1. Academic Validation
  2. Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

  • Int J Mol Sci. 2021 Mar 10;22(6):2820. doi: 10.3390/ijms22062820.
Stephan Altmann 1 Jürgen Mut 2 Natalia Wolf 2 Jutta Meißner-Weigl 1 Maximilian Rudert 1 Franz Jakob 1 Marcus Gutmann 3 Tessa Lühmann 3 Jürgen Seibel 2 Regina Ebert 1
Affiliations

Affiliations

  • 1 Bernhard-Heine-Center for Locomotion Research, University of Würzburg, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany.
  • 2 Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
  • 3 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Abstract

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated Click Chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

Keywords

click chemistry; glycocalyx; hMSC-TERT; metabolic glycoengineering; modified monosaccharides.

Figures
Products