Metabolic glycoengineering in hmsc-tert as a model for skeletal precursors by using modified azide/alkyne monosaccharides

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 mesen-chymal 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 (Ac4Man-NAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the gly-coengineering 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 flu-orescent 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-de-pendent 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 transi-ent cell surface modifications, and thus present a useful model to address different scientific ques-tions regarding glycosylation processes in skeletal precursors.