Sulfate Modification Research Articles

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Evaluation and improvement of the oxidative stability of leather fatliquors

Fatliquor oxidation may give leather unpleasant odor, and excessive amounts of Cr(VI) and volatile organic compounds. The accurate evaluation and improvement of the oxidative stability of fatliquors are of great significance to high-quality leather manufacturing. We proposed a set of practical methods for evaluating the oxidative stability of fatliquors on the basis of oxidation induction time, change in iodine value (∆ IV), and change in acid value (∆ AV) under accelerated oxidation conditions (at 100 °C with 10 L/h of air). Oxidation induction time is a highly sensitive marker for quantifying the oxidative stability of fatliquors, and ∆ IV and ∆ AV that are low cost and easy to operate are useful in evaluating the oxidative stability of fatliquors when the oxidation induction time is less than 22 h. The number of double bonds in fatliquors is an important factor affecting oxidative stability. The sulfation modification of fatliquors that greatly reduces double bonds and the addition of antioxidants, especially butylated hydroxyanisole and butylated hydroxytoluene, markedly improve oxidative stability of fatliquors.

Performance and poisoning analysis of organic sulfur resistance of Pd-Ce catalyst in catalytic oxidation of VOCs

In this paper, the effect of organic sulfur on the catalytic oxidation of toluene was explored. It was found that the low concentration of organic sulfur could cause rapid deactivation of the catalyst, and the activity of the catalyst could be restored to 100% after stopping the application of sulfur. The sulfation modification could significantly improve the organic sulfur resistance of the catalyst by changing the support and the sulfation modification of the support. There was a large amount of carbon deposition and a small amount of S-containing substances on the catalyst surface after the introduction of organic sulfur through the methods of TG, BET, FTIR, SEM (Mapping) and XPS. Then, through in-situ DRIFT and CH3SH-TPD, it was found that sulfation modification could inhibit the adsorption of organic sulfur on the catalyst surface and reduce the competitive adsorption. The decrease of catalyst efficiency due to the presence of organic sulfur in the reaction gas during VOCs catalytic oxidation is mainly due to the competitive adsorption of organic sulfur and VOCs molecules, and the temperature of VOCs catalytic oxidation of organic sulfur. Some polyhydrocarbons, ethyl sulfide, dimethyl sulfoxide and other substances were produced by incomplete oxidation, which covered the surface of the catalyst due to adsorption or deposition, resulting in catalyst poisoning.

Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands.

The immunomodulatory family of Siglecs recognizes sialic acid-containing glycans as "self", which is exploited in cancer for immune evasion. The biochemical nature of Siglec ligands remains incompletely understood, with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs─CHST1, CHST2, or CHST4─significantly enhance the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, a pharmacological blockade of N- and O-glycan maturation reveals a cell-type-specific pattern of importance for either class of glycan. Production of a highly homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-fold enhanced affinity for Neu5Acα2-3(6-O-sulfo)Galβ1-4GlcNAc and Neu5Acα2-3Galβ1-4(6-O-sulfo)GlcNAc, respectively, over Neu5Acα2-3Galβ1-4GlcNAc. CD33 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2-3(6-O-sulfo)Galβ1-4(6-O-sulfo)GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a general mechanism for tuning Siglec ligands on cells, including in cancer.

Sulfated modification enhances the immunomodulatory effect of Cyclocarya paliurus polysaccharide on cyclophosphamide-induced immunosuppressed mice through MyD88-dependent MAPK/NF-κB and PI3K-Akt signaling pathways

The present study investigated the effect of sulfation on the immunomodulatory effect of Cyclocarya paliurus polysaccharide (CP) through a Cyclophosphamide (CTX)-induced immunosuppression mice model. The results showed that sulfated Cyclocarya paliurus polysaccharide (SCP3) had stronger immunomodulatory ability than CP. Administration of SCP3 alleviated immune organ atrophy and restored hematopoiesis in immunosuppressed mice, enhanced splenocyte proliferation, and promoted cytokines and nitric oxide (NO) production in splenocyte supernatants, as well as the number of CD3+, CD4+ and CD8+ T lymphocytes. Meantime, SCP3 significantly improved oxidative stress via increasing the activities of antioxidant enzymes and decreasing the levels of malondialdehyde (MDA) in liver. In addition, SCP3 significantly upregulated the phosphorylation expression of JNK, Erk 1/2, p38 of MAPKs signaling pathway at a dose of 50 mg/kg and accordingly showed increased phosphorylation of Akt, NF-κB (p65), IκB-α, and promoted the degradation of IkB-α. Furthermore, SCP3 significantly increased the expression of the upstream signaling molecule MyD88. All results demonstrated that sulfation can be an effective way to enhance the immunomodulatory effect of polysaccharides. SCP3 has high potential to be a functional food supplement candidate for alleviating chemotherapy drug-induced immunosuppression.

