Galectins, the glycan binding proteins, and their respective carbohydrate ligands represent a unique fundamental regulatory network modulating a plethora of biological processes. The advances in galectin-targeted therapy must be based on a deep understanding of the mechanism of ligand-protein recognition. Carbosilane dendrimers, the well-defined and finely tunable nanoscaffolds with low toxicity, are promising for multivalent carbohydrate ligand presentation to target galectin receptors. The study discloses a synthetic method for two types of lactose-functionalized carbosilane glycodendrimers (Lac-CS-DDMs). Furthermore, we report their outstanding, dendritic effect-driven affinity to tandem-type galectins, especially Gal-9. In the enzyme-linked immunosorbent assay, the affinity of the third-generation multivalent dendritic ligand bearing 32 lactose units to Gal-9 reached nanomolar values (IC50 = 970 nM), being a 1400-fold more effective inhibitor than monovalent lactose for this protein. This demonstrates a game-changing impact of multivalent presentation on the inhibitory effect of a ligand as simple as lactose. Moreover, using DLS hydrodynamic diameter measurements, we correlated the increased affinity of the glycodendrimer ligands to Gal-3 and Gal-8 but especially to Gal-9 with the formation of relatively uniform and stable galectin/Lac-CS-DDM aggregates.

• MeSH
• Galectins * metabolism   MeSH
• Lactose * MeSH
• Ligands MeSH
• Polysaccharides MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• carbosilane MeSH Browser
• Galectins * MeSH
• Lactose * MeSH
• Ligands MeSH
• Polysaccharides MeSH

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.

• Keywords
• blood-group antigen, galectin-4, inhibitor, multivalency, oligosaccharide, transglycosylation,
• MeSH
• Galectin 4 * MeSH
• Galectins chemistry   MeSH
• Humans MeSH
• Ligands MeSH
• Neoplasms * MeSH
• Oligosaccharides chemistry   MeSH
• Carbohydrates MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galectin 4 * MeSH
• Galectins MeSH
• Ligands MeSH
• Oligosaccharides MeSH
• Carbohydrates MeSH

β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

• Keywords
• Pichia pastoris, Talaromyces flavus, site-directed mutagenesis, site-saturation mutagenesis, substrate specificity, β-N-acetylhexosaminidase,
• MeSH
• Acetylgalactosamine metabolism   MeSH
• Acetylglucosamine * metabolism   MeSH
• Acetylglucosaminidase MeSH
• beta-N-Acetylhexosaminidases * metabolism   MeSH
• Kinetics MeSH
• Mutation MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylgalactosamine MeSH
• Acetylglucosamine * MeSH
• Acetylglucosaminidase MeSH
• beta-N-Acetylhexosaminidases * MeSH

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

The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

• MeSH
• Killer Cells, Natural * chemistry   immunology   metabolism   MeSH
• Antigens, CD * chemistry   immunology   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte * chemistry   immunology   metabolism   MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B * chemistry   immunology   metabolism   MeSH
• Lectins, C-Type * chemistry   immunology   metabolism   MeSH
• Humans MeSH
• Oligosaccharides * chemistry   immunology   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD * MeSH
• CD69 antigen MeSH Browser
• Antigens, Differentiation, T-Lymphocyte * MeSH
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B * MeSH
• Lectins, C-Type * MeSH
• Oligosaccharides * MeSH

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

• MeSH
• Antigens, CD metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte metabolism   MeSH
• Rats MeSH
• Lectins, C-Type metabolism   MeSH
• Humans MeSH
• Oligopeptides chemical synthesis   pharmacology   MeSH
• Polysaccharides chemical synthesis   pharmacology   MeSH
• Receptors, Immunologic metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD MeSH
• CD69 antigen MeSH Browser
• Antigens, Differentiation, T-Lymphocyte MeSH
• Klrb1a protein, rat MeSH Browser
• Lectins, C-Type MeSH
• Oligopeptides MeSH
• Polysaccharides MeSH
• Receptors, Immunologic MeSH
• Recombinant Proteins MeSH