Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany
*Author for correspondence (e-mail: wmueller{at}mail.uni-mainz.de)
Accepted May 23, 2001
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SUMMARY |
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Key words: Geodia cydonium, Metazoa, Sponges, Aggregation factor, Aggregation receptor, Adhesion, Evolution
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INTRODUCTION |
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The major obstacle to the identification of the molecules in the complex and dynamic cell-cell and cell-matrix recognition in sponges was the fact that the underlying molecules involved had not been obtained by molecular cloning. Even until 1994, it remained uncertain if the sponge adhesion molecules display high sequence relationship to functionally related molecules present in higher Metazoa (Gamulin et al., 1994). With the isolation of a galectin (Pfeifer et al., 1993) as the first cell-cell adhesion molecule, and with integrin as the first cell-matrix adhesion receptor in G. cydonium (Pancer et al., 1997; Wimmer et al., 1999), it became obvious that sponges contain highly related molecules known to promote adhesion in Protostomia and Deuterostomia. This finding has been taken as one major clue to the now established view that Metazoa, including Porifera, are of monophyletic origin (Müller, 1995).
Earlier, the galectin of G. cydonium was cloned; sequence analysis revealed that those aa residues that are involved in galectins from mammalian species in binding to galactose are strikingly conserved in the sponge sequence (Pfeifer et al., 1993). The sponge galectin is one polypeptide that is associated with the adhesion system in sponges. The galectin links the AF complex to the membrane-associated AR (Wagner-Hülsmann et al., 1996). The sponge galectin occurs in at least three different sequence isoforms (Pfeifer et al., 1993; Wagner-Hülsmann et al., 1996; Müller, 2001; accession numbers X93925, AJ400908 and AJ400909), all of them present in a soluble and a membrane-associated form (Wagner-Hülsmann et al., 1996; Müller et al., 1997).
The putative AR was recently cloned from G. cydonium and found to comprise 14 scavenger receptor cysteine-rich (SRCR) domains, six short consensus repeats (SCR), a C-terminal transmembrane domain and a cytoplasmic tail (Blumbach et al., 1998). Competition experiments using recombinant AR or antibodies raised against this receptor suggested that the adhesion molecule present in the enriched AF (AF-Fraction 6B) binds to the AR. In addition, previous experiments indicated that the strength of binding of the AF-Fraction 6B to the cell surface AR is augmented by galectin (Wagner-Hülsmann et al., 1996). In the AF-complex from M. prolifera, Burgers group identified a proteoglycan-like core structure, a protein that had been termed MAFp3; it has subsequently been cloned (Fernandez-Busquets et al., 1996; Fernandez-Busquets and Burger, 1997). MAFp3 is likely to be entrapped into a polysaccharide cover (Fernandez-Busquets et al., 1996). Sequence analysis revealed that the gene encoding MAFp3 is highly polymorphic and might be involved in the cell adhesion system during sponge allogeneic reactions (Fernandez-Busquets and Burger, 1997); nevertheless the structure-function relationship of MAFp3 to sponge cell adhesion remains to be investigated (Fernandez-Busquets et al., 1996). Antibodies raised against the M. prolifera MAFp3 protein were found to identify, besides the core protein of the AF, a 68 kDa protein that does not belong to the AF (Fernandez-Busquets et al., 1998). Also, the cDNA libraries from G. cydonium were successfully screened for a sequence related to that from M. prolifera (Müller et al., 1999). Until now no further proteins have been cloned from the AF-complex even though a series of protein species have been demonstrated on protein level both in M. prolifera (Fernandez-Busquets and Burger, 1999) and in G. cydonium (Müller, 1982). It has even been suggested that more than one AF complex may exist, or that additional proteins are present on the cell membrane that contribute to the histocompatibility reaction of sponges, or that both scenarios co-exist (Fernandez-Busquets and Burger, 1999). In addition, evidence has been presented indicating that homologous AF complexes interact with each other (see Fernandez-Busquets and Burger, 1999). It should be stressed here that, besides protein-protein interactions, glycan-glycan binding reactions might contribute to AF-mediated cell-cell adhesion reactions (Misevic, 1999).
