Program

Natural products have been playing a key role in drug discovery. However, bioactive small molecules with new carbon backbones are still urgently required because drug-resistant diseases have become more widespread and the side effects of clinically-used drugs are problematic. Today, the most impressive remaining frontiers are largely microbial, and exploring unique or uninvestigated environments with suitably improved microbiological and chemical techniques. We have been focusing on bacteria inhabiting marine environments because secondary metabolites of bacteria in these habitats are not thoroughly studied.
LC/MS-based chemical analysis of symbiotic bacteria in insects led to the discovery of the structurally new bioactive secondary metabolites. For example, new cyclic peptides, ohmyungsamycins A and B, were discovered from a sand beach strain of Streptomyces. The ohmyungsamycins strongly inhibited Mycobacterium tuberculosis. Mohangamides A and B, new dilactone-tethered pseudo-dimeric peptides inhibiting Candida albicans isocitrate lyase, were discovered from an actinomycete strain collected in an intertidal mudflat. The discovery of structurally, biologically, and biosynthetically interesting secondary metabolites from actinomycetes inhabiting marine environments demonstrates that studying relatively-uninvestigated bacteria in search for new bioactive compounds could be a promising strategy for drug discovery.

A number of novel guanidine alkaloids have been found from wide variety of marine organisms ranging from microorganisms to fish. Most prominent examples are tetrodotoxin and saxitoxin, potent and selective sodium channel blockers. Many guanidine alkaloids, including oroidin family as exampled by palau’amine, and crambesidins and related compounds are known from sponges. We have recently reported isolation of novel guanidine alkaloids mellpaladines and dopargimines with potential neuronal activities form a Palauan Didemnidae tunicate. In search of biologically active molecules from the aqueous marine extract, we found a novel trisguanidine alkaloid form Epizoanthus sp. collected in Palau.
The sample of Epizoanthus sp. was extracted by water. The crude extract exhibited acute behavioral modulation in mice. Sephadex-LH20 fractions of the extracts had various activities including the acute convulsant activity or behavioral modulation with slow onset. We purified the fraction that caused slow death of mice by HPLC to afford isolation of active principle KB343 (1). The structure of 1 was determined on the basis of spectrometric and spectroscopic methods to be a novel trisguanidine alkaloid with unprecedented carbon skeletal structure. Here, chemical and biological studies regarding 1 and related metabolites will be presented.

The isolation and structure elucidation of secondary metabolites which were isolated through bioactivity directed fractionation of the extracts of marine invertebrates will be discusses.

Channa argus, a type of snakehead fish native to China, is a popular food fish in certain Asian countries. Given their lack of natural enemies outside their natural environment, snakeheads can cause ecological damage and it is a known destructive invasive species in the US. In this study, the two collagens (acid-soluble collagen and pepsin-solubilized collagen) were obtained from C. argus skin. The yields of ASC was 28.0% and that of PSC was 16.8% on the dry bases. The hydroxyproline-to-protein ratio in snakehead skin was 7.67%, which was close to that of silver carp skin. The collagens were identified as the collagen of type I by SDS-PAGE patterns. The denaturation temperature (Tds) were approximately 27.0 °C. Similar ultraviolet spectra of both collagens were observed. FTIR indicated PSC structure had a little change due to the loss of terminal domains by pepsin digestion. The results of X ray diffraction (XRD) proved that the two collagens retained their helical structures, The results suggest that the collagens isolated from C. argus can potentially be alternative sources of vertebrate collagens for use in the food and other industries.

