Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellidMonorhaphis chuni

Abstract

SUMMARYSilicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e.g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3·m and diameters of 10·mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27·kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70·kDa proteins. The 27·kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27·kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

Acquisition of Structure-guiding and Structure-forming Properties during Maturation from the Pro-silicatein to the Silicatein Form

“…Therefore, we used both E-64 and phenylmethylsulfonyl fluoride (PMSF) as inhibitors in the reaction assay. E-64 inhibited directly the processing of the cathepsin-related silicatein, as expected (data not shown) (31), while the maturation of prosilicatein to silicatein proceeded in the presence of PMSF. PMSF is known to be an effective inhibitor of thrombin protease (39), however, only for a very limited period of time (about 1 h) (32) and surely not for 24 h as seen in our assays 3 .…”

“…Silicatein cleavage has been proposed to proceed either by autolysis (29) or by a second protease (30). Since the processing of the S. domuncula pro-silicatein-␣ to silicatein-␣ can be prevented by the incubation with 5 M E-64 3 , an irreversible inhibitor of a wide range of cysteine peptidases, including silicatein from the hexactinellid Monorhaphis chuni (31), an autocatalytic cleavage process is most likely.…”

Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella , directs silica polycondensation

“…The silicateins are assembled into a linear fiber (28,29) that is found within the core of demosponge spicules and catalyze silica formation via a hydrolysis pathway from organically functionalized silicic acid precursors at neutral pH (8,9,11). In addition to their discovery in several other demosponge species (12-18), silicateins or silicatein-like proteins have also been reported from hexactinellid sponges such as M. chuni and C. meyeri (19)(20)(21). If silicatein exists in Euplectella, as is suggested based on partial cDNA data [GenBank accession no.…”

“…Müller et al (19)(20)(21) reported that antibodies against silicatein α from the demosponge Suberites domuncula cross-reacted with a 27-kDa protein in the giant anchor spicules of Monorhaphis chuni and with a 24-kDa protein in the spicules of Crateromorpha meyeri. Mass spectrometric analysis of a fragment from the M. chuni 27-kDa protein demonstrated that it contained sequences corresponding to the silicateins from demosponges (22).…”

“…Mass spectrometric analysis of a fragment from the M. chuni 27-kDa protein demonstrated that it contained sequences corresponding to the silicateins from demosponges (22). Silicatein sequences deduced from cDNA clones from M. chuni and C. meyeri contain the characteristic catalytic amino acid triad, serinehistidine-asparagine (19,21). The silicatein gene was also identified from Aulosaccus schulzei, although here, a catalytic cysteine was confirmed instead of the usual serine (23 In addition to the silicateins, other organic molecules proposed to be involved in spicule formation have been identified from hexactinellid sponges.…”