In the present study we identified one molecule of the AF complex in the G. cydonium system by raising antibodies against the enriched AF-Fraction 6B, which were found to inhibit cell-cell aggregation. The antibodies were used for immunoscreening of the G. cydonium cDNA expression library. A cDNA was isolated; the deduced polypeptide, termed putative aggregation factor (putative AF), displayed a distant relationship to amphiphysin II and the bridging integrator protein, two families of molecules that interact with integrin (Wixler et al., 1999). Functional studies revealed that the recombinant, putative AF abolishes the AF-complex-mediated cell adhesion in the homologous system. Consequently, it is concluded that the putative AF described here, is a component that is functionally involved in cell adhesion mediated by the AF-complex.
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MATERIALS AND METHODS |
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Buffers
The descriptions of Ca2+- and Mg2+-free seawater (CMFSW), of CMFSW containing EDTA (CMFSW-E) as well as of Ca2+ and Mg2+-containing artificial seawater (ASW) were given earlier (Rottmann et al., 1987).
Sponge and sponge components
Live specimens of G. cydonium (Porifera: Demospongiae: Tetractinomorpha: Astrophorida: Geodiidae) were collected near Rovinj, Croatia. The tissue samples were either immediately frozen in liquid nitrogen until use or were immediately processed for immunofluorescence analysis. The preparation of viable cells was described earlier (Müller and Zahn, 1973). The crude extract from G. cydonium tissue was obtained by homogenization of 5 g of sponge tissue with 15 ml of a 25 mM of Tris/HCl buffer (pH 7.5, 150 mM NaCl, 20 mM 2-mercaptoethanol, 50 µM phenylmethylsulfonyl fluoride); after centrifugation (90 minutes, 80,000 g), the supernatant obtained was collected. It contained 4.2 mg/ml of protein. Under these conditions the soluble galectins have been obtained (Müller et al., 1997).
Isolation and enrichment of the aggregation factor
The procedure for the isolation of the G. cydonium AF was as described (Müller and Zahn, 1973; Conrad et al., 1984; Wagner-Hülsmann et al., 1996). Briefly, crude extract was prepared from 50 g of tissue in CMFSW-E. After centrifugation the cell-free supernatant was supplemented with CaCl2 to precipitate the AF. The suspension was centrifuged and the resulting pellet was treated with CMFSW-E. After centrifugation the supernatant was applied to a Sepharose 6B column and elution was performed with CMFSW. The fractions eluting in the first peak, the void volume, was collected and termed AF-Fraction 6B. The protein concentration in the pooled AF-Fraction 6B was 4.8 mg/ml.
Antibodies
Polyclonal antibodies (PoAb) against the AF-Fraction 6B were raised in female rabbits (White New Zealand). Enriched AF-Fraction 6B (10 µg of protein) was injected at 4-week intervals. After three boosts, serum was collected and the antibodies prepared (Harlow and Lane, 1988). The PoAb selected for these studies were termed PoAb-AF. In control experiments, 100 µl of the PoAb-AF were adsorbed to 50 µg of enriched AF-Fraction 6B (30 minutes; 4°C) prior to its use. Preparation of Fab' fragments of PoAb-AF was performed as follows. PoAb-AF were purified by affinity chromatography using protein A agarose macrobeads (Harlow and Lane, 1988). IgG molecules were fragmented by enzymic digestion to (Fab')2 fragments using insoluble papain attached to agarose; Fab' fragments were obtained by subsequent reduction and alkylation (Acheson and Gallin, 1992).
