Systematical Ingredient Investigations of Ficus tikoua Bur. Fruit and Immunoregulatory and Antioxidant Effects of Different Fractions.

Although the fruit of Ficus tikoua Bur. has been consumed by montanic people in China for centuries, its chemical and biological composition was still unclear. A series of comprehensive investigations on its chemical constituents and bioactivities were carried out for the first time. As a result, six compounds were isolated and identified as the main components in this fruit. GC-MS analysis of the lipid components demonstrated that Ficus tikoua Bur. fruit contains some wholesome constituents such as fatty acids, vitamins, triterpenoids, and phytosterols. The fatty acids are mainly composed of linolenic acid (61.27%) and linoleic acid (22.79%). Furthermore, this fruit contains a relative high content of crude protein (9.41 ± 0.03%), total amino acids (9.28%), and total polyphenols (0.86 ± 0.01 g/100 g). The analysis of monosaccharide composition showed that the total polysaccharide mainly consists of glucose, glucuronic acid, xylose, arabinose, mannose, galactose, galacturonic acid, and rhamnose. The polysaccharide, polyphenol, water, ethanol, and flavonoid extracts exhibited prominent antioxidant activity determined by ABTS, DPPH, and FRAPS methods. Meanwhile, the total polysaccharide exhibited significant immunomodulatory effect by enhancing the release of cytokines and expression of iNOS and COX-2 in RAW264.7 cells, significantly decreasing the expression of c-Jun and p65 proteins in the cytoplasm; increasing the translocation of c-Jun and p65 to the nucleus; and regulating the phosphorylation level of Akt, PI3K, and PDK1 in the PI3K/AKT signaling pathway. This study proved that the fruit of F. tikoua is a reliable source of functional food.

The chemical structure and immunomodulatory activity of an exopolysaccharide produced by Morchella esculenta under submerged fermentation.

The extracellular polysaccharide of Morchella esculenta cultivated under submerged fermentation was extracted. A single polysaccharide was purified through DEAE-Cellulose 52 and Sephadex G 100, and named as MEP 2a. The molecular weight of MEP 2a was determined by HPGPC and it is about 1391.5 kDa. MEP 2a is composed of mannose and glucose as the monosaccharide unit with a molar ratio of 8.15 : 1.07. The main polysaccharide chemical structure was analyzed by 1D and 2D NMR. Methylation and NMR analysis revealed that the backbone of MEP 2a consists of 1,3,4-linked-Manp, 1,2-linked-Manp and 1,6-linked-Glcp. 1D and 2D NMR results indicated that the main chain is based on →1)-ß-D-Glcp-(6→, →1)-α-D-Manp-(3,4→, →1)-α-D-Manp-(2→) and the branch chain is composed of α-D-Manp-(1→, →1)-ß-D-Glcp-(6→ and α-D-Glcp-(1→). MEP 2a promoted the phagocytosis function and secretion of NO, IL-1ß, IL-6 and TNF-α of macrophages. In the present study, the chemical structure and immunomodulatory ability of an extracellular polysaccharide of Morchella esculenta was investigated which guarantees further research studies and promising applications.

Enzymatic extraction of pectic oligosaccharides from finger citron (Citrus medica L. var. sarcodactylis Swingle) pomace with antioxidant potential.

Finger citron pomace is a cheap and renewable by-product of the citrus processing industry, representing up to 60% of the fruit biomass. In this study, a pectinase-based and ultrasonic-assisted method was firstly used to extract pectic oligosaccharides (POS) from finger citron pomace. Using the orthogonal experiment design (OED), the maximum conversion rate of up to 64.5% from pomace to POS was obtained under the extraction conditions of 0.25 mg mL-1 pectinase and 50 mg mL-1 pectin at 45 °C and pH 4.5 for 2 h. The extracted POS was then fractionated and purified to homogeneous oligosaccharides (FCPOS-1) with a molecular weight of 2.15 kDa, and the analyses of monosaccharide composition, FTIR, NMR and ESI-MS indicated that FCPOS-1 consisted of GalA and a small amount of mannose, galactose and arabinose. Multiple antioxidant activity assays in vitro revealed that FCPOS-1 possessed remarkable antioxidant properties, especially scavenging activity against DPPH radicals up to 94.07%. FCPOS-1 has the potential to be an effective natural antioxidant for applications in the food and pharmaceutical industries.

Postnatal cadmium administration affects the presence and distribution of carbohydrates in the sperm membrane during maturation in the epididymis in adult Wistar rats.

