Figure 5. V44F/I45F double mutation of APP substrate blocks Aβ46 → Aβ43 and Aβ45 → Aβ42 cleavage steps by γ-Secretase and stabilizes E-S complexes.

(A) Transmembrane domain sequence alignment of WT versus V44F/I45F C99.

(B) Concentration of tri- or tetrapeptides detected by LC-MS/MS analysis from WT C100-FLAG and V44F/I45F C100-FLAG. Cleavage events along the Aβ40 pathway are represented by ITL → VIV → IAT (blue) and cleavage events along the Aβ42 pathway are represented by VIT → TVI → VVIA (red). Note that cleavage steps from Aβ46 → Aβ43 and Aβ45 → Aβ42 for the V44F/I45F mutation produce tripeptides FFV and TFF, respectively. “nd” indicates tri- or tetrapeptides unable to be detected by LC-MS/MS. Enzyme (30 nM) and 5 μM substrate were incubated at 37°C for 16 h for both trials. The average and range of both trials are represented in the graph.

(C) MALDI-TOF mass spectrometric analysis of AICD50–99 and AICD49–99 produced by γ-Secretase with WT versus V44F/I45F C100-FLAG. Note that the V44F/I45F double mutation leads to substantially reduced AICD50–99 production.

(D) Secreted Aβ40 and Aβ42 levels in the culture media of HEK293 cells stably expressing C99 and its mutants I45F and V44F/I45F were measured by specific ELISAs. Aβ42/Aβ40 ratios are also indicated. “NT” indicates the non-transfected control. Data are the mean ± SE from three independent experiments.

(E) Cellular C99 levels in HEK293 cells stably expressing C99 and its mutants were examined by western blotting using monoclonal antibody 6E10.

(F) Proteins (10 μg) from the various HEK293 cell lysates were subjected to blue native (BN)-PAGE (1% digitonin) or SDS-PAGE and analyzed by western blotting using antibodies specific for C99/Aβ, PS1-NTF, and GAPDH. Data are representative of three independent experiments.