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(The FASEB Journal. 2000;14:2022-2031.)
© 2000 FASEB

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula1 ,2

HEINZ C. SCHRÖDER*, ANATOLI KRASKO*, RENATO BATEL{dagger}, ALEXANDER SKOROKHOD*, SABINE PAHLER*, MICHAEL KRUSE{ddagger}, ISABEL M. MÜLLER* and WERNER E. G. MÜLLER*3

* Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, D-55099 Mainz, Germany;
{dagger} Center for Marine Research, ‘Ruder Boskovic’ Institute, HR-52210 Rovinj, Croatia; and
{ddagger} Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany

3Correspondence: Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz; Germany. E-mail: WMUELLER{at}mail.UNI-Mainz.DE


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.—Schröder, H. C., Krasko, A., Batel, R., Skorokhod, A., Pahler, S., Kruse, M., Müller, I. M., Müller, W. E. G. Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.


Key Words: Porifera • myotrophin-like molecule • collagen • primmorphs • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPONGES (PORIFERA) REPRESENT the lowest and simplest metazoan phylum still extant today. Since the introduction of molecular biological methods, it has become clear that sponges comprise 1) a series of extracellular matrix molecules, e.g., galectin (1) , 2) cell surface receptors, e.g., integrin (2) , receptor tyrosine kinases (3) , or a neuronal-like receptor (4) , 3) immune molecules, e.g., those comprising immunoglobulin (Ig) -like domains (5 , 6) or the (2'-5') oligoadenylate synthetase (7) , as well as 4) molecules involved in physiological and pathophysiological processes, e.g., apoptotic molecules (8) . These data altogether establish the view that all metazoans are of monophyletic origin (9 , 10) . Collagen can be considered another candidate of an autapomorphic molecule of Metazoa that had been cloned from the sponge Ephydatia muelleri (11) ; however, a collagen-like molecule was also identified in fungi (12) .

In sponges, the body wall consists of two layers of epithelial cells—the external formed by exopinacocytes and the internal by endopinacocytes; both surround the mesohyl (13) . Both types of pinacocytes are associated with collagen fibrils (14) . Until now it was unknown which chemical factors induce undifferentiated cells, the archaeocytes (13) , to become more specialized somatic cells. Recently growth factors have been identified in sponges that share sequence similarity to related molecules found in higher metazoan phyla, e.g., the endothelial-monocyte-activating polypeptide (15) . Until recently, investigations on the function of sponge molecules were hampered by the lack of proliferating cell cultures. From the sponge Suberites domuncula, such a system has now been established; it was demonstrated that dissociated cells retain their proliferation potency if they are assembled in the primmorph system (16 , 17) .

This paper describes the isolation of a cDNA encoding a myotrophin-like molecule from S. domuncula, SUBDOMYOL, that comprises high sequence similarity to the mammalian cardiac myotrophin (18 19 20) . For mammalian cardiac myotrophin it was demonstrated that it stimulates protein synthesis in myocytes (18) , suggesting a crucial role in the formation of cardiac hypertrophy (reviewed in ref 21 ).

It is known that myocardial hypertrophy is associated with a qualitative change in contractile protein composition in general (22) and of collagen specifically (reviewed in ref 23 ). Therefore, it was natural to speculate that recombinant sponge myotrophin may cause an increased expression primarily of those genes that code for structural and morphogenetic proteins. Besides myosin (24) , another candidate molecule, collagen, had already been cloned from a sponge (25) . The cDNA was cloned from the freshwater sponge E. muelleri (11) . The deduced polypeptide molecule was found to be homologous to vertebrate fibrillar collagen (26) ; the sponge collagen was classified to the group of short chain collagens (25) . The cDNA encoding collagen was now isolated from the marine sponge S. domuncula; its deduced protein sequence is similar that from E. muelleri. Using the S. domuncula cDNA as a probe, we show that homologous cells up-regulate the expression of the collagen gene in response to a treatment with recombinant sponge myotrophin.

