Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with ß-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Production of recombinant porin from Y. pseudotuberculosis in a water-soluble form for pseudotuberculosis diagnostics.

OmpF porin from the outer membrane of Yersinia pseudotuberculosis was cloned into pET-40b(+) plasmid. Using E. coli Rosetta (DE3) strain, MX medium, IPTG concentration of 0.2 mm and post-induction cultivation at 14°C overnight allowed us to obtain a water-soluble form of the recombinant protein (rs-OmpF). Rs-OmpF was shown to have the ordered spatial structure at the levels of secondary and tertiary structure. Rs-OmpF was found to be effective as diagnostic antigen in ELISA for pseudotuberculosis diagnostics.

Data supporting functional diversity of the marine bacterium Cobetia amphilecti KMM 296.

Data is presented in support of functionality of hyper-diverse protein families encoded by the Cobetia amphilecti KMM 296 (formerly Cobetia marina KMM 296) genome ("The genome of the marine bacterium Cobetia marina KMM 296 isolated from the mussel Crenomytilus grayanus (Dunker, 1853)" [1]) providing its nutritional versatility, adaptability and biocontrol that could be the basis of the marine bacterium evolutionary and application potential. Presented data include the information of growth and biofilm-forming properties of the food-associated isolates of Pseudomonas, Bacillus, Listeria, Salmonella and Staphylococcus under the conditions of their co-culturing with C. amphilecti KMM 296 to confirm its high inter-species communication and anti-microbial activity. Also included are the experiments on the crude petroleum consumption by C. amphilecti KMM 296 as the sole source of carbon in the presence of sulfate or nitrate to ensure its bioremediation capacity. The multifunctional C. amphilecti KMM 296 genome is a promising source for the beneficial psychrophilic enzymes and essential secondary metabolites.

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.

Genetically modified proteins: functional improvement and chimeragenesis.

This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit.

Recombinant production and characterization of a highly active alkaline phosphatase from marine bacterium Cobetia marina.

The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10- to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45 °C and 27 min at 40 °C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50 °C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg(2+) bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.

A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a ß/α-protein with the predominance of ß-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish.

Mannan-binding lectin of the sea urchin Strongylocentrotus nudus.

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/ß-protein with eight ß-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.

An extracellular S1-type nuclease of marine fungus Penicillium melinii.

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5-8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3'-5' mode to produce 5'-oligonucleotides and 5'-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

Terpenoid metabolites from Spongia spp. and their effects on nucleic acid biosynthesis in sea urchin eggs.

19-Norspongia-13(16),14-diene-3-one (1) was isolated for the first time from a natural source, along with a series of known spongiane diterpenoids (2-11) and sesquiterpene (12) from two unidentified species belonging to the genus Spongia. The effects of 1, 4, 5, 8-12 on biosynthesis of nucleic acids and embryonic development of the sea urchin Strongylocentrotus intermedius have been studied. All the compounds inhibit sea urchin embryo development at concentration of 20 microg/mL and above and DNA biosynthesis at the dose of 10 microg/mL. The inhibitory effect of diterpenoids at least partly may be explained by the inhibition of thymidine kinase activity.

Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase.

An alpha-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34 degrees C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular alpha-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The alpha-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric alpha-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This alpha-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

Differential induction of apoptosis of leukemic cells by rhizochalin, two headed sphingolipids from sponge and its derivatives.

Two-headed sphingolipids bear a certain similarity with sphingolipids in the structure and but differing from classical sphingolipids in alpha,omega-position of the basic groups. We analyzed the apoptotic effects of some two-headed sphingolipids including rhizochalin (Rhz) and its derivatives isolated from sponge Rhizochalina incrustata on human leukemia HL-60 cells. Direct addition of Rhz induced apoptosis of HL-60 cells. The aglycon of Rhz, which has no galactosyl residue, showed a stronger ability to induce apoptotic activity than Rhz. Rhz congeners with acetate derivatives only weakly induced apoptosis. The usual mitochondrial membrane permeability changes and the decrease of protein levels of procaspases-8, -9, and -3 correlated with the apoptotic activity of Rhzs. These results suggest that derivatives of two-headed sphingolipids potently induce apoptosis in mammalian cells when administered exogenously and this cell death was dependent on caspase activation pathways.

Differential effects of triterpene glycosides, frondoside A and cucumarioside A2-2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells.

Frondoside A is a pentaoside having an acetyl moiety at the aglycon ring and xylose as a third monosaccharide residue. Cucumarioside A(2)-2 is a pentaoside having glucose as a third monosaccahride unit. We compared the effects of frondoside A and A(2)-2 for cell death-inducing capability with close attention paid to structure-activity relationships. Both frondoside A and A(2)-2 strongly induced apoptosis of leukemic cells. Frondoside A-induced apoptosis was more potent and rapid than A(2)-2-induced apoptosis. A(2)-2-induced but not frondoside A-induced apoptosis was caspase-dependent. This suggests that holothurians may induce apoptosis of leukemic cells caspase-dependently or -independently, depending on the holothurian structure.

Isolation, characterization and molecular cloning of duplex-specific nuclease from the hepatopancreas of the Kamchatka crab.

BACKGROUND: Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking. RESULTS: Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 - 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact. CONCLUSION: We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.

Fucoidan inhibits UVB-induced MMP-1 expression in human skin fibroblasts.

Ultraviolet (UV)B irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signaling transduction pathways, which are responsible for the degradation or synthesis inhibition of collagenous extracellular matrix in connective tissues, causing skin photoaging. Using the human skin fibroblast (HS68) cell line in the present study, we investigated the inhibitory effects of fucoidan on MMP-1 expression by various in vitro experiments and elucidated the pathways of inhibition. Pretreatment with fucoidan inhibited UVB-induced MMP-1 expression in a dose-dependent manner. Extracellular signal regulated kinase (ERK) activation was markedly inhibited by treatment with fucoidan, though JNK activation was very slightly affected by fucoidan. We also found that fucoidan pretreatment significantly reduced MMP-1 mRNA expression in comparison with UVB irradiation only. In conclusion, our results demonstrate that fucoidan can mainly inhibit UVB-induced MMP-1 expression by inhibiting the ERK pathways. Therefore, fucoidan might be used as a potential agent for the prevention and treatment of skin photoaging.

Effect of 1,3;1,6-beta-D-glucans on developing sea urchin embryos.

The effect of 1,3;1,6-beta-D-glucooligo- and polysaccharides with different structures (from 1 to 10 kDa of molecular mass; from 10-25% of beta-1,6-linked glucose residues content) on the developing embryos of sea urchin, Strongylocentrotus intermedius, was evaluated for the screening of potential positive stimulants. 1,3;1,6-beta-D-glucans with a molecular mass of between 6-10 kDa and at concentrations of 0.05-0.25 mg/ml shown the best modulator effect on the sea urchin embryos. 1,3;1,6-beta-D-glucans increased the survival of the sea urchin embryos up to 2.5-fold compared with the control animals.