Structure of the Lipooligosaccharide from the Deep-Sea Marine Bacterium Idiomarina zobellii KMM 231T, Isolated at a Depth of 4000 Meters

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain

Structure of the Cell-Wall-Associated Polysaccharides from the Deep-Sea Marine Bacterium Devosia submarina KMM 9415T

Two cell-wall-associated polysaccharides were isolated and purified from the deep-sea marine bacterium Devosia submarina KMM 9415T, purified by ultracentrifugation and enzymatic treatment, separated by chromatographic techniques, and studied by sugar analyses and NMR spectroscopy. The first polysaccharide with a molecular weight of about 20.7 kDa was found to contain d-arabinose, and the following structure of its disaccharide repeating unit was established: →2)-α-d-Araf-(1→5)-α-d-Araf-(1→. The second polysaccharide was shown to consist of d-galactose and a rare component of bacterial glycans-d-xylulose: →3)-α-d-Galp-(1→3)-β-d-Xluf-(1→

Structure of the Lipooligosaccharide from the Deep-Sea Marine Bacterium <i>Idiomarina zobellii</i> KMM 231^T, Isolated at a Depth of 4000 Meters

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain

Structure of the Cell-Wall-Associated Polysaccharides from the Deep-Sea Marine Bacterium <i>Devosia submarina</i> KMM 9415^T

Two cell-wall-associated polysaccharides were isolated and purified from the deep-sea marine bacterium Devosia submarina KMM 9415T, purified by ultracentrifugation and enzymatic treatment, separated by chromatographic techniques, and studied by sugar analyses and NMR spectroscopy. The first polysaccharide with a molecular weight of about 20.7 kDa was found to contain d-arabinose, and the following structure of its disaccharide repeating unit was established: →2)-α-d-Araf-(1→5)-α-d-Araf-(1→. The second polysaccharide was shown to consist of d-galactose and a rare component of bacterial glycans-d-xylulose: →3)-α-d-Galp-(1→3)-β-d-Xluf-(1→

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium <i>Formosa haliotis</i>

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-&alpha;-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of &alpha;-(1,4)-glycosidic linkages in fucoidans built of alternating &alpha;-(1,3)-/&alpha;-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1&Delta;470 (71 kDa). Fhf1&Delta;470 has optimal activity at pH 8, 37&ndash;40 &deg;C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers

Immunomodulatory Properties of Polysaccharides from the Coral Pseudopterogorgia americana in Macrophages

Polysaccharides from marine organisms produce an important regulatory effect on the mammalian immune system. In this study, the immunomodulatory properties of a polysaccharide that was isolated from the coral Pseudopterogorgia americana (PPA) were investigated. PPA increased the expression levels of tumour necrosis factor-&alpha; (TNF-&alpha;), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), but not inducible nitric oxide synthase and nitric oxide, in macrophages. A mechanistic study revealed that PPA activated macrophages through the toll-like receptor-4 and induced the generation of reactive oxygen species (ROS), increased the phosphorylation levels of protein kinase C (PKC)-&alpha;, PKC-&delta; and mitogen-activated protein kinases (MAPK), and activated NF-&kappa;B. The inhibition of ROS and knockdown of PKC-&alpha; reduced PPA-mediated TNF-&alpha; and IL-6 expression; however, the knockdown of PKC-&delta; significantly increased PPA-mediated TNF-&alpha; expression. In addition, the inhibition of c-Jun N-terminal kinase-1/2 and NF-&kappa;B reduced PPA-mediated TNF-&alpha;, IL-6 and COX-2 expression. Furthermore, the inhibition of ROS, MAPK and PKC-&alpha;/&delta; reduced PPA-mediated NF-&kappa;B activation, indicating that ROS, MAPK and PKC-&alpha;/&delta; function as upstream signals of NF-&kappa;B. Finally, PPA treatment decreased the phagocytosis activity of macrophages and reduced cytokine expression in bacteria-infected macrophages. Taken together, our current findings suggest that PPA can potentially play a role in the development of immune modulators in the future

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 &ldquo;Gal&beta;1&ndash;4GlcNAc&beta; obligatory containing an additional fragment&rdquo;, like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues

Molecular Cloning and Characteristics of a Lectin from the Bivalve <i>Glycymeris yessoensis</i>

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 “Galβ1–4GlcNAcβ obligatory containing an additional fragment”, like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues