A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of Alzheimer's disease

Abstract

Amyloid deposits in the brains of patients with Alzheimer's disease (AD) contain a protein (beta A4) which is abnormally cleaved from a larger transmembrane precursor protein (APP). APP is believed to be normally released from membranes by the action of a protease referred to as APP secretase. Amyloid deposits have also been shown to contain the enzyme acetylcholinesterase (AChE). In this study, a protease activity associated with AChE was found to possess APP secretase activity, stimulating the release of a soluble 100K form of APP from HeLa cells transfected with an APP cDNA. The AChE-associated protease was strongly and specifically inhibited by soluble APP (10 nM) isolated from human brain. The AChE-associated protease cleaved a synthetic beta A4 peptide at the predicted cleavage site. As AChE is decreased in AD, a deficiency of its associated protease might explain why APP is abnormally processed in AD.

Gelatinase A possesses a β‐secretase‐like activity in cleaving the amyloid protein precursor of Alzheimer's disease

“…HeLa cells were transiently transfected by calcium phosphate coprecipitation with a plasmid (pAD-695) encoding the 695-amino acid form of APP [8,12]. The following day, cells were subcultured into 6-well plastic culture dishes at a density of 2 x 105 cells/well in Dulbecco's 0014-5793195/$9.50 © 1995 Federation of European Biochemical Societies.…”

“…HeLa cells were incubated with 200/A/well of serum-free medium containing 0.1% (w/v) Na-azide for 15 min at 37 °C to inhibit endogenous release of APP [12]. Activated 72 kDa gelatinase A was added at various concentrations in a total volume of 50/11 of buffer (20 mM Tris-HC1, pH 7.5 containing 10 mM CaCI2) and the cells were incubated for 30 min at 37 °C.…”

“…Activated 72 kDa gelatinase A was added at various concentrations in a total volume of 50/11 of buffer (20 mM Tris-HC1, pH 7.5 containing 10 mM CaCI2) and the cells were incubated for 30 min at 37 °C. The buffer was then removed and concentrated to 40 ml by filtration through Centricon-30 concentrators (Amicon, Beverly, MA) and then analysed by Western blotting as previously described [12].…”

“…The absorbance at 214 nm was monitored using an Applied Biosystems 1000S detector. Peptides were eluted with a linear gradient of 0-60% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid/H20 at a flow rate of 1 ml/min over 60 min [12]. Peptides eluting from the column were identified by amino acid sequencing using an Applied Biosystems model 470A sequencer with an on-line 120A PTH-amino acid analyser, as well as commercially by Chiron Mimotopes Pty.…”

“…q heir studies showed that a synthetic peptide homologous to r,~sidues [10][11][12][13][14][15][16][17][18][19][20] residue known to be the site of cleavage of the ~-secretase. However, as gelatinase A was activated by a procedure employing trypsin digestion and stromelysin activation, it was not conclusively demonstrated in that study that the activated gelatinase A was free of trypsin activity.…”