epiglucan and lipoteichoic-acid

In crustaceans, lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) plays an important role in innate immunity by mediating the recognition of pathogens to host cells. Hereby, LGBP was cloned from Fenneropenaeus merguiensis hepatopancreas. Its full-length cDNA (1280 bp) had an open reading frame of 1101 bp, encoding a peptide of 366 amino acids. The LGBP primary structure comprises a recognition motif for β-1,3-linkage of polysaccharides, two integrin binding motifs, a kinase C phosphorylation site and a bacterial glucanase motif. The LGBP mRNA was strongly expressed in hepatopancreas and significantly up-regulated to get the maximum at 12 h upon Vibrio harveyi challenge. Recombinant LGBP (rLGBP) could agglutinate Gram-negative and Gram-positive bacteria including yeast with Ca

Topics: Agglutination; Animals; beta-Glucans; Cells, Cultured; Cloning, Molecular; Hepatopancreas; Immunity, Innate; Lectins; Lipopolysaccharides; Pathogen-Associated Molecular Pattern Molecules; Penaeidae; Protein Binding; Receptors, Pattern Recognition; Teichoic Acids; Up-Regulation; Vibrio; Vibrio Infections

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and beta-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

Topics: Animals; beta-Glucans; Candida albicans; Escherichia coli; Flow Cytometry; Hemocytes; Insect Proteins; Larva; Lepidoptera; Lipopolysaccharides; Opsonin Proteins; Phagocytosis; Protein Binding; Receptors, Pattern Recognition; Reverse Transcriptase Polymerase Chain Reaction; Teichoic Acids; Tissue Culture Techniques

We have isolated and characterized a new beta-1,3-glucan recognition protein that is present in Manduca sexta cuticle and hemolymph. This 52 kDa protein, designated betaGRP-2, is 57% identical in sequence to betaGRP-1 from larval hymolymph of the same insect. BetaGRP-2 differs from betaGRP-1 in its absence of the naive larvae before the wandering stage begins. Transcription of the betaGRP-2 was up-regulated in larvae challenged with yeast or bacteria. BetaGRP-2 contains a region with sequence similarity to several glucanases but lacks glucanase activity. It aggregates yeasts and bacteria to, perhaps, limit the spread of invading cells and ensure a localized defense reaction. BetaGRP-2 binds laminarin and lipoteichoic acid, but not lipopolysaccharide. Laminarin-triggered prophenoloxidase activation was greatly enhanced in the induced larval hemolymph supplemented with purified betaGRP-2. Complementing other studies on pattern recognition molecules in M. sexta, these results indicate that a complex system of protein sensors is an integral component of the insect immune system and that different recognition molecules have overlapping binding specificity and functions.

Topics: Acute-Phase Proteins; Amino Acid Sequence; Animals; Bacteria; Base Sequence; beta-Glucans; Carrier Proteins; Catechol Oxidase; Cell Wall; DNA, Complementary; Enzyme Activation; Enzyme Precursors; Escherichia coli; Gene Expression Profiling; Glucans; Insect Proteins; Lipopolysaccharides; Manduca; Micrococcus luteus; Molecular Sequence Data; Recombinant Proteins; Saccharomyces cerevisiae; Sequence Alignment; Sequence Homology, Amino Acid; Staphylococcus aureus; Teichoic Acids

Invertebrates, like vertebrates, utilize pattern recognition proteins for detection of microbes and subsequent activation of innate immune responses. We report structural and functional properties of two domains from a beta-1,3-glucan recognition protein present in the hemolymph of a pyralid moth, Plodia interpunctella. A recombinant protein corresponding to the first 181 amino-terminal residues bound to beta-1,3-glucan, lipopolysaccharide, and lipoteichoic acid, polysaccharides found on cell surfaces of microorganisms, and also activated the prophenoloxidase-activating system, an immune response pathway in insects. The amino-terminal domain consists primarily of an alpha-helical secondary structure with a minor beta-structure. This domain was thermally stable and resisted proteolytic degradation. The 290 residue carboxyl-terminal domain, which is similar in sequence to glucanases, had less affinity for the polysaccharides, did not activate the prophenoloxidase cascade, had a more complicated CD spectrum, and was heat-labile and susceptible to proteinase digestion. The carboxyl-terminal domain bound to laminarin, a beta-1,3-glucan with beta-1,6 branches, but not to curdlan, a beta-1,3-glucan that lacks branching. These results indicate that the two domains of Plodia beta-1,3-glucan recognition protein, separated by a putative linker region, bind microbial polysaccharides with differing specificities and that the amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is responsible for stimulating prophenoloxidase activation.

Topics: Animals; beta-Glucans; Circular Dichroism; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Gene Deletion; Glucans; Immunity, Innate; Lipopolysaccharides; Moths; Polysaccharides; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Surface Plasmon Resonance; Teichoic Acids; Temperature; Time Factors