It is concluded that in sponges the myotrophin-related molecule causes the same/similar effect that has been reported for the mammalian cardiac myotrophin. In consequence, it can be assumed that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The sources of chemicals and enzymes used were given previously (27 , 28) . Natural, Ca2+- and Mg2+-containing seawater (SW) was obtained from Sigma (Deisenhofen, Germany), L-[4,5-3H] lysine (Lys; specific activity of 7.3 Ci/mmol) from Amersham (Amersham, England); DIG (digoxigenin) DNA labeling kit, DIG-11-dUTP, anti-DIG AP Fab fragments, CDP-Star (disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo(3.3.1.13,7)decan}-4-yl)phenyl phosphate) were obtained from Boehringer Mannheim (Mannheim, Germany).

Sponges
Specimens of the marine sponge S. domuncula (Porifera, Demospongiae, Hadromerida) were collected in the Northern Adriatic near Rovinj (Croatia) and then kept in aquaria in Mainz (Germany) at a temperature of 17°C.

Dissociation of cells and formation of primmorphs
The procedure for dissociation of sponge cells was described previously (16 , 17) . Primmorphs, the special form of sponge aggregates, reassociate from single cells after transferring them into medium composed of SW (16 , 17) . A cell suspension was adjusted to a concentration of 106 cells/ml and kept in SW supplemented with 0.1% (v/v) of Marine broth 2216 (DIFCO). After at least 2 days in culture, primmorphs of > 1 mm in diameter (average: 2 to 3 mm) are formed. Where indicated, either dissociated cells, after maintaining them for 1 day in culture, or primmorphs were incubated with recombinant myotrophin (rMYO); every second day the culture medium with the rMYO was changed.

PCR cloning of the S. domuncula myotrophin-like molecule
The complete sponge cDNA encoding the myotrophin-like molecule, termed SUBDOMYOL, was isolated from the S. domuncula cDNA library by polymerase chain reaction (PCR) (27) . The degenerate forward primer, directed against the conserved aa segments within the second ankyrin repeat found in the sequences from the chicken myotrophin V1 (29 , 30) aa65 to aa73 5'-GAT/CAAA/GCAT/CAAT/CATT/C/AACICCICTICT-3' (where I = inosine; double underlined in Fig. 1 ), in conjunction with the vector-specific primer, was used. The PCR reaction was carried out after an initial denaturation step at 95°C for 3 min, then 30 amplification cycles at 95°C for 30 s, 53.2°C for 45 s, 74°C for 1.5 min, and a final extension step at 74°C for 10 min. The reaction mixture was as described earlier (31) . The fragment of 300 bp was used to isolate the cDNA from the library (32) . The longest insert obtained had a size of 582 nt (excluding the poly(A) tail). The clone was termed SUBDOMYOL and was sequenced using an automatic DNA sequenator (Li-Cor 4200).



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  		Figure 1. Nucleotide sequence (nt) and deduced amino acid (aa) sequence of the cloned cDNA encoding S. domuncula myotrophin-like molecule MYOL_SUBDO. The nts are numbered in the 5' to 3' direction, starting with nt triplet encoding the start methionine; the putative start methionine is underlined and the stop codon is marked by an asterisk; the location of the primer is double underlined.

PCR cloning of the sponge collagen
In previous approaches such as heterologous screening using the E. muelleri cDNA (accession number A41207; ref 11 ), we failed to identify the S. domuncula cDNA for collagen in the library. Also, the described collagenous sequence (G-x-y) appeared not to be useful for a PCR screening with degenerate primers because of the high degeneracy of the codons for glycine. Since the deduced aa sequence of the E. muelleri cDNA (11) showed considerable sequence relationship at the noncollagenous carboxyl-terminal domain to the vertebrate type IV collagen (11) , a degenerate primer was selected from this region; more specifically, a forward primer that terminates at tryptophan for which only one codon can be expected. This approach was successful; the forward primer selected was 5'-CIAAT/CGGIGCIGTIGTITAT/CATIAGITGG-3'. The PCR reaction was performed as described for the cloning of the myotrophin cDNA using an annealing temperature of 54°C. A fragment of 800 bp was obtained and used for the isolation of the S. domuncula collagen cDNA SUBDOCOL1; the insert has a size of 1024 nt [without poly(A)].

