Lectin Sugar Research Articles

Single-molecule imaging of glycan-lectin interactions on cells with Glyco-PAINT.

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions.

Crystal structures of peanut lectin in the presence of synthetic β-N- and β-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for β-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of β-galactoside and β-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable β-N- and β-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of β-N- and β-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Tag-Free Site-Specific BMP-2 Immobilization with Long-Acting Bioactivities via a Simple Sugar-Lectin Interaction.

The construction of a biomaterial matrix with biological properties is of great importance to developing functional materials for clinical use. However, the site-specific immobilization of growth factors to endow materials with bioactivities has been a challenge to date. Considering the wide existence of glycosylation in mammalian proteins or recombinant proteins, we establish a bioaffinity-based protein immobilization strategy (bioanchoring method) utilizing the native sugar-lectin interaction between concanavalin A (Con A) and the oligosaccharide chain on glycosylated bone morphogenetic protein-2 (GBMP-2). The interaction realizes the site-specific immobilization of GBMP-2 to a substrate modified with Con A while preserving its bioactivity in a sustained and highly efficient way, as evidenced by its enhanced ability to induce osteodifferentiation compared with that of the soluble GBMP-2. Moreover, the surface with Con A-bioanchored GBMP-2 can be reused to stimulate multiple batches of C2C12 cells to differentiate almost to the same degree. Even after 4 month storage at 4 °C in phosphate-buffered saline (PBS), the Con A-bioanchored GBMP-2 still maintains the bioactivity to stimulate the differentiation of C2C12 cells. Furthermore, the ectopic ossification test proves the in vivo bioactivity of bioanchored GBMP-2. Overall, our results demonstrate that the tag-free and site (i.e., sugar chain)-specific protein immobilization strategy represents a simple and generic alternative, which is promising to apply for other glycoprotein immobilization and application. It should be noted that although the lectin we utilized can only bind to d-mannose/d-glucose, the diversity of the lectin family assures that a specific lectin could be offered for other sugar types, thus expanding the applicable scope further.

A sugar-lectin rich interface between soft tissue and the stiff byssus of Atrina pectinata.

Maintaining durable adhesion between soft tissues and relatively hard implant materials is one of the most elusive technological difficulties in bionic devices due to contact damage between mechanically mismatched materials. Although there are many examples of coexistence of soft and hard tissues in living organisms, relatively little is known about the mechanisms used to overcome mechanical mismatches occurring at the interface between soft and hard tissues. Among the various creatures possessing mechanically mismatched biological tissues, Atrina pectinata is a good model system where the interface between stiff byssal threads and soft tissues is distributed all over an extended organ. In this study, we found a wide distribution of various types of carbohydrates and lectins at the mechanically mismatched interface of the byssus of Atrina using histological methods and proteomics. Reversible and robust interactions between the carbohydrate and lectins at the interface would play a major role in mitigating the contact damage at the Atrina interface. Based on these results, the adhesion between sugar and lectin would be useful to overcome a wide range of contact damage observed in research studies on bionic devices.

Lectin-Recognizable MOF Glyconanoparticles: Supramolecular Glycosylation of ZIF-8 Nanocrystals by Sugar-Based Surfactants.

A strategy toward the integration of highly functional microporous materials, such as metal-organic frameworks (MOFs), in composites via biochemical recognition interactions is presented. Postsynthetic modification of zeolitic-imidazolate framework-8 MOF nanocrystals with a maltose-exposing biocompatible surfactant (the so-called “Glyco-MOFs”) was performed to confer affinity toward lectin protein concanavalin A. The addition of small amounts of concanavalin A to the colloidal Glyco-MOF dispersion triggers the aggregation of these units into self-limited size supramolecular architectures directed by specific sugar–lectin binding interactions.

Guided Cell Attachment via Aligned Electrospinning of Glycopolymers.

The creation of biomaterials with aligned fibers offers broad applications in tissue regeneration, guiding cell organization and physiological cues, and providing appropriate mechanical properties for many biomedical applications. Herein, for the first time, highly aligned electrospun membranes are designed and developed using glycopolymers. The membranes retain the strong mechanical properties of polycaprolactone, and fiber alignment facilitates the creation of anisotropic mechanical properties, enabling failure stress to be manipulated by an order of magnitude relative to randomly ordered fibers. Biocompatibility and cell attachment in these materials are characterized using tenocytes as a cell model. Both random and aligned fiber glycopolymers show promising biocompatibility, but aligned glycopolymer fibers additionally offer patterning to guide cell organization. These materials potentially provide a novel platform for tissue regeneration studies, demonstrating that the sugar-lectin interaction can produce materials capable of managing cell guidance.

Graphene oxide-based NIR fluorescence probe with aggregation-induced emission property for lectins detection and liver cells targeting

Probing lectin–sugar interactions is crucial to understand important biological process as well as develop clinical diagnostics and drug for the disease. Effective tools that target these interactions are thus in great demand. In this paper, two new aggregation-induced emission (AIE)-based glycosyl probes (PT-Man and PT-Gal) have been developed for the detection of lectin–sugar interactions in the near-infrared region (NIR). PT-Man and PT-Gal show significant fluorescence enhancement upon addition of a selective lectin with a relatively low detection limit. The fluorescence increment mechanism is ascribed to the aggregation of the AIE-based glycosyl probes after complexation with the selective lectin, which has been confirmed by the scanning electron microscope (SEM) measurement. Also, in order to further improve the sensitivity and selectivity of the probes, graphene oxide (GO) has been used as a platform to assemble with the two glycosyl probes. To our delight, the PT-Man@GO and PT-Gal@GO composites exhibit better sensitivity and selectivity for probing lectin–sugar interactions. In addition, due to the specific sugar-receptor interactions, the liver cancer cell line that over express the glycoprotein receptor can be specifically targeted by PT-Gal@GO.

Visual Detection of pH and Biomolecular Interactions at Micromolar Concentrations Aided by a Trivalent Diacetylene-Based Vesicle

A trivalent tris-(alkylenediynoic acid) is developed as a new monomer to prepare a poly(tris-(diacetylene)). Alkylenediynoic moieties, covalently linked to the gallic acid, lead to a high propensity for polymerization, resulting in blue poly(tris-(diacetylene)), within less than 20 s of UV irradiation as aq. vesicle solution and as thin film. The vesicle undergoes reversible thermochromism between room temperature and 95 °C. The corresponding chromism changes are blue-to-red-to-orange during heating and reverse on cooling. This reversible chromism also occurs to thin film between room temperature and 220 °C. The polymerized aq. vesicle exhibits pH-induced chromism in the alkaline range. In order to identify polymerization-induced chromatic transitions beneficial to a biosensing process, derivatization of the monomer with α-d-mannopyranoside moieties is conducted. Chromatic properties of sugar-derivatized tris-(alkylenediynoic acid) vesicle are established first. Poly(tris-(diacetylene)) vesicles, prepared from either pure or mixed monomer systems, exhibit blue phase, which do not undergo change in chromism when the sugar–lectin interaction is initiated. Whereas, when the monomer vesicle is prepared in the presence of the lectin and polymerized subsequently, chromism of the resulting poly(tris-(diacetylene)) solution is pink, as a result of ligand–lectin interaction. The chromism change can be conducted at ≈10 × 10−6 m each of both vesicle and the lectin.

Selective extraction of bioactive glycoprotein in neutral environment through Concanavalin A mediated template immobilization and dopamine surface imprinting

Inspired by the sugar–lectin interaction, Concanavalin A mediated glycoprotein pre-immobilization, combined with dopamine polymerization, is employed to fabricate a glycoprotein imprint that can work in physiological environments.

Stereoisomerism effect on sugar–lectin binding of self-assembled glyco-nanoparticles of linear and brush copolymers

Binding behavior of carbohydrate and protein is known to be crucial to the biological roles of sugars. Recently, we notice that, even to the same lectin, some reported binding results based on glycopolymers and/or self-assembled glyco-nanoparticles are different from those obtained on small molecular level. Some of such discrepancy could be associated with the different detection methods used. In this paper, by using self-assembled nanoparticles based on brush and linear glycopolymers, this stereoisomerism effect was evaluated systematically, both in solution and on solution-solid interface by different methods. All measurements led to the same conclusion that the stereoisomer of sugars determined the binding ability of glyco-nanoparticles with different lectins. The investigation also provided evidence on the different binding modes of nanoparticles of linear polymers and brush polymers.

Probing sugar–lectin recognitions in the near-infrared region using glyco-diketopyrrolopyrrole with aggregation-induced-emission

This study describes the construction of aggregation-induced-emission (AIE)-based glycosyl probes for the sensitive and selective detection of sugar–lectin interactions in the near-infrared (NIR) region. Mannosyl and galactosyl diketopyrrolopyrrole (DPP) derivatives were effectively synthesized by the Cu(I)-catalyzed azide-alkyne 1.3-dipolar cycloaddition reaction. We observed that these glycodyes had typical AIE behaviors in a semi-aqueous solution with a strong fluorescence (FL) emission in the NIR region. In a buffer solution, the glycosyl DPPs at the quenching state showed sharply increased FL upon addition of a selective lectin that recognizes the glycosyl moiety of the compounds with nanomolar limits of detection. In contrast, addition of unselective lectins, proteins and ions did not fluctuate the FL. Scanning electron microscopy analyses suggested that the FL generation could probably be a result of AIE of the glyco-DPPs upon complexation with lectins. These glyco-DPPs, to the best of our knowledge, represent the first fluorogenic AIE-based probes that can sense lectins in the NIR region, providing insights for the further extension towards low-background in vivo targeted imaging of tissues that express a lectin.

Protein crystalline frameworks with controllable interpenetration directed by dual supramolecular interactions.

Protein crystalline frameworks are attractive for biomimetic and nanotechnological studies because they could augment the useful functionalities of numerous proteins through dense packing and uniform orientation. However, their formation and precise structural control is challenging. Here we present novel protein crystalline frameworks with controllable interpenetration. The homotetrameric lectin concanavalin A is crosslinked by predetermined inducing ligands containing monosaccharide and rhodamine groups connected by an oligo(ethylene oxide) spacer. Two non-covalent interactions, that is, sugar-lectin binding and the dimerization of RhB, are responsible for the framework formation. The three-dimensional structure of the framework is fully characterized by X-ray crystallographic methods. For the first time, either interpenetrating or non-interpenetrating frameworks are obtained, and they are controlled by the spacer length of the inducing ligand. Further kinetics and mechanistic investigations reveal that, in the self-assembly process, the carbohydrate-protein binding occurs first, followed by RhB dimerization. This sequence favours rapid crystallization with a high yield when an excess amount of inducing ligand is used. In short, by using well-controlled dual non-covalent interactions, fast and versatile preparation of protein crystalline framework was achieved with high crystallization ratio of the proteins, which may shed light on protein crystallization in near future.

Isothermal calorimetric analysis of lectin-sugar interaction.

Isothermal titration calorimetry (ITC) is a powerful tool for analyzing lectin-glycan interactions because it can measure the binding affinity and thermodynamic properties such as ∆H and ΔS in a single experiment without any chemical modification or immobilization. Here we describe a method for preparing glycan and lectin solution to minimize the buffer mismatch, setting parameters, and performing experiments.

Comparison of Free-Solution and Surface-Immobilized Molecular Interactions Using a Single Platform

While it is generally accepted that surface immobilization affects the binding properties of proteins, it has been difficult to quantify these effects due to the lack of technology capable of making affinity measurements with species tethered and in free solution on a single platform. Further, quantifying the interaction of binding pairs with widely differing masses has also been challenging, particularly when it is desirable to tether the high molecular weight protein. Here we describe the use of backscattering interferometry (BSI) to quantify the binding affinity of mannose and glucose to concanavalin A (ConA), a 106 KDa homotetramer protein, in free solution using picomoles of the protein. Using the same platform, BSI, we then studied the effect on the binding constants of the ConA-carbohydrate interactions upon chemically immobilizing ConA on the sensor surface. By varying the distances (0, 7.17, and 20.35 nm) of the ConA tether and comparing these results to the free-solution measurements, it has been possible to quantify the effect that protein immobilization has on binding. Our results indicate that the apparent binding affinity of the sugar-lectin pair increases as the distance between ConA and the surface decreases. These observations could lend insight as to why the affinity values reported in the literature sometimes vary significantly from one measurement technique to another.

Mucin Multilayers Assembled through Sugar–Lectin Interactions

Multilayer films of biopolymers are attractive tools to exploit the extraordinary properties of certain biomacromolecules and introduce new functionalities to surfaces. Mucins, the gel-forming constituents of mucus, are versatile glycoproteins that have potential as new building blocks for biomaterial surface coatings. Multilayer films have mostly been assembled through the electrostatic pairing of polyelectrolytes, which results in limited pH and salt stability and screens charges otherwise available for useful payload binding. Here, we aim at assembling mucin multilayer films that differ from conventional paired polyelectrolyte assemblies to obtain highly stable and functional surface modifications. Using the lectin wheat germ agglutinin (WGA) to cross-link mucin-bound sugar residues, we show that (Mucin/WGA) films can grow into hydrated films and sustain exceptional resistance to extreme salt conditions and a large range of pH. Furthermore, we show that the addition of soluble N-acetyl-d-glucosamine can induce the controlled release of WGA from (Mucin/WGA) films. Last, we show that (Mucin/WGA) films can repeatedly incorporate and release a positively charged model cargo. The lubricating, hydration, barrier, and antimicrobial properties of mucins open multiple applicative perspectives for these highly stable mucin-based multilayer films.

The enzymatic lectin of field bean (Dolichos lablab): Salt assisted lectin–sugar interaction

Field bean seed contains a Gal/GalNAc lectin (DLL-II) that exhibits associated polyphenol oxidase (PPO) activity and does not bind to its sugar specific affinity matrix. The molecular basis for this lack of binding is not known. The DLL-II gene was therefore cloned and its sequence analyzed. A conserved aromatic residue in the sugar binding site required for a stacking interaction with the apolar backbone of Gal is replaced by His in DLL-II, which explains the lack of binding. However, specific sugar binding is achieved by including (NH₄)₂SO₄ in the buffer. Interestingly two other salts of the Hofmeister series, K₂HPO₄ and Na₂SO₄ also assist binding to immobilized galactose. In the presence of (NH₄)₂SO₄ the surface hydrophobicity of DLL-II and dissociation constant for 8-anilino 1-naphthalene sulfonic acid were enhanced three fold. This increased surface hydrophobicity in the presence of salt is probably the cause for assisted sugar binding in legume lectins that lack aromatic stacking interactions. Accordingly, two other lectins which lack the conserved aromatic residue show similar salt assisted binding. The salt concentrations required for Gal/GalNAc binding are not physiologically relevant in vivo, suggesting that the role of DLL-II per se in the seed is primarily that of a PPO purportedly for plant defense.

Label-free THz sensing of living body-related molecular binding using a metallic mesh

We have demonstrated label-free THz sensing of living body-related molecular binding using a thin metallic mesh and a polyvinylidene difluoride (PVDF) membrane. Metallic meshes in the THz region are designed for anomalous transmission phenomena derived from a resonant excitation of surface waves. Additionally, they are designed to have a sharp dip in transmittance. The metallic mesh is very sensitive to a change of the refractive index of materials attached to the metallic mesh. In this paper, we report sensing of interactions between lectin and sugar using this technique. We found that the dip frequency shift, transmittance attenuation of the dip frequency, and peak shift of the derivative spectrum of the phase shift depend on the bonding amount of lectin–sugar interactions. We also applied this technique to detect avidin–biotin interactions, leading to the detection of a small amount of biotin (0.17pg/mm2).

