Surface-Controlled Sialoside-Based Biosensing of Viral and Bacterial Neuraminidases.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein-electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

Sulfation of Heparan and Chondroitin Sulfate Ligands Enables Cell-Specific Homing of Nanoprobes.

Demystifying the sulfation code of glycosaminoglycans (GAGs) to induce precise homing of nanoparticles in tumor cells or neurons influences the development of a potential drug- or gene-delivery system. However, GAGs, particularly heparan sulfate (HS) and chondroitin sulfate (CS), are structurally highly heterogeneous, and synthesizing well-defined HS/CS composed nanoparticles is challenging. Here, we decipher how specific sulfation patterns on HS and CS regulate receptor-mediated homing of nanoprobes in primary and secondary cells. We discovered that aggressive cancer cells such as MDA-MB-231 displayed a strong uptake of GAG-nanoprobes compared to mild or moderately aggressive cancer cells. However, there was no selectivity towards the GAG sequences, thus indicating the presence of more than one form of receptor-mediated uptake. However, U87 cells, olfactory bulb, and hippocampal primary neurons showed selective or preferential uptake of CS-E-coated nanoprobes compared to other GAG-nanoprobes. Furthermore, mechanistic studies revealed that the 4,6-O-disulfated-CS nanoprobe used the CD44 and caveolin-dependent endocytosis pathway for uptake. These results could lead to new opportunities to use GAG nanoprobes in nanomedicine.

Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques.

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells.

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals. This parasite is an obligate intracellular pathogen and actively invades susceptible host cells, a process which is mediated by specific receptor-ligand interactions. Here, we have identified an unnatural 2,4-disulfated d-glucuronic acid (Di-S-GlcA), a hexuronic acid composed of heparin/heparan sulfate, as a potential carbohydrate ligand that can selectively bind to T. gondii parasites. More importantly, the gelatin conjugated Di-S-GlcA multivalent probe displayed strong inhibition of parasite entry into host cells. These results open perspective for the future use of Di-S-GlcA epitopes in biomedical applications against toxoplasmosis.

Microarray Analysis of Oligosaccharide-Mediated Multivalent Carbohydrate-Protein Interactions and Their Heterogeneity.

Carbohydrate-protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long-term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand-mediated interactions, we constructed a combinatorial library of mannose and galactose homo- and hetero-glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences.

Immobilization of multivalent glycoprobes on gold surfaces for sensing proteins and macrophages.

The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-ß-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using ß-cyclodextrin (ß-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-ß-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products.

Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells.

A host-guest interaction between Ru(ii)-complexes and sugar-capped ß-cyclodextrin was employed to synthesize metalloglycodendrimers. These glycodendrimers demonstrated selective carbohydrate-protein interactions and controlled the delivery of the Ru(ii) complexes into cancer cells, which may facilitate cell-specific apoptosis. Lectin binding assay revealed micromolar range IC50 values with different plant lectins. Cell viability assay and confocal imaging studies of Ru(ii) complexes exhibited cytotoxic activities in cancer cells compared to normal cells with IC50 values close to other literature Ru(ii) complexes. The cell death inducer was found to accumulate favorably to the endoplasmic reticulum (ER) and induced ER stress in cells. The upregulation of CHOP, caspase-3 and caspase-12 disturbed the ER morphology initiating the apoptosis pathway.

Screening of Neu5Acα(2-6)gal isomer preferences of siglecs with a sialic acid microarray.

Sialic acids (Sias) are important terminal sugars on cell surfaces involved in a wide range of protein-carbohydrate interactions. Hence, agents modulating sias-mediated protein interactions are promising inhibitors or vaccine candidates. Here, we report the synthesis of Neu5Acα(2-6)Gal structural analogs and their binding to a series of siglecs. The results showed distinct binding patterns with conserved siglecs (hCD22 and mCD22) compared to rapid evolving siglecs (Siglecs -3 & -10).

Correction: Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells.

Correction for 'Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells' by Harikrishna Bavireddi, et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob01546h.

Sialic Acid Hydroxamate: A Potential Antioxidant and Inhibitor of Metal-Induced ß-Amyloid Aggregates.

Current methods for Alzheimer's treatment require a three-component system: metal chelators, antioxidants, and amyloid ß (Aß)-peptide-binding scaffolds. We report sialic acid (Sia) hydroxamate as a potential radical scavenger and metal chelator to inhibit Aß aggregation. A cell viability assay revealed that Sia hydroxamate can protect HeLa and glioblastoma (LN229) cells from oxidative damage induced by the Fenton reaction. Sedimentation and turbidity assays showed profound protection of neuroblastoma SH-SY5Y cells from metal-induced Aß aggregation and neural toxicity.

Molecular recognition and proteoglycan mimic arrangement: modulating cisplatin toxicity.

We have demonstrated that cisplatin (CP), an anticancer drug, showed a preference for binding the sulfated-L-iduronic acid (S-L-IdoA) unit over the sulfated-D-glucuronic acid unit of heparan sulfate. The multivalency of S-L-IdoA, such as in the proteoglycan mimic, resulted in distinct modes of cell-surface engineering in normal and cancer cells, with these disparities having a significant impact on CP-mediated toxicity.

Amphiphilic Heparinoids as Potent Antiviral Agents against SARS-CoV-2.

Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.

Toxoplasma gondii secretory proteins bind to sulfated heparin structures.

Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most widespread infections in humans and animals, and is a major opportunistic pathogen in immunocompromised patients. Toxoplasma gondii is unique as it can invade virtually any nucleated cell, although the mechanisms are not completely understood. Parasite attachment to the host cell is a prerequisite for reorientation and penetration and likely requires the recognition of molecules at the host cell surface. It has been reported that the affinity of tachyzoites, the invasive form of T. gondii, for host cells can be inhibited by a variety of soluble-sulfated glycosaminoglycans (GAGs), such as heparan sulfate. Using heparin-functionalized zeolites in the absence of host cells, we visualized heparin-binding sites on the surface of tachyzoites by confocal and atomic force microscopy. Furthermore, we report that protein components of the parasite rhoptry, dense granule and surface bind GAGs. In particular, the proteins ROP2 and ROP4 from the rhoptry, GRA2 from the dense granules and the surface protein SAG1 were found to bind heparin. The binding specificities and affinities of individual parasite proteins for natural heparin and heparin oligosaccharides were determined by a combination of heparin oligosaccharide microarrays and surface plasmon resonance. Our results suggest that interactions between sulfated GAGs and parasite surface antigens contribute to T. gondii attachment to host cell surfaces as well as initiating the invasion process, while rhoptries and dense granule organelles may play an important role during the establishment of the infection and during the life of the parasite inside the parasitophorous vacuole.

Quantum dot nanometal surface energy transfer based biosensing of sialic acid compositions and linkages in biological samples.

Current methods for analyzing sialic acid diversity in modifications and linkages require multistep processing, derivatization, and chromatographic analyses. We here report a single-step optical method for identification and quantification of different compositions of sialoglycans on glycoproteins and in serum. This was achieved by measuring and quantifying nanometal surface energy transfer (NSET) signals between quantum dots and gold nanoparticles bound to specific sialic acid binding proteins (SBPs) and sialic acid moieties, respectively. The biosensing process is based on the NSET turn-on by external sialic acid species that compete for binding to the SBPs. Selectivity of the biosensor toward sialoglycans can be designed to detect the total amount, glycosylation linkages (α2-6 vs α2-3), and modifications (9-O-acetyl and N-glycolyl groups) in the samples. This nanobiosensor is a prototype expected to achieve limits of the detection down to the micromolar range for high-throughput quantification and analysis of different compositions of sialoglycans present in biological or biomedical samples.

The Impact of Nanomaterial Morphology on Modulation of Carbohydrate-Protein Interactions.

Carbohydrate-protein interactions (CPIs) play a crucial role in the regulation of various physiological and pathological processes within living systems. However, these interactions are typically weak, prompting the development of multivalent probes, including nanoparticles and polymer scaffolds, to enhance the avidity of CPIs. Additionally, the morphologies of glyco-nanostructures can significantly impact protein binding, bacterial adhesion, cellular internalization, and immune responses. In this review, we have examined the advancements in glyco-nanostructures of different shapes that modulate CPIs. We specifically emphasize glyco-nanostructures constructed from small-molecule amphiphilic carbohydrates, block copolymers, metal-based nanoparticles, and carbon-based materials, highlighting their potential applications in glycobiology.

Synergestic interplay of uronic acid and sulfation composition of heparan sulfate on molecular recognition to activity.

Heparan sulfate (HS) is ubiquitous polysaccharide on the surface of all mammalian cells and extracellular matrices. The incredible structural complexity of HS arises from its sulfation patterns and disaccharide compositions, which orchestrate a wide range of biological activities. Researchers have developed elegant synthetic methods to obtain well-defined HS oligosaccharides to understand the structure-activity relationship. These studies revealed that specific sulfation codes and uronic acid variants could synergistically modulate HS-protein interactions (HSPI). Additionally, the conformational flexibility of l-Iduronic acid, a uronic acid unit has emerged as a critical factor in fine-tuning the microenvironment to modulate HSPI. This review delineates how uronic acid composition in HS modulates protein binding affinity, selectivity, and biological activity. Finally, the significance of sulfated homo-oligo uronic acid as heparin mimics in drug development is also discussed.

