The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.

• Keywords
• 2D materials, carbohydrate, graphene, sensor, wheat germ agglutinin,
• MeSH
• Graphite chemistry   MeSH
• Horseradish Peroxidase MeSH
• Protein Structure, Quaternary MeSH
• Lectins analysis   metabolism   MeSH
• Polysaccharides chemistry   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Graphite MeSH
• Horseradish Peroxidase MeSH
• Lectins MeSH
• Polysaccharides MeSH

Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.

• Keywords
• ADSC, HUVEC, carbohydrate, galectin, integrin,
• MeSH
• Cell Adhesion * MeSH
• Human Umbilical Vein Endothelial Cells cytology   physiology   MeSH
• Galectins metabolism   MeSH
• Integrins metabolism   MeSH
• Blood Proteins metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mesenchymal Stem Cells cytology   physiology   MeSH
• Cell-Matrix Junctions metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galectins MeSH
• Integrins MeSH
• Blood Proteins MeSH
• LGALS3 protein, human MeSH Browser

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.

• Keywords
• galectin-3, molecular modeling, site-directed mutagenesis, solvent, substrate specificity, transglycosidase, β-N-acetylhexosaminidase,
• MeSH
• Enzyme Activation MeSH
• beta-N-Acetylhexosaminidases chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Kinetics MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Polysaccharides biosynthesis   chemistry   pharmacology   MeSH
• Protein Engineering MeSH
• Hydrogen Bonding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta-N-Acetylhexosaminidases MeSH
• Polysaccharides MeSH

BACKGROUND: Galectin-3 (Gal-3) is a promising target in cancer therapy with a high therapeutic potential due to its abundant localization within the tumor tissue and its involvement in tumor development and proliferation. Potential clinical application of Gal-3-targeted inhibitors is often complicated by their insufficient selectivity or low biocompatibility. Nanomaterials based on N-(2-hydroxypropyl)methacrylamide (HPMA) nanocarrier are attractive for in vivo application due to their good water solubility and lack of toxicity and immunogenicity. Their conjugation with tailored carbohydrate ligands can yield specific glyconanomaterials applicable for targeting biomedicinally relevant lectins like Gal-3. RESULTS: In the present study we describe the synthesis and the structure-affinity relationship study of novel Gal-3-targeted glyconanomaterials, based on hydrophilic HPMA nanocarriers. HPMA nanocarriers decorated with varying amounts of Gal-3 specific epitope GalNAcβ1,4GlcNAc (LacdiNAc) were analyzed in a competitive ELISA-type assay and their binding kinetics was described by surface plasmon resonance. We showed the impact of various linker types and epitope distribution on the binding affinity to Gal-3. The synthesis of specific functionalized LacdiNAc epitopes was accomplished under the catalysis by mutant β-N-acetylhexosaminidases. The glycans were conjugated to statistic HPMA copolymer precursors through diverse linkers in a defined pattern and density using Cu(I)-catalyzed azide-alkyne cycloaddition. The resulting water-soluble and structurally flexible synthetic glyconanomaterials exhibited affinity to Gal-3 in low μM range. CONCLUSIONS: The results of this study reveal the relation between the linker structure, glycan distribution and the affinity of the glycopolymer nanomaterial to Gal-3. They pave the way to specific biomedicinal glyconanomaterials that target Gal-3 as a therapeutic goal in cancerogenesis and other disorders.

• Keywords
• Carbohydrate, ELISA, Galectin-3, Glyconanomaterial, HPMA copolymer, Surface plasmon resonance,
• MeSH
• Acrylamides chemistry   metabolism   MeSH
• Galectin 3 metabolism   MeSH
• Galectins MeSH
• Glycoconjugates chemistry   metabolism   MeSH
• Blood Proteins MeSH
• Drug Delivery Systems * MeSH
• Humans MeSH
• Nanostructures chemistry   MeSH
• Drug Carriers chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acrylamides MeSH
• Galectin 3 MeSH
• Galectins MeSH
• Glycoconjugates MeSH
• Blood Proteins MeSH
• LGALS3 protein, human MeSH Browser
• N-(2-hydroxypropyl)methacrylamide MeSH Browser
• Drug Carriers MeSH