arginine kinase in the demosponge suberites domuncula: regulation

637

Sponges (phylum Porifera) are sessile filter feeders that insome marine biotopes represent the most dominant metazoantaxon on the benthos. Their growth rate is remarkably high, butonly a few exact measurements have been published. For somespecies, e.g. Haliclona loosanoffi, an increase in size duringthe summer period of up to tenfold has been described(Hartman, 1958). It is therefore conceivable that they consumeconsiderable amounts of organic matter to maintain theirmetabolism. Some sponges filtrate 0.002–0.84·cm3 ofwater·s–1·cm–3 of sponge tissue through their aquiferous canalsystem (see Osinga et al., 2003). Calculated from this immensefiltration capacity, sponges such as Pseudosuberites andrewsitake up approx. 3 million particles (mostly algae)per·cm3·tissue·min–1 from the surrounding aqueous milieu(Osinga et al., 2001). The particle/food uptake andconsumption depends strongly on the ambient physicalconditions, i.e. air, light, oxygen, salinity and temperature(Osinga et al., 1999; Gatti et al., 2002). But another factor,silica, also strongly influences the growth rate, the form of thespicules and the body size of these animals. Sponges from the

classes Demospongiae and Hexactinellida possess a skeletoncomposed of polymerized silicic acid, silica, while the thirdsponge class, Calcarea, build their skeleton from calciumcarbonate, a salt that is, in contrast to silicic acid, usually notrate limiting in the aqueous milieu.

Silica contributes over 60% of the dry demosponge biomass(Desqueyroux-Faundez, 1990), sometimes even reaching 90%(Barthel, 1995). Since the average concentration of silicain seawater is low, with values of less than 3·µmol·l–1

(Maldonado et al., 1999) close to the surface, it can beextrapolated that most of the energy generated by sponges isrequired to build the siliceous skeleton. This assumption issupported by a recent study, which demonstrated that spongesare provided with a (potential) silicic acid transporter thatdepends on the establishment of energy-requiring iongradients; this (potential) transporter comprises a 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) bindingsite (Schröder et al., 2004a). DIDS prevents uptake of silicicacid in sponge cells, indicating that the (potential) silicic acidtransporter is involved in the formation of silica in the spicules.

The Journal of Experimental Biology 208, 637-646Published by The Company of Biologists 2005doi:10.1242/jeb.01428

In Demospongiae (phylum Porifera) the formationof the siliceous skeleton, composed of spicules, is anenergetically expensive reaction. The present studydemonstrates that primmorphs from the demospongeSuberites domuncula express the gene for arginine kinaseafter exposure to exogenous silicic acid. The deducedsponge arginine kinase sequence displays the twocharacteristic domains of the ATP:guanidophosphotransferases; it can be grouped to the ‘usual’mono-domain 40·kDa guanidino kinases (argininekinases). Phylogenetic studies indicate that the metazoanguanidino kinases evolved from this ancestral spongeenzyme; among them are also the ‘unusual’ two-domain80·kDa guanidino kinases. The high expression level of thearginine kinase gene was already measurable 1 day after

addition of silicic acid by northern blot, as well as by insitu hybridization analysis. Parallel determinations ofenzyme activity confirmed that high levels of argininekinase are present in primmorphs that had been exposedfor 1–5 days to silicic acid. Finally, transmission electron-microscopical studies showed that primmorphs containinghigh levels of arginine kinase also produce siliceousspicules. These data highlight that silicic acid is aninorganic morphogenetic factor that induces theexpression of the arginine kinase, which in turn probablycatalyzes the reversible transfer of high-energyphosphoryl groups.

Key words: sponge, arginine kinase, siliceous spicule, Suberitesdomuncula, primmorph, energy metabolism.

Summary

Introduction

Arginine kinase in the demosponge Suberites domuncula: regulation of itsexpression and catalytic activity by silicic acid

Sanja Perovic-Ottstadt1, Matthias Wiens1, Heinz-C. Schröder1, Renato Batel2, Marco Giovine3,Anatoli Krasko1, Isabel M. Müller1 and Werner E. G. Müller1,*

1Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099Mainz, Germany, 2Center for Marine Research, ‘Ruder Boskovic’ Institute, HR-52210 Rovinj, Croatia and

3CNR-Direzione Progetto Finalizzato Biotecnologie, Via Leon Battista Alberti 4, I-16132 Genova, Italy*Author for correspondence (e-mail: [email protected])

Accepted 1 December 2004

THE JOURNAL OF EXPERIMENTAL BIOLOGY

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The silica of the spicules is synthesized enzymatically with thehelp of silicatein (Cha et al., 1999; Krasko et al., 2000). Thesubstrate required for silicatein to form silica is actively takenup by the tissue and finally by the cells, sclerocytes, whichform the spicules (Schröder et al., 2004a).

Since the studies of Roche et al. (Roche and Robin, 1954;Roche et al., 1957) it has been well established that spongescontain the creatine phosphate/creatine kinase system. Thesefindings were confirmed by molecular biological studies on thedemosponge Tethya aurantia (Ellington, 2000; Sona et al.,2004). Based on their data it was hypothesized that the creatinekinase system evolved early in metazoan evolution inorganisms composed of highly polarized cells (Ellington,2001). In line with these studies, we extend the availablebiochemical/molecular biological data for sponges toformulate the pathway involved in the formation of ATP in thecytoplasm.

In eukaryotic cells the major portion of ATP is generated inthe mitochondria via oxidative phosphorylation; the highenergy equivalents have then to be transported to thecytoplasm. The enzymes that catalyze the reversible transfer ofhigh-energy phosphoryl groups of ATP to naturally occurringguanidine compounds, e.g. creatine, glycocyamine,taurocyamine, lombricine and arginine, are the phosphagenkinases (see Suzuki et al., 1997). In vertebrates,phosphocreatine is the only phosphagen, and the correspondingphosphagen kinase is creatine kinase, while in invertebratesmore kinases corresponding to the respective phosphagen havebeen described (see Muhlebach et al., 1994). On the groundsof the high sequence similarity among these kinases it has beenproposed that members of the phosphagen kinases evolvedfrom one common ancestor (Suzuki and Furukohri, 1993);however, to date the evolutionary processes are not fullyunderstood (Ellington, 2001). Phosphagen kinases are missingin fungi and plants.

In the present study we show that the gene encoding argininekinase is differentially highly expressed during exposure ofsponges to silicic acid. We identified the cDNA from thedemosponge Suberites domuncula and determined itsexpression level. In parallel, the enzymic activity of argininekinase was monitored to establish the importance of thisenzyme during siliceous spicule formation. S. domuncula issuitable for such experimental studies since it can be maintainedin aquaria for over 3 years (Le Pennec et al., 2003) andproliferating cells from this sponge can be cultivated in 3D-cellcultures, termed primmorphs (Müller et al., 1999); furthermorean Expressed Sequence Tags (EST) library containing over15·000 cDNAs is available (http://spongebase.genoserv.de/).Searching this database revealed no creatine kinase or otherphosphagen kinases besides the arginine kinase.

The results reported here suggest that silicic acid causes aninduction of the expression of the arginine kinase gene and anincrease of enzyme activity in primmorphs after incubation.This effect could be abolished by coincubation with DIDS.Recently it was found in sponge cells that DIDS blocks theuptake of silicic acid (Schröder et al., 2004a).

Materials and methodsChemicals and enzymes

The sources of chemicals and enzymes used were as givenpreviously (Kruse et al., 1997; Krasko et al., 2000). Naturalsterile filtered seawater, hexokinase, glucose-6-phosphatedehydrogenase, DIDS [(4,4-diisothiocyanatostilbene-2,2-disulfonic acid disodium salt hydrate], phosphoarginine andsodium metasilicate were obtained from Sigma-Aldrich(Taufkirchen, Germany).

Sponges

Live specimens of Suberites domuncula Olivi (Porifera,Demospongiae, Hadromerida) were collected near Rovinj(Croatia) and then kept in aquaria in Mainz (Germany) formore than 2 years prior to their use.

Formation of primmorphs and incubation conditions

The procedure for the formation of primmorphs (3D-cellcultures) from single cells was as described previously(Custodio et al., 1998; Müller et al., 1999). Starting from singlecells, primmorphs of 3–7·mm are formed after 5 days. 7-day-old primmorphs were used for the experiments, cultivatedin natural seawater supplemented with 0.2% of RPMI1640medium. These 3D-cell cultures were transferred intoRPMI1640 medium. Incubation was performed in the presenceof the 2·µmol·l–1 silicic acid (ambient concentration, present inthe natural seawater) or in seawater adjusted to a silicic acidconcentration of 60·µmol·l–1 (Krasko et al., 2002) andincubated for up to 5 days. Then the samples were used for thedetermination of arginine kinase activity and for in situhybridization analysis. Where indicated, DIDS was added at afinal concentration of 100·µmol·l–1; the incubation studies wereperformed in darkness. In parallel, samples were taken forextraction of RNA from liquid-nitrogen-pulverized spongetissue using TRIzol Reagent (GibcoBRL, Grand Island, NY,USA), as recommended by the manufacturer.

Isolation of the cDNA for the arginine kinase

These studies were performed with primmorphs incubatedeither for 5 days in the absence of additional silicic acid in theRPMI1640/seawater medium, or cultivated in RPMI1640/seawater supplemented with 60·µmol·l–1 silicic acid.