Sequence comparisons
The sequences were analyzed using computer programs BLAST (33) and FASTA (34) . Multiple alignments were performed with CLUSTAL W Ver. 1.6 (35) . Phylogenetic trees were constructed on the basis of aa sequence alignments by neighbor 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 (37) . The degree of support for internal branches was further assessed by bootstrapping (36) . The graphic presentations were prepared with GeneDoc (38) .

Myotrophin cDNA expression
The insert from SUBDOMYOL was used for expression in Escherichia coli. The cDNA was inserted into the bacterial oligohistidine expression vector pQE-30 (Qiagen, Chatsworth, Calif.). E. coli strain XL1-blue was transformed with this plasmid and expression of fusion protein was induced for 3 h with 2 mM isopropyl 1-thio-ß-D-galactopyranoside (IPTG) (1) . Bacteria from 500 ml cultures were obtained by centrifugation and extracted with 4 ml of phosphate-buffered saline (PBS)/8 M urea. After sonication, the suspension was centrifuged; the supernatant is termed ‘bacterial crude extract’. It is used to purify the recombinant myotrophin. Purification of the fusion proteins, termed rMYO, was performed by metal-chelate affinity chromatography using Ni-NTA-agarose resin (Qiagen) according to Hochuli et al. (39) and the manufacturer’s instructions. One milliliter of the clear extract was applied to the column and after subsequent washing with 10 column volumes of PBS/urea, the native fusion protein was eluted from the column with 150 mM of imidazole in PBS/urea. After dialysis and concentrating to 0.3 mg protein/ml, rMYO was used for the experiments. The purity of the material was checked by 12% polyacrylamide gels containing 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (40) .

Incorporation studies
Assays (5 ml) of single cells or primmorphs from S. domuncula, containing 5 x 106 cells, were incubated with 25 µCi of the labeled DNA precursor [3H]Lys for 1 to 5 h (routinely for 3 h). After an additional incubation period of 12 h, the samples were analyzed for radioactivity in the acid-insoluble (protein) fraction as described (41 , 42) . The values for the radioactivity incorporated were correlated with the amount of protein from the cells used for the determination. Protein content was determined applying the described method (43) using bovine serum albumin as standard.

Northern blotting
RNA was extracted from liquid-nitrogen pulverized sponge tissue with TRIzol Reagent (GibcoBRL, Grand Island, N.Y.) as described in detail before (31) . An amount of 3 µg of total RNA was electrophoresed through a formaldehyde/agarose gel and blotted onto a Hybond N+ membrane, following the instructions of the manufacturer (Amersham). Hybridization experiments were performed with the following probes: the total cDNA inserts of both the S. domuncula myotrophin SUBDOMYOL and of collagen SUBDOCOL1 as well as the S. domuncula ß-tubulin (unpublished results) SDBTUB ({approx} 800 bp). These probes were labeled with DIG-11-dUTP and hybridization was performed as described (31) .

The hybridization signals were detected with anti-DIG Fab fragments (conjugated to alkaline phosphatase) and visualized by the chemiluminescence technique using CDP-Star, the chemiluminescence substrate of alkaline phosphatase, according to the manufacturer’s instructions. To quantitate the signals of the Northern blots, the chemiluminescence procedure was applied (44) . The screen was scanned with the GS-525 Molecular Imager (Bio-Rad, Hercules, Calif.). Relative values for expression of the SUBDOMYOL as well as the SUBDOCOL1 gene in S. domuncula cells were correlated with the intensities of the bands measured for the expression of the tubulin gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PCR cloning and sequencing of the cDNA encoding the sponge myotrophin-like molecule
The cDNA encoding the myotrophin-like polypeptide was isolated from the sponge S. domuncula as described in Materials and Methods. The S. domuncula nt sequence SUBDOMYOL is 582 nt long and has a potential open reading frame (ORF) of 360 nt encoding a 120 aa long deduced protein sequence (Fig. 1) . The putative translation initiation site is medium to strong and follows the sequence C-4-A-G-G-A-T-G- and G+4 (the start codon is underlined; 45 ). Northern blot analysis performed with the sponge SUBDOMYOL clone as a probe yielded one prominent band of {approx}0.7 kb, confirming that a full-length cDNA was isolated (see below).

