APP cleavage in live cells guided by C99 / 中华神经医学杂志

ABSTRACT

Objective To construct recombinant eukaryotic expression plasmid encoding Swedish and Flemish mutations of amyloid precursor protein (APP) fused with fluorescent protein and to investigate the APP cleavage progress. Methods The last 300 bases of APP (named as C99 containing Flemish mutation), together with cyan and yellow fluorescence sequence (named as CFP and YFP,respectively) were obtained by polymerase chain reaction (PCR). The 54 bases in the middle of APP sequence were synthesized (named as 54 bp containing Swedish mutation). The 4 fragments mentioned above (CFP, YFP, C99 as well as 54 bp) were inserted into the vector pcDNA3.0. By genetic engineering, the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 was constructed and identified by enzyme digestion, PCR and sequencing. Then the plasmid was transfected into SH-SY5Y cells. Its expression was examined by fluorescence confocal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (A) deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed the sequence of the constructed recombinant plasmid was correct. (2) FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. (3) The expression product of fusion gene was correct and cleaved by and secretases. (4) The A deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusion (1) The fusion protein can generate A by and γproteolytic processing. (2) It is for the first time to observe the APP cleavage by FRET. (3) It is also the first time to find that APP may be cleaved during its transportation from cell plasma to cell membrane. (4) C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal peptide. It might guide and direct the APP to the right location for the cleavage.