Insights into phosphatase-activated chemical defense in a marine sponge holobiont.

Marine sponges often contain potent cytotoxic compounds, which in turn evokes the principle question of how marine sponges avoid self-toxicity. In a marine sponge Discodermia calyx, the highly toxic calyculin A is detoxified by the phosphorylation, which is catalyzed by the phosphotransferase CalQ of a producer symbiont, "Candidatus Entotheonella" sp. Here we show the activating mechanism to dephosphorylate the stored phosphocalyculin A protoxin. The phosphatase specific to phosphocalyculin A is CalL, which is also encoded in the calyculin biosynthetic gene cluster. CalL represents a new clade and unprecedently coordinates the heteronuclear metals Cu and Zn. CalL is localized in the periplasmic space of the sponge symbiont, where it is ready for the on-demand production of calyculin A in response to sponge tissue disruption.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma.

Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by 'Entotheonella'.

Sponge metagenomes are a useful platform to mine cryptic biosynthetic gene clusters responsible for production of natural products involved in the sponge-microbe association. Since numerous sponge-derived bioactive metabolites are biosynthesized by the symbiotic bacteria, this strategy may concurrently reveal sponge-symbiont produced compounds. Accordingly, a metagenomic analysis of the Japanese marine sponge Discodermia calyx has resulted in the identification of a hybrid type I polyketide synthase-nonribosomal peptide synthetase gene (kas). Bioinformatic analysis of the gene product suggested its involvement in the biosynthesis of kasumigamide, a tetrapeptide originally isolated from freshwater free-living cyanobacterium Microcystis aeruginosa NIES-87. Subsequent investigation of the sponge metabolic profile revealed the presence of kasumigamide in the sponge extract. The kasumigamide producing bacterium was identified as an 'Entotheonella' sp. Moreover, an in silico analysis of kas gene homologs uncovered the presence of kas family genes in two additional bacteria from different phyla. The production of kasumigamide by distantly related multiple bacterial strains implicates horizontal gene transfer and raises the potential for a wider distribution across other bacterial groups.

Calyculin: Nature's way of making the sponge-derived cytotoxin.

Covering: up to 2015.Calyculin A is a major cytotoxic compound isolated from the Japanese marine sponge Discodermia calyx. Its potent cytotoxicity is attributable to the specific inhibition of protein phosphatases 1 and 2A, as in the case of okadaic acid and the microcystins. Its chemical structure is well-designed not only for enzyme inhibition but also for higher membrane permeability in order to impart its potent cytotoxicity. The biosynthetic gene cluster of this densely functionalized polyketide and nonribosomal peptide hybrid molecule was recently identified from the sponge-microbe association. The producer organism and the dynamic bioconversion process were also revealed. In this highlight, we focus on the recent studies addressing nature's design and biogenesis of the sponge-derived cytotoxin, calyculin A.

Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes.

Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 5'-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.

Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx.

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.

Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont.

The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.

Indole-porphyrin hybrids produced by metagenomics.

New indole-porphyrin hybrid molecules were isolated from Escherichia coli harboring metagenomic DNA from the Japanese marine sponge Discodermia calyx. The indole was appended to the reactive vinyl substituent of the harderoporphyrin chromophore, encoded by the insert DNA. Thus, the chimeric pathway between the heterologously expressed porphyrins and the endogenous indole generated new indole-conjugated chiral porphyrins in E. coli.

Heterologously expressed ß-hydroxyl fatty acids from a metagenomic library of a marine sponge.

Functional screening based on the antibacterial activity of a metagenomic library of the Japanese marine sponge, Discodermia calyx, afforded three ß-hydroxyl fatty acids: 3-hydroxypalmitic acid, 3-hydroxylauric acid and 3-hydroxymyristic acid, heterologously expressed in an antibacterial clone, pDC113. 3-Hydroxypalmitic acid showed moderate antibacterial activity against Bacillus cereus and Candida albicans. A sequence analysis of the insert DNA revealed 23 putative ORFs, with most sharing homology to bacterial fatty acid synthase II and lipid A biosynthesis enzymes. The other ORFs were probably transmembrane proteins involved in lipid A biosynthesis. Although lipid A was not detected under our experimental conditions, the production of ß-hydroxyl fatty acids as components of lipid A were enhanced in pDC113.

Porphyrins from a metagenomic library of the marine sponge Discodermia calyx.

Marine sponges harbouring uncultured symbiotic bacteria are important sources of biologically active compounds. Since they would be interesting resources to explore unknown functional genes by means of a metagenomic approach, we constructed a metagenomic library of the Japanese marine sponge Discodermia calyx. The functional screening afforded the two clones producing porphyrins as red pigments. The isolation and structural elucidation of the red pigments revealed that the major red pigment was Zn-coproporphyrin III. The sequence data of the clones identified genes encoding glutamyl-tRNA reductase along with other ORFs related to porphyrin biosynthesis.

Calyxamides A and B, cytotoxic cyclic peptides from the marine sponge Discodermia calyx.

Cyclic peptides containing 5-hydroxytryptophan and thiazole moieties were isolated from the marine sponge Discodermia calyx collected near Shikine-jima Island, Japan. The structures of calyxamides A (1) and B (2), including the absolute configurations of all amino acids, were elucidated by spectroscopic analyses and degradation experiments. The structures are similar to keramamides F and G, previously isolated from Theonella sp. The analysis of the 16S rDNA sequences obtained from the metagenomic DNA of D. calyx revealed the presence of Candidatus Entotheonella sp., an unculturable δ-proteobacterium inhabiting the Theonella genus and implicated in the biosynthesis of bioactive peptides.

Furan fatty acid as an anti-inflammatory component from the green-lipped mussel Perna canaliculus.

A lipid extract of Perna canaliculus (New Zealand green-lipped mussel) has reportedly displayed anti-inflammatory effects in animal models and in human controlled studies. However, the anti-inflammatory lipid components have not been investigated in detail due to the instability of the lipid extract, which has made the identification of the distinct active components a formidable task. Considering the instability of the active component, we carefully fractionated a lipid extract of Perna canaliculus (Lyprinol) and detected furan fatty acids (F-acids). These naturally but rarely detected fatty acids show potent radical-scavenging ability and are essential constituents of plants and algae. Based on these data, it has been proposed that F-acids could be potential antioxidants, which may contribute to the protective properties of fish and fish oil diets against chronic inflammatory diseases. However, to date, in vivo data to support the hypothesis have not been obtained, presumably due to the limited availability of F-acids. To confirm the in vivo anti-inflammatory effect of F-acids in comparison with that of eicosapentaenoic acid (EPA), we developed a semisynthetic preparation and examined its anti-inflammatory activity in a rat model of adjuvant-induced arthritis. Indeed, the F-acid ethyl ester exhibited more potent anti-inflammatory activity than that of the EPA ethyl ester. We report on the in vivo activity of F-acids, confirming that the lipid extract of the green-lipped mussel includes an unstable fatty acid that is more effective than EPA.