Bacterial expression, purification, and functional mapping of the amyloid beta/A4 protein precursor.
The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B. (1989) Cell 58, 615-622). Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695. The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation. Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active. Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts. These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.
Roch
J M
JM
Department of Neurosciences, University of California, San Diego, La Jolla 92093.
Shapiro
I P
IP
Sundsmo
M P
MP
Otero
D A
DA
Refolo
L M
LM
Robakis
N K
NK
Saitoh
T
T
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D016564:Amyloid beta-Protein Precursor; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011480:Protease Inhibitors / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D006133:Growth Substances; D011480:Protease Inhibitors; D011498:Protein Precursors