Designing and testing fluorescent sphingolipid-interacting peptides as probes of cell membrane organization.

Abstract

Lipid rafts have attracted much interest because of their roles in numerous pathological conditions. In-depth studies would thus shed light on rafts dynamics that is instrumental in future vaccines development. However, studies were impeded by the lack of probes available to trace sphingolipids trafficking pathways in cells. Recent discovery of a 25 amino acid long sphingolipid binding domain in amyloid beta peptide by Fantini can be conjugated to a fluorophore, and viewed under confocal microscope, thus paving a way for understanding uptake mechanisms of peptides into cells. Original sphingolipid binding domain probe described in Steinert et al and Hebbar et al includes a mutation from wild type lysine to glutamine at position 16. This was compared to new modified versions of the peptide that includes the reversion of the mutation. We also tested the effects of the presence of a new linker, 4 copies of polyethylene glycol instead of the original amino-ethoxy-ethoxy-acetyl linker used in the original publications. The results showed that SBD bind to and is taken up by human SH-SY5Y neuroblastoma cells in a time-temperature dependent manner. Peak uptake occurred 15 minutes after chase at 37 oC and the probe with polyethylene gycol linker and lysine at position 16 yield best uptake into cells.