Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as "Galß1-4GlcNAcß obligatory containing an additional fragment", like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.

Anti-Inflammatory Activity of 1,4-Naphthoquinones Blocking P2X7 Purinergic Receptors in RAW 264.7 Macrophage Cells.

P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and ELISA approaches, the ability of 1,4-naphthoquinone thioglucoside derivatives, compounds U-286 and U-548, to inhibit inflammation induced by ATP/LPS in RAW 264.7 cells via P2X7 receptors was demonstrated. It has been established that the selected compounds were able to inhibit ATP-induced calcium influx and the production of reactive oxygen species, and they also exhibited pronounced antioxidant activity in mouse brain homogenate. In addition, compounds U-286 and U-548 decreased the LPS-induced activity of the COX-2 enzyme, the release of pro-inflammatory cytokines TNF-α and IL-1ß in RAW 264.7 cells, and significantly protected macrophage cells against the toxic effects of ATP and LPS. This study highlights the use of 1,4-naphthoquinones as promising purinergic P2X7 receptor antagonists with anti-inflammatory activity. Based on the data obtained, studied synthetic 1,4-NQs can be considered as potential scaffolds for the development of new anti-inflammatory and analgesic drugs.

The Anti-Inflammatory Effect of Carrageenan/Echinochrom Complex at Experimental Endotoxemia.

The anti-inflammatory effects of the CRG/Ech complex in LPS-induced endotoxemia were investigated in vivo in mice and in vitro in LPS-stimulated RAW 264.7 cells and peritoneal macrophages. The results indicated that the CRG/Ech complex suppressed the LPS-induced inflammatory response by reducing the production of ROS and NO in the macrophages. Furthermore, the in vivo experiment indicated that the CRG/Ech complex minimized disorders of the physiological and metabolic processes in mice subjected to LPS intoxication and reduced the levels of proinflammatory cytokines in the mouse serum. The preventive administration of the CRG/Ech complex to mice prevented endotoxin-induced damage in the mouse model of endotoxemia, increased the mice's resistance to LPS, and prevented increases in the levels of proinflammatory cytokines (TNFα). In this work, we showed by the molecular docking that Ech interacted with carrageenan, and that H-donor and H-acceptor bonds are involved in the formation of the complex.

In Vitro and In Silico Studies of Human Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) Inhibition by Stereoisomeric Forms of Lipophilic Nucleosides: The Role of Carbohydrate Stereochemistry in Ligand-Enzyme Interactions.

Inhibition of human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) by different chiral lipophilic nucleoside derivatives was studied. New Tdp1 inhibitors were found in the series of the studied compounds with IC50 = 2.7-6.7 µM. It was shown that D-lipophilic nucleoside derivatives manifested higher inhibition activity than their L-analogs, and configuration of the carbohydrate moiety can influence the mechanism of Tdp1 inhibition.

Influence of the Structural Features of Carrageenans from Red Algae of the Far Eastern Seas on Their Antiviral Properties.

The structural diversity and unique physicochemical properties of sulphated polysaccharides of red algae carrageenans (CRGs), to a great extent, determine the wide range of their antiviral properties. This work aimed to compare the antiviral activities of different structural types of CRGs: against herpes simplex virus type 1 (HSV-1) and enterovirus (ECHO-1). We found that CRGs significantly increased the resistance of Vero cells to virus infection (preventive effect), directly affected virus particles (virucidal effect), inhibited the attachment and penetration of virus to cells, and were more effective against HSV-1. CRG1 showed the highest virucidal effect on HSV-1 particles with a selective index (SI) of 100. CRG2 exhibited the highest antiviral activity by inhibiting HSV-1 and ECHO-1 plaque formation, with a SI of 110 and 59, respectively, when it was added before virus infection. CRG2 also significantly reduced the attachment of HSV-1 and ECHO-1 to cells compared to other CRGs. It was shown by molecular docking that tetrasaccharides-CRGs are able to bind with the HSV-1 surface glycoprotein, gD, to prevent virus-cell interactions. The revealed differences in the effect of CRGs on different stages of the lifecycle of the viruses are apparently related to the structural features of the investigated compounds.

