Lipopolysaccharide And Beta-1,3-glucan Binding Protein Research Articles

Comparative transcriptome analysis reveals the different responding mechanisms of ovary and hepatopancreas following polyI:C challenge in Macrobrachium nipponense

The ovary in mammals has developed specialized mechanisms for protection against pathogen infections; however, the understanding of the innate immune system in the ovary of crustaceans is still limited. To elucidate the ovary's defense mechanisms in response to viral challenges, we subjected oriental river prawns (Macrobrachium nipponense) to poly I:C, a double-stranded RNA analog that emulates viral dsRNA, and analyzed the ovary's transcriptome profiles. Concurrently, RNA-seq analysis was performed on the hepatopancreas, a well-recognized immune-related tissue, following poly I:C challenge to investigate the distinct response mechanisms of the ovary and hepatopancreas and to gain a comprehensive understanding of the immune responses in both tissues. The results indicate that 1368 genes are differentially expressed in the ovary, with 903 genes upregulated and 465 genes downregulated. Subsequent analysis reveals that these differentially expressed genes (DEGs) include numerous genes associated with innate immunity, such as members of the C-type lectin, fibrinogen-related protein (Frep), Toll-like receptor, and NOD-like receptor (NLR) gene families, as well as acid phosphatase, scavenger receptor, crustin, Down syndrome cell adhesion molecule (Dscam), hemocyanin, and lipopolysaccharide and beta-1,3-glucan binding protein (LGBP). Furthermore, the DEGs include several genes related to ovary development, such as sox8, vitellogenin, progranulin, cyclin-dependent kinase, ecdysone receptor, frizzled, and members of the Fox gene family. In the hepatopancreas, a total of 729 DEGs were identified. Comparison of the DEGs in both tissues indicates that only 91 genes are common to both groups, highlighting significant tissue-specific responses to poly I:C stimulation. This study aims to enhance our understanding of the immune protective mechanisms employed by the ovary in response to pathogen exposure and establishes a foundation for investigating ovarian reproductive immunity in crustaceans.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120kDa which consists of a single polypeptide of ~39.5kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Effect of dietary date seed meal as an alternative carbohydrate source on immune-related gene expression of Pacific white shrimp Penaeus vannamei

Current study aimed to examine the effect of dietary date seed meal (DSM) as an alternative carbohydrate ingredient on immune-related genes expression of Pacific white shrimp (Penaeus vannamei). A total number of 750 shrimp (4.0 ± 0.1 g) were randomly assigned into five experimental treatments, each with three replicates. Experimental diets contained 0, 50, 100, 150 and 230 g/kg DSM substituted for both wheat and rice flour. Shrimp were fed at a rate of 5% body weight three times daily and the mRNA expression of α2-macroglobulin (α2-M), lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP), integrin β, and peroxinectin (PX) was studied on five shrimp per treatment after 8 weeks feeding trial. The results showed that replacement of DSM up to 100 g/kg in the diet of P. vannamei caused no significant changes in the expression of α2-M and integrin β, however both of these genes showed lower expression in shrimp fed diets containing 150 and 230 g/kg DSM compared to control group. Moreover, no significant differences were observed in the expression of PX and LGBP between shrimp fed diets containing different amounts of DSM and control group. These findings demonstrated that as an inexpensive source of carbohydrate, DSM could be substituted up to 100 g/kg for wheat and rice flour in the diet of farmed shrimp without suppressive effects on the immune status.

