Dynamic tracking of GD3 to GD2 gangliosides in T lymphoblasts via nuclear magnetic resonance spectroscopy

Gangliosides GD3 and GD2 are glycosphingolipids involved in key cellular processes and are overexpressed in various cancers, including T-cells leukemias. In this study, we investigated the temporal conversion of GD3 to GD2 in the human T lymphoblast cell line MOLT-4 by using flow cytometry and proton nuclear magnetic resonance (1H NMR) spectroscopy. Flow cytometry analysis revealed that at day 4 of culture, GD3 predominated on the cell surface, while GD2 showed low expression. By day 6, GD2 expression markedly increased, accompanied by a decrease in GD3, indicating a dynamic shift in ganglioside composition. Complementary 1H NMR analysis directly applied to cellular extracts identified diagnostic anomeric proton signals corresponding to GD3 at day 4 and GD2 at day 6, confirming the structural transition. This dual-platform approach demonstrates, for the first time, the NMR-mediated discrimination of GD3 and GD2 in unpurified human cell extracts, providing robust evidence of ganglioside remodeling during MOLT-4 cell proliferation and establishing a valuable methodology for functional and therapeutic studies involving gangliosides.