukins, secreted molecules from T lymphocytes [70]; the techylectins/tachylectins from the horseshoe crab, presumably proteins involved in non-selfrecognition [20]; the angiopoietins, which trigger the mechanism of blood vessel formation [67]; the ficolins, which are plasma proteins with lectin activity and are thought to be involved in host defense [71]; and, finally, the extracellular matrix proteins, the tenascins [72] – are derived molecules originating from the sponge fibrinogen-like protein In an earlier study it was shown that the deduced proteins from sponges, as the basal metazoans, are more similar to the related sequences from deuterostomians (humans), than to those from protostomians (D melanogaster and C elegans) [73] In line with this view is the assumption that the sponge fibrinogen-like protein gave rise to the blood clotting system in deuterostomians/vertebrates and to lectins and the extracellular matrix proteins, mentioned above Sponges not contain a blood circulation system; however, in previous studies it had been demonstrated that these animals comprise several morphogens and cytokines, e.g the allograft inflammatory factor-1, the glutathione peroxidase, the endothelial monocyte-activating polypeptide or the preB-cell colony-enhancing factor [74], which, in higher metazoan taxa, are transported in vascular systems Next, the level of expression of the fibrinogen-like protein was analysed and its spatial distribution determined It was demonstrated that the expression is strongly upregulated after exposure to curdlan Even after only day of incubation, upregulation is seen To strengthen the conclusions, in situ hybridization studies were performed, which again show that the expression of this gene is primarily seen in the cells lining the canals – the pinacoderm From these two series of experiments it can be concluded that curdlan initiates an activation circuit, from the surface Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1935 of the sponge cell to the gene expression level and back to a further molecule potentially involved in the recognition of the carbohydrate, the fibrinogen-like protein In line with previous data in human blood [66], it could be concluded that the sponge ancestral protein, with its fibrinogen domain, is also involved in binding to the carbohydrate The (1fi3)-b-D-glucans are known to activate a cell metabolism/defense system, both in protostomians and in deuterostomians In the horseshoe crab, this carbohydrate activates the prophenoloxidase activating system [75] In vertebrates, (1fi3)-b-D-glucans stimulate free radical production in macrophages and expression of those genes which are involved in inflammation [22] At present, our knowledge of sponge immune molecules, including cytokines and enzymes involved in blood coagulation and the complement system, is still at the very early stages One dominant domain, which is found in those molecules, and whose existence had already been established in sponges, is the EGF unit By applying PCR identification and subsequent cloning, one gene coding for an EGF precursor was identified The deduced polypeptide comprised, besides three low-density lipoprotein receptor repeats and one transmembrane region, three EGF-like domains A closer classification groups the sponge domain to the calciumbinding EGF-like domains, indicative that the molecule undergoes protein–protein interactions [76] The vertebrate EGF protein is synthesized as a prepro-EGF, which is subsequently processed to the active 53 amino acid EGF (P01133) Three intramolecular disulfide bonds, which are characteristic for EGF and required for biological activity [77], are also found in the sponge and are