Cadmium (Cd) is a heavy metal related to a decrease in sperm parameters. The transit of spermatozoa through the epididymis is necessary to generate changes in the sperm membrane, such as the assembly of various carbohydrates that are added to the spermatazoan's surface to prepare it for successful fertilisation of the oocyte. No studies have yet analysed whether Cd alters the presence and distribution of these carbohydrates. We aimed to evaluate the changes induced by Cd in the distribution pattern of N-acetylglucosamine, sialic acid, mannose and fucose on the sperm membrane in the epididymis (e.g. caput, corpus, cauda) and if it alters the epididymal epithelium. Male Wistar pups were treated with Cd doses (0.125, 0.25 and 0.5mg/kg) on postnatal days 1-49. At postnatal day 90, they were humanely killed, sperm samples were obtained from the epididymis and tissue samples were taken for histological analysis. Cd concentrations in the blood and epididymis increased in proportion to the dose administered and decreased the serum testosterone levels and sperm quality. Histological analysis revealed alterations in the epithelium in all Cd-treated groups. Cd altered the distribution patterns of carbohydrates and fluorescence indices. All these alterations affected the structure and functioning of sperm.

Structure, antioxidant and immunomodulatory activity of a polysaccharide extracted from Sacha inchi seeds.

In this study, a novel water-soluble polysaccharide (PVLP-1) was extracted and purified from Sacha inchi (Plukenetia volubilis L.) seeds and the structure, antioxidant and immunomodulatory activity of PVLP-1 were investigated. PVLP-1 (144 kDa) consisted of glucose (69.76%), mannose (14.86%), arabinose (10.53%), galactose (2.42%), ribose (1.23%), rhamnose (0.27%) and xylose (0.93%). PVLP-1 displayed characteristic polysaccharide bands in Fourier transform NMR spectra and infrared. The primary structure of PVLP-1 was a heteropolysaccharide with a backbone of (1 â†’ 6)-linked glucose, sidechains of (1 â†’ 4)-linked mannose, (1 â†’ 4)-linked glucose and (1 â†’ 3, 6)-linked mannose and a residue unit of →1)-linked arabinose as revealed the methylation analysis. PVLP-1 possessed good water-holding capacity (WHC), oil-holding capacity (OHC) and antioxidant capacities. Besides, PVLP-1 induced the proliferation of RAW264.7 cell and enhanced the expression of inflammatory cytokines IL-6, TNF-alpha(TNF-α) and IL-1 beta (IL-1ß). The present study indicated that PVLP-1 possessed immune-enhancing bioactivities and could be functional food or adjuvant drug to improve biological immunity of immunodeficiency diseases and hypoimmunity.

Characterization of Micronutrients, Bioaccessibility and Antioxidant Activity of Prickly Pear Cladodes as Functional Ingredient.

The Opuntia ficus indica (L.) (OFI) is used as a nutritional and pharmaceutical agent in various dietary and value added products. This study underlines the possible use of native prickly pear cladode powder as a functional ingredient for health-promoting food production. To summarise, chemical characterization of polyphenols, minerals and soluble dietary fibre was performed; furthermore, the antioxidant activity and bioaccessibility of polyphenols and minerals were assessed. Eleven compounds between phenolic acids and flavonoids were identified, with piscidic acid and isorhamnetin derivatives being the most abundant. Opuntia's dietary fibre was mainly constituted of mucilage and pectin, and was composed of arabinose, galactose, glucose, mannose, rhamnose, and xylose sugars. The polyphenols' bioaccessibility was very high: piscidic acid at 200%, eucomic and ferulic acids >110% and flavonoids from 89% to 100%. The prickly pear cladode powder is also a source of minerals, as cations (calcium, sodium, potassium and magnesium) and anions (sulphate and chloride), with high magnesium bioaccessibilty (93%). OFI powder showed good capacity of radical scavenging measured by DPPH and ABTS methods, with 740 and 775 µmol Trolox/100 g OFI, respectively. Finally, the presented results allow the consideration of this natural product as a source of several essential nutrients, with a possible use in the food industry as a functional ingredient.

Markers of malignant prostate cancer cells: Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of high mannose N-glycans on cell surface.

The ability to distinguish malignant from indolent prostate cancer cells is critically important for identification of clinically significant prostate cancer to minimize unnecessary overtreatment and sufferings endured by patients who have indolent cancer. Recently, we discovered that loss of giantin function as the primary Golgi targeting site for endoplasmic reticulum-derived transport vesicles in aggressive prostate cancer cells caused a shift of the Golgi localization site of α-mannosidase 1A to 130 KDa Golgi matrix protein (GM130)-65 KDa Golgi reassembly-stacking protein (GRASP65) site resulting in emergence of high mannose N-glycans on trans-Golgi enzymes and cell surface glycoproteins. To extend this observation, we isolated two cell clones (Clone 1 and Clone 2) from high passage LNCaP cells, which exhibited androgen refractory property missing in low passage LNCaP cells, and characterized their malignant property. We have found that comparing to Clone 2, which does not have cell surface high mannose N-glycans and exhibits localization of α-mannosidase 1A at giantin site, Clone 1 displays cell surface high mannose N-glycans, exhibits localization of α-mannosidase 1A at GM130-GRASP65 site, and shows a faster rate of closing the wound in a wound healing assay. The results indicate that Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of cell surface high mannose N-glycans may serve as markers of malignant prostate cancer cells.

The activation of NF-κB and MAPKs signaling pathways of RAW264.7 murine macrophages and natural killer cells by fucoidan from Nizamuddinia zanardinii.

Polysaccharides from Nizamuddinia zanardinii were extracted using water at elevated temperature and fractionated by a DEAE Sepharose FF column yielding four fractions (F1-F4). Crude and fractions were composed of neutral sugars (50.8-57.4%), proteins (10.8-18.1%), sulfates (7.5-17.3%) and uronic acids (3.5-7.7%). Various levels of galactose (13.4-44.4%), fucose (34.1-40.1%), mannose (14.1-33.2%) and xylose (7.4-15.2%) formed the building blocks of the polysaccharide structures. The weight average molecular weights (Mw) of polysaccharides varied between 40.3 and 1254.4 × 103 g/mol. F3 polysaccharide was the most active fraction stimulating RAW264.7 murine macrophage cells to secrete NO, TNF-α, IL-1ß and IL-6, and activating NK cells to release TNF-α, INF-γ, granzyme-B, perforin, NKG2D and FasL through NF-κB and MAPKs signaling pathways. Highly-branched F3 polysaccharide mainly consisted of (1 â†’ 2)-Fucp, (1 â†’ 2,3)-Manp, (1 â†’ 3)-Galp, (1 â†’ 2)-Manp, (1 â†’ 3)-Manp, (1 â†’ 2,3,4)-Manp and (1 â†’ 2,3,6)-Manp residues with great amount of (→1)-Fucp and (→1)-Xylp. Sulfates substituted at C-2 of fucose and galactose residues. Overall, fucoidan from N. zanardinii showed immense potency in boosting immune system through macrophages and NK cells activations and therefore suitable for further exploration in immune-mediated biomedical applications.

Nanostructured impedimetric lectin-based biosensor for arboviruses detection.

Arboviruses have been emerging as a significant global health problem due to the recurrent epidemics. Arboviruses require the development of new diagnostic devices due to the nonspecific clinical manifestations. Herein, we report a biosensor based on cysteine (Cys), zinc oxide nanoparticles (ZnONp), and Concanavalin A (ConA) lectin to differentiate between arboviruses infections. ConA is capable of interacting with the saccharide components of the viral capsid. In this study, we evaluated the reproducibility, sensitivity, and specificity of the sensor for the virus of Dengue type 2 (DENV2), Zika (ZIKV), Chikungunya (CHIKV), and Yellow fever (YFV). Atomic force microscopy measurements confirmed the electrode surface modification and revealed a heterogeneous topography during the biorecognition process. Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were used to characterize the biosensor. The blockage of the oxidation-reduction process is related to the formation of Cys-ZnONp-ConA system on the electroactive area and its subsequent interaction with viral glycoproteins. The sensor exhibited a linear response to different concentrations of the studied arboviruses. Our study demonstrates that ConA lectin recognizes the structural glycoproteins of the DENV2, ZIKV, CHIKV, and YFV. DENV2 is the most structurally similar to ZIKV. Our results have shown that the impedimetric response correlates with the structural glycoproteins, as follow: DENV2 (18.6 kΩ) > ZIKV (14.6 kΩ) > CHIKV (6.86 kΩ) > YFV (5.98 kΩ). The homologous structural regions contribute to ConA-arboviruses recognition. Our results demonstrate the use of the proposed system for the development of biosensors for arboviruses infections.

Structural properties and antioxidant activities of polysaccharide from fruit bodies of Pholiota nameko.

