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 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.

Improved Protocol to Study Osteoblast and Adipocyte Differentiation Balance

“…Among human cells, most of them are MSCs immortalized through TERT, and among mice cell lines, the highest number comes from C3H mice. Therefore, we selected two cell lines, mouse C3H10T1/2 and human hMSC-TERT, with proven capacity to differentiate to either osteoblast [36,[47][48][49][50] or adipocyte. In contrast to hMSC-TERT, mouse C3H10T1/2 cells were efficiently differentiated to osteoblast and adipocyte cell fates, as confirmed by the differentiation marker genes.…”

Metabolic glycoengineering – exploring glycosylation with bioorthogonal chemistry

“…Telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) were successfully labeled with different azide-modified aminosugars without interrupting the cell differentiation behavior in vitro . 224 This allowed a more detailed investigation of glycosylation processes during skeletal development. MGE was also used to protect insulin-producing pancreatic β cells.…”

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylated glycoconjugates: a comparative study

“…The reaction crude was purified by silica flash column chromatography (50 µm, 40 g, dry load, CH 2 Cl 2 /MeOH 95:5 v/v), fractions containing the product were gathered and concentrated under reduced pressure, affording ManNAz as a white powder (175 mg, 0.67 mmol, 57%). 13 C NMR (75 MHz, D 2 O) δ = 171.87 (s, C7 α), 170.97 (s, C7 β), 92.89 (s, C1 α), 92.83 (s, C1 β), 76.40 (s, C5 β), 72.03 (s, C3 β), 71.97 (s, C5 α), 68.82 (s, C3 α), 66.76 (s, C4 α), 66.52 (s, C4 β), 60.41 (s, C6 β), 60.39 (s, C6 α), 54.28 (s, C2 β), 53.37 (s, C2 α), 51.72 (s, C8 β), 51.65 (s, C8 α).…”

“…1 H NMR (300 MHz, CDCl 3 ) δ = 6.64 (d, J = 8.9 Hz, 1H, H1), 6.56 (d, J = 9.2 Hz, 1H, H1′), 6.05 (d, J = 1.9 Hz, 1H, NH′), 5.89 (d, J = 1.6 Hz, 1H, NH), 5.35 (dd, J = 10.1 Hz, 4.2 Hz, 1H, H3′), 5.22 (t, J = 10.1 Hz, 1H, H4′), 5.17 (t, J = 9.8 Hz, 1H, H4), 5.06 (dd, J = 9.8 Hz, 3.8 Hz, 1H, H3), 4.73 (ddd, J = 8.9 Hz, 3.8 Hz, 1.6 Hz, 1H, H2), 4.62 (ddd, J = 9.3, 4.2, 1.9, 1H, H2′), 4.30-4.00 (m, 5H, H5' + H6 + H6' + CH 2 -N 3 + CH 2 -N 3 ′), 3.82 (ddd, J = 9.8 Hz, 4.5 Hz, 2.5 Hz, 1H, H5), 2.24-1.93 (m, 12H, OAc + OAc′). 13 C NMR (75 MHz, CDCl 3 ) δ = 170.67-166.55 (m, CO OAc+C1+C1′), 91.31 (s, CN), 90.26 (s, CN′), 73.40 (s, C5), 71.45 (s, C3), 70.27 (s, C5′), 68.86 (s, C3′), 65.10 (s, C4′), 64.93 (s, C4), 61.75 (s, C6′), 61.67 (s, C6), 52.62 (s, CH 2 -N 3 ), 52.45 (s, CH 2 -N 3 ′), 49.72 (s, C2), 49.26 (s, C2'), 20.90-20.56 (m, 4 × CH3 OAc).…”

“…Among the bioorthogonal ligations used to label the modified sialylated GCs with a fluorescent or biotinylated probe, cycloadditions including the Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and the strainpromoted [3 + 2] alkyne-azide cycloaddition (SPAAC), have been widely used in vitro and in vivo [7][8][9]12,13,[17][18][19][20][21][22] . CuAAC is the only bioorthogonal reaction in which alkynes and azides can be introduced indifferently either on the metabolic analog or on the probe for the ligation, making this reaction very flexible and adaptable to various labeling strategies.…”