Vascular endothelial growth factor-A₁₆₅ (VEGF-A₁₆₅) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A₁₆₅ and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A₁₆₅ stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A₁₆₅ on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A₁₆₅ have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

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.

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.

We report the synthesis and testing of a novel type (new lead structure) of powerful and highly selective human O-N-acetyl-β-d-glucosaminidase (enzyme associated with Alzheimer's disease) inhibitors that are not based on transition state mimetics.

Inhibition of the human O-linked β-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure–activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO₂ oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH₃/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, K_i = 27 nM). This compound had a K_i of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein–ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds.