Discovery of a fucoidan endo-4O-sulfatase: Regioselective 4O-desulfation of fucoidans and its effect on anticancer activity in vitro.

Fucoidans are a class of sulfated fucose-containing bioactive polysaccharides produced by brown algae. The biological effects exhibited by fucoidans are thought to be related to their sulfation. However, the lack of methods for sulfation control does not allow for a reliable conclusion about the influence of the position of certain sulfate groups on the observed biological effects. We identified the gene encoding the endo-acting fucoidan sulfatase swf5 in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. This is the first report on the sequence of fucoidan endo-sulfatase. Sulfatase SWF5 belongs to the subfamily S1_22 of the family S1. SWF5 was shown to remove 4O-sulfation in fucoidans composed from the alternating α-(1→3)- and α-(1→4)-linked residues of sulfated L-fucose but not from fucoidans with the α-(1→3)-linked backbone. The endo-sulfatase was used to selectively prepare 4O-desulfated fucoidan derivatives. It was shown that the 4O-desulfated fucoidans inhibit colony formation of DLD-1 and MCF-7 cells less effectively than unmodified fucoidans. Presumably, 4O-sulfation makes a significant contribution to the anticancer activity of fucoidans.

Expression and biochemical characterization of two recombinant fucoidanases from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T.

Genomic analysis of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T revealed the presence of four fucoidanase genes fwf1, fwf2, fwf3, fwf4 that belonged to the glycoside hydrolase family 107 (GH107, CAZy), which is located in one gene cluster putatively involved in fucoidan catabolism. Genes encoding two fucoidanases fwf1 and fwf2 were cloned, and the proteins FWf1 and FWf2 were produced in Escherichia coli cells. The recombinant fucoidanases were purified and the biochemical properties of these enzymes were studied. The amino acid sequences of FWf1 and FWf2 showed 41 and 51% identity respectively with a fucoidanase FcnA from the marine bacterium Mariniflexile fucanivorans, with the established 3D structure. Structures of the oligosaccharides produced during enzymatic hydrolysis of fucoidan by FWf1 and FWf2 have been determined by NMR spectroscopy. Detailed substrate specificities of FWf1 and FWf2 were studied using fucoidans and sulfated fucooligosaccharides with different structures. Both fucoidanases catalyzed hydrolysis of 1→4-glycosidic bonds between sulfated α-l-fucose residues but had different specificities regarding sulfation patterns of the fucose residues in fucoidan molecules. Specific cleavage sites recognizable by the fucoidanases in fucoidan molecules were determined. The obtained results provide new knowledge about differences between specificities of the fucoidanases belonging to the GH107 family.

Effect of Sulfated Polysaccharides from Marine Hydrobionts on Humoral Immune Response to Ovalbumin in Mice.

Adjuvant activity of fucoidans in experimental vaccine compositions with ovalbumin was studied on a mouse model. Compositions with sulfated polysaccharides from brown alga Fucus evanescens (native fucoidan in combination with polyphenols, and a product of fucoidan enzymatic hydrolysis) induced multiple productions of antigen-specific antibodies - total IgG, its isotypes IgG1 and, especially, IgG2a, in comparison with an individual ovalbumin. The adjuvant effect of native and structurally modified fucoidans is slightly inferior to that of the traditional licensed aluminum hydroxide adjuvant. The results indicate the prospects of using sulfated polysaccharides from F. evanescens as adjuvants in vaccines.

Radiosensitizing effect of the fucoidan from brown alga Fucus evanescens and its derivative in human cancer cells.

Fucoidan from brown alga Fucus evanescens and its product of enzymatic hydrolysis have precisely established structure and possess significant biological activities. The aim of present study was to determine radiosensitizing activity of fucoidan from brown alga F. evanescens and its derivative in human melanoma, breast adenocarcinoma, and colorectal carcinoma cell lines and elucidate mechanism of their action. The fucoidan from F. evanescens and its derivative had a comparable radiosensitizing activity and increased the inhibiting effect of X-ray radiation on proliferation and colony formation of human cancer cells, with significant inhibition of melanoma cells. The molecular mechanism of this action was associated with the induction of apoptosis by activating the initiator and effector caspases, suppressing the expression of the anti-apoptotic protein, and enhancing the fragmentation of DNA. The obtained data confirm the prospects of using fucoidan's derivative in combination with radiation therapy for the improvement of the schemes of cancer therapy.

