Polysaccharide Chains Research Articles

The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase, Wzz, from enterobacterial common antigen biosynthesis form a complex.

The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.

Effects of polysaccharides from Lyophyllum decastes (Fr.) Singer on gut microbiota via in vitro-simulated digestion and fermentation.

Lyophyllum decastes (Fr.) Singer polysaccharides (LDSPs) have been verified to possess strong biological properties. However, the effects of LDSPs on intestinal microbes and their metabolites have rarely been addressed. The in vitro-simulated saliva-gastrointestinal digestion and human fecal fermentation were used to evaluate the effects of LDSPs on non-digestibility and intestinal microflora regulation in the present study. The results showed a slight increase in the content of the reducing end of the polysaccharide chain and no obvious change in the molecular weight during in vitro digestion. After 24 h in vitro fermentation, LDSPs were degraded and utilized by human gut microbiota, and LDSPs could be transformed into short-chain fatty acids leading to significant (p < 0.05) decrease in the pH of the fermentation solution. The digestion did not remarkably affect the overall structure of LDSPs and 16S rRNA analysis revealed distinct shifts in the gut microbial composition and community diversity of the LDSPs-treated cultures, compared with the control group. Notably, the LDSPs group directed a targeted promotion of the abundance of butyrogenic bacteria, including Blautia, Roseburia, and Bacteroides, and an increase in the n-butyrate level. These findings suggest that LDSPs might be a potential prebiotic to provide a health benefit.

Physicochemical and Molecular Properties of Spelt and Wheat Starches Illuminated with UV Light

Scope: Aqueous suspensions of spelt and wheat starch were irradiated with ultraviolet radiation for 5–50 h. The changes in the molecular structure of the starch induced by UV radiation were related to the photodegradation of starch chains and the subsequent recombination of the resulting polysaccharide radicals during prolonged irradiation. Methods and Results: Aqueous suspensions of spelt and wheat starch were irradiated with ultraviolet radiation for 5–50 h. The HPSEC-MALLS-RI method was used to measure the weight-average molecular weights of the starch polysaccharide chains and the distribution of the amylopectin side chains. FTIR spectroscopy of the modified samples and a study of the starch surface morphology were conducted using a scanning electron microscope. However, an increase in both the rate constant of the first stage of hydrolysis (k1) and its final level were demonstrated. The changes in the molecular structure of the starch induced by UV radiation were related to the photodegradation of starch chains and the subsequent recombination of the resulting polysaccharide radicals during prolonged irradiation. Conclusions: Changes in starch granules could influence some of their functional properties. Rearrangement in the polysaccharide chains could obtain novel starches with defined properties.

Physicochemical and Structural Characterization of Alkali-Treated Biopolymer Sphingan WL Gum from Marine Sphingomonas sp. WG.

Sphingan WL gum (WL), a kind of exopolysaccharide, is produced by Sphingomonas sp. WG, which was screened from sea mud samples of Jiaozhou Bay by our group. The solubility of WL was investigated in this work. First, 1 mg/mL of WL solution was stirred at room temperature for at least 2 h to obtain a uniform opaque liquid, and further the solution became clear with the increased NaOH and stirring time. Subsequently, the structural features, solubility, and rheological properties of WL before and after alkali treatment were compared systematically. FTIR, NMR, and zeta potential results indicate that the alkali causes acetyl group hydrolysis and carboxyl group deprotonation. XRD, DLS, GPC, and AFM results suggest that the alkali destroys the ordered arrangement and inter- and intrachain entanglement of polysaccharide chains. In the same case, 0.9 M NaOH-treated WL presents better solubility (stirring for 15 min to obtain a clarified solution) but, unsurprisingly, worsens rheological properties. All results demonstrated that the good solubility and transparency of alkali-treated WL will help promote its postmodification and application.

Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites in IsPETase and the efficient post-translational modification in endoplasmic reticulum, IsPETase is heavily glycosylated during secretory expression with P. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of IsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated IsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated IsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of IsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable IsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.

Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

Free energy landscape of wrapping of lipid nanocluster by polysaccharides

Wrapping of a 20-mer cholesterol nano-cluster (CHL-nanoC) by two widely different types of β-glucan polysaccharides (23–25 mers) having significantly varying glycosidic linkage patterns and side chains is studied by Well-Tempered MetaDynamics (WT-MetaD) simulations. The problem has its relevance in the faecal sterol and bile acid excretion in humans and the role of dietary fibres in aiding the process and combating dyslipidemia. Additionally, the distinctive collective variables studied here can be extended for modeling of polymer wrapped soft clusters/nano-particles in general. The wrapping ability is observed to be significantly correlated to the bending of the polysaccharide chain, an attribute of the glycosidic linkage type. By biasing two unique collective variables, the radius of gyration of the polysaccharide (Rg, poly) and the second order Legendre polynomial of the segment orientation parameter, θ, we could successfully observe the wrapping process. This work compares in detail the physical properties of the polysaccharide encapsulated CHL-nanoC by probing the radius of curvature (Rcurv, poly) of the polysaccharides, their coordination number with respect to the CHL-nanoC (CN), fractional CHL-nanoC surface coverage and the electrostatic surface potentials of the complex assembly. Results indicate that the β-glucan having 1–4 glycosidic linked monomers with intermittent 1–3 linkage is able to wrap the CHL-nanoC more effectively. The 1–3 glycosidic linked β-glucan with 1–6 glycosidic bonds in side chains is significantly curled up and appears to be less efficient in wrapping the nanoC. This work provides a comparative molecular level picture of mutual interaction between two major dietary polysaccharide variants and lipid globules as indicated by numerous clinical level studies involving mice and human models.

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag+/Cl− colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔHITC upon interacting with free Ag+ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag+ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag+/Cl− colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag+/Cl− colloids. Biomineralization of Ag+/Cl− colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-β-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

HPLC-MS Characterization of Tissue-Derived Heparan Sulfate and Chondroitin Sulfate.

Glycosaminoglycans (GAGs) are built up of repeating disaccharide units resulting in long, linear polysaccharide chains. In most classes of GAGs, sulfation and epimerization complicate the structure of the chain and influence biochemical functions. The most widespread way of their investigation by instrumental analytical techniques is to degrade them into the constituent disaccharide building blocks, followed by capillary electrophoresis or high-performance liquid chromatography (HPLC) separation. The analysis of GAG disaccharides with varying sulfation degrees poses a real challenge both from chromatographic and mass spectrometric (MS) points of view. This necessitates the constant improvement of their analytical methodology. In this chapter, an optimized workflow will be discussed for the sample preparation and subsequent HPLC-MS characterization of tissue-derived chondroitin sulfate and heparan sulfate.

Evaluation of Immunohistochemical Expression of Heparanase in Helicobacter pylori-Associated Chronic Gastritis.

Chronic gastritis (CG) is a very common disease. More than half of the worldwide population suffers from symptoms of CG. This disease has received great attention since the discovery of H. pylori as the most important cause of CG. Symptoms experienced by patients with CG are attributed to H. pylori-induced inflammatory reactions. Heparanase (HPSE) is a mammalian β-endoglucoronidase. In inflammation; HPSE degrades and remodels the extracellular matrix's heparan sulfate polysaccharide chains liberating heparan sulfate-bound cytokines and chemokines, HPSE also facilitates movement of inflammatory cells. This study aimed to detect the function of HPSE in CG by correlating levels of HPSE expression with histopathological features of CG, including H. pylori infection, acute and chronic inflammatory cells, mucosal atrophic and/or metaplastic features. Ninety-five upper endoscopic-guided gastric punch biopsies were enrolled in this study. From each specimen, formalin-fixed and paraffin-embedded tissue blocks were prepared. Tissue sections were stained by Hematoxylin and eosin, Giemsa, and anti-heparanase antibody. HPSE expression was statistically associated with H. pylori infection (P-value < .000), and intensity of chronic lymphocytic inflammatory infiltrate in the gastric mucosal tissues (P = .004). High levels of HPSE expression were also related to the presence of neutrophils in the gastric surface epithelium and lamina propria (P-value < .009). HPSE expression was upregulated in H. pylori-associated chronic gastritis. Thus, future therapeutic agents that could specifically inhibit HPSE enzyme activity, may aid in the reduction of sequelae of H. pylori infection.