Western blotting
Gel electrophoresis of the protein extracts was performed in polyacrylamide gels (the percentage is given with the respective experiments) containing 0.1% NaDodSO4 (PAGE), as previously descibed (Laemmli, 1970). Protein samples were subjected to gel electrophoresis in the presence of 2-mercaptoethanol and stained with Coomassie brilliant blue. Semi-dry electrotransfer was performed onto PVDF-Immobilon as described (Kyhse-Andersen, 1984). Membranes were processed (Bachmann et al., 1986) and incubated with PoAb-AF (diluted 1:500) for 90 minutes at room temperature. After blocking the membranes with 5% bovine serum albumin, the immune complexes were visualized by incubation with anti-rabbit IgG (alkaline phosphatase conjugated), followed by staining with 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate. In one control experiment, the PoAb-AF (100 µg) was treated with 20 µg of enriched AF-Fraction 6B prior to use in western blots.
Cell aggregation and aggregation inhibition assay
In the standard incubation assay (3 ml volume) a suspension of 25±5x106 cells per ml of ASW was placed into glass tubes and rolled by 35 rpm at 20°C (Müller and Zahn, 1973). Enriched AF-Fraction 6B, galectin and/or Fab' fragments (PoAb-AF) were added at the indicated concentrations. The suspension was incubated for 60 minutes. The size of the aggregates formed was determined optically and is given in micrometers (Müller et al., 1979).
Following a previously described procedure (Brackenbury et al., 1977), the AF-Fraction 6B (in a 10-fold higher concentration than used in the aggregation assay) was pre-incubated for 30 minutes at 20°C together with 100 µg of the Fab' fragments and subsequently added to the cells.
In one series of experiments the single cells at a density of 1±5x108 were treated with 0-3 µg/ml of recombinant AF, rAF_GEOCY, in CMFSW for 60 minutes (4°C). The cells were then washed in CMFSW by centrifugation (10 minutes at 800 g), adjusted to the cell concentration required for the aggregation assay and incubated in ASW with 100 µg AF-Fraction 6B per assay.
Screening of cDNA library
The cDNA library from G. cydonium (Pfeifer et al., 1993) was screened with PoAb-AF, according to a described procedure (Young and Davis, 1983). Positive clones were isolated, rescreened twice and converted into the plasmid vector (pBK-CMV, Stratagene) using the rapid excision kit (Stratagene) according to the manufacturers instructions. The sequence obtained, termed GEOCYAF, was confirmed by screening the library using PCR.
Sequence analysis
The sequences were analyzed using computer programs BLAST (http://.www.ncbi.nlm.nih.gov/BLAST/) and FASTA (http://www.ebi.ac.uk/fasta3). Multiple alignments were performed with CLUSTAL W Ver. 1.6 (Thompson et al., 1994). Phylogenetic trees were constructed on the basis of aa sequence alignments by neighbour-joining, as implemented in the Neighbor program from the PHYLIP package (J. Felsenstein, University of Washington, Seattle). The distance matrices were calculated using the 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 (K. B. Nicholas and H. B. Nicholas Jr (1997). GeneDoc: a tool for editing and annotating multiple sequence alignments. Version 1.1.004. http://www.psc.edu/biomed/genedoc). Hydropathicity analysis (window size 15 aa) was performed as described (Klein et al., 1985).
Protein expression
Expression of the GEOCYAF gene was performed in E. coli using the GST (glutathione-S-transferase) Fusion system (Amersham) as described (Ausubel et al., 1995; Coligan et al., 2000) and following the instructions of the manufacturer. The GEOCYAF clone was introduced into the pGEX2T plasmid containing the Schistosoma japonicum glutathione S-transferase gene and expressed with IPTG. The fusion protein was purified by affinity chromatography on glutathione Sepharose 4B (Coligan et al., 2000). If not mentioned otherwise, this recombinant fusion protein, rAF_GEOCY, was used for the experiments. In one series of experiments, the fusion protein was cleaved with thrombin (10 units/mg) to separate glutathione-S-transferase from the recombinant sponge putative AF.