Sulfated Modification, Characterization and Potential Bioactivities of Polysaccharide From Ziziphus jujuba cv. Jinsixiaozao

In this study, jujube polysaccharide (JP) was extracted from Ziziphus jujuba cv. Jinsixiaozao and sulfated JP (SJP) was prepared. The optimum preparation conditions were as follows: reaction temperature 75°C, reaction time 1 h, ratio of chlorosulfonic acid-to-pyridine ( VCSA/ VPyr) 1. The degree of substitution of SJP was 0.664 ± 0.014. JP and SJP were typical heteropolysaccharides, which were composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose, but the molar ratio of monosaccharides was different. Fourier transform infrared spectra showed that JP was sulfated successfully. Compared with JP, the molecular weight of SJP increased to 3.17 × 105 Da, its water solubility increased significantly, and its viscosity decreased significantly. When the microstructure of SJP was examined, it was found that the surface of the polysaccharides became loose and porous after sulfation. SJP had a higher hydroxyl radical scavenging activity than the unsulfated polysaccharide. Moreover, sulfation enhanced the antibacterial activity of the polysaccharides against Escherichia coli and Bacillus subtilis. Therefore, sulfation is an effective way to improve the biological activity of the polysaccharide, and SJP can be used as a potential antioxidant and antimicrobial agent in the field of food and medicine.

Sulfated Alginate Hydrogels Prolong the Therapeutic Potential of MSC Spheroids by Sequestering the Secretome.

Cell-based approaches to tissue repair suffer from rapid cell death upon implantation, limiting the window for therapeutic intervention. Despite robust lineage-specific differentiation potential in vitro, the function of transplanted mesenchymal stromal cells (MSCs) in vivo is largely attributed to their potent secretome comprising a variety of growth factors (GFs). Furthermore, GF secretion is markedly increased when MSCs are formed into spheroids. Native GFs are sequestered within the extracellular matrix (ECM) via sulfated glycosaminoglycans, increasing the potency of GF signaling compared to their unbound form. To address the critical need to prolong the efficacy of transplanted cells, alginate hydrogels are modified with sulfate groups to sequester endogenous heparin-binding GFs secreted by MSC spheroids. The influence of crosslinking method and alginate modification is assessed on mechanical properties, degradation rate, and degree of sulfate modification. Sulfated alginate hydrogels sequester a mixture of MSC-secreted endogenous biomolecules, thereby prolonging the therapeutic effect of MSC spheroids for tissue regeneration. GFs are sequestered for longer durations within sulfated hydrogels and retain their bioactivity to regulate endothelial cell tubulogenesis and myoblast infiltration. This platform has the potential to prolong the therapeutic benefit of the MSC secretome and serve as a valuable tool for investigating GF sequestration.

Sulfation modification of dopamine in brain regulates aggregative behavior of animals.

Behavioral plasticity and the underlying neuronal plasticity represent a fundamental capacity of animals to cope with environmental stimuli. Behavioral plasticity is controlled by complex molecular networks that act under different layers of regulation. While various molecules have been found to be involved in the regulation of plastic behaviors across species, less is known about how organisms orchestrate the activity of these molecules as part of a coherent behavioral response to varying environments. Here we discover a mechanism for the regulation of animal behavioral plasticity involving molecular sulfation in the brain, a modification of substrate molecules by sulfotransferase (ST)-catalyzed addition of a sulfonate group (SO3) from an obligate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the substrates. We investigated aggregation behaviors of migratory locusts, which are well-known for extreme phase change plasticity triggered by population density. The processes of PAPS biosynthesis acted efficiently on induction of locust behavioral transition: Inhibition of PAPS synthesis solicited a behavioral shift from gregarious to solitarious states; external PAPS dosage, by contrast, promoted aggregation in solitarious locusts. Genetic or pharmacological intervention in the sulfation catalyzation resulted into pronounced solitarizing effects. Analysis of substrate-specific STs suggests a widespread involvement of sulfated neurotransmitters in the behavioral response. Dopamine in the brain was finally identified to be actively sulfate conjugated, and the sulfate conjugation enhanced the free DA-mediated behavioral aggregation. Similar results in Caenorhabditis elegans and mice indicate that sulfation may be involved more broadly in the modulation of animal aggregation. These findings reveal a general mechanism that effectively regulates animal social-like behavioral plasticity, possibly through sulfation-mediated modification of neural networks.