A polysaccharide named PNP was extracted and purified from Pholiota nameko. The total sugar content of PNP was 95.29% and the molecular weight was 1.89 × 103 kDa. The structural features of PNP were investigated by the combination of chemical and instrumental analysis such as UV spectrophotometer, specific rotation determination, FT-IR, methylisation analysis and Congo red. The results showed that the optical rotation of PNP was +120° and that it had a triple-helical structure. Besides, PNP was mainly composed of glucose and mannose at the molar ratio of 4.24:1.00. The backbone of PNP was composed of (1→3)-linked-Glc and (1→3)-linked-Man whereas the branches of (1→3,6)-linked- Glc, (1→3,6)-linked-Man and T- Glc. Consistenting with the results of UV-Vis spectra, FT-IR spectroscopy and 1H NMR, indicated that PNP was a complex of polysaccharides and polyphenols. In vitro antioxidant results suggested that PNP was processed with certain scavenging capacity.

Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling.

Chemical characterization and immunomodulatory activity of acetylated polysaccharides from Dendrobium devonianum.

The chain conformation, chemical characters and immunomodulatory activity of polysaccharide from Dendrobium devonianum (DDP) were investigated. Results showed that molecular weights, polydispersity index, radius of gyrations of DDP were 3.99×105 Da1.27, 74.1nm, respectively. By applying the polymer solution theory, the exponent (v) values of z1/2=kMwv was calculated as 0.38, which revealed that DDP existed as a globular shape in aqueous solution, and further confirmed by AFM analysis. Furthermore, the main monosaccharide compositions were Man and Glc with the ratio of 29.61:1.00. Indeed, the main glycosidic linkages were ß-1,4-Manp, and substituted with acetyl groups at O-2 and O-3 position. Notably, DDP could promote the immune functions of macrophages including NO release and phagocytosis. Thus, DDP could be explored as a natural immune-stimulating agent in the health and functional food area as well as pharmaceutical industries.

Transdifferentiation of mucous neck cells into chief cells in fundic gastric glands shown by GNA lectin histochemistry.

The epithelium of the gastric mucosa and its glands in the corpus of rat stomach contains mucous surface cells (MSCs), parietal cells, mucous neck cells (MNCs), zymogenic or chief cells (ZCs), several types of enteroendocrine cells, and intermediate cells with characteristics between MNCs and ZCs also called transitional or prezymogenic cells (pre-ZCs). The aim of our work was to analyze the expression of Mannose (Man) in the rat gastric glands by means of Galanthus nivalis lectin (GNA) histochemistry to identify the differences between MNC, pre-ZCs and ZCs and to establish the relationships between these cells. Most of the cytoplasm of MNCs was negative for GNA histochemistry. Intensity of GNA labeling in the gastric gland showed a graduation from pre-ZCs (weak labeling) to ZCs (moderate labeling). Labeling of ZCs was stronger at the perinuclear and apical cytoplasm. In the last years, strong evidence has been reported supporting that ZCs differentiate from MNCs. Our work also supports the origin of ZCs from MNCs, because the GNA labeling graduation might be due to oligosaccharides which are not expressed in MNCs, start to express in pre-ZCs and are more abundant in ZCs, indicating that differentiation from MNCs to ZCs is a process in which glycans with Man moieties are synthesized.

Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma.

Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics. We developed a targeted MS platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas. PD-L1 abundance ranged 50-fold, from ∼0.03 to 1.5 fmol/microgram protein and the parallel reaction monitoring (PRM) data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody. PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r2 = 0.062, p = 0.264). PD-1 abundance was weakly correlated (r2 = 0.3057, p = 0.009) with the fraction of lymphocytes and histiocytes in sections. PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58. In 10 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation. We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples. Extent of PD-L1 glycan modification varied by ∼10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function. Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA, and CD40, which all displayed distinct expression independent of PD-1, PD-L1, and PD-L2. Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics.

Microwave-assisted deglycosylation for rapid and sensitive analysis of N-glycans via glycosylamine derivatization.

A rapid and sensitive N-glycan profiling strategy for MALDI-MS incorporating the use of deglycosylation with microwave assistance and the co-derivatization of glycosylamine labeling with tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) and methylamidation has been developed in this work. Notably, highly efficient release and tagging of N-glycans from ribonuclease B was achieved in less than 90 min, providing up to 35-fold enhancement of MALDI-MS sensitivity with comparison to underivatized N-glycans. After further validation with other two standard glycoproteins (ovalbumin and bovine fetuin), the proposed strategy was applied to human serum for preliminary pathological analysis of N-glycans between healthy and lung cancer individuals. As a result, significant differences (T test p value <0.01) of 6 glycan structures were determined from 54 detected N-glycan structures with only 50 nL of loading amount and further confirmed through PCA and ROC (AUC) analyses between two sample sets. Subsequently, the trend of each lung cancer stage and controls in expression of the selected glycans was implemented with T test and box-plots. Accordingly, these structures can be used as potential lung cancer glycan-based biomarkers and for further definition of cancer progression highlighting the ability of proposed method to rapidly and efficiently analyze N-glycome present in human serum. Graphical abstract MALDI-TOF MS analysis of N-glycans by microwave-assisted deglycosylation and glycosylamine derivatization.