Histological analysis
Fresh tissue from G. cydonium was transferred to isopentane and cooled to -80°C. After transfer for 1 hour into 6% sodium fluoride to dissolve the spicules, the samples were washed in ASW. Then the tissue was fixed in 4% (w/v) paraformaldehyde in CMFSW-E, which was supplemented with 1% (w/v) sodium borohydrate to suppress autofluorescence (Pancer et al., 1996). After a further wash in CMFSW-E the samples were transferred into 20% (w/v) sucrose in CMFSW-E for 10 minutes and then immediately frozen at -80°C in isopentane. The frozen tissue was sectioned in a cryostat at -20°C. Sections, measuring 8 µm, were collected on 3-aminopropyltriethoxy-silane-coated slides (Ohno et al., 1994). After fixation to the slides the cells were made permeable with 0.1% saponin (Hafen et al., 1983), washed in PBS and incubated with PoAb-AF (1:500 dilution) for 30 minutes. The slides were then incubated with FITC-conjugated goat anti-rabbit IgG for 2 hours. The sections were inspected by immunofluorescence with an Olympus AHBT3 microscope.
Binding of recombinant putative AF to G. cydonium galectin
To demonstrate that the recombinant putative AF binds to the homologous galectin, rAF_GEOCY was either used directly as fusion protein with the glutathione S-transferase or the fusion protein was used after digestion (see above). The recombinant proteins were size separated on a 12% polyacrylamide gel containing 0.1% NaDodSO4. Subsequently, the proteins were transferred to PVDF-Immobilon membrane and incubated for 2 hours with 400 µg/ml of the crude sponge extract. The blots were washed and finally incubated with the mouse monoclonal antibody (McAb) IIIc8 (1:500 dilution) raised against the G. cydonium galectin (Wagner-Hülsmann et al., 1996), anti-galectin McAb. After incubation, the immunocomplex was detected as described above.
Protein content was determined with the Lowry method (Lowry et al., 1951).
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RESULTS |
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Immunohistological analysis
Sections through tissue from of G. cydonium were prepared and reacted with antibodies against the AF-Fraction 6B, PoAb-AF. The immunocomplexes were visualized with a secondary FITC-labeled antibody. As shown in Fig. 4A the cells in the mesohyl of the sponge were brightly stained, whereas, in the control sections that were treated with only the secondary antibody (Fig. 4B), a scattered, faint staining was visible, due to residual autofluorescence. The autofluorescence could be largely abolished by sodium borohydrate, as described under Materials and Methods.
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The 1710 bp long nucleotide sequence, termed GEOCYAF, has an open reading frame (ORF) of 1242 nt (accession number AJ311598). Northern blot analysis revealed a single band of 1.7 kb, indicating that the clone is of full length (not shown). The translation product of GEOCYAF has 414 aa and was named AF_GEOCY, (Fig. 5A) has a calculated size of Mr 46,558 and a pI of 4.75; according to its computed instability index of 65.7 it is a predicted to be an unstable protein (PC/GENE (1995). Data Banks CD-ROM; Release 6.85. (A. Bairoch, University of Geneva, Switzerland, IntelliGenetics, Inc. Mountain View, CA). One potential N-glycosylation site is found at residue Asn61 (Isrec-Server (2001) http://www.isrec.isb-sib.ch/cgi-bin/PFSCAN_form_parser).
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Phylogenetic analyses of the putative AF
Searching the databank (BLAST databank) with the deduced G. cydonium putative AF revealed highest similarity with the deuterostomian (Homo sapiens) amphiphysin and BIN1 sequences with significance scores of 10-55 and alignment scores (in bits) of >200, as found in the respective BLASTP report (Coligan et al., 2000). A moderate similarity is also present between the sponge putative AF and the protostomian sequences of Drosophila melanogaster amphiphysin (10-45, 178; accession number AJ242855.1) and of the Caenorhabditis elegans amphiphysin-like protein (10-37, 152; Z68217). In this context it must be mentioned that, in contrast to the sponge sequence, the D. melanogaster amphiphysin contains an SH3 domain. Only distantly related are the viability/starvation protein RVS161 from Saccharomyces cerevisiae (10-10, 64; NP_009935), and the hypothetical protein MDF20 from Arabidopsis thaliana (10-4, 44; AB009050-BA000015).