Fluorescent glyco-gold nanocluster induced EGFR mediated targeting of cancer cells.

A lot of attention has been focused on the functionalization of carbohydrate ligands on specific sizes and shapes of gold nanoparticles (AuNPs), where ultrasmall fluorescent AuNPs have not been well explored for direct imaging. Herein, we have engineered fluorescent gold nanoclusters with sulfated oligo-iduronic acid ligands (I34), which strongly bind to the HB-EGF receptor over FGF2, and regulate EGF receptor-mediated cancer cell homing in both two- and three-dimensional (2D and 3D) cell culture systems. These results offer a new practical and direct imaging tool for carbohydrate research.

Biocatalysis versus Molecular Recognition in Sialoside-Selective Neuraminidase Biosensing.

Sialic acid recognition and hydrolysis are essential parts of cellular function and pathogen infectivity. Neuraminidases are enzymes that detach sialic acid from sialosides, and their inhibition is a prime target for viral infection treatment. The connectivity and type of sialic acid influence the recognition and hydrolysis activity of the many different neuraminidases. The common strategies to evaluate neuraminidase activity, recognition, and inhibition rely on extensive labeling and require a large amount of sialylated glycans. The above limitations make the effort of finding viral inhibitors extremely difficult. We used synthetic sialylated glycans and developed a label-free electrochemical method to show that sialoside structural features lead to selective neuraminidase biosensing. We compared Neu5Ac to Neu5Gc sialosides to evaluate the organism-dependent neuraminidase selectivity-sensitivity relationship. We demonstrated that the type of surface and the glycan monolayer density direct the response to either binding or enzymatic activity. We proved that while the hydrophobic glassy carbon surface increases the interaction with the enzyme hydrophobic interface, the negatively charged interface of the lipoic acid monolayer on gold repels the protein and enables biocatalysis. We showed that the sialoside monolayers can serve as tools to evaluate the inhibition of neuraminidases both by biocatalysis and molecular recognition.

Optimization of localized surface plasmon resonance transducers for studying carbohydrate-protein interactions.

Noble metal nanostructures supporting localized surface plasmons (SPs) have been widely applied to chemical and biological sensing. Changes in the refractive index near the nanostructures affect the SP extinction band, making localized surface plasmon resonance (LSPR) spectroscopy a convenient tool for studying biological interactions. Carbohydrate-protein interactions are of major importance in living organisms; their study is crucial for understanding of basic biological processes and for the construction of biosensors for diagnostics and drug development. Here LSPR transducers based on gold island films prepared by evaporation on glass and annealing were optimized for monitoring the specific interaction between Concanavalin A (Con A) and D-(+)-mannose. The sugar was modified with a PEG-thiol linker and immobilized on the Au islands. Sensing assays were performed under stationary and flow conditions, the latter providing kinetic parameters for protein binding and dissociation. Ellipsometry and Fourier transform-infrared (FT-IR) data, as well as scanning electron microscopy (SEM) imaging of fixated and stained samples, furnished independent evidence for the protein-sugar recognition. Enhanced response and visual detection of protein binding was demonstrated using Au nanoparticles stabilized with the linker-modified mannose molecules. Mannose-coated transducers display an excellent selectivity toward Con A in the presence of a large excess of bovine serum albumin (BSA).

Glyco-ß-cyclodextrin capped quantum dots: synthesis, cytotoxicity and optical detection of carbohydrate-protein interactions.

Highly fluorescent water soluble glyco-quantum dots were synthesized using a sonochemical procedure. The synthetic approach is based on specific host-guest interactions between ß-cyclodextrin (ß-CD) and trioctylphosphine oxide (TOPO) surfactant on quantum dots. The modified QDs were analyzed by a combination of FT-IR, (1)H-NOESY NMR spectroscopy and by TEM. The high sugar density on QDs resulted in selective colloidal aggregation with ConcanavalinA (ConA), Galanthus nivalis lectin (GNA) and Peanut agglutinin (PNA) lectins. Subsequently, in vitro studies indicated that ß-CD modification of QDs enabled good cell viability of human hepatocellular carcinoma cell line (HepG2) cells. Finally, flow cytometry and confocal imaging studies revealed that ßCDgal capped QDs undergo preferential binding with HepG2 cells. These results clearly demonstrate that ß-CD capped QDs could be a promising candidate for further carbohydrate-based biomedical applications.