Marine algae get attention as the health food ingredient year by year. An edible red alga Meristotheca papulosa contains the high-mannose (HM-) N-glycan binding lectin, MPL-1, which belongs to the jacalin-related lectin (JRL) family. MPL-1 consists of a disulfide bonded homodimer of a 15 kDa polypeptide. Carbohydrate binding analyses revealed that MPL-1 did not bind to any monosaccharides tested, including mannose. In a detailed oligosaccharide binding assay using 27 pyridylaminated (PA-) oligosaccharides by centrifugal ultrafiltration HPLC method, MPL-1 represented an extremely strict binding specificity to α1-2 linked mannose in D3 arm of HM-glycans; MPL-1 did not show any binding to complex type glycans, oligomannosides, glycolipid type oligosaccharides, and HM-glycans bearing exposed α1-6 linked mannose in D3 arm. These features reveal that MPL-1 is an unprecedented HM-glycan specific lectin. By E. coli expression system, an active form of recombinant MPL-1 (rMPL-1) was successfully produced using a pCold expression vector. Kinetic binding analysis for rMPL-1 with its most preferable PA-oligosaccharide, Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-PA, elucidated that MPL-1 had two binding sites in a molecule and that the association constant to the PA-decasaccharide was 2.43×10⁸ (M^-1). As predicted from the binding specificity, MPLs showed a high association constant (2.22×10⁸ M^-1) with an HIV envelope gp120, which is heavily glycosylated with HM-glycans, and potent anti-HIV activities with low nanomolar IC₅₀ values. To evaluate the behavior of MPL-1 after intake, digestion tests using artificial gastric and/or intestinal juices were examined. Interestingly, MPL-1 was little degraded after these treatments. In further digestion tests using papain, proteinase K, and Actinase E, in non-denaturing conditions, MPL-1 was also little digested. These results suggested that MPL-1 could be delivered in the active form to the intestines.

In gonadal maturation process, the ovary of scallop Mizhopecten yessoensis accumulate carotenoids, mainly pectenorone, thus turning it a characteristic pink color. This conspicuous color, however, turns to pale orange by heating. Because carotenoids can bind to proteins and that bathochromically shift its color, proteins responsible for this function are collectively called carotenoproteins. We thus presumed that presence of a carotenoprotein in the ovary. The mechanism of accumulation of and biological function of carotenoids in the ovary are unclear mainly because of lack of information on the carotenoprotein. In the present study, we separated and characterized the protein towards the goal of further understanding for the mechanism of accumulation of the carotenoids in the ovary.
Efficient extraction of the pink-colored intact protein has been difficult. We however found that use of KBr-containing buffer efficiently extracted the pinkish solution from the ovary, where carotenoids, such as pectenolone, were presented. The extract was treated with ammonium sulfate series and the precipitation was separated on a gel-filtration column. A fraction colored slightly in red was eluted as a single peak. We therefore named this reddish protein pectenovarin.
The fraction containing pectenovarin, however, exhibited multiple ladder-like bands between about 90 kDa and 130 kDa in SDS-PAGE. Because these multiple polypeptides were conceivable to be subunits of pectenovarin, we extracted the bands from the acrylamide gel and the enzyme digests were analyzed for its amino acid sequence using a Q-TOF ESI-MS/MS. The results of a high resolution de novo sequencing of the enzyme digests indicated that the amino acid sequences of the peptide fragments from the pectenovarin-containing fraction were highly homologous to that of vitellogenin, a precursor of egg yolk proteins.

Many corals thrives at tropical and subtropical areas by harboring symbiotic algae Symbiodinium, but corals often acquire Symbiodinium from environment every generation．We purified two lectins, N-acetyl-galactosamine (GalNAc)/N-acetyl-glucosamine (GlcNAc) binding lectin AtTL-2, and GlcNAc binding lectin ActL, from a hard coral Acropora tenuis, and the lectins were involved in acquisition of Symbiodinium culture strain NBRC102920, as Symbiodinium acquisition was inhibited by anti-lectin antibody or sugars the lectins recognize. We also found lectins attract Symbiodinium. In the present study, we examined the effect of these lectins to the motion of Symbiodinium. First, we classified motions of Symbiodinium NBRC102920 strain to five, "circle", "flat", "line", "movement", and "reverse". AtTL-2 was prepared by using E. coli BL21 Gold (DE3), and the recombinant AtTL-2 was 31 kDa, and bound to GalNAc and GlcNAc. When a capillary containing AtTL-2 was inserted to NBRC102920 culture, it slowly attracts NBRC102920 like "movement," although NBRC102920 usually rotate same place as "circle". When a capillary containing crude extract was inserted, crude extract also attracts the NBRC102920, but the motion of NBRC102920 away from the crude extract was "line", and that close to crude extract was "movement". When crude extract was preincubated with GalNAc, NBRC102920 was moved as "line" but to random direction. These results suggest that AtTL-2 controls direction of movement and ActL changes swimming motion to “line” without direction. ActL with small amount of AtTL-2 attract NBRC102920 like crude extract, suggesting that collaboration of ActL and AtTL-2 attracts Symbiodinium to the coral.