Ganglioside GM1 binding the N-terminus of amyloid precursor protein

Abstract 

Secreted amyloid precursor protein (APPs) plays a role in several neuronal functions, including the promotion of synaptogenesis, neurite outgrowth and neuroprotection. Previous study has demonstrated that ganglioside GM1 inhibits the secretion of APPs; however the underlying mechanism remains unknown. Here we reported that GM1 can bind cellular full length APP and APPs secreted from APP₆₉₅ stably-transfected SH-SY5Y cells. To characterize the GM1–APP interaction further, we expressed and purified recombinant fragments of the N-terminal APP. Immunoprecipitation experiments revealed that GM1 was able to bind the recombinant APP_18–81 fragment. Moreover, the synthetic peptide APP_52–81 could inhibit the binding. Therefore, the binding site for GM1 appears to be located within residues 52–81 of APP. Furthermore, we found that only GM1, but not GD1a, GT1b and ceramide, binds APP-N-terminus, indicating that the specific binding depends on the sugar moiety of GM1. Fluorescent studies revealed a decrease in the intrinsic fluorescence intensity of the APP_52–81 peptide in phosphatidylcholine (PC)/GM1 vesicles. By using FTIR techniques, we found that the major secondary structure of the APP_52–81 peptide was altered in PC/GM1 vesicles. Our results demonstrate that GM1 binds the N-terminus of APP and induces a conformational change. These findings suggest that secreted APP is decreased by membrane GM1 binding to its precursor protein and provide a possible molecular mechanism to explain the involvement of GM1 in APP proteolysis and pathogenesis of Alzheimer's disease.

Abbreviations: AD, Alzheimer's disease, Aβ, β-amyloid peptide, APP, amyloid β-protein precursor, APPs, secreted APP derivatives, APP-NTFs, APP N-terminal fragments, GAL-CER, gal-ceramide, CTB, cholera toxin B subunit, anti-APP_CT, the anti-C-terminal APP polyclonal antibody, CTB-HRP, cholera toxin B subunit conjugated to peroxidase, PC, phosphatidylcholine, PG, phosphatidylglycerol, HRP, horseradish peroxidase, SY5Y695, the human neuroblastoma cell line SH-SY5Y transfected with human APP₆₉₅ cDNA, FCS, fetal calf serum, PBS, phosphate-buffered saline, RT, reverse transcription, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, TBS, tris-buffered saline, ECL, enhanced chemiluminescence, SUVs, small unilamellar vesicles, FTIR, Fourier transform infrared

Keywords: Amyloid precursor protein, GM1, Gangliosides, Glycosphingolipid, Fluorescence, Fourier transform infrared spectroscopy