Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

Con A lectin binding by synthetic bivalent arabinomannan tri- and pentasaccharides reveals connectivity-dependent functional valencies.

Lectin Con A, with specificity to interact with α-d-mannopyranoside, achieves tight binding affinity with the aid of optimal multivalent ligand valencies, distances and orientations between the ligands. A series of synthetic arabinomannans, possessing arabinan core and mannan at the non-reducing ends, is studied to assess the above constraints involved with lectin binding in this report. Trisaccharides, with (1 â†’ 2)(1 â†’ 3), (1 â†’ 2)(1 â†’ 5) and (1 â†’ 3)(1 â†’ 5) glycosidic bond connectivities, and a pentasaccharide with mannopyranosides at the non-reducing ends are synthesized. The binding affinities of the mannose bivalent ligands are studied with tetrameric Con A lectin by isothermal titration calorimetry (ITC). Among the derivatives, trisaccharide with (1 â†’ 2)(1 â†’ 3) glycosidic bond connectivity and the pentasaccharide undergo lectin interaction, clearly fulfilling the bivalent structural and functional valencies. Remaining oligosaccharides exhibit only a functional monovalency, defying the bivalent structural valency. The trisaccharide fulfilling the structural and functional valencies represent the smallest bivalent ligand, undergoing the lectin interaction in a trans-mode.

Selective interactions of Co2+-Ca2+-concanavalin A with high mannose N-glycans.

Concanavalin A (ConA) has an intrinsic binding affinity to carbohydrates. Here, we obtained Co2+-Ca2+-ConA (2.83 Å, PDB: 8I7Q) via X-ray crystallography by substituting native ConA (Mn2+-Ca2+); it has binding selectivity for high-mannose N-glycan similar to native ConA. Our findings may thus inform antiviral reagent design.

Elucidating Structures of Protein Complexes by Collision-Induced Dissociation at Elevated Gas Pressures.

Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes. We show that CID of the homotetrameric protein complexes streptavidin (53 kDa), neutravidin (60 kDa), and concanavalin A (110 kDa) provides access to all subunits of the investigated protein complexes, including structurally informative dimers. We report on an "atypical" dissociation pathway, which for concanavalin A proceeds via symmetric partitioning of the precursor charges and produces dimers with the same charge states that were previously reported from surface induced dissociation. Our data suggest a correlation between the formation of subunits by CID in tandem-TIMS/MS, their binding strengths in the native tetramer structures, and the applied activation voltage. Ion mobility spectra of in situ-generated subunits reveal a marked structural heterogeneity inconsistent with annealing into their most stable gas phase structures. Structural transitions are observed for in situ-generated subunits that resemble the transitions reported from collision-induced unfolding of natively folded proteins. These observations indicate that some aspects of the native precursor structure is preserved in the subunits generated from disassembly of the precursor complex. We rationalize our observations by an approximately 100-fold shorter activation time scale in comparison to traditional CID in a collision cell. Finally, the approach discussed here to conduct CID at elevated pressures appears generally applicable also for other types of tandem-ion mobility spectrometers.

Targeted delivery of a short antimicrobial peptide (CM11) against Helicobacter pylori gastric infection using concanavalin A-coated chitosan nanoparticles.

Helicobacter pylori is the cause of most cases of stomach ulcers and also causes some digestive cancers. The emergence and spread of antibiotic-resistant strains of H. pylori is one of the most important challenges in the treatment of its infections. The present study aims to develop a concanavalin A (ConA) coated chitosan (CS) nanocarrier-based drug delivery for the targeted release of peptides to the site of H. pylori infection. Accordingly, chitosan was used as an encapsulating agent for CM11 peptide delivery by applying ionotropic gelation method. Con-A was used for coating CS nanoparticles to target H. pylori. The CS NPs and ConA-CS NPs were characterized by FTIR, dynamic light scattering (DLS), and scanning electron microscopy (SEM). The MIC of CM11-loaded ConA-CS NPs against H. pylori SS1 strain was analyzed in vitro. In order to evaluate the treatment efficiency in vivo, a gastric infection model of H. pylori SS1 strain was established in mice and histopathological studies and IL-1ß cytokine assay were performed. Based on the results, the size frequency for CS NPs and ConA-CS NPs was about 200 and 350 nm, respectively. The prepared CM11-loaded ConA-CS NPs exhibited antibacterial activity against H. pylori SS1 strain with a concentration of 32 µg/ml. The highest healing process was observed in synthesized CM11-loaded ConA-CS NPs treatments and a significant decrease in IL-1ß was observed. Our findings highlight the potential of chitosan nanoparticles as a drug delivery vehicle in the treatment of gastric infection model of H. pylori SS1 strain.

Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media.

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 µm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster.

Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 µg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 µg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 e 70 µg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. We believe that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage.

Binding Affinity of Concanavalin A to Native and Acid-Hydrolyzed Phytoglycogen Nanoparticles.

Phytoglycogen (PG) is a polysaccharide produced in the kernels of sweet corn as soft, highly branched, compact nanoparticles. Its tree-like or dendritic architecture, combined with a high-safety profile, makes PG nanoparticles attractive for use in biological applications, many of which rely on the association or binding of small biomolecules. We have developed a methodology to functionalize surface plasmon resonance (SPR) sensor surfaces with PG nanoparticles, and we demonstrate the utility of the PG-functionalized SPR sensor by measuring the binding affinity of the tetrameric concanavalin A (ConA) protein to both native PG nanoparticles and smaller, softer acid-hydrolyzed PG nanoparticles. We measure comparable values of the equilibrium association constant K for native and acid-hydrolyzed PG, with a slightly smaller value for the acid-hydrolyzed particles that we attribute to unfavorable lateral interactions between the tetrameric subunits of ConA due to the increase in surface curvature of the smaller acid-hydrolyzed PG particles. We also use infrared reflection-absorption spectroscopy (IRRAS) to show that ConA maintains a large fraction of its native conformation, and thus its bioactivity, upon binding to PG, representing an important step toward the realization of PG as a novel bioactive delivery vehicle.

Computer simulation of the Receptor-Ligand Interactions of Mannose Receptor CD206 in Comparison with the Lectin Concanavalin A Model.

Computer modeling of complexation of mono- and oligosaccharide ligands with the main (fourth) carbohydrate-binding domain of the mannose receptor CD206 (CRD4), as well as with the model receptor concanavalin A (ConA), was carried out for the first time, using methods of molecular dynamics and neural network analysis. ConA was shown to be a relevant model of CD206 (CRD4) due to similarity of the structural organization of the binding sites and high correlation of the values of free energies of complexation between the literature data and computer modeling (r > 0.9). Role of the main factors affecting affinity of the ligand-receptor interactions is discussed: the number and nature of carbohydrate residues, presence of Me-group in the O1 position, type of the glycoside bond in dimannose. Complexation of ConA and CD206 with ligands is shown to be energetically caused by electrostatic interactions (E) of the charged residues (Asn, Asp, Arg) with oxygen and hydrogen atoms in carbohydrates; contributions of hydrophobic and van der Waals components is lower. Possibility of the additional stabilization of complexes due to the CH-π stacking interactions of Tyr with the Man plane is discussed. The role of calcium and manganese ions in binding ligands has been studied. The values of free energies of complexation calculated in the course of molecular dynamics simulation correlate with experimental data (published for the model ConA): correlation coefficient r = 0.68. The Pafnucy neural network was trained based on the set of PDBbind2020 ligand-receptor complexes, which significantly increased accuracy of the energy predictions to r = 0.8 and 0.82 for CD206 and ConA receptors, respectively. A model of normalization of the complexation energy values for calculating the relevant values of ΔGbind, Kd is proposed. Based on the developed technique, values of the dissociation constants of a series of CD206 complexes with nine carbohydrate ligands of different structures were determined, which were not previously known. The obtained data open up possibilities for using computer modeling for the development of optimal drug carriers capable of active macrophage targeting, and also determine the limits of applicability of using ConA as a relevant model for studying parameters of the CD206 binding to various carbohydrate ligands.

Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility-Mass Spectrometry and Theory.

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge-dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: ß-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term "native-like" to describe results from native ion mobility-mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.

Concanavalin A: coordination diversity to xenobiotic metal ions and biological consequences.

The binding ability of lectins has gained attention owing to the carbohydrate-specific interactions of these proteins. Such interactions can be applied to diverse fields of biotechnology, including the detection, isolation, and concentration of biological target molecules. The physiological aspects of the lectin concanavalin A (ConA) have been intensively studied through structural and functional investigations. X-ray crystallography studies have proven that ConA has two ß-sheets and a short α-helix and that it exists in the form of a metalloprotein containing Mn2+ and Ca2+. These heterometals are coordinated with side chains located in a metal-coordinated domain (MCD), and they affect the structural environment in the carbohydrate-binding domain (CBD), which interacts with carbohydrates through hydrogen bonds. Recent studies have shown that ConA can regulate biophysical interactions with glycoproteins in virus envelopes because it specifically interacts with diverse polysaccharides through its CBD (Tyr, Asn, Asp, and Arg residues positioned next to the MCD). Owing to their protein-protein interaction abilities, ConA can form diverse self-assembled complexes including monomers, dimers, trimers, and tetramers, thus affording unique results in different applications. In this regard, herein, we present a review of the structural modifications in ConA through metal-ion coordination and their effect on complex formation. In recent approaches, ConA has been applied for viral protein detection, on the basis of the interactions of ConA. These aspects indicate that lectins should be thoroughly investigated with respect to their biophysical interactions, for avoiding unexpected changes in their interaction abilities.

Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag.

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

Surface Density of Ligands Controls In-Plane and Aggregative Modes of Multivalent Glycovesicle-Lectin Recognitions.

Glycovesicles are ideal tools to delineate finer mechanisms of the interactions at the biological cell membranes. Multivalency forms the basis which, in turn, should surpass more than one mechanism in order to maintain multiple roles that the ligand-lectin interactions encounter. Ligand densities hold a prime control to attenuate the interactions. In the present study, mannose trisaccharide interacting with a cognate receptor, namely, Con A, is assessed at the vesicle surface. Synthetic (1→3)(1→6)-branched mannose trisaccharides tethered with a diacetylene monomer and glycovesicles of varying sugar densities were prepared. The polydiacetylene vesicles were prepared by maintaining uniform lipid concentrations. The interactions of the glycovesicles with the lectin were probed through dynamic light scattering and UV-Vis spectroscopy techniques. Binding efficacies were assessed by surface plasmon resonance. Aggregative and in-plane modes of interactions show ligand-density dependence at the vesicle surface. Vesicles with sparsely populated ligands engage lectin in an aggregative mode (trans-), leading to a cross-linked complex formation. Whereas glycovesicles embedded with dense ligands engage lectin interaction in an in-plane mode intramolecularly (cis-). Sub-nanomolar dissociation constants govern the intramolecular interaction occurring within the plane of the vesicle, and are more efficacious than the aggregative intermolecular interactions.

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase.

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Monose-modified organic electrochemical transistors for cell surface glycan analysis via competitive recognition to enzyme-labeled lectin.

A competitive strategy for glycan determination on cell surface with organic electrochemical transistors (OECTs) has been developed. The carboxylic multi-wall carbon nanotubes were firstly immobilized on the gate interface to cross-link the specific monose with adipic dihydrazide as the linker, which could then competitively recognize horseradish peroxidase (HRP)-labeled lectin with the target monose on the cell surface. The HRP captured on the gate interface through the affinity of lectin to monose finally catalyzed the reduction of hydrogen peroxide to produce the output current signal for detection of cell surface monose under the optimal gate voltage of 0.9 V. Using mannose and galactose groups as the target models, HRP-labeled concanavalin A and peanut agglutinin were used to competitively recognize these groups on both cell surface and gate interface, respectively. The amounts of mannose and galactose on HeLa cells were measured to be 3.41 × 108 and 2.92 × 108 molecules per cell, respectively. The changes of the mannose and galactose expressions upon external stimulation were also observed with the proposed biosensors, which showed consistent results with flow cytometric analysis, indicating that the OECT-based biosensor is suitable for analysis of different glycan expressions on cell surface. Graphical abstract.

Redefining Protein Interfaces within Protein Single Crystals with DNA.

Proteins are exquisite nanoscale building blocks: molecularly pure, chemically addressable, and inherently selective for their evolved function. The organization of proteins into single crystals with high positional, orientational, and translational order results in materials where the location of every atom can be known. However, controlling the organization of proteins is challenging due to the myriad interactions that define protein interfaces within native single crystals. Recently, we discovered that introducing a single DNA-DNA interaction between protein surfaces leads to changes in the packing of proteins within single crystals and the protein-protein interactions (PPIs) that arise. However, modifying specific PPIs to effect deliberate changes to protein packing is an unmet challenge. In this work, we hypothesized that disrupting and replacing a highly conserved PPI with a DNA-DNA interaction would enable protein packing to be modulated by exploiting the programmability of the introduced oligonucleotides. Using concanavalin A (ConA) as a model protein, we circumvent potentially deleterious mutagenesis and exploit the selective binding of ConA toward mannose to noncovalently attach DNA to the protein surface. We show that DNA association eliminates the major PPI responsible for crystallization of native ConA, thereby allowing subtle changes to DNA design (length, complementarity, and attachment position) to program distinct changes to ConA packing, including the realization of three novel crystal structures and the deliberate expansion of ConA packing along a single crystallographic axis. These findings significantly enhance our understanding of how DNA can supersede native PPIs to program protein packing within ordered materials.

