Dominance of amyloid precursor protein sequence over host cell secretases in determining beta-amyloid profiles studies of interspecies variation and drug action by internally standardized immunoprecipitation/mass spectrometry.
beta-Amyloid peptides, tentatively regarded as the principal neurotoxins responsible for Alzheimer's Disease, make up a set of products that varies significantly among different biological systems. The full implications of this complexity and its variations have yet to be defined. In this work, Abeta peptide populations were extracted from animal brain tissue or cell-conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. (15)N-Substituted Abeta internal standards were added to gauge variations in the profile of captured peptides. Results from a range of species, including guinea pig, dog, rabbit, and wild-type and transgenic mice, showed that the Abeta peptide population in each system was mainly determined by the species of origin of the amyloid precursor protein (APP) and not by the host tissue or cell line. The same method was used to gauge the effect on the Abeta peptide profile of an inhibitor of gamma-secretase, one of the two proteinases that excises Abeta peptides from the precursor protein with different effects on specific peptides. Overall, the results demonstrate that the species of origin of the APP substrate dictates the outcome of APP processing to a greater extent than the origin of the processing enzymes, an important consideration in rationalizing the properties of different model systems.
Du
Ping
P
Pfizer Global Research and Development, Groton, CT 06340, USA.
Wood
Kathleen M
KM
Rosner
Michele H
MH
Cunningham
David
D
Tate
Barbara
B
Geoghegan
Kieran F
KF
D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D001921:Brain / Q000201:enzymology* / Q000378:metabolism*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D001381:Azepines; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; C484278:N2-((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-L-alaninamide; D010446:Peptide Fragments; D053829:Amyloid Precursor Protein Secretases; D000409:Alanine