Deduced aa sequence of sponge myotrophin and phylogenetic analysis
Besides the sequences from vertebrates, until now only the myotrophin-related molecule from Caenorhabditis elegans (46) has been identified. The S. domuncula myotrophin MYOL_SUBDO, with a calculated Mr of 12,837, shares high sequence similarity with the hitherto known metazoan myotrophin (related) sequences (Fig. 2A ). The sponge sequence shows the characteristic features known from the vertebrate myotrophins (30) : one-half of an ankyrin repeat is located at the NH2 terminus of the protein (aa10 to aa28), followed by two complete repeats toward the COOH terminus (aa30 to aa62 and aa63 to aa95); Fig. 2A .



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  		Figure 2. Sponge myotrophin. A) Alignment of the deduced sponge myotrophin from S. domuncula MYOL_SUBDO with the following proteins: the Myotrophin-related molecule deduced from the cDNA yk109a3.3 from Caenorhabditis elegans (109a-CAEEL; accession number AAA96086; ref 46 ), myotrophin V1 protein homologue from chicken (ViP_CHICK; BAA05379; ref 29 ), and myotrophin V1P from mouse (V1P_MOUSE; P80144; ref 19 ). The alignment was performed using CLUSTAL W program. Residues of aa, identical among all sequences, are shown in inverted type; those present in at least three sequences are shaded. The characteristic ankyrin motifs, one-half repeat (half ANK) and two complete repeats (ANK repeat-1 and -2), are indicated. B) Phylogenetic tree using the sponge myotrophin MYOL_SUBDO and the three sequences listed in panel A. The human FEM-1-like death receptor binding protein (FEM1L_HUMAN; AAF05314; ref 47 ) was used as outgroup. The tree was calculated by neighbor-joining. The numbers at the nodes refer to the level of confidence as determined by bootstrap analysis (1000 bootstrap replicates). Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.

The closest similarity of the sponge protein was found to the corresponding molecules from mouse and chicken, mouse myotrophin V1P (19) and chicken myotrophin V1 protein homologue (29) , with 50% (73%) identical (identical plus similar) aa residues. Somewhat less is the sequence relationship to the myotrophin-related molecule yk109a3.3 from C. elegans (46) with 45% (65%). A rooted phylogenetic tree was constructed (Fig. 2B ) with the distantly related human FEM-1-like death receptor binding protein (47) , 30% (47%) identical (identical plus similar) aa, which was chosen as outgroup. The tree revealed that the sponge and the nematode myotrophins fall in one branch while the vertebrate sequences form a second one.

Expression of SUBDOMYOL in S. domuncula
The level of expression of the SUBDOMYOL gene was determined semiquantitatively by Northern blotting. In single cells, the expression of the gene is very low and amounts to {approx} 0.2-fold (Fig. 3 ) with respect to the expression observed in 5-day-old primmorphs. Comparing the level measured in primmorphs with those in intact animals, the level of myotrophin expression is slightly higher in vivo than in the in vitro formed primmorphs (Fig. 3) . For the in vivo study, the SUBDOMYOL expression was determined in tissue from the oscule region, that part of the body where the excurrent canals are joined together and through which the water current is ejected, as well as in tissue taken from the surface of the sponge body where inhalant canals originate. The experiments showed that the myotrophin expression is {approx} 30% higher around the oscule if compared with the other surface of the sponge body.



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  		Figure 3. Level of expression of SUBDOMYOL in S. domuncula cells and primmorphs formed in vitro, and the expression of this gene in tissue taken from an animal. Total RNA (3 µg each) from isolated cells (Cells), from 5-day-old primmorphs (Prim), from tissue samples taken from two areas of the surface of the animals, the oscule region (Osc) as well as the sponge body where inhalant canals originate (Body). The Northern blot analysis to estimate the level of expression of the myotrophin gene has been performed using the SUBDOMYOL probes as described in Materials and Methods. The transcript size of the myotrophin gene is indicated.

