Influence of Aβ on GD3S activity.

<p>(<i>A</i>) Effect of Aβ on GD3S activity. MEF lacking Aβ because of PS- or APP-deficiency (MEF PS1/2-/- and MEF APP/APLP2-/-, respectively) were incubated with physiological Aβ40 or Aβ42 concentrations or the solvent used for the Aβ peptides (control). The level of the GD3S activity of the corresponding wildtype cells are indicated (horizontal dotted black line); corresponding wildtype/control cells for MEF PS1/2-/-: PS1/2 deficient MEF retransfected with PS1 (MEF PS1r); corresponding wildtype/control cells for MEF APP/APLP2: mouse embryonic fibroblasts of wildtype mice (MEF wt). Both Aβ species partially rescued the increased GD3S activity in PS- or APP-deficient MEF. No significant differences were observed between Aβ40 and Aβ42 peptides. (<i>B</i>) Direct effect of Aβ40 and Aβ42 on GD3S activity. Homogenates of PS1/2-/- cells incubated with Aβ40 and Aβ42 show decreased GD3S activity. Similar results are obtained with Aβ40 and Aβ42 in a cell-free assay containing only purified GD3S and the substrate GM3. (<i>C</i>) Influence of aggregated Aβ and inverted Aβ on GD3S activity. Inverted Aβ peptides and aggregated Aβ showed no influence on GD3S activity. (<i>D</i>) GM3, the substrate for GD3S, binds to Aβ: Physiological concentrations of cellular derived Aβ bind GM3 analyzed by co-immunoprecipitation (co-IP) of Aβ in presence of GD3 or GM3. GM3, but not GD3, binds to Aβ. After IP, Aβ bound gangliosides were detected via TLC. GM3 binds to synthetic Aβ40 and Aβ42 (shown for equimolar concentrations), and cellular derived Aβ40 and Aβ42 (shown for physiological concentrations, approx. 10∶1 ratio). Thin black vertical lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034095#pone-0034095-g002" target="_blank">Fig. 2D</a> left) indicate that the TLC plates were scraped to separate lines. (<i>E</i>) Co-IP of Aβ and GD3S in dependence of GM3. Aβ only binds to GD3S in presence of the substrate GM3. Data are represented as mean +/− SEM.