The technique of differential display for identification of thearginine kinase cDNA was as described (Müller et al., 2002,2003a). RNA (1·µg) from controls and from specimens treatedwith silicic acid was reverse-transcribed using the T11CColigonucleotide as 3′ primer. The resulting cDNA was addedto the polymerase chain reaction (PCR) using the arbitraryprimer GTGATCGCAG and the T11CC primer in the assay,together with [α32P]-dATP. After amplification the radioactivefragments were separated on a 6% polyacrylamide sequencinggel and autoradiographed. The major DNA bands werecollected and subsequently reamplified in 50·µl reactions.Finally, the products were subcloned in pGEM-T vector(Promega, Madison, WI, USA) and sequenced. Among theover 50 sequences obtained, five partial cDNAs were isolated

S. Perovic-Ottstadt and others


639Activation of arginine kinase in sponge cells

whose deduced polypeptide sequence showed similarity to thatof arginine kinase. The complete cDNA was obtained byprimer walking (Ausubel et al., 1995; Wiens et al., 1998). Thearginine kinase cDNA was 1355 nucleotides (nt) long and wastermed SDAK.

Sequence analysis

Sequences were analyzed using computer programsBLAST 2003 (http://www.ncbi.nlm.nih.gov/blast/blast.cgi)and FASTA 2003 (http://www.ncbi.nlm.nih.gov/BLAST/fasta.html). Multiple alignments were performed with CLUSTAL WVer. 1.6 (Thompson et al., 1994). Phylogenetic trees wereconstructed on the basis of amino acid (aa) sequencealignments by neighbour-joining, as implemented in the‘Neighbor’ program from the PHYLIP package (Felsenstein,1993). The distance matrices were calculated using theDayhoff PAM matrix model as described (Dayhoff et al.,1978). The degree of support for internal branches was furtherassessed by bootstrapping (Felsenstein, 1993). The graphicpresentations of the alignments were prepared with GeneDoc(Nicholas and Nicholas, 1997).

RNA preparation and northern blot analysis

RNA was extracted from liquid-nitrogen-pulverized tissueusing TRIzol Reagent (GibcoBRL) as described (Grebenjuk etal., 2002). Then 5·µg of total RNA was electrophoresed andblotted onto Hybond-N+ nylon membrane (Amersham; LittleChalfont, Bucks, UK). Hybridization was performed using a350·nt segment of the SDAK cDNA; a 400·nt segment ofthe house-keeping gene β-tubulin of S. domuncula, SDTUB(EMBL/GenBank accession number AJ550806), was used asan internal standard. The probes were labeled using the PCR-digoxigenin (DIG)-Probe-Synthesis Kit (Roche, Mannheim,Germany). After washing, DIG-labeled nucleic acid wasdetected with anti-DIG Fab fragments and visualized bychemiluminescence technique using CDP (Roche). Forsemiquantitative analysis of the expression levels, the bands onthe film were scanned using the GS-525 Molecular Imager(Bio-Rad, Hercules, CA, USA).

In situ localization studies

The method used was based on a described procedure (Polakand McGee, 1998; Perovic et al., 2003). Frozen sections(8·µm) were prepared, fixed, treated with Proteinase K andsubsequently fixed again with paraformaldehyde. To removethe sponge color the sections were washed with increasingconcentrations of ethanol and decreasing concentrations ofacetone. After rehydration the sections were hybridized withthe labeled probe, a 250·nt long SDAK cDNA portion. Afterblocking, the sections were incubated with an anti-digoxigeninantibody conjugated with alkaline phosphatase. The dyereagent NBT/X-Phosphate was used for visualization of thesignals. Antisense and sense ssDNA DIG-labeled probes weresynthesized by PCR using the PCR-DIG-Probe-Synthesis Kit(Roche). Sense probes were used in parallel as negativecontrols in the experiments.

Determination of arginine kinase activity

Primmorphs were exposed to silicic acid in the absence orpresence of DIDS as indicated. Samples were homogenized inlysis-buffer [1� TBS (Tris-buffered saline), pH·7.5,1·mmol·l–1 EDTA and protease inhibitor cocktail (1tablet/10·ml; Roche)], centrifuged and the supernatantssubjected to enzyme activity determination.

Arginine kinase activity was determined according to amodified procedure (Nealon and Herderson, 1976). Thereaction mixture (final volume 1·ml) contained 10·mmol·l–1

Tris-maleate buffer (pH·7.0) supplemented with 0.4·mmol·l–1

phosphoarginine, 10·mmol·l–1 glucose, 5·mmol·l–1 MgSO4,1·mmol·l–1 NADP+, 5·units·ml–1 hexokinase, 1·unit·ml–1

glucose-6-phosphate dehydrogenase and 10·µl of enzymepreparation. The appearance of NADPH was monitored in aspectrophotometer at 340·nm (25°C) and standardized usingknown amounts of ATP. Arginine kinase activity wasexpressed on the basis of protein content and given asnmol·ATP·min–1·µg–1·protein. Five parallel experiments wereperformed.

Microscopical inspections

Transmission electron-microscopic (TEM) observationswere performed using a Zeiss (Aalen, Germany) EM 9Aelectron microscope. Primmorphs were pre-fixed in 2%glutaraldehyde in 0.05·mol·l–1 sodium cacodylate buffer(pH·7.4) with 0.2·mol·l–1 sucrose. After 3 days the specimenswere post-fixed in 1% osmium tetroxide in 0.05·mol·l–1

cacodylate buffer (pH·7.4) at 4°C for 2·h. After dehydrationthe specimens were embedded in araldite; thin sections (700·Å)were double stained in uranyl acetate and lead citrate (Mülleret al., 1986).

Analytical techniques

Protein concentration was measured according to Lowry etal. (1951) using bovine serum albumin as standard. Theconcentration of silicate in the natural seawater used for theexperiments was determined, applying the molybdate (SiliconTest) method (Simpson et al., 1985).

ResultsCloning of the cDNA encoding arginine kinase from

S. domuncula by differential display

The differential display of mRNA was examined inprimmorphs grown in the absence or presence of silicic acid(60·µmol·l–1) for 5 days. RNA was isolated, reverse transcribedand the cDNA was used for PCR. The fragments wereseparated on sequencing gels. A comparison of the mRNAexpression of non-treated versus silicic acid-treatedprimmorphs showed that among the >50 fragments sequenced,10% encoded a putative arginine kinase. The completesequence was obtained by primer walking. The arginine kinasecDNA, SDAK, had one open reading frame ranging from nt89-91

to nt1229-1231 (stop). The 380 aa long deduced polypeptide,AK_SUBDO, has a putative size (Mr) of 43282 (Fig.·1A).


640

Northern blot analysis performed with the sponge SDAK cloneas a probe yielded one prominent band of ≈1.4·kb, indicatingthat the full-length cDNA had been isolated (see below).

The highest similarity of the S. domuncula AK_SUBDO wasfound with arginine kinase cDNA from the cnidarian (Anthozoa)

Anthopleura japonica (Suzuki et al., 1997), with an ‘Expectvalue [E]’ (Coligan et al., 2000) of 6e–65 and an overall score ofsimilar and identical aa residues of 51% and 35%, respectively.Therefore, the sponge sequence was termed arginine kinase. Thesponge polypeptide displays the two characteristic domains of


A

0.1

AK_SUBDO

GLYCAM_NEREIS

CK_HUMAN

AK_STICJA

8511000

918

KARGn_ANTJAKARGc_ANTJA

1000

991

AK_CRASGI

AKn_CORBJAAKn_SOLSTRI

AKn_ENSIS 521

1000

AKc_ENSIS

AKc_SOLSTRIAKc_CORBJA

556

1000

998

997990

AK_CAEELKARG_ARTSF AK_DROME

AK_SCHIAM

880

AK_ERISI

1000

1000

KARG_HOMGA

B

D

D

Fig. 1.



the ATP:guanido phosphotransferases (ISREC server; http://hits.isb-sib.ch/cgi-bin/PFSCAN_parser), the N-terminal (betweenaa29 to aa109; Pfam PF02807 [http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?]) and C-terminal catalytic domains (aa120 toaa380; Pfam PF00217). These domains are implicated in thereversible transfer of high energy phosphate from ATP to thevarious phosphogens. The aa residues coordinating transfer ofMg2+ to ATP (Suzuki et al., 1997) are also present (Fig.·1A).

Phylogenetic analysis of S. domuncula arginine kinase

Arginine kinases, like all other members of the guanidinokinases, are restricted primarily to Metazoa; so no rooting couldbe performed when constructing a phylogenetic tree. Thephylogenetic tree comprised the ‘usual’ mono-domain 40-kDaguanidino kinases (arginine kinases) and the ‘unusual’ two-domain 80·kDa guanidino kinases (arginine kinases). The two-domain kinases identified in the Cnidarian Anthopleura japonica(Suzuki et al., 1997) and the mollusks (Bivalvia) Solen strictus(Suzuki et al., 2002), Corbicula japonica (Suzuki et al., 2002)and Ensis directus (Compaan and Ellington, 2003), weredissected into two parts and both parts were aligned in parallelwith the mono-domain guanidino kinases from mollusks, oysterCrassostrea gigas and Solen strictus, crustaceans Homarusgammarus (KARG_HOMGA, 538542), Eriocheir sinensis andArtemia franciscana, and insects, Drosophila melanogaster andSchistocerca americana. In addition, glycocyamine kinase fromthe annelid Nereis virens, human creatine kinase (Mariman etal., 1987) and the holothurian enzyme from Stichopus japonicuswere included in the alignment. The tree shows very strikinglythat the duplication of the arginine kinases occurred in somemollusk species (e.g. S. strictus), but not in others (e.g. C. gigas;Fig.·1B). It had been proposed recently (Takeuchi et al., 2004)that gene duplication and their subsequent fusion in mollusksoccurred separately from events that took place in hydrozoa (A.japonica).

Induction of arginine kinase gene expression in response tosilicic acid

In order to clarify whether the S. domuncula arginine kinase

gene is upregulated after incubation with silicic acid,primmorphs were cultured in the presence or absence of60·µmol·l–1 additional silicic acid. Northern blot analyses wereperformed with the labeled cDNA probe for arginine kinase(SDAK) and the house-keeping gene β-tubulin (SDTUB).These studies revealed that after addition of silicic acid to theprimmorphs, within 1 day a significant (threefold) increase ofthe steady-state level of arginine kinase transcripts couldalready be measured, a value which further increased afteradditional 2–4 days (Fig.·2, left). In contrast, if the primmorphsremained in culture without additional silicic acid, nosignificant change of the expression level could be seen (Fig.·2,right). Control blots, performed with the house-keeping geneβ-tubulin, confirmed that the same amount of RNA was loadedonto the gels. In parallel, expression studies were performed inthe presence of 100·µmol·l–1 DIDS for 0–5 days, and in thepresence of this inhibitor the silicic acid-mediated upregulationof arginine kinase expression was not seen (data not shown).

In situ hybridization analyses

As further proof that in primmorphs gene expression ofarginine kinase is positively affected by silicic acid, in situhybridization studies were performed. Primmorphs wereincubated for 5 days in the absence (Fig.·3A,B) or presence of60·µmol·l–1 of silicic acid (Fig.·3C,D). Then cryosections werehybridization with the labeled probe for arginine kinase(SDAK). Sections from primmorphs that remained for 5 daysin the absence of silicic acid did not show high signals usingantisense SDAK ssDNA probes (Fig.·3A,B). However, if theprimmorphs had been kept in the presence of silicic acid, highexpression of this gene was seen, especially in the center ofthese 3D-cell cultures (Fig.·3C,D). In a further series, theprimmorphs were cultivated in the presence of silicic acidtogether with the inhibitor DIDS (100·µmol·l–1; Fig.·3E,F). Insections from these primmorphs the expression level wasalmost identical to that seen in cultures without additionalsilicic acid. No reaction was observed when the cells weretreated with sense probes (not shown).

Fig.·1. S. domuncula arginine kinase. (A) The sponge deduced protein (AK_SUBDO) is aligned with the arginine kinase from the CnidarianAnthopleura japonica (KARG_ANTJA, O15992; Suzuki et al., 1997), and Drosophila melanogaster (AK_DROME, AAA68172), glycocyaminekinase from Nereis virens (GLYCAM_NEREIS, AAL26699) and human creatine kinase (CK_HUMAN, AAC31758; Mariman et al., 1987).Residues conserved (similar or related with respect to their physico-chemical properties) in all sequences are shown in white on black and thosein at least three sequences in black on gray. The two characteristic domains for the ATP:guanido phosphotransferases, the N-terminal domain(ATP_GUA_N) and the C-terminal catalytic domain (ATP_GUA_C), are marked. The A. japonica arginine kinase is a two-domain enzyme,so the two phosphotransferase domains are also marked for this protein. The residues involved in coordinating Mg2+ to ATP are marked [ATP].(B) Radial, unrooted phylogenetic tree, which includes the above mentioned sequences; the two domain enzyme from A. japonica has been splitinto the N-terminal and C-terminal segments (KARGn_ANTJA, aa1 to aa367; KARGc_ANTJA, aa368 to aa715). Shown in addition are thedeuterostomian related sequence from (1) the Holothuria Stichopus japonicus (AK_STICJA, BAA76385), (2) the insect Schistocerca americana(AK_SCHIAM, AAC47830.1), (3) the mollusks Solen strictus (Bivalvia) (AK_SOLSTRI, BAB91358; N terminus aa1 to aa372 and C terminusaa373 to aa724; Suzuki et al., 2002), Corbicula japonica (Bivalvia) (AK_CORBJA, BAB91357.1; N terminus aa1 to aa372 and C terminus aa373

to aa723; Suzuki et al., 2002) and Ensis directus (Bivalvia) (AK_ENSIS, AAM90698.1; N terminus aa1 to aa372 and C terminus aa373 to aa723;Compaan and Ellington, 2003), (4) the oyster Crassostrea gigas (Bivalvia) (AK_CRASGI, BAD11950.1), (5) Crustaceans Homarus gammarus(KARG_HOMGA, 538542), Eriocheir sinensis (AK_ERISI, AAF43437) and Artemia franciscana (KARG_ARTSF, Q95V58), and (6) thenematode Caenorhabditis elegans (AK_CAEEL, NP_492714.1). Gene duplications within the mollusks and the Cnidarian taxa are indicated(D). The scale bar indicates an evolutionary distance of 0.1aa substitutions per position in the sequence.


