Galectins are small human proteins participating in inflammation processes, immune response, and cancerogenesis. Tandem-repeat galectins comprising Gal-4, Gal-8, and Gal-9 are a vital yet less studied part of the galectin fingerprint in cancer-related processes. The present work studies a library of prepared multivalent neo-glycoproteins decorated with poly-N-acetyllactosamine and human-milk-type oligosaccharides as ligands of this underexplored family of tandem-repeat galectins. A thorough binding evaluation by ELISA and biolayer interferometry was complemented with a detailed epitope mapping both from the galectin and the glycoconjugate viewpoints by nuclear magnetic resonance. The found interactions in the galectin binding site were correlated to in silico data from molecular modeling. The present work reveals pioneer information on the binding of tandem-repeat galectins to multivalent glycoconjugates carrying complex carbohydrate ligands and represents an invaluable starting point for the development of new high-affinity tailored ligands of tandem-repeat galectins, needed both for diagnosis and therapy.

• MeSH
• Galectins * chemistry   metabolism   MeSH
• Glycoproteins * chemistry   metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Milk, Human * chemistry   MeSH
• Oligosaccharides * chemistry   metabolism   MeSH
• Polysaccharides MeSH
• Tandem Repeat Sequences MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galectins * MeSH
• Glycoproteins * MeSH
• Ligands MeSH
• Oligosaccharides * MeSH
• poly-N-acetyllactosamine MeSH Browser
• Polysaccharides MeSH

INTRODUCTION: The immunosuppressive roles of galectin-3 (Gal-3) in carcinogenesis make this lectin an attractive target for pharmacological inhibition in immunotherapy. Although current clinical immunotherapies appear promising in the treatment of solid tumors, their efficacy is significantly weakened by the hostile immunosuppressive tumor microenvironment (TME). Gal-3, a prominent TME modulator, efficiently subverts the elimination of cancer, either directly by inducing apoptosis of immune cells or indirectly by binding essential effector molecules, such as interferon-gamma (IFNγ). METHODS: N-(2-Hydroxypropyl)methacrylamide (HPMA)-based glycopolymers bearing poly-N-acetyllactosamine-derived tetrasaccharide ligands of Gal-3 were designed, synthesized, and characterized using high-performance liquid chromatography, dynamic light scattering, UV-Vis spectrophotometry, gel permeation chromatography, nuclear magnetic resonance, high-resolution mass spectrometry and CCK-8 assay for evaluation of glycopolymer non-toxicity. Pro-immunogenic effects of purified glycopolymers were tested by apoptotic assay using flow cytometry, competitive ELISA, and in vitro cell-free INFγ-based assay. RESULTS: All tested glycopolymers completely inhibited Gal-3-induced apoptosis of monocytes/macrophages, of which the M1 subtype is responsible for eliminating cancer cells during immunotherapy. Moreover, the glycopolymers suppressed Gal-3-induced capture of glycosylated IFNγ by competitive inhibition to Gal-3 carbohydrate recognition domain (CRD), which enables further inherent biological activities of this effector, such as differentiation of monocytes into M1 macrophages and repolarization of M2-macrophages to the M1 state. CONCLUSION: The prepared glycopolymers are promising inhibitors of Gal-3 and may serve as important supportive anti-cancer nanosystems enabling the infiltration of proinflammatory macrophages and the reprogramming of unwanted M2 macrophages into the M1 subtype.

• Keywords
• carbohydrate, galectin-3, glycopolymer, interferon-gamma, monocyte, tumor microenvironment,
• MeSH
• Acrylamides chemistry   pharmacology   MeSH
• Apoptosis drug effects   MeSH
• Galectin 3 * antagonists & inhibitors   MeSH
• Galectins MeSH
• Interferon-gamma * metabolism   MeSH
• Blood Proteins MeSH
• Humans MeSH
• Macrophages drug effects   MeSH
• Monocytes * drug effects   MeSH
• Tumor Microenvironment drug effects   MeSH
• Polymers * chemistry   pharmacology   MeSH
• Antineoplastic Agents * pharmacology   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acrylamides MeSH
• Galectin 3 * MeSH
• Galectins MeSH
• Interferon-gamma * MeSH
• Blood Proteins MeSH
• LGALS3 protein, human MeSH Browser
• Polymers * MeSH
• Antineoplastic Agents * MeSH

Pulmonary hypertension is a cardiovascular disease with a low survival rate. The protein galectin-3 (Gal-3) binding β-galactosides of cellular glycoproteins plays an important role in the onset and development of this disease. Carbohydrate-based drugs that target Gal-3 represent a new therapeutic strategy in the treatment of pulmonary hypertension. Here, we present the synthesis of novel hydrophilic glycopolymer inhibitors of Gal-3 based on a polyoxazoline chain decorated with carbohydrate ligands. Biolayer interferometry revealed a high binding affinity of these glycopolymers to Gal-3 in the subnanomolar range. In the cell cultures of cardiac fibroblasts and pulmonary artery smooth muscle cells, the most potent glycopolymer 18 (Lac-high) caused a decrease in the expression of markers of tissue remodeling in pulmonary hypertension. The glycopolymers were shown to penetrate into the cells. In a biodistribution and pharmacokinetics study in rats, the glycopolymers accumulated in heart and lung tissues, which are most affected by pulmonary hypertension.

• MeSH
• Pulmonary Artery drug effects   metabolism   MeSH
• Biomarkers MeSH
• Fibroblasts drug effects   metabolism   MeSH
• Galectin 3 * antagonists & inhibitors   metabolism   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Hypertension, Pulmonary * drug therapy   metabolism   MeSH
• Polymers chemistry   pharmacology   MeSH
• Tissue Distribution 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
• Biomarkers MeSH
• Galectin 3 * MeSH
• Polymers MeSH

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

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.

• Keywords
• enzymatic synthesis, glycosidase, human milk oligosaccharide, mutagenesis,
• MeSH
• Bifidobacterium bifidum * metabolism   MeSH
• Glycoside Hydrolases metabolism   MeSH
• Glycosyltransferases metabolism   MeSH
• Humans MeSH
• Milk, Human * metabolism   MeSH
• Infant, Newborn MeSH
• Oligosaccharides chemistry   MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycoside Hydrolases MeSH
• Glycosyltransferases MeSH
• Oligosaccharides MeSH

The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.

• Keywords
• HPMA copolymer, galectin, glycomimetic, glycopolymer, inhibition, molecular recognition, multivalency,
• MeSH
• Acrylamides chemistry   pharmacology   MeSH
• Cryoelectron Microscopy MeSH
• Galectin 1 chemistry   genetics   MeSH
• Galectins chemistry   genetics   MeSH
• Blood Proteins chemistry   genetics   MeSH
• Humans MeSH
• Ligands MeSH
• Methacrylates chemistry   pharmacology   MeSH
• Polymers chemistry   pharmacology   MeSH
• Carbohydrates chemistry   MeSH
• Protein Binding drug effects   MeSH
• Binding Sites drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acrylamides MeSH
• Galectin 1 MeSH
• Galectins MeSH
• hydroxypropyl methacrylate MeSH Browser
• Blood Proteins MeSH
• LGALS1 protein, human MeSH Browser
• LGALS3 protein, human MeSH Browser
• Ligands MeSH
• methacrylamide MeSH Browser
• Methacrylates MeSH
• Polymers MeSH
• Carbohydrates 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