Preparation of recombinant myotrophin
SUBDOMYOL was expressed in E. coli. as recombinant oligohistidine-rMYO fusion protein. The bacteria remained either uninduced or the ß-galactoside promotor was induced by IPTG. The extracts were analyzed by PAGE (see Materials and Methods). The staining pattern for protein is shown in Fig. 4 , from uninduced (lane a) and induced cultures (lane b). Subsequently, the fusion protein was purified by affinity chromatography using Ni-NTA-agarose resin; the recombinant protein preparation is almost completely pure with a size of 13.8 kDa (Fig. 4 , lane c). The calculated Mr for the deduced aa sequence of the cDNA MYOL_SUBDO (including the histidine tag) is 13,767.



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  		Figure 4. Analysis of the recombinant oligohistidine-rMYO fusion protein. Lanes a and b: Proteins in the crude extract were prepared either from bacteria that remained untreated (lane a; -) or had been IPTG-treated (lane b; +) were size separated and stained with Coomassie brilliant blue. Lane c: The recombinant rMYO was purified by affinity chromatography and size separated by 12% PAGE. In lane M the protein size markers are given.

Effect of recombinant myotrophin on protein synthesis
One day after dissociation, cells were incubated with rMYO as described in Materials and Methods. In the first series of experiments rMYO was added at a concentration of 0 or 1 µg/ml to the assays and incubated for 1 to 5 h (Fig. 5A ). After 12 h, the incorporation rate of [3H]Lys into the acid-insoluble fraction was determined. The results revealed that the incorporation rates in the cells incubated with 1 µg/ml of myotrophin were fivefold higher than those that remained untreated. After an incubation period of 3 h, 840 ± 110 cpm of [3H]Lys was found to be incorporated into the protein fraction (1 mg); In contrast, only 110 ± 15 cpm of the precursor per mg of protein was incorporated in the absence of myotrophin.



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  		Figure 5. Effect of rMYO on protein synthesis in sponge cells. A) Incorporation of [3H]Lys into acid-insoluble fraction from cells of S. domuncula. Single dissociated cells were incubated with [3H]Lys for up to five h as described in Materials and Methods. The acid-insoluble fraction was determined after an additional incubation period of 12 h and the incorporation values were correlated with the protein content in the sample. The samples remained either untreated (open boxes) or were incubated in the presence of 1 µg/ml of recombinant myotrophin closed boxes). The results are expressed as mean values ± SD; n=10. B) Effect of different concentrations of rMYO (5 h exposure) on the incorporation rate of [3H]Lys into protein fraction. Parallel assays of cells were incubated for 17 h each with the indicated final concentration of rMYO in the assay as indicated.

Next, rMYO was added at different concentrations to cell suspensions (Fig. 5B ). During the 17 h incubation period chosen, a plateau of maximal incorporation was reached within the concentration range of 1 to 10 µg/ml of rMYO; the incorporation rate was ~800 cpm/mg protein.

Effect of myotrophin on the shape of primmorphs
As described before (17) , first irregular aggregates (Fig. 6B ) are formed from dissociated cells (Fig. 6A ) after an incubation period of 1 day. After further incubation (longer than 2 days), round-shaped, ball-like primmorphs, with a diameter of {approx} 2 to 3 mm are formed (Fig. 6C ). In 5-day-old cultures, the ratio between the longest and the smallest axis of the primmorphs varies between the values 1.5 and 1. The calculated volume of the spheres is between 3 and 6 mm3 (n=15).



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  		Figure 6. Effect of myotrophin on the shape of the primmorphs. Starting from dissociated cells (A), first irregular aggregates (B) are formed after an incubation period of 1 day. C) From these aggregates primmorphs are formed after further incubation; here a primmorph after 5 days incubation is shown. D) In the presence of rMYO (1 µg/ml) elongated oval-shaped primmorphs are formed (incubation period: 5 days). Magnification: A, x50 (bar: 0.1 mm); B, x30 (1 mm); C, x12 (1 mm); D, x5 (1 mm).

In a parallel experiment, dissociated cells were immediately treated with 1 µg/ml of myotrophin. Incubation of cells and subsequently of primmorphs in the presence of rMYO yielded predominantly elongated oval-shaped assemblies (Fig. 6D ). The size of the primmorphs varied after an incubation period of 5 days between 3 and 6 mm with respect to the longer axis; the ratio between the longest and the smallest axis of the primmorphs varied between 2.2 and 3.4, leading to a calculated volume between 4 and 8 mm3 (n=15).

PCR cloning and sequencing of the cDNA encoding sponge collagen
The full-length sponge cDNA encoding collagen was obtained by PCR cloning as described in Materials and Methods.

The insert of the cDNA, termed SUBDOCOL1, has a size of 1024 nt. The potential ORF spans from the start codon for methionine (nt33 to nt35) and the stop codon at nt879 to nt881; Fig. 7 . The deduced 282 aa long polypeptide, termed COL1_SUBDO, has a putative size of 29,553. Northern blot analysis performed with the sponge SUBDOCOL1 clone as a probe yielded one prominent band of ~1.1 kb, indicating that the full-length cDNA was isolated (see below). According to the calculation of the physico-chemical parameters (48 ; Physchem), the S. domuncula collagen polypeptide has an instability index of 44.5, classifying it as an unstable protein.



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  		Figure 7. Sponge collagen. Nucleotide and deduced aa sequence (COL1_SUBDO) from the S. domuncula cDNA SUBDOCOL1. The potential start codon for methionine (underlined) and the location of the degenerate primer used to identify the sponge collagen cDNA (double underlined) are shown.

The S. domuncula collagen COL1 SUBDO comprises three segments: 1) the noncollagenous amino-terminal domain (NC1; aa1 to aa26), 2) the collagenous internal domain (COL; aa27 to aa102), and 3) the noncollagenous carboxyl-terminal domain (NC2; aa103 to aa282); Fig. 8A . The collagenous internal domain is unusually short and comprises only 24 G-x-y collagen triplets; only one internal block diverges and comprises the amino acid residues G-P-A-N. In contrast, the deduced collagen polypeptide from E. muelleri internal collagen domain is composed of 57 triplets (11) . The noncollagenous amino-terminal domain of the S. domuncula collagen comprises no significant homology to any protein listed in the data banks, whereas the noncollagenous carboxyl-terminal domain, as expected, shares significant sequence similarity (31%) to the noncollagenous carboxyl-terminal domain to the E. muelleri collagen (Fig. 8A ). No cell attachment site, RGD, is present in the S. domuncula collagen; such a signature is usually present in metazoan collagens (reviewed in ref 49 ).



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  		Figure 8. Phylogenetic analysis of sponge collagen. A) Comparison of the sponge collagen sequence COL1_SUBDO with the related collagen from the freshwater sponge Ephydatia muelleri (COL4_EPHMU; A41207; ref 11 ). The noncollagenous amino-terminal domain (NC1), the collagenous internal domain (COL), and the noncollagenous carboxyl-terminal domain (NC2) are indicated. B) Unrooted phylogenetic tree constructed from the two sponge collagens (COL1_SUBDO and COL4_EPHMU) with two invertebrate sequences, the collagen-related protein 3 precursor from Hydra magnipapillata (COL3_HYDRA; C41132, ref 58 ), and the cuticular collagen from the nematode Brugia pahangi (COL1_BRUGIA; CAA63070, ref 59 ) as well as the vertebrate collagens, the human collagen alpha 1 (V) (COL5A1_HUMAN; NM000093.1, ref 60 ), the chicken collagen alpha 2 (IX) (COLA2_CHICK; S23296, ref 61 ), the collagen alpha 1 (I) from the bullfrog Rana catesbeiana (COLA1_RANA;BAA29028; ref 62 ), and the collagen alpha 1 (I) from the Japanese common newt Cynops pyrrhogaster (COLA1_CYNOPS; BAA36973, ref 63 ). Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.

Phylogenetic analysis of sponge collagen
With this report two collagen sequences are known from sponges, sharing 18% of identical and 25% of similar aa to each other. They are included in a phylogenetic study with most homologous collagen sequences known from members of higher metazoan phyla. The sequences were aligned and an unrooted tree was constructed (Fig. 8B ); this tree cannot be rooted since a suitable outgroup is not available. The tree shows that the invertebrate sequences from Hydra magnipapillata and from the nematode Brugia pahangi fall together with the sponge sequences into one branch, while the most similar collagen sequences from vertebrates (< 13% of identical and < 20% of similar aa) form a second branch (Fig. 8B ).

Expression of SUBDOCOL1 in cells after treatment with myotrophin
The technique of Northern blotting was applied to determine the expression of SUBDOMYOL in cells from S. domuncula semiquantitatively. Single cells remained either untreated or were incubated with 1 µg/ml of recombinant myotrophin. In the absence of myotrophin the expression of SUBDOMYOL was low and remained unchanged during the 5 day incubation period (Fig. 9A ). In contrast, cells that had been exposed to myotrophin showed after 1 day (0.3-fold increase [correlated with the level of expression of tubulin; Fig. 9B ]) and especially after 3 (0.7-fold) and 5 days (1.1-fold) a strong up-regulation of the expression of the gene encoding collagen (Fig. 9A ).



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  		Figure 9. Expression of collagen gene in response to myotrophin. A) Northern blot analyses to estimate the level of expression of the gene has been performed using the sponge collagen SUBDOCOL1 as a probes. Dissociated cells remained either without myotrophin treatment (minus myotrophin) or were treated with 1 µg/ml of myotrophin (plus) for 0 day (control [Con]; first lane), 1 day, 3 days, or 5 days (second to fourth lane). Then RNA was extracted and 3 µg of total RNA each was size separated; after blot transfer, hybridization was performed with the probe. B) The intensities of the transcripts for SUBDOCOL1 correlated with the expression of ß-tubulin (parallel samples of the expression of SUBDOCOL1 in untreated samples) are shown.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Until the introduction of molecular biological techniques, it could not be foreseen that sponges (Porifera) are provided with the basic molecules and signal transduction pathways previously known only from members of higher metazoan phyla, the Eumetazoa. After having cloned cDNAs encoding putative polypeptides, which were found to be similar in sequence composition to those previously identified in Eumetazoa, the classification of Metazoa into 1) Parazoa that include the Porifera and 2) Eumetazoa (50) became questionable. These new data replaced the view that animals (Eumetazoa) with the exception of Porifera are monophyletic (51) ; it is now generally accepted that all Metazoa, including Porifera, have a monophyletic origin (9 , 10 , 52) . Since the integration of molecular biological with cell (tissue) biological techniques, it became likely that the functional properties of these ‘metazoan’ molecules are shared within all metazoans, as has been pointed out for the integrin-mediated pathway (2 , 28) .

The consequences of the new insights in sponge biology lead to reconsideration of whether other pathways and recognition systems so far assumed to be characteristic only for higher Metazoa would not also have their origin in the simplest Metazoa, the sponges. First, focusing on the immune system (reviewed in ref 6 ), studies in the past 2 years revealed that sponges are provided with elements of the mammalian innate immune system, such as molecules containing scavenger receptor cysteine-rich domains or cytokine-like molecules or the (2'-5')oligoadenylate synthetase system. Furthermore, ‘precursors’ of the second type of immune response in mammals, the adaptive immune system, have been traced in sponges (reviewed in ref 6 ); it has been shown that 1) the expression of a lymphocyte-derived cytokine from mammals is up-regulated during non-self recognition (53) or 2) receptors are present that comprise polymorphic immunoglobulin-like domains (54) .

In the present study it is shown that sponges contain a myotrophin-related molecule that shares high sequence similarity to vertebrate cardiac myotrophin. In vertebrates, myotrophin has been implicated in cardiac growth due to an increase in the protein content of myocytes (reviewed in ref 21 ). The signaling involves increase of the levels of a series of proto-oncogenes (55) and an interaction of myotrophin with NF-{kappa}B (20) , processes that are mediated by tyrosine kinase-coupled pathway(s) or are the consequences of a translocation of protein kinase C from the cytosol to the cell membrane (21) . It was suggested that these steps occur in parallel with an increased expression of genes coding for contractile proteins and collagen (21) . Like the other metazoan molecules (30) , the sponge myotrophin polypeptide comprises two complete and one-half ankyrin repeats. These are critical for the demonstrated protein–protein and protein–nucleic acid interactions (30) .

The expression level of the gene encoding myotrophin was determined in S. domuncula cells in vitro as well as in vivo. As shown, the level of expression was low in dissociated single cells whereas in both primmorphs and tissue samples taken from intact animals, the expression was significantly higher. This finding demonstrates again that the survival of sponge cells is crucially dependent on the functional maintenance of an intact cell–cell interaction; as described before, dissociated single cells undergo cell death if they have lost cell–cell contact (56) . Focusing on the expression in vivo, the level is higher in the oscule region compared to other parts of the sponge body. It should be stressed here that from the oscule region originates a primordial organizer activity (57) .

The sponge myotrophin was expressed and the 13.8 kDa oligohistidine-rMYO fusion protein was tested for biological activity. Incubation of cells for a period of 5 days with recombinant myotrophin resulted in a change of the shape of the primmorphs formed. Whereas those aggregates are round shaped and ball-like in the absence of myotrophin, the majority of primmorphs that had been formed in the presence of myotrophin are elongated and oval-shaped assemblies. Their size is slightly larger than measured for primmorphs formed after incubation in the absence of myotrophin.

In the homologous cell system, the sponge recombinant myotrophin displayed an up-regulation of protein synthesis, as demonstrated by incorporation studies with a labeled amino acid precursor. The fivefold increase in incorporation seen in vitro is similarly high, as determined for the effect of the rat cardiac myotrophin on myocytes in vitro (18) . The optimal concentration for the myotrophin effect is seen in both systems at around 1 µg/ml. The finding that the sponge myotrophin causes the same response in homologous system is important. It proves that an extracellular signaling factor, hitherto known to exist only in Metazoa and for the first time during evolution in the phylum Porifera, causes a defined cellular reaction throughout the metazoan kingdom.

To be more specific and to exceed the observations with mammalian cells it was determined whether the recombinant myotrophin causes in sponge cells an up-regulation of the expression of the collagen gene. As a first prerequisite, the sponge collagen cDNA had to be isolated from the S. domuncula system. Applying PCR, a cDNA for a short chain collagen was identified that comprises the characteristic collagen stretch flanked by two noncollagenous terminal domains (11) .

Using the S. domuncula collagen gene expression as one end point marker for an effect of myotrophin on cells in vitro, they were incubated with this factor at a concentration of 1 µg/ml for 5 days. The Northern blot experiments unequivocally show that this polypeptide causes a strong increase of collagen gene expression in dissociated S. domuncula cells. These data show that, as suggested for vertebrate cells, myotrophin causes a stimulatory effect on at least some genes supposed to be expressed also during ventricular hypertrophy in mammalians (22) . The expression of other genes, e.g., the cytokine-related gene for pre-B cell colony-enhancing factor or for glutathione peroxidase, has not been found to be up-regulated during the incubation in the presence of myotrophin under the conditions used (unpublished results).

From these data it can be concluded that sponges are provided with factors (here, Myotrophin) that cause an effect on cells and gene expression comparable or even identical to the corresponding humoral stimuli in mammalian systems. There, myotrophin is involved in the initiation of cardiac hypertrophy. This finding sheds also first light on potential molecular markers of diseases in invertebrates.


   ACKNOWLEDGMENTS
 
We thank Ms. Renate Steffen for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Mü 348/12–3), the Commission of the European Communities (project ‘SPONGE’, and the International Human Frontier Science Program (RG-333/96-M).


   FOOTNOTES
 
1 The sequences reported here are being deposited in the EMBL data base, Suberites domuncula gene coding the myotrophin-like molecule (accession number AJ252240) and the collagen-1 (AJ252241).

2 This paper is dedicated to our close friend and colleague, Dr. Annie-Pierre Sève (Hôpital Saint-Louis, Paris), in commemoration.

Received for publication January 20, 2000. Revision received April 20, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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