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Arginine kinase activity in sponge primmorphs after silicicacid incubation

Primmorphs were obtained as described under ‘Materialsand methods’. After formation of the 3D-cell cultures,cultivation in seawater was continued in the absenceof additional silicic acid for 5 days. Under theseconditions, arginine kinase activity was approximately2.5·nmol·ATP·generated·min–1·10·µg–1·protein. This value didnot change significantly when primmorphs were incubatedwith 100·µmol·l–1 DIDS (Fig.·4). However, if the cultures were

incubated with an additional 60·µmol·l–1 of silicic acid theenzyme activity had already increased after 1 day to6·nmol·ATP·min–1·10·µg–1·protein, a value which rose to27·nmol·ATP·min–1·10·µg–1·protein on further incubation.Coincubation with DIDS reduced the enzyme activity by morethan 70% (Fig.·4). These data show that the arginine kinaseactivity increased strongly in primmorphs in response to silicicacid.

Formation of spicules in primmorphs from S. domuncula

We have previously demonstrated that primmorphs start toform spicules after incubation with 60·µmol·l–1 silicic acid(Krasko et al., 2000). Here we cut cross sections throughprimmorphs that had been cultivated for 5 days in the absenceor presence of 60·µmol·l–1 silicic acid. TEM analysis ofprimmorphs grown in the absence of silicic acid did not showany cells that synthesized spicules. However, in primmorphskept in the presence of silicic acid, sclerocytes that containednewly growing spicules were frequently observed (Fig.·5). InFig.·5A two sclerocytes are seen, each surrounding a spicule.At higher magnification it can be observed that the blunt endof one spicule is tightly associated with two fibrils, which arevery likely collagen fibers (Fig.·5B,D); however such fiberswere not always present at the blunt end (Fig.·5C). Those fiberssurrounded the spicules at all phases (Fig.·5B–D). Note that thesharp, likewise growing, tip of the spicule showed an


Plus

Tubulin

Argininekinase

1.4

Minus

0 1 3 5 0 1 3 5 days

Silicic acid

1.5

kb

Fig.·2. Expression of the arginine kinase gene in primmorphs isdependent on addition of silicic acid. Primmorphs were incubated inthe presence (plus) or absence of silicic acid (minus) for 0–5 days.Then RNA was extracted, and identical amounts of total RNA weresize-separated. After blot transfer, hybridization was performed usingthe probes for arginine kinase or the housekeeping gene β-tubulin.

Fig.·3. Expression of the arginine kinase gene is dependent on the presence of silicic acid. (A–D) Primmorphs were cultivated for 5 days in theabsence (–; A,B) or presence of 60·µmol·l–1 silicic acid (SA+; C,D). (E,F) Primmorphs were treated with silicic acid and 100·µmol·l–1 DIDSand in situ hybridization performed with SDAK as the probe. Hybridized cells are stained by brown/black deposits. Magnification: �10 (A,C,E);�20 (B,D,F).



inhomogeneous structure, suggesting that the material in thisregions is not densely packed (Fig.·5D).

DiscussionThe phylum Porifera comprises species of sizes between

2–4·mm and up to 2·m; their bodies are structured into organ-like tissues, which are not connected through a nervouscoordination network (see Müller et al., 2004b). Evencontraction of channels and pumping of the water through theaqueous system are not mediated by muscle organs. Musclesevolved first in the second diploblastic phylum, theCoelenterata. Therefore it might be expected that in spongesphosphagen (creatine), which is the characteristic energysource in muscles for the generation of ATP, is not present,nor the corresponding energy transferring enzyme, creatinekinase. The latter assumption is supported by database searchesin the S. domuncula EST library, which does not contain acDNA for this enzyme. However, in invertebrates otherphosphorylated high-energy guanidine phosphagens, e.g.phospho-arginine or phospho-glycocyamine, are known toserve as energy donors for the formation of ATP in thecytoplasm. As in most cells, in sponges ATP is also generatedby oxidative phosphorylation (see Sona et al., 2004). Thephosphagen kinases are localized at the sites of ATP hydrolysis

and/or synthesis, allowing a temporal and spatial ATP buffersystem as well as a tuned regulation of the intracellularphosphate concentration (reviewed in Ellington, 2001). In thepresent study the arginine kinase activity in S. domuncula was

