Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.

•SERP1 has complicated structure due to its composition of nine monosaccharides.•SERP1 shows high hypoglycemic properties in diabetic mice.•SERP1 could be explored as a functional food or supplement from industrial waste.An acidic polysaccharide (SERP1) was isolated from the residue of Sarcandra glabra (Thunb.) Nakai by water extraction and purified by decoloration and ion exchange chromatography. The structure of SERP1 was determined by HPLC, HPSEC-MALLS, FT-IR, and NMR. The results indicated that SERP1 was a homogeneous heteropolysaccharide with the absolute molecular weight of 4.208 × 104 Da in the aqueous phase. 1,4-linked α-d-galacturonic acid, methyl esterified 1,4-linked α-d-galacturonic acid, 1,4-linked α-d-glucuronic acid, 1,5-linked α-l-arabinose, 1,3-linked β-d-galactose 1,4-linked α-d-glucose, 1,4,6-linked β-d-glucose, 1,6-linked β-d-glucose, and 1,2-linked rhamnose existed in SERP1. In vitro α-glucosidase inhibition assay indicated that SERP1 had a low IC50 value of 49.01 μg/mL, which exhibited stronger α-glucosidase enzyme inhibitory activity than acarbose at the same concentration. The treatment of SERP1 to type 2 diabetes mellitus mice alleviated the hyperglycemia, increased glucose utilization of peripheral tissues of the liver and inhibited the liver injury. This study provides a possible exploration to use valuable industrial waste.

•A purified acidic polysaccharide was obtained from fruits of Lycium barbarum L.•The structural characteristics were investigated by a combination of chemical and instrumental analysis approaches along with a degradation pattern study.•The fine primary structure and chain conformation of this polysaccharide was elucidated.An acidic polysaccharide, named as p-LBP, was isolated from Lycium barbarum L. by water extraction and purified by decoloration, ion exchange chromatography, dialysis and gel chromatography, successively. The primary structure analysis was determined by HPAEC-PAD, HPSEC, FT-IR, GC–MS, and NMR. The results showed p-LBP was a homogeneous heteropolysaccharide as a pectin molecule with an average molecular weight of 64 kDa p-LBP was an approximately 87 nm hollow sphere in 0.05 mol/L sodium sulfate solution determined by HPSEC-MALLS, DLS and TEM. A discussion of degradation patterns gave the detailed structural information of p-LBP. Therefore, the results from degraded fragments elucidated that the backbone of p-LBP was formed by → 4-α-GalpA-(1 →, repeatedly. Partial region was connected by → 4-α-GalpA-(1 → and → 2-α-Rhap-(1 →, alternatively. On the C-4 of partial → 2-α-Rhap-(1 → residues existed branches forming by → 4-β-Galp-(1 →, → 3-β-Galp-(1 → or → 5-α-Araf-(1 →, while on the C-6 of partial → 3-β-Galp-(1 → residues existed secondary branches forming by terminal-α-Araf, terminal-β-Galp or → 3-α-Araf-(1 →.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

We have previously constructed an engineered anti-diabetic fusion protein using glucagon-like peptide-1 and the globular domain of adiponectin. Herein, we evaluated the therapeutic effects of this fusion protein (GAD) on high-fat diet (HFD)-fed ApoE−/− mice. The lipid-lowering effect of GAD was determined in C57BL/6 mice using a lipid tolerance test. The effects of GAD on HFD-induced glucose intolerance, atherosclerosis, and hepatic steatosis were evaluated in HFD-fed ApoE−/− mice using glucose tolerance test, histological examinations and real-time quantitative PCR. The anti-inflammation activity of GAD was assessed in vitro on macrophages. GAD improved lipid metabolism in C57BL/6 mice. GAD treatment alleviated glucose intolerance, reduced blood lipid level, and attenuated atherosclerotic lesion in HFD-fed ApoE−/− mice, which was associated with a repressed macrophage infiltration in the vessel wall. GAD treatment also blocked hepatic macrophage infiltration and prevented hepatic inflammation. GAD suppressed lipopolysaccharide-triggered inflammation responses on macrophages, which can be abolished by H89, an inhibitor of protein kinase A. These findings demonstrate that GAD is able to generate a variety of metabolic benefits in HFD-fed ApoE−/− mice and indicate that this engineered fusion protein is a promising lead structure for anti-atherosclerosis drug discovery.

Translational efficiency in Escherichia coli is strongly influenced by mRNA secondary structure of translational initiation region (TIR). We have previously reported that the expression of heterologous protein is directly related to the minimal folding free energy (ΔG) of the local secondary structure. However, identifying biologically relevant maximum and minimum levels of expression, or exploring the optimal level between them, is a key to successful optimization of heterologous protein expression. To systematically search a large range of the ΔG of TIR, we now present a quantitative analysis of the relationship between expression level and these ΔGs. The ΔG of TIR in green fluorescent protein is found to be linearly correlated with the fluorescence intensity over a range of tenfold change. The result demonstrates that the increasing ΔG of TIR can enhance the expression level linearly with no threshold or plateau.

Thymosin alpha 1 (Tα1) is commonly used for treating several diseases; however its usage has been limited because of poor penetration of the target tissue, such as tumor cells. In the present study, Tα1-iRGD, a peptide by conjugating Tα1 with the iRGD fragment, was evaluated its performance in MCF-7 and MDA-MB-231 human breast cancer cells. Compared with the wild-type peptide, Tα1-iRGD was more selective in binding tumor cells in the cell attachment assay. Furthermore, the MTT assay confirmed that Tα1-iRGD proved more effective in significantly inhibiting the growth of MCF-7 cells in contrast to the general inhibition displayed by Tα1. Further, conjugation of Tα1 with iRGD preserved the immunomodulatory activity of the drug by increasing the proliferation of mouse spleen lymphocytes. Further, compared with Tα1 treatment, Tα1-iRGD treatment of MCF-7 cells considerably increased the number of cells undergoing apoptosis, resulting in a dose-dependent inhibition of cancer cell growth, which was associated with a much better effect on up-regulation of the expression of BCL2-associated X protein (Bax), caspase 9, etc. More importantly, treatment with Ta1-iRGD was more efficacious than treatment with Ta1 in vivo. This study highlights the importance of iRGD on enhancement of cell penetration and tumor accumulation. In summary, our findings demonstrate that the novel modified Tα1 developed in this study has the potential to be used for treating breast cancer.

•A miniature library of homogenous low-molecular-weight α-glucans was obtained.•We studied the molecular size-immunomodulating activity relationship of α-glucans.•We have indicated for the first time the probable functional unit of YCP, a marine α-glucan.YCP, an α-d-glucan with molecular weight of 2.4 × 103 kDa, was isolated from the mycelium of marine fungus Phoma herbarum YS4108. It interacted with TLR2 and TLR4 to induce B cells proliferation and activation. Here, a series of homogenous LMWYCPs (namely LMWYCP-1 to LMWYCP-6) were obtained by acid degradation. LMWYCP-3 and LMWYCP-4 were capable of inducing the proliferation of B cells dramatically. Further research involved TLR defunctionalization and competitive binding assay demonstrated that the immunomodulating activities of LMWYCP-4 was TLR4-dependent but TLR2-independent, while that mediated by LMWYCP-3 was neither TLR2- nor TLR4-dependent. Together with the structural information that LMWYCPs shared the similar structure features with YCP, we deduced that LMWYCP-4 may be the functional unit of YCP responsible for YCP/TLR4 interaction. This is the first time that the probable functional unit of glucan with the structure of α-d-(1–4)-linked residues in main chain and α-d-(1–6)-linked residues in branches is reported.

•A method is utilized for immobilizing natural polysaccharide as affinity absorbent.•An optimal solution of the synthesis of YCP-Sepharose is found.•The ligand density of YCP-Sepharose is impressive.•TLR2 and TLR4 are verified as receptors of YCP in B cells.YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)4 was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate–protein interaction.

An acidic polysaccharide (SGP-2), with a molecular weight of 1880 kDa, was purified from the defatted whole-plant of Sarcandra glabra (Thunb.) Nakai. SGP-2 is mainly composed of glucose, galactose, mannose, arabinose and galacturonic acid in a molar ratio of 12.19:8.68:6.03:1.00:15.24. The primary structure analysis reveals that SGP-2 consists of 1,4-linked α-D-galacturonic acid, methyl-esterified 1,4-linked α-D-galacturonic acid, 1,5-linked α-L-arabinose, 1,4-linked α-D-mannose, 1,6-linked β-D-glucose and 1,3-linked β-D-galactose with branch chains of 1,4,6-linked β-D-glucose, 1,3,6-linked α-D-mannose and 1,4,6-linked α-D-galactose. The results of a cell viability assay and colony formation assay indicate that SGP-2 has a potent anti-proliferation activity on human osteosarcoma MG-63 cells. SGP-2 increases the proportion of apoptotic cells and activates caspase-3. In addition, the anti-proliferation effect induced by SGP-2 is blocked by the pan-caspase inhibitor. Moreover, SGP-2 inhibits the migratory capacity of MG-63 cells accompanied with the inhibition of receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB). Taken together, these results suggest that SGP-2 has anti-cancer potential in the treatment of human osteosarcoma.

•A novel lncRNA, 1700040D17Rik, was found to be down-regulated in EAE model.•After the treatment of rhIL23R-CHR, 1700040D17Rik was significantly increased.•1700040D17Rik was revealed to associate with the differentiation of Th17 cells.•1700040D17Rik was confirmed to regulate RORγt to affect Th17 cells.IL-23/STAT3 signaling pathway is a key process in Th17 cell differentiation, and Th17 cells are closely related to the development of autoimmune diseases. We previously designed and prepared rhIL23R-CHR protein to antagonize endogenous IL-23, showing effectiveness in the treatment of experimental autoimmune encephalomyelitis (EAE) in mice. To further elucidate the mechanism of action, mouse lncRNA microarray was used to screen expression profiles of lncRNAs, and a particular lncRNA, 1700040D17Rik was found to down-regulate in EAE model and its expression was significantly increased after the treatment by rhIL23R-CHR. The function of 1700040D17Rik was revealed to associate with the differentiation of Th17 cells through the regulation of the key transcription factor RORγt. Together, regulation of Th17 cells through lncRNA is responsible for the effects of rhIL23R-CHR to balance the immune responses, and 1700040D17Rik has the potential to serve as a therapeutic target or a biomarker for autoimmune diseases.

•Different linker peptides were introduced into the fusion protein construction.•The optimized fusion protein showed high potency in receptors activation.•The optimized fusion protein showed high potency in glucose- and lipid-lowering.Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.Download full-size image

•We report a soluble receptor, tIL12rβ1 for the treatment of lupus nephritis.•We present a new therapeutic strategy targeting IL-12/IL-23 p40 for lupus nephritis.•Both Th1 and Th17 cells are involved in the pathogenesis of cGVHD-induced SLE.Th1 and Th17 cells have been strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-12 and IL-23 respectively drive the polarization of Th1 and Th17 cells and share a common p40 subunit. In this study, the protective and therapeutic effects of a truncated human IL-12rβ1 receptor (tIL12rβ1) targeting IL-12/IL-23 p40 were evaluated in chronic graft-versus-host disease (cGVHD)-induced SLE-like model. The results indicated that tIL12rβ1 treatment effectively delayed the proteinuria onset and induced a significant remission of proteinuria, autoantibody production, and immune complex deposition in the mouse model. Remarkably, the therapeutic effects of tIL12rβ1 were predominantly dependent on the suppression of pathogenic Th1 and Th17 cell commitment through the reduction of RORγt and T-bet expression. Collectively, this receptor molecule may offer a new treatment option for SLE.

•De novo generation of antibody-secreting cells from naïve lymphocytes in vitro.•Convert in vitro immunization of unfractionated PBMCs to step-by-step cell culture.•Succeed to differentiate naïve CD4+ T cells to Tfh cells for in vitro immunization.•Detected AID and Blimp-1 indicate class-switch recombination might be initiated.In vitro immunization with antigens and cytokines triggers specific human B-cell response in short periods and is therefore superior to conventional in vivo immunization for antibody development. However, this new technology is limited by low efficiency, poor reproducibility, and requirement of pre-immunized lymphocytes. In this study, we demonstrate a novel method for de novo inducing antigen-specific human B cells in vitro. Unlike previous in vitro immunization of unfractionated PBMCs, we firstly optimized the conditions for inducing monocyte-derived dendritic cells (DCs) to efficiently capture, process, and present antigens. Instead of using the conventional method to activate Th2 cells for in vitro immunization, we succeeded to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells using antigen-sensitized DCs and cytokine cocktail. We discovered the differentiated T cells expressed ICOS, PD-1, BCL-6, and IL-21 at high levels. After 12 days of T-B co-culture, we observed induced T cells efficiently promoted naïve B cells to differentiate into plasmablasts secreting antigen-specific antibodies, with expression of Blimp-1 and AID related to affinity maturation and class switching. Thus, we established a new co-culture system with naïve lymphocyte populations for de novo acquisition of specifically in vitro immunized B cells potentially for development of therapeutic antibodies, which also provides novel insights into understanding the complex interactions among immune cells in lymph nodes.Download high-res image (184KB)Download full-size image