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) stimulates prophenoloxidase activating system in Chinese mitten crab (Eriocheir sinensis)

Melanization mediated by prophenoloxidase (proPO) activating system play an essential role in killing invading microorganisms in invertebrates. Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) as a pattern recognition protein have been demonstrated to active the proPO cascade in insect and shrimp. In this study, we investigated the role of LGBP in prophenoloxidase cascade-induced melanization in Chinese mitten crab (Eriocheir sinensis). By RT-PCR analysis, EsLGBP was detected in all tested tissues, and showed highest expression in hemocytes, gill, intestine and brain. The expression of EsLGBP was up-regulated in the hemocytes following injections of LPS and β-1, 3-glucan. The recombinant EsLGBP protein (rEsLGBP) was produced via prokaryotic expression system and affinity chromatography. By western blotting, rEsLGBP was discovered to exhibit the ability to bind to all tested microorganisms, including Gram-negative bacteria, Gram-positive bacteria and yeast (Pichia pastoris). Meanwhile we found rEsLGBP has a high binding activity towards microbial immune elicitors such as LPS and β-1, 3-glucan whereas no binding activity is detected with peptidoglycan. Moreover, the effects of RNAi-mediated blockade of EsLGBP were investigated on bacterial counts in the hemolymph and cumulative mortality rate of crabs infected with Vibrio parahaemolyticus in vivo. Further experiments demonstrate that rEsLGBP can trigger the whole hemolymph dependent melanization and stimulate to proPO cascade in vitro. Taken together, these results provide experimental evidence for role of LGBP in innate immunity, especially in the activation of prophenoloxidase activating system.

Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston) (Pectinoida, Pectinidae).

Zhikong scallop Chlamys farreri (Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

Molecular cloning and characterization of the lipopolysaccharide and β-1, 3-glucan binding protein in Chinese mitten crab (Eriocheir sinensis)

The lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a pattern recognition protein which is fundamental for the innate immune response of crustaceans. A partial cDNA produced by the random sequencing of cDNA clones from a haemocyte cDNA library of Eriocheir sinensis showed similarity to the LGBP gene of the Chinese shrimp (Fenneropenaeus chinensis). The full-length cDNA was subsequently cloned and sequenced by the technique of rapid amplification of cDNA ends (RACE). The E. sinensis LGBP gene (designated as Es-LGBP) was 1236 bases long and was capable of encoding a polypeptide of 362 amino acids showing significant similarity to homologous genes in shrimp. The crab LGBP deduced amino acid sequence carrying conserved features of this gene family including a potential recognition motif for beta-1, 3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp)cell adhesion sites. Real-time quantitative reverse transcription-PCR (RT-PCR) analysis showed that LGBP gene expresses in haemocytes, hepatopancreas, muscles, gills, stomachs, and intestines with the highest level in haemocytes and the lowest in the stomach. The LGBP gene expression is up-regulated upon bacterial infection and the binding of lipopolysaccharide and beta-1, 3-glucan to LGBP could induce a series of immune reactions. The temporal expression of the LGBP gene after bacterial challenge was measured by real-time quantitative RT-PCR. The result demonstrated that the LGBP gene expression in crab was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 and 24 h. Our data suggest that the crab LGBP is an inducible acute-phase protein that could be critical in crab immunity.

CDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri

Lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP) play a crucial role in the innate immune response of invertebrates as a pattern recognition protein (PRP). The scallop LGBP gene was obtained from Chlamys farreri challenged by Vibrio anguillarum by randomly sequencing cDNA clones from a whole body cDNA library, and by fully sequencing a clone with homology to known LGBP genes. The scallop LGBP consisted of 1876 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 440 amino acids with the estimated molecular mass of 47.16 kDa and a predicted isoelectric point of 5.095. The deduced amino acid sequence showed a high similarity to that of invertebrate recognition proteins from blue shrimp, black tiger shrimp, mosquito, freshwater crayfish, earthworms, and sea urchins, with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. The temporal expression of LGBP genes in healthy and V. anguillarum-challenged C. farreri scallop, measured by real-time semiquantitative reverse transcription polymerase chain reaction (PCR), showed that expression was up-regulated initially, followed by recovery as the stimulation cleared. Results indicated that scallop LGBP was a constitutive and inducible acute-phase protein that could play a critical role in scallop–pathogen interaction.

A Lipopolysaccharide- and β-1,3-Glucan-binding Protein from Hemocytes of the Freshwater Crayfish Pacifastacus leniusculus: PURIFICATION, CHARACTERIZATION, AND cDNA CLONING

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.