CASB: a concanavalin A-based sample barcoding strategy for single-cell sequencing.

Sample multiplexing facilitates single-cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost-effective strategies are rather limited. Here, we reported a concanavalin A-based sample barcoding strategy (CASB), which could be followed by both single-cell mRNA and ATAC (assay for transposase-accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA-seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound-specific heterogeneous response; 2) CASB together with both snATAC-seq and scRNA-seq to illustrate the IFN-γ-mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN-γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.

Dectin-2-mediated initiation of immune responses caused by influenza virus hemagglutinin.

Antigen-presenting cells express pattern recognition receptors (PRRs), which sense pathogen-associated molecular patterns from microorganisms and lead to the induction of inflammatory responses. C-type lectin receptors (CLRs), the representative PRRs, bind to microbial polysaccharides, among which Dectin-2 and Mincle recognize mannose-containing polysaccharides. Because influenza virus (IFV) hemagglutinin (HA) is rich in mannose polysaccharides, Dectin-2 or Mincle may contribute to the recognition of HA. In this study, we addressed the possible involvement of Dectin-2 and Mincle in the viral recognition and the initiation of cytokine production. Interleukin (IL)-12p40 and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) upon stimulation with HA was significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice whereas there was no difference between WT mice and Mincle KO mice. BM-DCs that were treated with Syk inhibitor resulted in a significant reduction of cytokine production upon stimulation with HA. The treatment of BM-DCs with methyl-α-D-mannopyranoside (ManP) also led to a significant reduction in cytokine production by BM-DCs that were stimulated with HA, except for the A/H1N1pdm09 subtype. IL-12p40 and IL-6 synthesis by BM-DCs was completely diminished upon stimulation with HA treated with concanavalin A (ConA)-bound sepharose beads. Finally, GFP expression was detected in reporter cells that were transfected with the Dectin-2 gene, but not with the Mincle gene, when stimulated with HA derived from the A/H3N2 subtype. These data suggested that Dectin-2 may be a key molecule as the sensor for IFV to initiate the immune response and regulate the pathogenesis of IFV infection.

Enhanced Concanavalin†A Binding to Preorganized Mannose Nanoarrays in Glycodendrimersomes Revealed Multivalent Interactions.

The effect of the two-dimensional glycan display on glycan-lectin recognition remains poorly understood despite the importance of these interactions in a plethora of cellular processes, in (patho)physiology, as well as its potential for advanced therapeutics. Faced with this challenge we utilized glycodendrimersomes, a type of synthetic vesicles whose membrane mimics the surface of a cell and offers a means to probe the carbohydrate biological activity. These single-component vesicles were formed by the self-assembly of sequence-defined mannose-Janus dendrimers, which serve as surrogates for glycolipids. Using atomic force microscopy and molecular modeling we demonstrated that even mannose, a monosaccharide, was capable of organizing the sugar moieties into periodic nanoarrays without the need of the formation of liquid-ordered phases as assumed necessary for rafts. Kinetics studies of Concanavalin A binding revealed that those nanoarrays resulted in a new effective ligand yielding a ten-fold increase in the kinetic and thermodynamic constant of association.

Tracking of Glycans Structure and Metallomics Profiles in BRAF Mutated Melanoma Cells Treated with Vemurafenib.

Nearly half of patients with advanced and metastatic melanomas harbor a BRAF mutation. Vemurafenib (VEM), a BRAF inhibitor, is used to treat such patients, however, responses to VEM are very short-lived due to intrinsic, adaptive and/or acquired resistance. In this context, we present the action of the B-Raf serine-threonine protein kinase inhibitor (vemurafenib) on the glycans structure and metallomics profiles in melanoma cells without (MeWo) and with (G-361) BRAF mutations. The studies were performed using α1-acid glycoprotein (AGP), a well-known acute-phase protein, and concanavalin A (Con A), which served as the model receptor. The detection of changes in the structure of glycans can be successfully carried out based on the frequency shifts and the charge transfer resistance after interaction of AGP with Con A in different VEM treatments using QCM-D and EIS measurements. These changes were also proved based on the cell ultrastructure examined by TEM and SEM. The LA-ICP-MS studies provided details on the metallomics profile in melanoma cells treated with and without VEM. The studies evidence that vemurafenib modifies the glycans structures and metallomics profile in melanoma cells harboring BRAF mutation that can be further implied in the resistance phenomenon. Therefore, our data opens a new avenue for further studies in the short-term addressing novel targets that hopefully can be used to improve the therapeutic regiment in advanced melanoma patients. The innovating potential of this study is fully credible and has a real impact on the global patient society suffering from advanced and metastatic melanomas.