[Effect of Sulfated Polysaccharides from Brown Alga Fucus evanescens and Their Enzymatic Transformation Product on Functional Activity of Innate Immunity Cells.]

The effect of sulfated polysaccharides (PS) from brown alga Fucus evanescens and their enzymatic transformation and low molecular weight product on the functional activity of the innate immunity cells, i.e. polymorphonuclear leukocytes of human peripheral blood (NF) was comparatively studied. The in vitro NF contact with PS resulted in significant changes in the functional activity of NF, evident from higher density of molecules CD69, CD14, CD11b on the cell membranes with simultaneous lowering of that of CD62L and increased phagocytic and bactericidal activity of NF. The low molecular weight product resulting from fucoidan transformation with fucoidanases showed a higher effect on the level of the molecules CD14, CD11b and CD62L expression vs. the high molecular weight PS.

Antitumor activity of cucumarioside A2-2.

BACKGROUND: The cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. However, the molecular mechanism(s) of the antitumor action of triterpene glycosides on cancer cells remain unclear. This article reports a continuation of investigations of triterpene glycoside cucumarioside A2-2 isolated from the Far-Eastern sea cucumber Cucumaria japonica. It describes a study of glycoside anticancer activity in vivo and glycoside interaction with mouse Ehrlich carcinoma cells in vitro. METHODS: The cytotoxicity of cucumarioside A2-2 and its effect on apoptosis, the cell cycle, DNA biosynthesis and p53 activity, and glycoside anticancer action against Ehrlich carcinoma cells were studied. RESULTS: Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by nonspecific esterase assay and MTT assay was 2.1 and 2.7 µM, respectively. Cucumarioside A2-2 at a subcytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells in a caspase-dependent way, bypassing the activation of the p53-dependent segment. CONCLUSION: The anticancer and proapoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. The in vivo anticancer effect of cucumarioside A2-2 may be associated with the ability of the drug to arrest the cell cycle in the synthetic phase and induce programmed tumor cell death.

Determination of cucumarioside A2-2 in mouse spleen by radiospectroscopy, MALDI-MS and MALDI-IMS.

The distribution of triterpene glycoside cucumarioside A2-2, the main compound of medical lead Cumaside in immunodeficiency diseases, in mouse spleen was determined. For this purpose the stability and dynamics of glycoside content changes over time in Balb/c mouse spleen tissue homogenate as well as the study of the cucumarioside A2-2 spatial distribution in tissue sections were investigated using radiospectroscopy, MALDI-MS and MALDI Imaging Mass Spectrometry (IMS), correspondingly. Cucumarioside A2-2 is reliably detected by MALDI-MS in the mouse spleen tissue after single intraperitoneal (i.p.) injection at a dosage of 5 mg/kg. The glycoside is stable in the spleen and does not undergo metabolic transformation in either tissue homogenates or in the intact organ within 24 h after i.p. injection. The cucumarioside A2-2 was absorbed fairly rapidly: the glycoside maximum concentration (Cmax) in tissue homogenate was observed in the first 30 min after injection; the minimum values were registered in 3 h. These results are in agreement with those obtained in the pharmacokinetic study of (3)H-cucumarioside A2-2. It was established by MALDI-IMS that glycoside was mainly located in the tunica serosa part of the spleen and only a small amount was detected within the red and white pulp of the organ. MALDI MS images obtained 15-30 min post dosage clearly reflect high drug concentrations in the regions surrounding the organ followed by its decline in the surface part and a very slight redistribution to the internal part of the spleen.

Catalytic properties and amino acid sequence of endo-1→3-ß-D-glucanase from the marine mollusk Tapes literata.

A specific 1→3-ß-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45°C. The 1→3-ß-D-glucanase catalyzes hydrolysis of ß-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K(m) for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-ß-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-ß-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-ß-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.

Immunomodulatory properties of frondoside A, a major triterpene glycoside from the North Atlantic commercially harvested sea cucumber Cucumaria frondosa.

Frondoside A, a major triterpene glycoside from North Atlantic commercially harvested sea cucumber Cucumaria frondosa, possesses strong immunomodulatory properties in subtoxic doses. Frondoside A stimulates lysosomal activity of mouse macrophages in vivo at a maximal effective stimulatory dose of 0.2 microg per mouse and is maintained over 10 days. This glycoside also shows a 30% stimulation of lysosomal activity in mouse macrophages in vitro at concentrations of 0.1-0.38 microg/mL. Frondoside A enhances macrophage phagocytosis of the bacterium Staphylococcus aureus in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates reactive oxygen species formation in macrophages in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates an increase in the number of antibody plaque-forming cells (normally B-cells in spleen) in vivo with a maximal stimulatory effect at a concentration of 0.2 microg per mouse (stimulatory index, 1.86). Frondoside A has a weak effect upon immunoglobulin (Ig) M production after immunization with sheep erythrocytes in mice. Frondoside A does not stimulate Ig production in mice and does not significantly enhance the ovalbumin-stimulated IgM and IgG antibody levels in ovalbumin-immunized mice. Hence frondoside A is an immunostimulant of cell-based immunity including phagocytosis without a significant effect on amplification of humoral immune activity or adjuvant properties. Therefore, frondoside A may provide curative and/or preventive treatment options against diseases wherein a depleted immune status contributes to the pathological processes.

Estrogenic activity of triterpene glycosides in yeast two-hybrid assay.

Estrogenic potency of six triterpene glycosides, Holothurin A, Holotoxin A1, Frondoside A, Cucumarioside A2-2 and Cauloside C, that are natural products and semi-synthesized Ginsenoside-Rh2, were examined with yeast two-hybrid system, including expressed genes of human estrogen receptor, hERalpha, the co-activator TIF2 and lacZ as a reporter gene. Only Ginsenoside-Rh2 exhibited significant moderate estrogenic activity in the concentration range of 10(-7) to 10(-6)M. Its effect was approximately 30% of the activity of 17beta-estradiol applied at half-effective concentration. This indicates Ginsenosides-Rh2 is a weak phytoestrogen. The sea cucumber triterpene glycosides, Holothurin A, Holotoxin A1, Cucumarioside A2-2 and Frondoside A, and plant glycoside Cauloside C had no appreciable estrogenic activity. Data obtained by yeast two-hybrid assay reflect structure-activity relationship between tested compounds and 17beta-estradiol. Only Ginsenoside-Rh2 has some similarity in chemical structure with 17beta-estradiol that might explain affinity of this glycoside to the hERalpha receptor.

Immunomodulatory properties of Cumaside.

The medical lead, so-called Cumaside, was created on the basis of triterpene oligoglycosides from the Far-Eastern edible sea cucumber (holothurian) Cucumaria japonica and its immunomodulatory properties were studied. The haemolytic activity of Cumaside was significantly reduced in comparison with original glycosides due to the glycoside-cholesterol complex formation. The influence of Cumaside on mouse macrophages in low doses was accompanied by more then two-fold stimulation of lysosomal activity. This preparation was found to increase significantly the animal resistance against bacterial infections elicited by various pathogens. It stimulated phagocytosis, ROS formation, IL6 and TNF-alpha production in lymphocytes, increased the number of antibody producing cells and amplified the expression of several cell surface molecules (CD3, CD4, CD8) preliminary cultured with hydrocortisone. At the same time the preparation did not affect the delayed-type hypersensitivity, proliferative activity of lymphocytes, cytotoxic activity of NK-cells and cytokine IFNgamma and IL12p70 release. The mechanism of Cumaside action is discussed.