0 1 2 3 4 50

5

10

15

20

25

30

35

Incubation (days)

–SA+SA+DIDS, –SA+DIDS, +SA

Fig.·4. Arginine kinase activity in primmorphs from S. domuncula inresponse to silicic acid. The primmorphs were incubated for 0–5 daysin the absence (white bars) of presence of 60·µmol·l–1 silicic acid (SA;black bars). In a parallel series of experiments the primmorphs werecoincubated with 100·µmol·l–1 of DIDS in assays without (hatched)and with SA (cross-hatched). The enzyme activity is given innmoles·ATP·generated·min–1·10·µg–1·protein. Values are means ±S.E.M., N=5.

Fig.·5. Growing spicules in sclerocytes obtained from primmorphs cultured in the presence of 60·µmol·l–1 silicic acid; sequence of TEM images.(A) Two sclerocytes (scl) in the primmorph start to synthesize spicules (sp). The blunt end of one newly growing spicule (B) is associated withcollagen-related fibrils (col), but not the second spicule (shown enlarged in C). The sharp end of the spicule shows inhomogeneous structure(D), suggesting less dense packing of silica in the spicule. Collagen fibers (col) surround the two spicules.


644

determined to be approximately 2.5·nmol·ATP·generatedmin–1·10·µg–1·protein, a value that is within the range ofarginine and creatine kinase activities measured in theprimitive-type sperm of certain marine invertebrates (Tombesand Shapiro, 1989).

The technique of differential display of mRNA was used toidentify which energy transferring kinase is involved in onemajor energy-consuming reaction in sponges, the formation ofthe skeleton. Primmorphs were exposed to silicic acid and theirmRNA expression patterns were compared with those in 3D-cell aggregates, which grew in the low-silicic acid medium.Silicic acid is the inorganic component of the spicules inDemospongiae. As outlined in the Introduction, the spiculesare the major skeletal elements of sponges; their framework isconnected by filamentous organic fibrils (Garrone, 1978).Furthermore, it should be mentioned that the growth ofspicules is a fast process (Elvin, 1972). The synthesis andformation of this scaffold require considerable energy reserves.Analytical studies revealed that arginine is one dominantamino acid in sponges, reaching intracellular concentrations of2·mmol·l–1 (Marco Giovine; unpublished observations). Thedifferential display analyses revealed that 10% of the transcriptfragments contribute to the arginine kinase cDNA. The full-length cDNA was identified. It comprises, as with all othermetazoan phosphagen kinases, the two domains characteristicfor these kinases, the N-terminal domain and the C-terminalcatalytic domain. Also the Mg2+/ATP coordinating residueswithin the C-terminal domain are conserved.

To study the phylogenetic aspect of the sponge argininekinase was equally interesting. In yeast (Saccharomycescerevisiae) or plants (e.g. Arabidopsis thaliana) no argininekinases, or related enzymes, exist. Hence the sponge moleculeis evolutionarily one of the oldest phosphagen kinases. Thisfact is also reflected by the radial phylogenetic tree, whichshows that the human/vertebrate creatine kinases, and theglycocyamine kinase (Nereis virens) branched off from thesponge common ancestor molecule (Fig.·1B). Arginine kinasesemerged in the other direction, comprising the ‘usual’ mono-domain 40-kDa guanidino kinases (arginine kinases) and the‘unusual’ two-domain 80·kDa guanidino kinases (argininekinases). Interestingly, the next closest arginine kinase familymember, the cnidarian A. japonica (Suzuki et al., 2002;Takeuchi et al., 2004) has already undergone an independentgene duplication in another taxon, the mollusks (see Fig.·1).While the guanidino kinase from the mollusk C. gigas belongsto the mono-domain kinases, other molluskan kinases, from S.strictus, C. japonica and E. directus, are two-domain 80·kDaguanidino kinases. It is amazing that within the Bivalvia,and even in the same subclass Lammellibranchia, thisduplication proceeded from mono-domain, C. gigas(superorder Filibranchia), to two-domain kinases, S. strictus(Eulamellibranchia), C. japonica (Eulamellibranchia) and E.directus (Eulamellibranchia). It was suspected that this geneduplication parallels the higher enzyme activity (Takeuchi etal., 2004). Crystal structure classification (Cheek et al., 2002)supports the grouping of the kinases deduced from alignment

studies with protein sequence data. The enzyme activity ofarginine kinase has been detected in the flagellate protozoaTrypanosoma cruzi (Alonso et al., 2001) and Parameciumcaudatum (Noguchi et al., 2001), indicating that argininekinases evolved before the emergence of multicellular animals.Recent phylogenetic analyses also indicates that the ciliatearginine kinases show a high phylogenetic relationship to thoseof sea anemones (Ellington and Suzuki, 2005).

The primmorph system was established in order to obtainmolecular biological insights into the proliferation (Le Pennecet al., 2003), differentiation (Müller et al., 2004b) andimmunological (Müller et al., 2002) capacities of primordialsponge cells. Here, this system was used to demonstrate thatthe expression level of the gene encoding the arginine kinasestrongly depends on the presence of silicic acid in the medium.The gene induction is fast; after 1 day a strong upregulation isalready seen. This fast response is characteristic for theprimmorph system. Previously it was shown that the genes forsilicatein, the enzyme that causes the formation of silica in thespicules, and for collagen, the organic cover around the skeletalelements, also already show strong induction 1 day after silicicacid incubation (Krasko et al., 2000). One piece of evidencethat the increased expression of the arginine kinase gene isindeed dependent on the exogenous silicic acid, came frominhibition studies using DIDS, an established inhibitor of band3-mediated anion exchanger through covalent binding to lysineresidue(s) (Kopito, 1990). The inhibition is not restricted to thetransport of the Cl–/HCO3

– exchanger in vitro but also affectstransport systems in vivo, e.g. in rats (Horie et al., 1993;Kubota et al., 2003). Note that DIDS has been shown toprevent uptake of silicic acid via the silica transport in thisspecies (Schröder et al., 2004a).

One major differentiation pathway is the transition from thetotipotent stem cells, the archaeocytes, into the spicule-formingsclerocytes (Müller et al., 2003b). Archaeocytes are the majorcell fraction in primmorphs (Müller et al., 1999) and theinducer of this differentiation direction is silicic acid; in thearchaeocyte cell lineage noggin and MSCP expression arecrucial for the fate of the sclerocyte differentiation (Schröderet al., 2004b). In the present study it is seen that the expressionof the arginine kinase gene in primmorphs, incubated in thepresence of silicic acid, occurs primarily in the center of these3D-cell aggregates. This localized expression is in accordancewith previous findings that differentiation of cells, e.g. for theformation of aqueous canals, is first detected in the center ofthe primmorphs (Müller et al., 2004a). It interesting to observethis specific localization in the choanocyte-forming region ofprimmorphs. In fact, these peculiar cells need high amounts ofenergy for cilia movement and phosphoarginine should beprimarily involved as energy reservoir. It is intriguing to noticethat in ciliates (Protozoa) arginine kinase activity also plays afundamental role in cilia movement (Noguchi et al., 2001).The specificity of the silicic acid-mediated induction of thearginine kinase gene is, again, shown by inhibition studies withDIDS; this stilbene analogue almost completely prevents thisexpression.




The increased expression of arginine kinase also correlateswith a strong enhancement of arginine kinase enzyme activity.In primmorphs a strong increase of activity was measured after1 day of exposure to silicic acid, again a process that could beblocked by DIDS. Since this inhibitor blocks both spiculeformation and expression of arginine kinase gene, it can beassumed that under these conditions a steep electrochemicalgradient for silicic acid transport results. An association ofphosphagen kinases with membrane-associated ATPase is welldocumented (reviewed in Ellington, 2001). Final support forthe differentiation-mediating capacity of silicic acid, whichallows formulation of a chain of reactions from increasedexpression of the arginine kinase gene to enhanced enzymeactivity and finally to the onset of spicule formation inprimmorphs, was documented by transmission electron-microscopy. This analysis revealed that spicules are formed inthose primmorphs that were exposed to silicic acid.

Taken together, the results of the present work show thatarginine kinase is a crucial enzyme in the high energy-consuming reaction of spicule formation in primmorphs. Inaddition, the data demonstrate that the evolutionary oldestmetazoan phylum, the Porifera, comprise the ancestralphosphagen kinase, an arginine kinase, for all other kinases ofhigher taxa. Studies are in progress to elucidate the role ofarginine kinase in adult specimens; there the highest energyconsumption should be localized in the choanocytes, which arethe ‘motor’ cells that drive the water through the aqueous canalsystem. A similar role for creatine kinase in choanocytefunction has recently been suggested for another sponge (Sonaet al., 2004).

This work was supported by grants from the DeutscheForschungsgemeinschaft, the Bundesministerium für Bildungund Forschung Germany (project: Center of ExcellenceBIOTECmarin) and the International Human Frontier ScienceProgram (RG-333/96-M). Note that the cDNA sequence forarginine kinase from Suberites domuncula has been deposited(EMBL/GenBank) under the accession number AJ744770(AK_SUBDO).

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arginine kinase in the demosponge suberites domuncula: regulation

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