Layer-by-layer construction of protein architectures through avidin–biotin and lectin–sugar interactions for biosensor applications

In this review, the preparation and properties of protein architectures constructed by layer-by-layer (LbL) deposition through avidin-biotin and concanavalin A (Con A)-sugar interactions are discussed in relation to their use for optical and electrochemical biosensors. LbL films can be constructed through the alternate deposition of avidin and biotin-labeled enzymes on the surfaces of optical probes and electrodes. The enzymes retain their catalytic activity, resulting in the formation of optical and electrochemical biosensors. Alternatively, Con A can be used to construct enzyme-containing LbL films and microcapsules using sugar-labeled enzymes. Some enzymes such as glucose oxidase and horseradish peroxidase can be used for this purpose without labeling with sugar, because these enzymes contain intrinsic hydrocarbon chains on their molecular surfaces. The Con A/enzyme LbL architectures were successfully used to develop biosensors sensitive to specific substrates of the enzyme. In addition, Con A-based films can be used for the optical and electrochemical detection of sugars.

Evidence for the involvement of surface carbohydrates in the recognition of Haematococcus pluvialis by the parasitic blastoclad Paraphysoderma sedebokerensis

The unicellular green alga Haematococcus pluvialis (Chlorophyta, Volvocales) is currently the best commercial source of the natural red ketocarotenoid astaxanthin. Paraphysoderma sedebokerensis (Blastocladiomycota), a parasitic blastoclad that is specific for this microalga, was recently isolated and identified in our laboratory. In this study, we investigated the recognition process between the parasite and H. pluvialis. Obligatory requirements for recognition were identified as an ion concentration in the medium of 20 mM, the presence of calcium ions, and neutral to basic conditions; these requirements imply that a protein is involved in the process. In a search for potential lectin–sugar interactions as a major event in the recognition process, we screened for exposed glycosidic moieties on the cell wall of the alga and on the parasite zoospore surface. Competition experiments with the appropriate lectins and monosugars identified Ricinus communis agglutinin (RCA 120) as the lectin that recognizes Gal- N-acetyl- d-glucosamine, an oligosaccharide located on the host. We propose that an RCA 120-like lectin–sugar interaction mediates the highly specific interaction between the blastocladian parasite and its algal host.

Study of the biosensor based on platinum nanoparticles supported on carbon nanotubes and sugar–lectin biospecific interactions for the determination of glucose

Highly sensitive electrochemical platform based on Pt nanoparticles supported on carbon nanotubes (Pt nano-CNTs) and sugar–lectin biospecific interactions is developed for the direct electrochemistry of glucose oxidase (GOD). Firstly, Pt nano-CNTs nanocomposites were prepared in the presence of carbon nanotubes (CNTs), and then the mixture was cast on a glassy carbon electrode (GCE) using chitosan as a binder. Thereafter, concanavalin A (Con A) was adsorbed onto the precursor film by the electrostatic force between positively charged chitosan and the negatively charged Con A. Finally, the multilayers of Con A/GOD films were prepared based on biospecific affinity of Con A and GOD via layer-by-layer (LBL) self-assembly technique. The electrochemical behavior of the sensor was studied using cyclic voltammetry and chronoamperometry. The electrochemical parameters of GOD in the film were calculated with the results of the electron transfer coefficient ( α) and the apparent heterogeneous electron transfer rate constant ( k s) as 0.5 and 5.093 s −1, respectively. Experimental results show that the biosensor responded linearly to glucose in the range from 1.2 × 10 −6 to 2.0 × 10 −3 M, with a detection limit of 4.0 × 10 −7 M under optimized conditions.