Chemistry, histochemistry and microscopy of the organic matrix of spicules from a gorgonian coral. Relationship to alcian blue staining and calcium binding

Alcian blue dye normally binds to polyanionic, polymeric substances. Such structures are often associated with calcium binding portions of the organic matrix in calcifying tissues. The organic matrix of spicules prepared from the gorgonian Pseudoplexaura flagellosa (Houttuyn) is alcianophilic. The dye is very tightly bound to the lipoid portion of the insoluble spicule matrix. No acidic substances (sulfated or acidic polysaccharides or phospholipids) were demonstrable in this material, suggesting an unusual but unknown interaction between dye and substrate. On a microscopical basis, inclusion of Alcian blue (or Ruthenium red) is an essential co-requisite to glutaraldehyde fixation. Without the dye the morphological integrity of the spicule is lost on decalcification. The fragmented matrix is still alcianophilic suggesting that the dye may substitute for material solubilized by the decalcifying agents. Examination of post-decalcification supernatants demonstrate that approximately 13% of the matrix is solubilized on demineralization, releasing 93% of the carbohydrate but less than 20% of the protein. Liberated protein takes the form of peptides ranging from 1100-1500 daltons. The composition of these peptides is a function of the demineralizing agent. Acidic demineralizers produce peptides proportionately high in acidic amino acids, that do not bind calcium. Peptides produced by chelator decalcification appear to bind calcium but other evidence strongly suggests that the binding is due to adsorbed chelator rather than by soluble matrix.     

Seasonal localization of a collagenous protein in the organic matrix of spicules from the gorgonian Leptogorgia virgulata (Cnidaria: Gorgonacea)

Spicules of the gorgonian Leptogorgia virgulata possess an insoluble matrix fraction that is predominantly collagenous in summer months. This collagenous component is largely absent in winter months. Using an antibody directed against the 140 kD collagenous protein (CP) of the insoluble matrix, immuno-gold labelling was employed to localized this protein at the transmission electron-microscopy level throughout the year, and in different areas of the gorgonian colonies. Within the tip regions, the 140 kD CP varied throughout the year in the spicules, electron-dense bodies (EDBs) of scleroblasts, polyp vesicles, desmocytes and axes. In the mid and base regions, the 140 kD CP varied throughout the year in the spicules, EDBs and lysosomes of scleroblasts, desmocytes and axes. This variation in the location and density of the label suggests a dynamic annual cycling of the collagenous component of the insoluble matrix. EDBs may transport a collagenous component of the matrix to the spicule-forming vacuole. A component of the 140 kD CP may be transported and/or degraded by polyp vesicles and lysosomes, respectively. The pattern of labelling of the axial region suggests that translocation and storage of a component of the collagenous protein may occur. Environmental factors may be responsible for the triggering of matrix cycling.     

Synthesis and transport of the organic matrix of the spicules in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The sequence of the synthesis and transport of the organic matrix of spicules has been elucidated in the gorgonian Leptogorgia virgulata by use of ³H-aspartic acid as the tracer in electron-microscopic autoradiography. The entire process of matrix synthesis and transport takes approximately 2 h. It seems that the protein moiety of the organic matrix is synthesized in the RER prior to 5 min following the initial 10 min incubation in the tracer. At the 5 min chase the label is moving from the RER to the Golgi complexes where the carbohydrate moiety of the matrix is presumed to be synthesized. At the 5 to 15 min chases the label is transported out of the Golgi complexes via Golgi vesicles. This phase continues for 30 min. From 60 to 120 min the ³H-aspartic acid moves to the spicules. After 120 min the majority of the label has moved into the spicules. Silver grain counts over both multivesicular and electron-dense bodies remain at relatively low and constant levels over 4 h indicating that neither organelle is involved in the synthesis and transport of the organic matrix.Contribution No 512; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208, USA     

Synthesis of (-)-isishippuric acid A, a 4,5-secoquadranoid from a gorgonian coral

The first synthesis of (-)-isishippuric acid A was accomplished in two steps from a bicyclic synthetic intermediate for isishippuric acid B, which confirmed the structure of isishippuric acid A, including its absolute stereochemistry.     

Pregnane steroids from the South China Sea gorgonian coral Subergorgia suberosa

Chemical investigation on gorgonian coral Subergorgia suberosa collected from the South China Sea led to the isolation and identification of eight 17-acetyl pregnane steroids (1–8), of which 1, 6 and 7 were obtained for the first time from this species, as well as from the genus Subergorgia. Compounds 1 and 7 are new natural products. Compared with other species in the genus Subergorgia, pregnane steroids with 17-acetyl group (1–8) were only achieved from S. suberosa, suggesting that the 17-acetyl pregnane steroids might be characteristic constituents of S. suberosa, and may be useful as chemotaxonomic markers for the species S. suberosa.     