Arabinogalactan protein polysaccharide chains are required for normal biogenesis of plasmodesmata.

Arabinogalactan proteins (AGPs) are a plant-specific family of extracellular proteoglycans characterized by large and complex galactose-rich polysaccharide chains. Functional elucidation of AGPs, however, has been hindered by the high degree of redundancy of AGP genes. To uncover as yet unexplored roles of AGPs in Arabidopsis, a mutant of Hyp O-galactosyltransferase (HPGT), a critical enzyme that catalyzes the common initial step of Hyp-linked arabinogalactan chain biosynthesis, was used. Here we show, using the hpgt1,2,3 triple mutant, that a reduction in functional AGPs leads to a stomatal patterning defect in which two or more stomata are clustered together. This defect is attributed to increased and dysregulated symplastic transport following changes in plasmodesmata structure, such that highly permeable complex branched plasmodesmata with cavities in branching parts increased in the mutant. We also found that the hpgt1,2,3 mutation causes a reduction of cellulose in the cell wall and accumulation of pectin, which controls cell wall porosity. Our results highlight the importance of AGPs in the correct biogenesis of plasmodesmata, possibly acting through the regulation of cell wall properties surrounding the plasmodesmata.

Effect of polyol plasticizers on properties and microstructure of soluble soybean polysaccharide edible films

Similar to most of the other films based on water-soluble natural polymers, pure soluble soybean polysaccharide (SSPS) films are rigid, fragile and brittle in nature, which make them difficult to process and limit their practical applications in food packaging. In this work, the effects of three commonly used polyol plasticizers, i. e. glycerol, sorbitol and polyethylene glycol 400 (PEG400), on properties of SSPS films were investigated. The SSPS films were prepared by solvent-casting and plasticized at three plasticizer concentrations (15 wt%, 30 wt% and 45 wt% based on SSPS). The addition of plasticizers significantly improved the ductility and water content of the SSPS films, thus overcoming the inherent brittleness of pure SSPS matrix. SSPS films plasticized with PEG400 exhibit the highest optical transparency, thermal stability and the lowest water solubility. Sorbitol-plasticized SSPS films show better tensile strength and water vapor barrier properties compared to other plasticized SSPS films. The use of glycerol as a plasticizer resulted in SSPS films with the highest elongation at break, moisture content and water solubility. X-ray diffraction, Fourier transform infrared spectroscopy and scanning electron microscopy analyses suggested that the changed interactions between adjacent polysaccharide chains with the addition of various polyol plasticizers can be responsible for the observed changes in the properties of the SSPS films.

Differential MMP-14 targeting by biglycan, decorin, fibromodulin, and lumican unraveled by in silico approach.

Small leucine-rich proteoglycans (SLRPs) are major regulators of extracellular matrix assembly and cell signaling. Lumican, a member of the SLRPs family, and its derived peptides were shown to possess antitumor activity by interacting directly with the catalytic domain of MMP-14 leading to the inhibition of its activity. The aim of the present report was to characterize by in silico three-dimensional (3D) modeling the structure and the dynamics of four SLRPs including their core protein and their specific polysaccharide chains to assess their capacity to bind to MMP-14 and to regulate its activity. Molecular docking experiments were performed to identify the specific amino acids of MMP-14 interacting with each of the four SLRPs. The inhibition of each SLRP (100 nM) on MMP-14 activity was measured and the constants of inhibition (Ki) were evaluated. The impact of the number of glycan chains, structures, and dynamics of lumican on the interaction with MMP-14 was assessed by molecular dynamics simulations. Molecular docking analysis showed that all SLRPs bind to MMP-14 through their concave face, but in different regions of the catalytic domain of MMP-14. Each SLRPs inhibited significantly the MMP-14 activity. Finally, molecular dynamics showed the role of glycan chains in interaction with MMP-14 and shielding effect of SLRPs. Altogether, the results demonstrated that each SLRP exhibited inhibition of MMP-14 activity. However, the differential targeting of MMP-14 by the SLRPs was shown to be related not only to the core protein conformation but also to the glycan chain structures and dynamics.