Protein-Bound Polysaccharide from Corbicula fluminea Inhibits Cell Growth in MCF-7 and MDA-MB-231 Human Breast Cancer Cells.

A novel protein-bound polysaccharide, CFPS-1, isolated from Corbicula fluminea, is composed predominantly of mannose (Man) and glucose (Glc) in a molar ratio of 3.1:12.7. The polysaccharide, with an average molecular weight of about 283 kDa, also contains 10.8% protein. Atomic force microscopy, high-performance liquid chromatography, Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that CFPS-1 has a backbone of 1,6-linked and 1,4,6-linked-α-D-Glc, which is terminated with a 1-linked-α-D-Man residue at the O-4 position of 1,4,6-linked-α-D-Glc, in a molar ratio of 3:1:1. Preliminary in vitro bioactivity tests revealed that CFPS-1 effectively and dose-dependently inhibits human breast cancer MCF-7 and MDA-MB-231 cell growth, with an IC50 of 243 ± 6.79 and 1142 ± 14.84 µg/mL, respectively. In MCF-7, CFPS-1 produced a significant up-regulation of p53, p21, Bax and cleaved caspase-7 and down-regulation of Cdk4, cyclin D1, Bcl-2 and caspase-7. These effects resulted in cell cycle blockade at the S-phase and apoptosis induction. In contrast, in MDA-MB-231, with limited degree of change in cell cycle distribution, CFPS-1 increases the proportion of cells in apoptotic sub-G1 phase executed by down-regulation of Bcl-2 and caspase-7 and up-regulation of Bax and cleaved caspase-7. This study extends our understanding of the anticancer mechanism of C. fluminea protein-bound polysaccharide.

Water-Soluble Compounds from Lentinula edodes Influencing the HMG-CoA Reductase Activity and the Expression of Genes Involved in the Cholesterol Metabolism.

A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and ß-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.

Structural Characterization and Immunomodulatory Activity of a Novel Polysaccharide from Lepidium meyenii.

A novel polysaccharide named as MC-1 was isolated from the roots of Lepidium meyenii using a water extraction method. Structural characterization revealed that MC-1 had an average molecular weight of 11.3 kDa and consisted of arabinose (26.21%), mannose (11.81%), glucose (53.66%), and galactose (8.32%). The main linkage types of MC-1 were proven to be (1 → 5)-α-L-Ara, (1 → 3)-α-L-Man, (1 → 2,6)-α-L-Man, (1 → )-α-D-Glc, (1 → 4)-α-D-Glc, (1 → 6)-α-D-Glc and (1 → 6)-ß-D-Gal by methylation analysis, periodate oxidation-Smith degradation and NMR analysis. The immunostimulating assay indicated that MC-1 could significantly enhance the pinocytic and phagocytic capacity and promote the NO, TNF-α, and IL-6 secretion of RAW 264.7 cells, involving toll-like receptor 2, complement receptor 3, and mannose receptor mainly. These results suggested the potential utilization of MC-1 as an attractive functional food supplement candidate for hypoimmunity population.

Carbohydrate derivative-functionalized biosensing toward highly sensitive electrochemical detection of cell surface glycan expression as cancer biomarker.

Accurate and highly sensitive detection of glycan expression on cell surface is extremely important for cancer diagnosis and therapy. Herein, a carbohydrate derivative-functionalized biosensor was developed for electrochemical detection of the expression level of cell surface glycan (mannose used as model). Thiomannosyl dimer was synthesized to design the thiomannosyl-functionalized biosensor by direct and rapid one-step protocols. The biosensing surface-confined mannose could effectively mimic the presentation of cell surface mannose and was responsible for competing with mannose on cancer cells in incubation solution. Greatly enhanced sensitivity was achieved by exploiting the excellent conductivity of multiwalled carbon nanotube/Au nanoparticle (MWNT/AuNP), the amplification effect of MWNTs, and the favorable catalytic ability of horseradish peroxidase (HRP). Using competitive strategy, the developed biosensor exhibits attractive performances for the analysis of mannose expression with rapid response, high sensitivity and accuracy, and possesses great promise for evaluation of cell surface glycan expression by using a greater variety of lectins.

Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.