Biologically active polyphenolic compounds from Lespedeza bicolor.

We investigated the ability of six prenylated prerocarpans, stilbenoid, and a new dimeric flavonoid, lespebicolin B, from stem bark as well as two 3-O-rutinosides and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol from flowers of Lespedeza bicolor to inhibit HSV-1 replication in Vero cells. Pretreatment of HSV-1 with polyphenolic compounds (direct virucidal effect) showed that pterocarpans lespedezol A2 (1), (6aR,11aR)-6a,11a-dihydrolespedezol A2 (2), (6aR,11aR)-2-isoprenyldihydrolespedezol A2 (4), and (6aR,11aR,3'R)-dihydrolespedezol A3 (5) significantly inhibited viral replication, with a selective index (SI) ≥10. Compound 4 possessed the lowest 50% - inhibiting concentration (IC50) and the highest SI values (2.6 µM and 27.9, respectively) in this test. (6aR,11aR)-2-Isoprenyldihydrolespedezol A2 (4) also had a moderate effect under simultaneous treatment of Vero cells with the tested compound and virus (IC50 and SI values were 5.86 µM and 12.4, respectively). 3-O-rutinosides of quercetin and kaempferol and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol (10 and 12) also showed significant virucidal activity, with SI values of 12.5, 14.6, and 98.2, respectively, and IC50 values of 8.6, 12.2, and 3.6, respectively. We also performed a quantitative structure-activity relationship (QSAR) analysis of data on the virucidal activity of polyphenolics with 4 < pIC50 < 6. It was found that the virucidal activity of these compounds depended on both the structure of the aromatic part and the conformation of geranyl and isoprenyl side chains of their molecules. These findings are correlated with the largest value of the principal moment of inertia (pmi) descriptor describing the geometry of molecules.

In Vitro Anti-Orthohantavirus Activity of the High-and Low-Molecular-Weight Fractions of Fucoidan from the Brown Alga Fucus evanescens.

The Hantaan orthohantavirus (genovariant Amur-AMRV) is a rodent-borne zoonotic virus; it is the causative agent of haemorrhagic fever with renal syndrome in humans. The currently limited therapeutic options require the development of effective anti-orthohantavirus drugs. The ability of native fucoidan from Fucus evanescens (FeF) and its enzymatically prepared high-molecular-weight (FeHMP) and low-molecular-weight (FeLMP) fractions to inhibit different stages of AMRV infection in Vero cells was studied. The structures of derivatives obtained were determined using nuclear magnetic resonance (NMR) spectroscopy. We found that fucoidan and its derivatives exhibited significant antiviral activity by affecting the early stages of the AMRV lifecycle, notably virus attachment and penetration. The FeHMP and FeLMP fractions showed the highest anti-adsorption activity by inhibiting AMRV focus formation, with a selective index (SI) > 110; FeF had an SI of ~70. The FeLMP fraction showed a greater virucidal effect compared with FeF and the FeHMP fraction. It was shown by molecular docking that 2O-sulphated fucotetrasaccharide, a main component of the FeLMP fraction, is able to bind with the AMRV envelope glycoproteins Gn/Gc and with integrin ß3 to prevent virus-cell interactions. The relatively small size of these sites of interactions explains the higher anti-AMRV activity of the FeLMP fraction.

Relationship between the structure of a highly regular fucoidan from Fucus evanescens and its ability to form nanoparticles.

Chitosan/fucoidan nanoparticles were created using two fucoidans from the Fucus evanescens algae. One of them was a regular fucoidan obtained for the first time from the alga harvested at the reproductive growth stage, using only standard extraction methods, without additional modifications. Its structure was established via NMR spectroscopy to consist of the repeating →3)-α-L-Fucp-(2,4SO3-)-(1 â†’ 4)-α-L-Fucp-(2SO3-)-(1→ fragment. Such fragment also coustituted 55% of the other fucoidan's structure, however it also included long sequences of α-L-fucopyranose residues sulfated only at C2. The nanoparticles were re-dispersed in water and the influence of fucoidan/chitosan mass ratio on the nanoparticles' size and zeta potential was investigated. 3D models of the regular fucoidan and chitosan's sections were created and their molecular docking was performed, showing that either polymer could occupy the exterior of the complex, depending on their ratio. Thermodynamic parameters of fucoidan-chitosan binding process were accessed, with the results indicating that significant conformational changes of fucoidan and chitosan molecules take place during the interaction, presumably to allow for more effective binding.

Antiviral Potential of Sea Urchin Aminated Spinochromes against Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) is one of the most prevalent pathogens worldwide requiring the search for new candidates for the creation of antiherpetic drugs. The ability of sea urchin spinochromes-echinochrome A (EchA) and its aminated analogues, echinamines A (EamA) and B (EamB)-to inhibit different stages of HSV-1 infection in Vero cells and to reduce the virus-induced production of reactive oxygen species (ROS) was studied. We found that spinochromes exhibited maximum antiviral activity when HSV-1 was pretreated with these compounds, which indicated the direct effect of spinochromes on HSV-1 particles. EamB and EamA both showed the highest virucidal activity by inhibiting the HSV-1 plaque formation, with a selectivity index (SI) of 80.6 and 50.3, respectively, and a reduction in HSV-1 attachment to cells (SI of 8.5 and 5.8, respectively). EamA and EamB considerably suppressed the early induction of ROS due to the virus infection. The ability of the tested compounds to directly bind to the surface glycoprotein, gD, of HSV-1 was established in silico. The dock score of EchA, EamA, and EamB was -4.75, -5.09, and -5.19 kcal/mol, respectively, which correlated with the SI of the virucidal action of these compounds and explained their ability to suppress the attachment and penetration of the virus into the cells.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Sponge-derived polybrominated diphenyl ethers and dibenzo-p-dioxins, irreversible inhibitors of the bacterial α-d-galactosidase.

An integrated in vitro and in silico approach was applied to evaluate the potency of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and spongiadioxins (OH-PBDDs) isolated from Dysidea sponges on the activity of the recombinant α-d-galactosidase of the GH36 family. It was revealed for the first time that all compounds rapidly and apparently irreversibly inhibited the bacterial α-d-galactosidase. The structure-activity relationship study in the series of OH-PBDEs showed that the presence of an additional hydroxyl group in 5 significantly enhanced the potency (IC50 4.26 µM); the increase of bromination in compounds from 1 to 3 increased their potency (IC50 41.8, 36.0, and 16.0 µM, respectively); the presence of a methoxy group decreased the potency (4, IC50 60.5 µM). Spongiadioxins 6, 7, and 8 (IC50 16.6, 33.1, and 28.6 µM, respectively) exhibited inhibitory action comparable to that of monohydroxylated diphenyl ethers 1-3. Docking analysis revealed that all compounds bind in a pocket close to the catalytic amino acid residues. Molecular docking detected significant compound-enzyme interactions in the binding sites of α-d-galactosidase. Superimposition of the enzyme-substrate and the enzyme-inhibitor complexes showed that their binding sites overlap.

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.

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.

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.

Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis.

The retaining endo-1,3-beta-D-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (Km approximately 600 microg/mL). The 1,3-beta-D-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 degrees C, respectively. cDNA encoding the endo-1,3-beta-D-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-beta-D-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-beta-D-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and beta-1,3-glucan-binding proteins. The fold of the LV was more closely related to kappa-carrageenase, agarase and 1,3;1,4-beta-D-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-beta-D-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of kappa-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.