Phagocytic activities of the gorgonian coral Swiftia exserta

The cellular response component of body defense in gorgonians and other cnidarians is thought to be carried out by cells with phagocytic capabilities. To test for the phagocytic character of cells, the introduction of foreign particles was employed and observed in both living cells and histological preparations of the gorgonian coral Swiftia exserta. Observations of untreated tissues revealed normal cells and tissue morphologies. A microscopic observation of living cells following the introduction of particles in a cut revealed that only a mixed population of colorless cells phagocytized the particles. Also particles or clumps of particles were seen on the surface of the colorless cells. Subsequent histological observations allowed identity of colorless cells to be inferred as granular amoebocytes, ectodermal cells, and gastrodermal cells. Cells stained for localization of peroxidase (indicative of phagocytic activity) demonstrated the presence of peroxidase-positive cells. Histological preparations revealed that major phagocytosis of particles was associated with tissue trauma. When particles were introduced by means of a cut or inserted thread, phagocytic activity was detected within 2 h. However, it was confined to the granular amoebocytes in the immediate site of trauma. After 24 h, extensive phagocytosis spread throughout a relatively large area surrounding the wound. At that later time, phagocytic cell types included granular amoebocytes, epidermal cells, sclerocytes, mesogleal cells, and gastrodermal cells of the solenia. Observations suggest that trauma induces phagocytosis in cells not normally phagocytic in S. exserta. No localization of phagocytic cells and no mitotic cells were observed at either 2 or 24 h after particle introduction.     

Characterization of proteins from the matrix of spicules from the alcyonarian, Lobophytum crassum

The organic matrix of spicules of the alcyonarian coral, Lobophytum crassum, was studied to investigate its molecular characteristics and functional properties. The shape of the spicules was identified using scanning electron microscopy. The soluble organic matrix comprised 0.03% of the spicule weight. The SDS-PAGE analysis of the preparation showed four protein bands with apparent molecular weights of 37, 48, 67 and 102 kDa. The 67- and 102-kDa proteins appeared to be calcium binding proteins, detected as radioactive bands by ⁴⁵Ca autoradiography. The 67-kDa protein appears to be glycosylated. The N-terminal amino acid sequence of the 67 kDa was determined; 7 of 20 residues were acidic. A database search for homologous proteins did not give a clear indication of the function of the 67-kDa protein. The isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formation, indicating that organic matrix is not only structural protein but also a catalyst. An interpretation of these results is that the spicule of alcyonarian corals has a proteinaceous organic matrix related to the calcification process.     

Molecular cloning and characterization of first organic matrix protein from sclerites of red coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.     

Calcium associated with a fibrillar organic matrix in the scleractinian coral Galaxea fascicularis

Summary.  Field emission scanning electron microscopy of frozen-hydrated preparations of the scleractinian coral Galaxea fascicularis revealed organic fibrils which have a diameter of 26 nm and are located between calicoblastic ectodermal cells and the underlying CaCO₃ skeleton. Small (37 nm in diameter) nodular structures observed upon this fibrillar organic material possibly correspond to localised Ca-rich regions detected throughout the calcifying interfacial region of freeze-substituted preparations by X-ray microanalysis. We propose that these Ca-rich regions associated with the organic material are nascent crystals of CaCO₃. Significant amounts of S were also detected throughout the calcifying interfacial region, further verifying the likely presence of organic material. However, the bulk of this S is unlikely to be derived from mucocytes within the calicoblastic ectoderm. It is suggested that in the scleractinian coral G. fascicularis, nodular crystals of CaCO₃ establish upon a fibrillar, S-containing, organic matrix within small but distinct extracellular pockets formed between calicoblastic ectodermal cells and skeleton. This arrangement conforms with the criteria necessary for biomineralisation and with the long-held theory that organic matrices may act as templates for crystal formation and growth in biological mineralising systems. Received April 30, 2002; accepted September 11, 2002; published online March 11, 2003     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Development of microsatellite markers for the Mediterranean gorgonian coral Corallium rubrum

Corallium rubrum, an endemic Mediterranean gorgonian coral, has undergone an intensive exploitation leading to the extinction of local commercial banks and changes in the structure and dynamics of coastal populations. Management and conservation of this species requires a better understanding of the genetic structuring and connectivity among populations. With this aim, seven microsatellite loci have been isolated. All loci were polymorphic with allele numbers ranging from five to 26 and observed heterozygosity ranging from 0.18 to 0.68. Significant deviations from Hardy–Weinberg expected genotype frequencies due to heterozygote deficiency were detected at all loci.     

Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization

Soluble organic matrix (SOM) synthesis and secretion were investigated in two scleractinian corals using antibodies raised against this organic matrix. Results demonstrate that even if other cell types, including zooxanthellae, can supply precursors for SOM synthesis, only calicoblastic cells facing the skeleton are directly responsible for the synthesis and secretion of the SOM components. Results also indicate that, as is the case for other biominerals, skeleton formation is biologically controlled and not chemically dominated as originally believed. In addition to advancing the understanding of mechanisms of coral biomineralization, these antibodies could have numerous applications: for example as markers of skeletogenesis, as tools for cell culture, and in comparative studies among calcifying organisms.     

Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla

Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.     

Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.