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

BackgroundSulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans.ResultsOur screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated.ConclusionThe present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

Chemically Sulfated Polysaccharides from Agaricus blazei Murill: Synthesis, Characterization and Anti-HIV Activity.

AIDS, caused by HIV-1, is one of the most dangerous infectious diseases in the world. Therefore, it is necessary to develop new drugs with more potent bioactivities, less toxicity and higher tolerability for controlling the viral load, particularly by using the raw materials that are widely available. Agaricus blazei Murill (AbM), known in China as jisongrong, is of great importance as a food source and as a health-promoting supplement for immunomodulation. The polysaccharides of AbM exhibit various biological activities, such as regulating cellular immunity and providing anti-oxidative, anti-infective, and anti-inflammatory effects. At present, to our knowledge, no report has explored the chemically sulfated and anti-HIV-1 activity of AbM polysaccharides. Herein, the sulfated AbM polysaccharides with different sulfur contents were prepared by the chlorosulfonic acid-pyridine method. The characteristics of sulfated derivatives were established by the determination of the sulfur content, the relative molecular weight, and the Fourier-transform infrared spectroscopy. The anti-HIV activities of the sulfated AbM polysaccharides were evaluated by CCK-8 and the single-cycle pseudovirus infection (TZM-bl) assay. The sulfated AbM polysaccharides had strong antiviral properties, and the half-maximal inhibitory concentrations approached that of the positive control, azidothymidine. Sulfated modification of AbM polysaccharides can increase their anti-HIV pharmacological activity, which makes them promising alternative candidates as bioactive macromolecules for biomedical applications in HIV/AIDS.

Carbon-Based Stable CsPbIBr2 Solar Cells with Efficiency of over 10% from Bifunctional Quinoline Sulfate Modification

Carbon-Based Stable CsPbIBr₂ Solar Cells with Efficiency of over 10% from Bifunctional Quinoline Sulfate Modification

Sulfated Modification of Polysaccharides from Sweet Corncob and Its Antiglycation Activity in Streptozotocin Induced Diabetic Rats

Polysaccharides extracted from sweet corncob (SCP) were modified by sulfuric acid to sulfated sweet corncob (SSCP) with a molecular weight of 13.412 kDa, and their antiglycation activity was studied. SSCP had high inhibitory effects on glycation and showed antiglycation activity stronger than that of SCPin vitro.The maximum inhibition rates of the Amadori products, dicarbonyl compounds, caboxymethyl-lysine (CML), and advanced glycation end products (AGEs) were 76.35, 73.78, 52.79, and 76.36%, respectively. SSCP effectively increased body weight, reduced blood glucose, and increased oral glucose tolerance in streptozotocin (STZ)-induced diabetic ratsin vivo.Furthermore, SSCP inhibited AGE formation in liver tissue and repaired pancreatic injury. The resultsin vivoandin vitroreflect that SSCP has antiglycation effects, which may be closely related to its antidiabetic effects.

Sulfated modification enhances the modulatory effect of yam polysaccharide on gut microbiota in cyclophosphamide-treated mice

Our previous study has demonstrated that sulfated yam polysaccharide (SCYP) had a stronger immunomodulatory activity than yam polysaccharide (CYP). In order to investigate how the sulfated modification influence the immunological activity of CYP, this research was mainly focused on the study of gut microbiotal. The results showed that SCYP treatment could increase the digestive enzyme activities of colon contents and restore the production of short-chain fatty acids (SCFAs) in mice that were decreased by cyclophosphamide (Cy) treatment. Furthermore, SCYP treatment could modulate the structure of the gut microbiota, which was mainly manifest in the result of an increased abundances of Lactobacillus, Bacteroidetes and Akkermansia, and the decrease of the proportion of Proteobacteria and Verrucomicrobia in the microbial community. Diversity and overall structure of microbial community were also improved by SCYP treatment based on alpha-diversity and beta-diversity analysis results. The biomarkers of the gut microbial that were regulated by SCYP have been identified by linear discriminant analysis (LDA) effect size (LEfSe). These findings further indicate that there is great potential for SCYP to be developed into prebiotics or functional foods.

Effect of sulfated modification on rheological and physiological properties of oat β-glucan oligosaccharides prepared by acid or oxidative degradation

Two kinds of oat β-glucan oligosaccharides (βG-H and βG-O) were obtained by acid and oxidative degradation methods, respectively. Further, six kinds of sulfated oat β-glucan oligosaccharides (βG-H-S1, βG-H-S2, βG-H-S3, βG-O-S1, βG-O-S2, and βG-O-S3) with different degree of substitution were prepared by controlling the reaction time. The structure analysis showed that the molecular weights (Mw) and degree of substitution (DS) of sulfated oat β-glucan oligosaccharides increased with the prolonged reaction time. After sulfated modification, the apparent viscosity of oat β-glucan oligosaccharides increased and their viscoelasticity was changed. Sulfated modification endowed βG-H with enhanced fat binding capacity and bile acid binding capacity, and the above-mentioned properties increased with the increasing Mw and DS. Meanwhile, sulfated modification endowed βG-O with enhanced fat binding capacity and attenuated bile acid binding capacity, and the capacities of βG-O-S decreased with the increasing Mw and DS. Sulfated modification enhanced hypoglycemic effect of oat β-glucan oligosaccharides, it showed ability to decrease the glucose availability and inhibit α-glucosidase activity. The results revealed that the physiological properties of sulfated β-glucan oligosaccharides depend on their Mw, DS, and degradation method of β-glucan oligosaccharides.

Sulfated modification, structures, antioxidant activities and mechanism of Cyclocarya paliurus polysaccharides protecting dendritic cells against oxidant stress

In this study, Cyclocarya paliurus polysaccharides purified by DEAE-cellulose columns (CPP0.05) were modified with chlorosulfonic acid–pyridine (CSA–Pyr) method to obtain the sulfation derivative (S-CPP0.05), whose structures were characterized by methylation and NMR analysis. The backbone of S-CPP0.05 was composed of →3)-β-D-Galp-(1→4)-α-D-Glcp-(1→ substituted by α-L-Araf-(1→, →4, 6)-β-D-Galp-(1→ and α-D-Glcp-(1→ at O-4/5/6 position, terminated with α-L-Araf-(1→ and occasionally by α-D-Glcp-(1→. Sulfate groups were substituted at C6 of →4)-α-D-Glcp-(1→. CPP0.05 and S-CPP0.05 had the potential to scavenge DPPH, ABTS, hydroxyl and radical showing reducing power in vitro. CPP0.05 and S-CPP0.05 were found to suppress oxidant stress by improving the enzyme activities of SOD and CAT in dendritic cells (DCs) in the presence of H2O2. Results indicated that S-CPP0.05 showed stronger antioxidant activity compared with CPP0.05. Meanwhile, we found that Nrf2 proteins were significantly increased by the stimulus of polysaccharides with Keap1 decreasing which proved that the antioxidant effect was possibly performed via the Keap1-Nrf2 pathway. The results confirmed that C6 of Cyclocarya paliurus polysaccharides is an effective substitution site for sulfate groups.

Cellulose sulfate/EMIMAc solution: rheological properties and shaping into polyelectrolyte complexes for protein adsorption

Cellulose sulfates (CS) with low degree of substitution (DS) and high degree of polymerization (DP) were prepared via a controlled sulfation modification. The obtained CS with DS 0.42 and DP 293 showed an enhanced solubility of 14.7 g/100 g in the ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIMAc) compared to unmodified cellulose. The rheological study revealed that the CS/EMIMAc solution behaved as polymers in a θ solvent with the unique three-region flow curves under shear stress. It also showed a property of typical polyelectrolyte solution with the overlap concentration (c*) of 0.2 wt% and the entanglement concentration (ce) of 3.0 wt%, respectively. The CS/EMIMAc solution with a concentration of 5 wt% was selected to develop polyelectrolyte complex (PEC) with chitosan using extrusion-spherization and emulsification-solvent evaporation. The produced PEC beads and magnetic microspheres had an average size of 1.0 mm and 22.1 µm, respectively. The PEC microspheres showed greater absorption capability for bovine serum albumin than the beads with the maximum adsorption value of 780 mg/g.

Improvement of ion conductivity and selectivity of heterogeneous membranes by sulfated zirconia modification

Inorganic-organic composites based on the foil and standard RALEX® cation-exchange heterogeneous membranes (Mega a.s., Czech Republic) were prepared by in situ modification with sulfated zirconia (S-ZrO2). The composite membranes were characterized by SEM, TGA, X-ray diffraction, and FTIR spectroscopy. The effect of S-ZrO2 doping on membrane transport properties was studied using measurements of water uptake, ion-exchange capacity, conductivity, cation diffusion, hydrogen permeability, current-voltage characteristics, and membrane specific permselectivity (Ca2+/Na+). The S-ZrO2 incorporation leads to an increase in conductivity and permselectivity of the composite membranes. The proton conductivity of the S-ZrO2-doped foil membrane (0.0316 S/cm at 30°С) is 4 times higher than that of the pristine membrane. The Ca2+/Na+ permselectivity of the standard RALEX® CM membrane doped by S-ZrO2 reaches 3.8 at low current densities. Moreover, the composite membranes retain their selectivity during the long-term tests (> 50 h continuous electrodialysis). The sulfated zirconia doping of heterogeneous membranes demonstrated an excellent separation efficiency that can be used in wastewater treatment, desalination, and related electromembrane separation processes as well as to reduce scaling of electrodialysis modules.

Sulfated modification, characterization and bioactivities of an acidic polysaccharide fraction from an edible mushroom Pleurotus eous (Berk.) Sacc.

The acidic fraction (P3a) of Pleurotus eous was successfully sulfated by sulphur trioxide-pyridine complex method. The effect of sulfate modification (SP3a) on the structure, physicochemical properties and in vitro biological activity of P3 was studied. The structural characteristics were established by UV absorption, FT-IR, HPGPC and GC-MS. Biological studies were carried out, such as in vitro antioxidant, anticoagulant, anti-tumour and antibacterial activities. The sulfation process changed its physicochemical and biological characteristics. Compared with P3a, the molecular weight of SP3a is reduced. P3a and SP3a are composed of galactose, xylose, arabinose with different molar percentages. Sulfated derivatives have strong antioxidant and anticoagulant properties. Compared with P3a, SP3a showed obvious cytotoxicity to Jurkat and HeLa cells. SP3a showed a higher inhibition zone for Gram-positive and Gram-negative bacteria. This article demonstrates that sulfation is an effective way to enhance biological activity, especially SP3a is a promising candidate for bioactive macromolecules and has great potential for industrial and biomedical applications.

Biological and microbiological activities of isolated Enterobacter sp. ACD2 exopolysaccharides from Tabuk region of Saudi Arabia

Several forms of bacterial exopolysaccharides (EPSs) have been reported as having industrial and medical benefits. Previously, our group had achieved a high yield of EPS production (8.6 gm/L) from Enterobacter sp. isolated from the marine environment of Haqel Beach, in the Tabuk region of Saudi Arabia, by optimizing its culture conditions. Then, the strain selected was identified using conventional methods and confirmed by molecular characterization that used 16S Ribosomal RNA (16S rRNA) gene sequencing. In the present study, we determined the composition of this EPS and evaluated its anticoagulation, fibrinolytic, antimicrobial, and prebiotic activities. We determined the monosaccharide moieties of this EPS using acid hydrolysis by qualitative and quantitative paper chromatography (PC). The monosaccharide composition of this EPS was almost 25% glucose, 25% galactose, 40% fucose, in addition to 10% uronic acid, with traces of fructose. Sulfation modification was applied to the EPS produced. The biological and microbiological (anticoagulation, fibrinolytic, antimicrobial, and prebiotic) activities of both the native and sulfated EPS were investigated and both exhibited higher antibacterial activity against Staphyllococcus aureus and Escherichia coli, although neither form displayed antifungal activity against Candida albicans. The minimum inhibitory concentration (MIC) of the native and sulfated EPS that sustained the highest bacterial inhibition zone was 15 mg/dl. No prebiotic activities were recorded for either EPS. In conclusion, the biopolymer produced has significant biological activities that require further adaptation for practical and industrial applications.

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role.

Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.