After alignment, a phylogenetic tree was constructed, using the plant sequence as an outgroup (Fig. 5B). It shows that the amphiphysin/BIN molecules from Metazoa form one branch and exclude the yeast sequence. The tree suggests that the sponge sequence displays a higher similarity to the human sequences; however, this overall higher similarity is due to the closer similarity of the sponge putative AF to the two human sequences with respect only to the N-terminal part of the sequence. With respect to the C-terminus, the sponge sequence is ancestral since the protostomian sequences share the functional important SH3 domain with the human amphiphysin/BIN molecules.
Recombinant putative AF
A 1133 nt-long segment, corresponding to nt148 to nt1281 (the start ATG of ORF is located at nt40-42), of the cDNA GEOCYAF was expressed as a GST fusion protein in E. coli. After induction of the ß-galactoside promotor with IPTG, one band of 62 kDa became strongly visible in the bacterial lysate after NaDodSO4-PAGE (Fig. 6A, lanes b,c versus lane a). After purification, the 62 kDa fusion protein was obtained (Fig. 6A, lane c), comprising the 26 kDa GST moiety (Coligan et al., 2000) and the 36 kDa GEOCYAF fragment.
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Functional study with the recombinant putative AF
Incubation of the rAF_GEOCY polypeptide with single cells from G. cydonium prior to the addition of the AF-Fraction 6B resulted in a strong reduction of the size of the aggregates formed in the presence of the high concentration of AF-Fraction 6B (100 µg/assay). In the absence of rAF_GEOCY, the aggregates measure 2,300±250 µm is measured, whereas, after a preincubation with 3 µg of rAF_GEOCY per assay and a subsequent incubation with 100 µg AF-Fraction 6B/assay, the size of the aggregates is reduced to 430±65 µm (Fig. 2A; Fig. 3D). In a further series of experiments the cells were pretreated with increasing concentrations of rAF_GEOCY. After one washing step the cells were incubated for 60 minutes with AF-Fraction 6B. The results show that the recombinant rAF_GEOCY at a concentration of 0.3 µg/assay and higher, significantly reduced the size of the aggregates formed after 60 minutes (Fig. 2B).
From these data we conclude that the recombinant putative AF from G. cydonium competes with the binding protein present in the AF complex (AF-Fraction 6B) for the cell membrane-associated AR/galectin molecule(s).
Binding of the G. cydonium galectin to the recombinant putative AF
A modified western blotting approach was chosen to clarify if the recombinant putative AF, AF_GEOCY binds to the sponge galectin. Therefore, the undigested (Fig. 7, lane b) as well as the digested rAF_GEOCY (Fig. 7, lane c) was applied onto the gel. After PAGE and subsequent incubation of the blot with crude extract that contains the galectin, the binding of the rAF_GEOCY to the galectin was demonstrated by the anti-galectin McAb IIIc8. In this approach the antibody recognized the galectin:full-size fusion protein (Mr 62 kDa; Fig. 7, lane b) or the processed form (Mr 36 kDa; Fig. 7, lane c).
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DISCUSSION |
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In G. cydonium the AF is known to associate with the AR; the strength of this binding is enhanced by galectin (Pfeifer et al., 1993; Wagner-Hülsmann et al., 1996). In the present study, antibodies were raised against the AF complex; the high molecular weight fraction AF-Fraction 6B was used to identify the molecule that binds to galectin. This approach resulted in the isolation of a cDNA encoding a protein termed putative AF, AF_GEOCY. The molecule was expressed and the recombinant protein shown to inhibit the AF-Fraction 6B-mediated cell-cell interaction. This finding indicates, first, that the cloned putative AF is involved in cell adhesion and, second, that the recombinant, putative AF competes with the particle (core structure)-associated adhesion molecule for the binding site at the cell surface. The latter result can be explained by the assumption that the recombinant putative AF has bifunctional activity: to bind to the AR/galectin at the cell surface and to associate with the AF core structure.
In an approach to demonstrate that the putative AF binds to the cell surface-AR via galectin, western blot studies have been performed. These studies revealed that the putative AF binds to galectin present in a crude extract obtained from the same sponge species. Hence, the following schematic model for the AF-mediated cell-cell interaction can be outlined. The core structure of the sponge AF is associated with at least two adhesion-promoting proteins: the 86 kDa selectin-related molecule and the 36 kDa putative AF. For the latter molecule, it is demonstrated in the present study that it associates with galectin, which in turn, as reported earlier, binds to the AR (Wagner-Hülsmann et al., 1996) (Fig. 8). The G. cydonium galectin molecules have only one carbohydrate binding site that is specific for galactans (Müller et al., 1997); galectin-galactan interaction requires Ca2+ for full activity (Diehl-Seifert et al., 1985). Consequently, it might be adopted that two galectins are involved in the bridging of the AR to the putative AF. Both the putative AF (one N-glycosylation site) and the aggregation receptor (24 N-glycosylation sites of the large membrane-spanning isoform) (accession number Y14953) from G. cydonium have potential glycosylation sites that could harbor oligosaccharide side-chains. However, it is more likely that only one molecule of galectin is involved in this binding since the putative AF, obtained in a recombinant form from E. coli, which has been applied here, can bind to the galectin. It must be stressed that there is very limited knowledge of the type of glycosylation of sponge proteins as well as of the composition of the carbohydrate chains in Porifera.
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Sequence analysis of the putative AF indicates that the N-terminal part is highly similar to the human amphiphysin II and BIN1 protein. However, in the C-terminal portion of the sponge molecule, the SH3 domain, likely to be involved in amphiphysin/BIN1 protein-protein interaction, is lacking. In this region the putative AF shares no obvious relationship to non-sponge molecules. It should be noted that the sponge putative AF with its distant relationship to amphiphysin/BIN1 shares only a low similarity to a S. cerevisiae molecule and only a very distant similarity to an A. thaliana molecule. This result corroborates earlier data that indicate a common ancestry of Metazoa and Yeast and only a distant relationship with Viridiplantae (Müller, 2001). As outlined in the Introduction, the sponge AF is assumed to be released from the cells via exocytosis. Hence it might be assumed that the putative AF and the amphiphysin/BIN1 molecules share a common ancestor molecule that might be involved in plasma membrane fusion processes. The size of the putative AF, after identification by PAGE/western blots of the protein both in the AF-Fraction 6B and in its recombinant form is 36 kDa. By contrast, the Mr for the predicted polypeptide corresponding to the ORF from the cDNA GEOCYAF is 47 kDa. Since the antibody used for the detection of the native as well as the recombinant proteins was the same as the one used for the identification of the cDNA, it can be assumed that either the protein is processed after translation, or the PAGE migration behavior of the putative AF is unusual. The latter explanation appears more likely in view of the fact that over 55% of the putative polypeptide exists in the stable helical form, as predicted by secondary structure analysis (Garnier et al., 1978).
In conclusion, it is demonstrated that the sponge putative AF is a relevant molecule involved in the heterophilic cell-cell interaction in sponges. This molecule comprises a sequence similarity to molecules of other metazoan phyla (e.g. to amphiphysin/BIN1), but no significant relationship to yeast or plant molecules. This finding supports the view that the adhesion molecules in Metazoa, with Porifera as the phylogenetically oldest phylum, represent evolutionary novelties that contributed to the successful transition to Metazoa. It remains to be studied if the adhesion molecules or the subsequent molecules, involved in the signal transduction pathway (Rottmann et al., 1987) evolved separately or in parallel. Recent data with immune molecules, the ITAM/ITiM-motif-containing receptors and their regulatory kinase Syk, again autapomorphic characters of Metazoa, suggest a co-evolution (W.E.G.M., unpublished). Taken together, the data reported here demonstrate that the putative AF of G. cydonium is a component which is functionally involved in cell adhesion, and is associated with the AF-complex. These data complement earlier research activities with the sponge M. prolifera, which revealed that the core structure of the AF-complex is composed of a polymorphic, apparently sponge-specific, MAFp3 protein (Fernandez-Busquets and Burger, 1999).
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ACKNOWLEDGMENTS |
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