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.

• Klíčová slova
• enzymatic synthesis, glycosidase, human milk oligosaccharide, mutagenesis,
• MeSH
• Bifidobacterium bifidum * metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• glykosyltransferasy metabolismus   MeSH
• lidé MeSH
• mateřské mléko * metabolismus   MeSH
• novorozenec MeSH
• oligosacharidy chemie   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glykosidhydrolasy MeSH
• glykosyltransferasy MeSH
• oligosacharidy MeSH

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, Ki = 27 nM). This compound had a Ki 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.

• Klíčová slova
• Prime refinement, QM/MM optimization, glyconolactone sulfonylhydrazone, hHexB, hOGA, inhibitor,
• MeSH
• antigeny nádorové chemie   metabolismus   MeSH
• beta-hexosaminidasa, beta řetězec chemie   metabolismus   MeSH
• histonacetyltransferasy chemie   metabolismus   MeSH
• hyaluronoglukosaminidasa chemie   metabolismus   MeSH
• hydrazony chemická syntéza   chemie   farmakologie   MeSH
• inhibitory enzymů chemická syntéza   chemie   farmakologie   MeSH
• laktony chemie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• oxidy chemie   MeSH
• sloučeniny manganu chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• beta-hexosaminidasa, beta řetězec MeSH
• HEXB protein, human MeSH Prohlížeč
• histonacetyltransferasy MeSH
• hyaluronoglukosaminidasa MeSH
• hydrazony MeSH
• inhibitory enzymů MeSH
• laktony MeSH
• manganese dioxide MeSH Prohlížeč
• OGA protein, human MeSH Prohlížeč
• oxidy MeSH
• sloučeniny manganu MeSH

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.

• Klíčová slova
• adult stem cells, basic fibroblast growth factor (bFGF), cell adhesion, cell proliferation, endothelial cells, heterologous expression, recombinant vascular endothelial growth factor (VEGF), regenerative medicine, tissue engineering, vascular replacements,
• MeSH
• buněčná adheze účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   farmakologie   MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• prasata MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny chemie   farmakologie   MeSH
• vaskulární endoteliální růstový faktor A chemie   genetika   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• rekombinantní proteiny MeSH
• vaskulární endoteliální růstový faktor A MeSH
• VEGFA protein, human MeSH Prohlížeč