Characterization of Azospirillum brasilense Lipopolysaccharides

Azospirillum brasilense is a gram-negative, nitrogen-fixing bacterium that colonizes the rhizosphere of various types of grasses and cereals. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell membrane of gramnegative bacteria and mediate plant-bacteria interactions. Although the effects of LPS of pathogenic plant bacteria on the induction of plant defense mechanisms have been characterized, the role of LPS of beneficial rhizobacteria on plant growth is less clear. Therefore, a very important point is the study of the chemical, biological, and functional activities of A. brasilense LPS, which was the aim of this work. Methods. A. brasilense LPSs were isolated from dry bacterial mass by the phenol-water method. The carbohydrates were analyzed by the Dubois method, nucleic acids — by Spirin, protein content — by Lowry and 2-keto-3-deoxyoctonic acid (KDO) — by Osborn. Pyrogenicity of LPS was tested observing the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromatomass spectrometry system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. LPS of 3 strains of A. brasilense were isolated from dry bacterial mass and purifi ed from nucleic acids by ultracentrifugation. The purified LPSs were characterized by different relative yields from 2.44% to 4.75%, which is slightly higher than other strains of the A. brasilense (1—3%). The studied preparations were characterized by a rather high content of carbohydrates from 50.1% to 72.1%. All LPS contained up to 0.17% KDO, which is a specific component of the LPS of gram-negative bacteria. Analysis of the monosaccharide composition indicates that the LPSs of the studied A. brasilense strains turned out to be heterogeneous. At the same time, such monosaccharides as mannose, galactose, glucose, and heptose were recorded in the LPS of all tested strains. The study of the fatty acid composition of LPS showed the presence of fatty acids containing from 14 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids and their cis isomers were found. In the investigated LPS, the dominant fatty acids were 16:0, 18:1, 14:0(3-OH), and 16:0(3-OH), which coincides with the literature data. The research of the pyrogenic effect of LPS of A. brasilense studied strains showed that LPS solutions are apyrogenic. The double immunodiffusion reaction in Ouchterlon agar showed that all tested LPS in homologous systems exhibited ancultitigenic activity. Serological cross-reactions can be used as an approach in classifying different bacteria. Thus, we found that antisera to A. brasilense 18-2 and 61 react with all LPSs of the studied strains, which may indicate the presence of common antigenic determinants in them and that these strains belong to the same serogroup. The electrophoretic distribution data indicate that A. brasilense produces S-forms of LPS which differ in the length of O-specifi c polysaccharide chains. Conclusions. For the first time, LPS were isolated from cells of A. brasilense 10/1, 18-2 and 61. A characteristic feature of these LPS is their heterogeneity in monosaccharide and fatty acid composition, all of them were apyrogenic. The results obtained during biological-functional studies of three strains of A. brasilense LPS contribute to the biological characteristics of this species.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1–16 mol % smooth LPS (corresponding to 16–79 wt %), while vesicles with inner leaflet LPS reach coverages of 2–7 mol % smooth LPS (28–60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Water-Based Synthesis of Novel Hybrid Material of Sulfated Polysaccharide Through Radical Polymerization Process

ABSTRACT Water-soluble “sulfated polysaccharides of Chaetomorpha antennina” (CMsps) seaweed were isolated and fractionated (charged & neutral) using column chromatography followed by modification with acrylamide (AAm) to get novel hybrid materials. This polysaccharide comprises 6.356 polydispersity which ascribes to their branching behavior. The crude polysaccharides consist of ribose, arabinose, xylose, and galactose, carbohydrates units. The peak that appeared in the FT-IR spectrum at 1250 cm−1 confirms the sulfated nature of polysaccharides. The charged polysaccharides, pronounced “control CMsps” before the modification, were modified with AAm under microwave irradiation and characterized to investigate potential grafting. FT-IR, SEM, C13NMR, TGA, XRD, CD, and optical rotation were applied for control CMsps and CMsps-g-PAAm (grafted product). The FT-IR and SEM tools have significant capability to prove occurring successful potential grating with fluctuating peaks and morphology, respectively. XRD graphs for materials “control CMsps and CMsps-g-PAAm” reaffirm the successful grafting, leading to symmetrical molecular construction. TGA graphs support the exclusive thermal stability of CMsps-g-PAAm rather than control CMsps. The varying peak/trough ratio was evaluated by the CD spectrum, which identified the reversal change in the chirotopical profile with the insertion of polyacrylamide (PAAm) in polysaccharide chains. The materials, control CMsps and CMsps-g-PAAm were capable to develop hydrogel hence it can be exploited as a promising rheological modifier for food industry applications. The materials can also be useful in the biomedical field as demulcent drug formulation, dental impression product, and drug delivery for local therapy.

Boronate Affinity-Amplified Electrochemical Aptasensing of Lipopolysaccharide.

As lipopolysaccharide (LPS) is closely associated with sepsis and other life-threatening conditions, the point-of-care (POC) detection of LPS is of significant importance to human health. In this work, we illustrate an electrochemical aptasensor for the POC detection of low-abundance LPS by utilizing boronate affinity (BA) as a simple, efficient, and cost-effective amplification strategy. Briefly, the BA-amplified electrochemical aptasensing of LPS involves the tethering of the aptamer receptors and the BA-mediated direct decoration of LPS with redox signal tags. As the polysaccharide chain of LPS contains hundreds of cis-diol sites, the covalent crosslinking between the phenylboronic acid group and cis-diol sites can be harnessed for the site-specific decoration of each LPS with hundreds of redox signal tags, thereby enabling amplified detection. As it involves only a single-step operation (∼15 min), the BA-mediated signal amplification holds the significant advantages of unrivaled simplicity, rapidness, and cost-effectiveness over the conventional nanomaterial- and enzyme-based strategies. The BA-amplified electrochemical aptasensor has been successfully applied to specifically detect LPS within 45 min, with a detection limit of 0.34 pg/mL. Moreover, the clinical utility has been validated based on LPS detection in complex serum samples. As a proof of concept, a portable device has been developed to showcase the potential applicability of the BA-amplified electrochemical LPS aptasensor in the POC testing. In view of its simplicity, rapidness, and cost-effectiveness, the BA-amplified electrochemical LPS aptasensor holds broad application prospects in the POC testing.

Conjugation Mechanism for Pneumococcal Glycoconjugate Vaccines: Classic and Emerging Methods.

Licensed glycoconjugate vaccines are generally prepared using native or sized polysaccharides coupled to a carrier protein through random linkages along the polysaccharide chain. These polysaccharides must be chemically modified before covalent linking to a carrier protein in order to obtain a more defined polysaccharide structure that leads to a more rational design and safer vaccines. There are classic and new methods for site-selective glycopolysaccharide conjugation, either chemical or enzymatic modification of the polysaccharide length or of specific amino acid residues of the protein carrier. Here, we discuss the state of the art and the advancement of conjugation of S. pneumoniae glycoconjugate vaccines based on pneumococcal capsular polysaccharides to improve existing vaccines.

Chemoenzymatic synthesis of sulfur-linked sugar polymers as heparanase inhibitors

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond (‘S-link’) between sugar residues in place of a natural ‘O-linked’ bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions.