Mitsuru Jimbo

Long‐Chain Polyamines (LCPAs) from Marine Sponge: Possible Implication in Spicule Formation

Two distinct marine organisms, diatoms and sponges, deposit dissolved silicates to construct highly architectural and species‐specific body supports. Several factors such as proteins, long‐chain polyamines (LCPAs), or polypeptides modified with LCPAs are known to be involved in this process. The LCPAs contained in the silica walls of diatoms are thought to play pivotal roles in the silica deposition. In sponges, however, a protein called silicatein and several other proteins have been reported to be the factors involved in the silica deposition. However, no other factors involved in this process have been reported. We have identified the LCPAs from the marine sponge Axinyssa aculeata and present here some evidence that sponge‐derived LCPAs can deposit silica and that the LCPA derivatives are associated with spicules. The results indicate a common chemistry between sponges and diatoms, the two major players in the biological circulation of silicon in the marine environment. A wide variety of organisms are known to utilize silica in their biological processes. Polyamines or other functional molecules might be involved, in combination with proteins, in their biosilicification process.

Octocoral chemical signaling selects and controls dinoflagellate symbionts.

Symbioses between zooxanthellae (Symbiodinium spp.) and marine invertebrates, including corals, are common in shallow marine environments. The zooxanthellae contribute to host nutrition by translocating photosynthetic products and enabling them to effloresce in oligotrophic conditions. Coral mainly acquire Symbiodinium spp. by capturing freeswimming cells from the environment (1). Cultured Symbiodinium cells show a diel growth cycle with alternation between motile and non-motile cell stages once a day (2, 3), and the cell divides only during the latter stage (2). When associated with a host, however, cells are arrested in a non-motile stage while healthy cell division is maintained (4). We deduced that host-directed and chemical-based mechanisms are responsible for this phenomenon since SLL-2, a lectin that binds to carbohydrate chains with a D-galactosyl moiety, produced by the octocoral Sinularia lochmodes, is localized on the cell surface of the Symbiodinium harbored in the host (5). Here we describe SLL-2 as the key chemical factor for arresting Symbiodinium in the cell-dividing, non-motile stage, while some nonsymbiotic microalgae were even destroyed by SLL-2. Symbiotic associations between photosynthetic dinoflagellates (Symbiodinium spp.) and invertebrate hosts such as corals are crucial for the survival of the host animals since Symbiodinium supplies organic compounds to them and enables them to prosper in the oligotrophic environment

The d-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates

A D-galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. D-Galactose, N-acetyl-D-galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80 degrees C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes.

Cellular Origin of Dysiherbaine, an Excitatory Amino Acid Derived from a Marine Sponge

The cellular origin of dysiherbaine, a marine‐sponge toxin, was investigated immunohistochemically by using an anti‐dysiherbaine antibody. Dysiherbaine‐like immunoreactivity was found to be localized in spherical cells harbored in the sponge mesohyl. A combination of ribosomal RNA gene (rDNA) analysis and cell‐morphology analysis revealed that the spherical cells were Synechocystis cyanobacteria. However, the sponge, identified as Lendenfeldia chondrodes on the basis of its rDNA sequence, appeared to contain two different chemotypes—dysiherbaine‐producing (DH+) and nondysiherbaine‐producing (DH−)—both of which inhabited the same region. Synechocystis cells in the DH− sponge were not labeled with antibody, although the 16S rDNA gene profile of the cyanobacteria in the DH− sponge was indistinguishable from that of the cyanobacteria in the DH+ sponge. On the basis of these results, we hypothesize that dysiherbaine is a metabolite of certain varieties of endosymbiotic Synechocystis sp.

Modulating effect of acorn barnacle C-type lectins on the crystallization of calcium carbonate

The modulating effect of invertebrate C-type lectins on the crystallization of calcium carbonate was investigated. The multiple C-type lectins, named BRA-1, −2 and −3, isolated from the acorn barnacle Megabalanus rosa inhibited the nucleation and growth of calcium carbonate cyrstals. Among BRA, BRA-2 most efficiently inhibited the crystal nucleation of aragoite and calcite at the concentrations of >3.3 and >26 ⧎g/mL, respectively. The inhibitory activities of BRA were enhanced by the addition of various biomolecules such as D-glucosamine, D-galactosamine, chitosan oligosaccharides, L-arginine and L-aspartic acid. Although the crystals of aragonite and calcite were formed in the presence of lower concentrations of BRA, the shape and size of the crystals were changed. These results further indicate the participation of the lectins in biomineralization.

Diverse Sugar-Binding Specificities of Marine Invertebrate C-Type Lectins

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized α2-6 sialylation but not α2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with α-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewisx and Lewisa epitopes.

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as “granule bodies”, which were found only in the pericentral cells of the thallus.

Synthesis and Biological Evaluation of the Forssman Antigen Pentasaccharide and Derivatives by a One‐Pot Glycosylation Procedure

The synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives thereof by using a one-pot glycosylation and polymer-assisted deprotection is described. The Forssman antigen pentasaccharide, composed of GalNAcα(1,3)GalNAcβ(1,3)Galα(1,4)Galβ(1,4)Glc, was recently identified as a ligand of the lectin SLL-2 isolated from an octocoral Sinularia lochmodes. The chemo- and α-selective glycosylation of a thiogalactoside with a hemiacetal donor by using a mixture of Tf(2)O, TTBP and Ph(2)SO, followed by activation of the remaining thioglycoside, provided the trisaccharide at the reducing end in a one-pot procedure. The pentasaccharide was prepared by the α-selective glycosylation of the N-Troc-protected (Troc=2,2,2-trichloroethoxycarbonyl) thioglycoside with a 2-azide-1-hydroxyl glycosyl donor, followed by glycosidation of the resulting disaccharide at the C3 hydroxyl group of the trisaccharide acceptor in a one-pot process. We next applied the one-pot glycosylation method to the synthesis of pentasaccharides in which the galactosamine units were partially and fully replaced by galactose units. Among the three possible pentasaccharides, Galα(1,3)GalNAc and Galα(1,3)Gal derivatives were successfully prepared by the established method. An assay of the binding of the synthetic oligosaccharides to a fluorescent-labeled SLL-2 revealed that the NHAc substituents and the length of the oligosaccharide chain were both important for the binding of the oligosaccharide to SLL-2. The inhibition effect of the oligosaccharide relative to the morphological changes of Symbiodinium by SLL-2, was comparable to their binding affinity to SLL-2. In addition, we fortuitously found that the synthetic Forssman antigen pentasaccharide directly promotes a morphological change in Symbiodinium. These results strongly indicate that the Forssman antigen also functions as a chemical mediator of Symbiodinium.

Dynamic changes in the accumulation of metabolites in brackish water clam Corbicula japonica associated with alternation of salinity

The brackish water clam Corbicula japonica inhabits rivers and brackish waters throughout Japan where the major fishing grounds in the Ibaraki Prefecture, Japan, are located at the Hinuma Lake and Hinuma River. Water salinity in the Lake Hinuma is low and stable due to the long distance from the Pacific Ocean, whereas that in the downstream of the river varies daily due to a strong effect of tidal waters. In the present study, we dissected the gill and foot muscle of brackish water clam collected from these areas, and subjected them to metabolome analysis by capillary electrophoresis-time-of-flight mass spectrometry. More than 200 metabolites including free amino acids, peptides and organic acids were identified, and their amounts from the foot muscle tend to be higher than those from the gill. The principal component analysis revealed that the amount of each metabolite was different among sampling areas and between the gill and foot muscle, whereas no apparent differences were observed between male and female specimens. When the metabolites in the female clam at high salinity were compared with those at low salinity, concentrations of β-alanine, choline, γ-aminobutyric acid, ornithine and glycine betaine were found to be changed in association with salinity. We also compared various metabolites in relation to metabolic pathways, suggesting that many enzymes were involved in their changes depending on salinity.

Crystal structure of a symbiosis-related lectin from octocoral

D-Galactose-binding lectin from the octocoral, Sinularia lochmodes (SLL-2), distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellate into a non-motile (coccoid) symbiotic state. SLL-2 binds with high affinity to the Forssman antigen (N-acetylgalactosamine(GalNAc)α1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-ceramide), and the presence of Forssman antigen-like sugar on the surface of Symbiodinium CS-156 cells was previously confirmed. Here we report the crystal structures of SLL-2 and its GalNAc complex as the first crystal structures of a lectin involved in the symbiosis between coral and dinoflagellate. N-Linked sugar chains and a galactose derivative binding site common to H-type lectins were observed in each monomer of the hexameric SLL-2 crystal structure. In addition, unique sugar-binding site-like regions were identified at the top and bottom of the hexameric SLL-2 structure. These structural features suggest a possible binding mode between SLL-2 and Forssman antigen-like pentasaccharide.