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First published online January 18, 2008
Journal of Experimental Biology 211, 300-309 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.008193
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Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni

Werner E. G. Müller1,*, Alexandra Boreiko1,{dagger}, Ute Schloßmacher1,{dagger}, Xiaohong Wang2, Carsten Eckert1,3, Klaus Kropf1, Jinhe Li4 and Heinz C. Schröder1

1 Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany
2 National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, People's Republic of China
3 Museum für Naturkunde, Institut für Systematische Zoologie, Invalidenstraße 43, D-10155 Berlin, Germany
4 Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, CHN-266071 Qingdao, People's Republic of China


Figure 1
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  		Fig. 1. Megascleres of M. chuni (A–D,G–I) and M. intermedia (E,F); scanning electron microscopic analysis. (A) The tauactins are covered by a collagen (col) sheath, which is interspersed with 5 µm holes (h). (B) Cross-section through a giant basal spicule showing the outer lamellar zone (la) and the central axial cylinder (cy), which surrounds the axial canal (ac). (C,D) The tauactins are composed of a maximum of 20 lamellae. (E) A stack of lamellae of the outer zone of a giant basal spicule. (F) Cross-section through a giant basal spicule; the surface has been etched with HF, disclosing the composition of the lamellae by silica nanoparticles. (G) The tip of a tauactin, allowing inspection of the axial canal with its axial filament (af). (H) Cut-through section of a tauactin at the position where two rays/spines originate, exposing two axial canals with their axial filaments. (I) Tip of a tauactin, harbouring three axial canals.

 

Figure 2
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  		Fig. 2. Analysis of proteins in giant basal spicules from M. chuni. (A) Protein extracts were prepared from the outer lamellar region of the spicules (la; lane a) or from total spicule (to; lane b) and size separated by 10% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue. Size markers are given (M). (B) Corresponding western blot from the separation of a protein extract obtained from lamellae; the blot was reacted with polyclonal antibodies raised against silicatein (PoAb-aSILIC) and then with labelled secondary antibodies (lane a). In a separate series, the blot was first incubated with PoAb-aSILIC that had been adsorbed with recombinant silicatein (ads), and then incubated with the labelled secondary antibodies (lane b). (C) Analysis of a total spicule extract by two-dimensional gel electrophoresis (first isoelectric focusing and then size separation). The arrows mark the positions of the two sets of proteins in the total spicule extract, the 27/30 kDa molecules and the 70 kDa polypeptides. The gel was stained with Coomassie Brilliant Blue. Further details are given under Materials and methods.

 

Figure 3
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  		Fig. 3. Sponge cystatin, a cysteine proteinase inhibitor. (A) Alignment of the sponge cystatin (CYTA_SUBDO) with the corresponding cystatins from humans, cystatin A (CYTAA_HUMAN; accession number NP_005204) and cystatin B (CYTAB_ HUMAN; NP_000091). Amino acids that are similar among all three sequences are in white type. The characteristic cystatin domain spans amino acids 11 to 66 (CysD). (B) A phylogenetic tree constructed after alignment of the above-mentioned three sequences as well as the pig leukocyte cysteine proteinase inhibitor 1 (CYSPROINH_PIG; P35479), pig stefin A8 (STEFA8; NP_999025), pig stefin A5 (CYTAA5_PIG; Q28986), cattle proteinase inhibitor stefin-C from Bos taurus (CYTAC_BOVIN; P35478), the insect putative protein CG31016 from Drosophila melanogaster (CG31016_DROME; NP_733393), the putative protein from Caenorhabditis elegans (C17H1_CAEEL; NP_493295), the yeast enzyme DNA topoisomerase III from Saccharomyces cerevisiae (DNATOPO_YEAST; NP_013335), and the plant cysteine proteinase inhibitor from Arabidopsis thaliana (CYSPROINH_ARATH; NP_850570). The protein from A. thaliana was used as an outgroup to root the tree. Scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position in the sequence. (C) The cloned cystatin cDNA from S. domuncula was used for transfection of E. coli. After treatment of the bacteria with IPTG, the protein (cystatin; lane a) was extracted and purified as described under Materials and methods. M, size markers.

 

Figure 4
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  		Fig. 4. Proteolytic activity of silicatein, present in spicule extract from S. domuncula. Extracts were prepared and separated by electrophoresis. After size separation the gel was incubated overnight at room temperature to identify the zones of proteolytic digestion as outlined under Materials and methods. The migration distance of the cleared zone (24/25 kDa) is characteristic for silicatein (lane a). If the sample from the spicules had been pre-incubated with E-64 (lane b) or with the recombinant sponge cystatin (lane c), no clearance zone could be seen. Form these data we conclude that silicatein, present in the spicule extract, still retains proteolytic activity. M, size markers.

 

Figure 5
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  		Fig. 5. Effect of cysteine proteinase inhibitors E-64 and cystatin on the proteolytic activity of cathepsin L (left), as well as spicule extracts from S. domuncula (middle) and M. chuni (right). The activity of the samples was determined either directly (black columns), or following pretreatment with E-64 (dark grey columns) or cystatin (light grey columns). The proteolytic activity was measured with the synthetic substrate Z-Phe-Arg-AMC, and is given as nmol AMC released mg–1 protein min–1. Means (±s.d.) of five independent experiments are given.

 

Figure 6
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  		Fig. 6. Identification of the 27 kDa protein species in the total extract obtained from M. chuni giant basal spicules. (A) An extract from lamellae (la) of giant basal spicule was obtained and subjected to 10% SDS-PAGE. The 27 kDa protein is stained with Coomassie Brilliant Blue (lane a). M, size markers. (B) For the reaction with the labelled inhibitor (biotinylated E-64) the Monorhaphis spicule proteins were blot transferred and reacted with biotinylated E-64 as described under Materials and methods. The binding reaction with this reagent was performed either by direct addition of biotinylated E-64 to the blot (lane a, –; the 27 kDa protein is labelled) or after a pretreatment of the blot with a surplus of unlabelled E-64 (lane b, +).

 

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© The Company of Biologists Ltd 2008