highly conserved in domain Likewise, amino acids, which are involved in ligand-receptor binding [52] and required for biological activity [51], are present, especially in EGF domain-2 In order to elucidate whether binding of (1fi3)-b-D-glucan to sponge cells changes the steady-state expression level of the sponge EGF precursor gene, Northern blot studies were performed They revealed that the expression level strongly increased during the course of exposure to the carbohydrate The human prepro-EGF contains, like the sponge polypeptide, a transmembrane region, which anchors the molecule into the membrane [78] During activation, the 53 amino acid EGF becomes released In order to establish whether the sponge putative EGF precursor molecule also undergoes processing during incubation with curdlan, an antibody was raised against the recombinant sponge protein This antibody was used to determine the size of the mature peptide in the sponge tissue, before and after exposure to curdlan The data revealed that, almost exclusively, a protein was identified with a size of kDa The signal of this peptide was higher in Western blots of extracts from curdlan-exposed sponge tissue Based on this finding, it is concluded that in the sponge an EGF molecule of similar size to that found in mammals exists Furthermore, the synthesis of this processed protein is upregulated in the presence of curdlan As first proof that the (1fi3)-b-D-glucan-mediated changes in the expression level of the genes studied here might be controlled by the same (or a coupled) pathway, inhibition studies were performed First, it was shown that at least one protein undergoes phosphorylation at tyrosine residue(s) in response to curdlan Therefore, it was then studied whether inhibition of tyrosine kinase-mediated phosphorylation resulted also in a reduction of expression of the three genes studied here As a tool, the secondary metabolite, aeroplysinin, isolated from the sponge V aerophoba [15,16], was applied Aeroplysinin has previously been suggested to be an inhibitor of tyrosine kinase [79] Inhibition studies, with low concentrations of this bioactive compound, almost completely blocked the expression of the genes coding for the (1fi3)-b-D-glucan-binding protein, fibrinogen-like protein and the EGF precursor This result is taken as a strong indication that the curdlan-influenced expression of these three genes, in response to the carbohydrate, are linked No toxicity of aeroplysinin on sponge cells in vitro could be detected following application of the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide viability assay (R Steffen, unpublished results) Taken together, the data presented here demonstrate that sponges, in this study using S domuncula as the model, comprise a (1fi3)-b-D-glucan-binding protein, which bind to (1fi3)-b-D-glucans, e.g curdlan or laminarin After binding of the carbohydrate to the receptor, the expression of the gene coding for the binding protein increases In turn, the potential scavenger molecule, a fibrinogen-related polypeptide, is synthesized Finally, the production of a low-molecular-weight EGF is accelerated, which might result in a further stimulation of cell metabolism Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (Mu/14–3), the Bundesministerium fur Bildung und ă ă Forschung Germany (project: Center of Excellence BIOTECmarin) and the International Human Frontier Science Program (RG-333/ 96-M) References Sarma, A.S., Daum, T & Muller, W.E.G (1993) Secondary ¨ Metabolites from Marine Sponges Akademie gemeinnutziger ¨ Wissenschaften zu Erfurt-Ullstein-Mosby Verlag, Berlin Fusetani, N (2000) Drugs from the Sea S Karger, Basel Proksch, P., Edrada, R.A & Ebel, R (2002) Drugs from the seas – current status and microbiological implications Appl Microbiol Biotechnol 59, 125–134 Wiens, M., Luckas, B., Brummer, F., Ammar, M.S.A., Steen, R., ă Batel, R., Diehl-Seifert, B., Schroder, H.C & Muller, W.E.G ă ¨ (2003) Okadaic acid: a potential defense molecule for the sponge Suberites domuncula Mar Biol 142, 213–223 Muller, W.E.G & Muller, I.M (2003) Origin of the metazoan ă ă immune system: identification of the molecules and their functions in sponges Integr Comp Biol 43, 281–292 Schroder, H.C., Ushijima, H., Krasko, A., Gamulin, V., Schutze, ă ă J., Muller, I.M & Muller, W.E.G (2003) Emergence and disă ă appearance of an immune molecule, an antimicrobial lectin, in basal Metazoa: the tachylectin family J Biol Chem 278, 32810–32817 Bohm, M., Hentschel, U., Friedrich, A., Fieseler, L., Steen, R., ă Gamulin, V., Muller, I.M & Muller, W.E.G (2001) Molecular ă ¨ response of the sponge Suberites domuncula to bacterial infection Mar Biol 139, 1037–1045 Riggi, S.J & Luzio, N.R (1961) Identification of a reticuloendothelial simulating agent in zymosan Am J Physiol 200, 297– 305 ´ 1936 S Perovic-Ottstadt et al (Eur J Biochem 271) Matsushita, M (1990) Curdlan, (1fi3)-b-D-glucan from Alcaligenes faecalis var myxogenes IFO13140, activates the alternative complement pathway by heat treatment Immunol Lett 26, 95–97 10 Masihi, K.N., Madaj, K., Hintelmann, H., Gast, G & Kaneko, Y (1997) Down-regulation of tumor necrosis factor-a, moderate reduction of interferon-1b, but not interleukin-6 or interleukin-10, by glucan immunmodulators curdlan sulfate and lentinan Int J Immunopharmacol 19, 463–468 11 Black, W.A.P., Cornhill, W.J., Dewar, E.T & Woodward, F.N (1951) Manufacture of algal chemicals III Laboratory-scale isolation of laminarin from brown marine algae J Appl Chem 1, 505–517 12 Beschin, A., Bilej, M., Hanssens, F., Raymakers, J., Van Dyck, E., Revets, H., Brys, L., Gomez, J., De Baetselier, P & Timmermans, M (1998) Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade J Biol Chem 273, 24948–24954 13 Lee, S.Y., Wang, R & Soderhall, K (2000) A lipopolysaccharideă and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus Purification, characterization, and cDNA cloning J Biol Chem 275, 1337–1343 14 Engstad, C.S., Engstad, R.E., Olsen, J.O & Østerud, B (2002) The effect of soluble b-1,3-glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood Int Immunopharmacol 2, 1585–1597 15 Kreuter, M.H., Robitzki, A., Chang, S., Steffen, R., Michaelis, M., Kljajic, Z., Bachmann, M., Schroder, H.C & Muller, W.E.G ¨ ¨ (1992) Production of the cytostatic agent, aeroplysinin by the sponge Verongia aerophoba in in vitro culture Comp Biochem Physiol 101C, 183–187 16 Ebel, R., Brenzinger, M., Kunze, A., Gross, H & Proksch, P (1997) Wound activation of prototoxins in the marine sponge Aplysina aerophoba J Chem Ecol 23, 1451–1462 17 Roux, M.M., Pain, A., Klimpel, K.R & Dhar, A.K (2002) The lipopolysaccharide and beta-1,3-glucan binding protein gene is upregulated in white spot virus-infected shrimp (Penaeus stylirostris) J Virol 76, 7140–7149 18 Dimopoulos, G., Richman, A., Muller, H.M & Kafatos, F.C (1997) Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites Proc Natl Acad Sci USA 94, 11508–11513 19 Bachman, E.S & McClay, D.R (1996) Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus Proc Natl Acad Sci USA 93, 6808–6813 20 Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S.N., Iwanaga, S & Kawabata, S (1999) Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen Proc Natl Acad Sci USA 96, 10086–10091 21 Kenjo, A., Takahashi, M., Matsushita, M., Endo, Y., Nakata, M., Mizuochi, T & Fujita, T (2001) Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi J Biol Chem 276, 19959–19965 22 Kataoka, K., Muta, T., Yamazaki, S & Takeshige, K (2002) Activation of macrophages by linear (1fi3)-b-D-glucans J Biol Chem 277, 36825–36831 23 Skorokhod, A., Gamulin, V., Gundacker, D., Kavsan, V., Muller, ă I.M & Muller, W.E.G (1999) Origin of insulin receptor tyrosine ă kinase: cloning of the cDNAs from marine sponges Biol Bull 197, 198–206 24 Kruse, M., Muller, I.M & Muller, W.E.G (1997) Early evolution ă ă of metazoan serine/threonine- and tyrosine kinases: identification of selected kinases in marine sponges Mol Biol Evol 14, 1326–1334 Ó FEBS 2004 25 Krasko, A., Batel, R., Schroder, H.C., Muller, I.M & Muller, ă ă ă W.E.G (2000) Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin Eur J Biochem 267, 4878–4887 26 Novotna, V., Mikes, L., Horak, P., Jonakova, V & Ticha, M (1996) Preparation of water-soluble and water-insoluble poly(acrylamide-allylamine) derivatives of polysaccharides Int J Bio-Chromatogr 2, 37–47 27 Castro, R., Couso, N., Obach, A & Lamas, J (1999) Effect of different b-D-glucans on the respiratory burst of turbot (Psetta maxima) and gilthead seabream (Sparus aurata) phagocytosis Fish Shellfish Immunol 9, 529–541 28 Bohm, M., Muller, I.M., Muller, W.E.G & Gamulin, V (2000) ă ă ă The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula Biol Cell 29, 95–104 29 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 30 Bachmann, M., Mayet, W.J., Schroder, H.C., Pfeifer, K., Meyer ă zum Buschenfelde, K.-H & Muller, W.E.G (1986) Association of ă ă La and Ro antigen with intracellular structures in HEp-2 carcinoma cells Proc Natl Acad Sci USA 83, 7770–7774 31 Wiens, M., Koziol, C., Hassanein, H.M.A., Batel, R & Muller, ă W.E.G (1998) Expression of the chaperones 14–3)3 and HSP70 induced by PCB 118 (2,3¢,4,4¢,5-pentachlorobiphenyl) in the marine sponge Geodia cydonium Mar Ecol Prog Series 165, 247–257 32 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidmann, J.G & Struhl, K (1995) Current Protocols in Molecular Biology John Wiley and Sons, New York 33 BLAST (2003) http://www.ncbi.nlm.nih.gov/blast/blast.cgi 34 FASTA (2003) http://www.ncbi.nlm.nih.gov/BLAST/fasta.html 35 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673– 4680 36 Felsenstein, J (1993) PHYLIP, Version 3.5 University of Washington, Seattle 37 Dayhoff, M.O., Schwartz, R.M & Orcutt, B.C (1978) A model of evolutionary change in protein In Atlas of Protein Sequence and Structure (Dayhoff, M.O., ed.), pp 345–352 National Biomedical Research Foundation, Washington, DC 38 Nicholas, K.B & Nicholas, H.B Jr (1997) GENEDOC: a Tool for Editing and Annotating Multiple Sequence Alignments, Version 1.1.004 Distributed by the author; http://www.psc.edu/biomed/ genedoc 39 Schutze, J., Krasko, A., Diehl-Seifert, B & Muller, W.E.G ă ă (2001) Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium J Cell Sci 114, 3189– 3198 40 Grebenjuk, V.A., Kuusksalu, A., Kelve, M., Schutze, J., Schroder, ă ă H.C & Muller, W.E.G (2002) Induction of (2Â)5Â) oligoadenylate ă synthetase in the marine sponges Suberites domuncula and Geodia cydonium by the bacterial endotoxin lipopolysaccharide Eur J Biochem 269, 1382–1392 41 Polak, J.M & McGee, J.D (1998) In Situ Hybridization Oxford University Press, Oxford ´ 42 Perovic, S., Schroder, H.C., Sudek, S., Grebenjuk, V.A., Batel, R., ă Stifaniổ, M., Muller, I.M & Muller, W.E.G (2003) Expression of ă ă one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation Evol Dev 5, 240–250 43 Rao, J & Argos, P (1986) A conformational preference parameter to predict helices in integral membrane proteins Biochem Biophys Acta 869, 197–214 Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-D-glucan (Eur J Biochem 271) 1937 44 PC/GENE (1995) Data Banks CD-ROM; Release 14.0 Intelligenetics, Inc., Mountain View, CA 45 Doolittle, R.F (1984) Fibrinogen and fibrin Annu Rev Biochem 53, 195–229 46 Spraggon, G., Applegate, D., Everse, S.J., Zhang, J.Z., Veerapandian, L., Redman, C., Doolittle, R.F & Grieninger, G (1998) Crystal structure of a recombinant alphaEC domain from human fibrinogen-420 Proc Natl Acad Sci USA 95, 9099–9104 47 Barrett, A.J., Rawlings, N.D & Woessner, J.F (2002) Handbook of Proteolytic Enzymes Academic Press, Amsterdam 48 Ohashi, T & Erickson, H.P (1998) Oligomeric structure and tissue distribution of ficolins from mouse, pig and human Arch Biochem Biophys 360, 223–232 49 Isrec-Server (2003) http://hits.isb-sib.ch/cgi-bin/hits_motifscan 50 Boonstra, J., Rijken, P., Humbel, B., Cremers, F., Verkleij, A & van Bergen en Henegouwen, P (1995) The epidermal growth factor Cell Biol Int 19, 413–430 51 Ray, P., Mey, F.J., Montelione, G.T., Liu, J.F., Narang, S.A., Scheraga, H.A & Wu, R (1988) Structure–function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene Biochemistry 27, 7289–7295 52 Engler, D.A., Montelione, G.T & Niyogi, S.K (1990) Human epidermal growth factor Distinct role of tyrosine 37 and arginine 41 in receptor binding as determined by site-directed mutagenesis and nuclear magnetic resonance spectroscopy FEBS Lett 271, 47–50 53 Cimino, G & Ghiselin, M.T (2001) Marine natural products chemistry as an evolutionary narrative In Marine Chemical Ecology (McClintock, J.B & Baker, B.J., eds), pp 115–154 CRC Press, Boca Raton 54 Muller, W.E.G., Brummer, F., Batel, R., Muller, I.M & Schroder, ¨ ¨ ¨ ¨ H.C (2003) Molecular biodiversity Case study: Porifera (sponges) Naturwissenschaften 90, 103–120 55 Thakur, N.L., Hentschel, U., Krasko, A., Pabel, C.T., Anil, A.C & Muller, W.E.G (2003) Antibacterial activity of the sponge ă Suberites domuncula and its primmorphs: potential basis for epibacterial chemical defense Aquat Microbiol Ecol 31, 77–83 56 Muller, W.E.G., Klemt, M., Thakur, N.L., Schroder, H.C., Aiello, ă ă A., DEsposito, M., Menna, M & Fattorusso, E (2003) Molecular/ chemical ecology in sponges: evidence for an adaptive antibacterial response in Suberites domuncula Mar Biol 144, 19–29 57 Bringmann, G., Lang, G., Muhlbacher, J., Schaumann, K., ¨ Steffens, S., Rytik, P.G., Hentschel, U., Morschhauser, J., Brun, ¨ R & Muller, W.E.G (2003) Sorbicillactone A, a structurally ună precedented bioactive novel-type alkaloid from a sponge-derived fungus In Marine Molecular Biotechnology (Muller, W.E.G., ed.), ă pp 231253 Springer-Press, Berlin 58 Ma, C & Kanost, M.R (2000) A b1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade J Biol Chem 277, 36825–36831 59 Sritunyalucksana, K., Lee, S.Y & Soderhall, K (2002) The betaă ¨ 1,3-glucan binding protein from the black tiger shrimp, Penaeus monodon Dev Comp Immunol 26, 237–245 60 Cerenius, L., Liang, Z., Duvic, B., Keyser, P., Hellman, U., Palva, E.T., Iwanaga, S & Soderhall, K (1994) A b-D-glucan binding ă ă protein in crustacean blood Structure and biological activity of a fungal recognition cascade J Biol Chem 269, 29462–29467 61 Umemoto, N., Kakitani, M., Iwamatsu, A., Yoshikawa, M., Yamaoka, N & Ishida, I (1997) The structure and function of a soybean b-glucan-elicitor-binding protein Proc Natl Acad Sci USA 94, 1029–1034 62 Kawabata, S.I., Osaki, T & Iwanaga, S (2003) Innate immunity in the horseshoe crab In Innate Immunity (Ezekowitz, R.A.B & Hoffmann, J.A., eds), pp 109–125 Humana Press, Totowa 63 Gundacker, D., Leys, S.P., Schroder, H.C., Muller, I.M & Mulă ă ¨ ler, W.E.G (2001) Isolation and cloning of the C-type lectin from the hexactinellidian sponge Aphrocallistes vastus: a putative aggregation factor Glycobiology 11, 21–29 64 Wiens, M., Mangoni, A., D’Esposito, M., Fattorusso, E., Korchagina, N., Schroder, H.C., Grebenjuk, V.A., Krasko, A., ă Batel, R., Muller, I.M & Muller, W.E.G (2003) The molecular ă ă basis for the evolution of the metazoan bodyplan: extracellular matrix-mediated morphogenesis in marine demosponges J Mol Evol 57, S60–S75 65 Xu, X & Doolittle, R.F (1990) Presence of a vertebrate fibrinogen-like sequence in an echinoderm Proc Natl Acad Sci USA 87, 2097–2101 66 Le, Y., Lee, S.H., Kon, O.L & Lu, J (1998) Human L-ficolin: plasma levels, sugar specificity and assignment of its lectin activity to the fbrinogen-like (FBG) domain FEBS Lett 425, 367–370 67 Davis, S., Aldrich, T.H., Jones, P.F., Acheson, A., Compton, D.L., Jain, V., Ryan, T.E., Bruno, J., Radziejewski, C., Maisonpierre, P.C & Yancopoulos, G.D (1996) Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning Cell 87, 1161–1169 68 Pan, Y & Doolittle, R.F (1992) cDNA sequence of a second fibrinogen a chain in lamprey: an archetypal version alignable with full length b and c chains Proc Natl Acad Sci USA 89, 2066– 2070 69 Patthy, L (1999) Protein Evolution Blackwell Science, Oxford 70 Marazzi, S., Blum, S., Hartmann, R., Gundersen, D., Schreyer, M., Argraves, S., von Fliedner, V., Pytela, R & Ruegg, C (1998) Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes J Immunol 161, 138–147 71 Ichijo, H., Hellman, U., Wernstedt, C., Gonez, L.J., ClaessonWelsh, L., Heldin, C.H & Miyazono, K (1993) Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen- and collagen-like domains J Biol Chem 268, 14505–14513 72 Gulcher, J.R., Nies, D.E., Alexakos, M.J., Ravikant, N.A., Sturgill, M.E., Marton, L.S & Stefansson, K (1991) Structure of the human hexabrachion (tenascin) gene Proc Natl Acad Sci USA 88, 9438–9442 73 Gamulin, V., Muller, I.M & Muller, W.E.G (2000) Sponge ă ă proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans Biol J Linnean Soc 71, 821–828 74 Muller, W.E.G., Blumbach, B & Muller, I.M (1999) Evolution of ¨ ¨ the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum (Porifera) and those in vertebrates Transplantation 68, 1215–1227 75 Soderhall, K & Cerenius, L (1998) Role of the prophenoloxidase ă ă activating system in invertebrate immunity Curr Opin Immunol 10, 23–28 76 Downing, A.K., Knott, V., Werner, J.M., Cardy, C.M., Campbell, I.D & Handford, P.A (1996) Solution structure of a pair of calcium-binding epidermal growth factor-like domain: implication for the Marfan syndrome and other genetic disorders Cell 85, 597–605 77 Taylor, J.M., Mitchell, W.M & Cohen, S (1972) Epidermal growth factor: physical and chemical properties J Biol Chem 247, 5928–5934 78 Rall, L.B., Scott, J., Bell, G.I., Crawford, R.J., Penshow, J.D., Niall, H.D & Coghlan, J.P (1985) Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues Nature 313, 228–231 ´ 79 Rodrigues-Nieto, S., Gozales-Iriarte, M., Carmona, R., Munoz´ Chapuli, R., Medina, M.A & Quesada, A.R (2001) Anti-angiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge FASEB J doi: 10.1096.fj.01-0427fje ... from the Diptera Anopheles gambiae (ENSAN1_ANGA, XP_312118.1), (ENSAN5_ANGA, XP_312116.1) and (BACBP_ANGA, CAA04496.1), as well as the GLUBP from the lobster Homarus gammarus (GLUBP_HOGAM, CAE47485.1)... curdlan Then, extracts were prepared and subjected to PAGE in the presence of a low concentration of SDS and in the absence of b-mercaptoethanol After size separation (Fig 2; lane a) , the proteins... exist to the nonmetazoan and the protostomian/nematode putative proteins present in the database After alignment of all similar sequences, a radial tree was constructed which shows that the sponge