Qingsong Xu

Isolation and structural characterization of the polysaccharide LRGP1 from Lycium ruthenicum.

A novel water-soluble glycoconjugate, designated as Lycium ruthenicum glycoconjugate polysaccharide 1 (LRGP1), was isolated from the fruits of Lycium ruthenicum Murr. The crude polysaccharide was obtained by hot water extraction and purified by ion-exchange and gelfiltration chromatography. Its molecular weight was 56.2 kDa determined by HPGPC (high performance gel permeation chromatography). Monosaccharide composition analysis revealed that it was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.65:10.71:0.33:0.67:1:10.41. The existence of O-type carbohydrate-peptide linkage in LRGP1 was demonstrated by β-elimination reaction. On the basis of monosaccharide composition, partial acid hydrolysis, methylation analysis and ESI-MS analysis, LRGP1 was characterized as a branched polysaccharide rich in arabinose and galactose with a backbone composed of (1→3)-linked Gal. The branches were composed of (1→5)-linked Ara, (1→2)-linked Ara, (1→6)-linked Gal, (1→3)-linked Gal, (1→4)-linked Gal and (1→2,4)-linked Rha. Arabinose, xylose, mannose, and glucose were located at the terminal of the branches.

Chitosan oligosaccharides block LPS-induced O-GlcNAcylation of NF-κB and endothelial inflammatory response.

It is known that chitosan oligosaccharides (COS) suppress LPS-induced vascular endothelial inflammatory response by mechanism involving NF-κB blockade. It remains unknown how COS inhibit NF-κB. We provided evidence both in cultured endothelial cells and mouse model supporting a new mechanism. Regardless of the endothelial cell types, the LPS-induced NF-κB-dependent inflammatory gene expression was suppressed by COS, which was associated with reduced NF-κB nucleus translocation. LPS enhanced O-GlcNAc modification of NF-κB/p65 and activated NF-κB pathway, which could be prevented either by siRNA knockdown of O-GlcNAc transferase (OGT) or pretreatment with COS. Inhibition of either mitogen-activated protein kinase or superoxide generation abolishes LPS-induced NF-κB O-GlcNAcylation. Consistently, aortic tissues from LPS-treated mice presented enhanced NF-κB/p65 O-GlcNAcylation in association with upregulated gene expression of inflammatory cytokines in vascular tissues; however, pre-administration of COS prevented these responses. In conclusion, COS decreased OGT-dependent O-GlcNAcylation of NF-κB and thereby attenuated LPS-induced vascular endothelial inflammatory response.

Chitosan Oligosaccharides Inhibit the Expression of Interleukin-6 in Lipopolysaccharide-induced Human Umbilical Vein Endothelial Cells Through p38 and ERK1/2 Protein Kinases

Chitosan oligosaccharides (COS) have been reported to exert anti-fungal activities, antitumour activities and immuno-enhancing effects. However, the potential roles of COS in the treatment of vascular inflammations remain unknown. In the present study, we examined the effects of COS on interleukin-6 (IL-6) production in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Induction of HUVECs with LPS (100 ng/ml) increased the mRNA expression and protein secretion of IL-6 (versus the vehicle-treated group, p < 0.01), which were significantly reverted by the pre-treatment with COS (50-200 microg/ml) for 24 hr before LPS exposure (versus the LPS-treated group, p < 0.05 or 0.01). Signal transduction studies showed that the pre-treatment of HUVECs with COS (50-200 microg/ml) for 24 hr markedly inhibited the LPS-induced over-expression of phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2 and nuclear factor kappaB (NF-kappaB). Moreover, the LPS-induced NF-kappaB activation was suppressed by the specific ERK1/2 inhibitor PD98059 (30 microM) (versus the LPS-treated group, p < 0.01), but not by the specific p38 MAPK inhibitor SB203580 (25 microM). Additionally, both MAPK inhibitors markedly suppressed LPS-induced IL-6 mRNA expression in HUVECs (versus the LPS-treated group, p < 0.01). In conclusion, our results suggest that COS inhibit LPS-induced up-regulation of IL-6 in HUVECs, and this can be regulated by at least two parallel signalling pathways: one via p38 MAPK pathway independent of NF-kappaB activation and one via ERK1/2 pathway dependent on NF-kappaB activation.

Characterization of a new endo-type alginate lyase from Vibrio sp. W13

A gene, encoding a new alginate lyase Algb, was identified and cloned from marine bacterium Vibrio sp. W13. The recombinant alginate lyase was characterized followed by being purified on Ni-NTA Sepharose. It exhibited the highest activity (457 U/mg) at pH 8.0 and 30 °C. Interestingly, Algb possessed broader substrate specificity. It showed activities toward both polyM (poly β-D-mannuronate) and polyG (poly α-L-guluronate). Furthermore, K(m) values of Algb toward alginate (0.67 mg/ml) and polyMG (0.50 mg/ml) are lower than those toward polyG (1.04 mg/ml) and polyM (6.90 mg/ml). The TLC and ESI-MS analysis suggested that Algb mainly released oligosaccharides with DP of 2-5 from the four kinds of substrates in an endolytic manner. Therefore, it may be a potent tool to produce alginate oligosaccharides with low DP.

Characterization of an immunologically active pectin from the fruits of Lycium ruthenicum

An immunologically active pectin, named LRGP5, was firstly isolated from the fruits of Lycium ruthenicum Murr. It contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:2.2:0.5:1.2:4.7. Its molecular weight was estimated to be approximately 1.37 × 10(5)Da by high performance gel permeation chromatography. The structure was elucidated using methylation analysis, partial acid hydrolysis, NMR and ESI-MS analysis. Results showed that LRGP5 consisted of a (1 → 4)-linked galacturonic acid backbone occasionally interrupted by (1 → 2)-linked rhamnose. The side chains were attached to position 4 of the rhamnosyl units, including (1 → 3)-linked arabinose, (1 → 3)-linked galactose, (1 → 3,6)-linked galactose, (1 → 4)-linked galacturonic acid, (1 → 2)-linked rhamnose and (1 → 2,4)-linked rhamnose, and the termini were arabinose and rhamnose. Immunological assay results demonstrated that LRGP5 could significantly promote macrophage proliferation and enhance the secretion of nitrogen monoxide in vitro.

Chitosan oligosaccharides downregulate the expression of E-selectin and ICAM-1 induced by LPS in endothelial cells by inhibiting MAP kinase signaling

The expression of adhesion molecules in endothelial cells elicited by lipopolysaccharide (LPS) is involved in the adhesive interaction between endothelial cells and monocytes in inflammation. In this study, in order to characterize the anti-inflammatory effects of chitosan oligosaccharides (COS) on LPS‑induced inflammation and to elucidate the underlying mechanisms, the mRNA levels of E-selectin and intercellular adhesion molecule-1 (ICAM-1) were measured in porcine iliac artery endothelial cells (PIECs). When these cells were treated with COS, the LPS-induced mRNA expression of E-selectin and ICAM-1 was reduced through the inhibition of the signal transduction cascade, p38 mitogen‑activated protein kinase (MAPK)/extracellular regulated protein kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Moreover, through the inhibition of p38 MAPK and ERK1/2, COS suppressed the LPS-induced NF-κB p65 translocation. We found that COS suppressed the phosphorylation of p38 MAPK and the translocation of NF-κB p65 into the nucleus in a dose-dependent manner, and inhibited the adhesion of U973 cells to PIECs. Based on these results, it can be concluded that COS downregulate the expression of E-selectin and ICAM-1 by inhibiting the phosphorylation of MAPKs and the activation of NF-κB in LPS-treated PIECs. Our study demonstrates the valuable anti-inflammatory properties of COS.

Crustacean hyperglycemic hormones directly modulate the immune response of hemocytes in shrimp Litopenaeus vannamei

ABSTRACT A robust immune response against invading pathogens is crucial for host to survive, which depends greatly on the well balance of metabolism. Increasing evidence has indicated that some metabolic hormones, such as insulin, could modulate immune responses directly. Crustacean hyperglycemic hormone (CHH) family is a group of ecdysozoans‐specific peptide hormone involved in glucose metabolism and other biological events. In the present study, two members of CHH family (designated as LvCHH I and LvCHH II) in shrimp Litopenaeus vannamei with one and two crustacean neurohormone domains respectively were chosen to investigate their putative modulatory roles in both glucose metabolism and immune response. LvCHH I and LvCHH II were both expressed in the sinus gland and lamina ganglionalis of eyestalks and were significantly induced after white spot syndrome virus (WSSV) infection. Meanwhile, significant increases of hemolymph glucose levels were observed in shrimp at 12 and 24 h after WSSV infection while the glucose inside the hemocytes decreased at 6 h and then increased at 12 h. Gain‐of‐function of rLvCHHs was subsequently conducted in vivo by injecting the recombinant proteins (rLvCHH I and rLvCHH II). The hemolymph glucose increased significantly from 0.5 h to 3 h after the shrimps received an injection of rLvCHH I, while it decreased at 0.5 h and increased afterward at 3 h post rLvCHH II injection. At the meantime, significant decreases of reactive oxygen species level in hemocytes were observed at 3 h and 6 h post rLvCHH I injection, while it remained unchanged in rLvCHH II injection group. rLvCHH I and rLvCHH II could bind to the cytomembrane of primary shrimp hemocytes in vitro, and the expressions of superoxide dismutase and LvRelish increased when the hemocytes were incubated with rLvCHH I for 3 h. Meanwhile, the expression of antimicrobial peptides, crustin and penaeidin‐4, were also induced by rLvCHH I and rLvCHH II. These results demonstrated that host immune response, in addition to glucose metabolism, could be directly modulated by LvCHH family, and the present study provided new insights into the immunomodulation role of metabolic hormones in invertebrate. HighlightsGlucose level and LvCHH transcripts changed vigorously during WSSV infection.Endogenous LvCHHs were located in the lamina ganglionalis and sinus gland of eyestalks.LvCHH I modulated glucose and ROS level in vivo.LvCHHs modulated immune‐related genes by binding to shrimp hemocytes directly.

Chitosan oligosaccharide inhibits EGF-induced cell growth possibly through blockade of epidermal growth factor receptor/mitogen-activated protein kinase pathway.

Chitosan oligosaccharide (COS) has been shown to regulate various cellular and biological functions. Epidermal growth factor (EGF) plays a significant role in tumorigenesis and invasiveness of some cancers. The aim of this study was to evaluate the effects of COS on EGF-induced cell growth and the further mechanisms. The results demonstrated that COS could inhibit EGF-induced epithelial GE11 cells proliferation in a dose dependent manner. In addition, EGF stimulated the epithelial cells to undergo morphological alteration, exhibiting mesenchymal cells higher metastatic and invasive potential, however, COS could partly suppress aforementioned morphological change. Signal transduction studies indicated COS repressed epidermal growth factor receptor (EGFR) phosphorylation and mitogen-activated protein kinase (MAPK) activation, but not Grb2, Ras, and Raf. Taken together, chitosan oligosaccharide inhibited EGF-induced cell growth and migration through blockade of the EGFR/MAPK signal transduction pathway.

Characterization of a new family 75 chitosanase from Aspergillus sp. W-2.

A new chitosanase gene of glycoside hydrolase (GH) family 75, csnw2, was cloned from an isolated strain Aspergillus sp. W-2 (CGMCC7018). The mature CsnW2 protein, fused to His-tag at C-terminus, was expressed in Pichia pastoris X-33 and purified with the affinity chromatography of Ni(2+)-NTA. The novel recombinant CsnW2 showed maximal activity with chitosan at pH 6.0 and 55°C. Moreover, it had good pH stability and thermostability at a broad pH range of 3.0-10.0 and a temperature range of 30-70°C, respectively. The enzymatic activity of the CsnW2 could be significantly enhanced by Ca(2+), Mn(2+) and Mg(2+) at a concentration of 1mM, but strongly inhibited by Fe(2+), Zn(2+), Ge(2+), Ni(2+) and Cu(2+) above 1mM. The CsnW2 showed specific hydrolytic activity against chitosan and preferred to hydrolyze chitosan with high degree of deacetylation. The main products of chtiosan (92% deacetylation) were chitosan oligosaccharides (COS) with degree of polymerization (DP) arranging from 2 to 6. Combined with the hydrolysis of COS from DP2 to DP6, CsnW2 was considered to be an endo-acting chitosanase.

Two short peptidoglycan recognition proteins from Crassostrea gigas with similar structure exhibited different PAMP binding activity.

ABSTRACT Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in specifically recognizing bacterial peptidoglycan (PGN) and eliminating the invading bacteria. In the present study, the full length cDNA of two PGRP genes, CgPGRPS2 and CgPGRPS4, were cloned from oyster Crassostrea gigas. Their amino acid sequences both contained one signal peptide, one typical PGRP/amidase domain with conserved catalytic residues responsible for amidase activity (55H, 90Y, 164H, 172C in CgPGRPS2, and 98H, 133Y, 207H, 215C in CgPGRPS4), and specific PGN recognition (84R, 85W, 104R, 109V in CgPGRPS2, and 127G, 128W, 147R, 152V in CgPGRPS4), and they shared 55.9% sequence similarity. The mRNA transcripts of CgPGRPS2 and CgPGRPS4 were constitutively expressed in all the examined tissues, including haemocytes, hepatopancreas, mantle, gonad, heart, adductor muscle and gill, with the highest expression level in adductor muscle and hepatopancreas, respectively. Both CgPGRPS2 and CgPGRPS4 proteins were mainly localized in the cytoplasma. The recombinant protein of CgPGRPS2 (rCgPGRPS2) could bind lipopolysaccharide (LPS), PGN and mannan (Man), as well as various microorganisms including Gram‐negative bacteria Escherichia coli, Vibrio anguillarum, Gram‐positive bacteria Staphylococcus aureus and fungi Yarrowia lipolytica. The recombinant protein of CgPGRPS4 (rCgPGRPS4) exhibited higher binding affinity to PGN, lower binding affinity to LPS, while no binding activity to Man and Y. lipolytica. The results indicated that CgPGRPS2 and CgPGRPS4 could function as pattern recognition receptors (PRR) in the innate immune response of oyster, and they exhibited a certain degree of functional differentiation in recognition of Man. HIGHLIGHTSCgPGRPS2 and CgPGRPS4 were identified as short type PGRP genes from Pacific oyster.CgPGRPS2 and CgPGRPS4 showed highest mRNA expression in adductor muscle and hepatopancreas, respectively.CgPGRPS2 and CgPGRPS4 proteins were mainly localized in cytoplasma of oyster haemocytes.rCgPGRPS2 could bind LPS, PGN, Man, E. coli, V. anguillarum, S. aureus and Y. lipolytica.rCgPGRPS4 could bind PGN, LPS, but not Man and Y. lipolytica.