title: Mass spectrometric characterization of amyloid-β species in the 7PA2 cell model of Alzheimer's disease.
abstract: The Chinese hamster ovary cell line 7PA2, stably transfected with the 751 amino acid isoform of amyloid-β protein precursor (AβPP) containing the Val → Phe mutation at residue 717, is one of the most used models to study the biochemistry and toxicity of secreted amyloid-β (Aβ) peptides, particularly Aβ oligomers, which are considered to be of relevance to the pathogenesis of Alzheimer's disease. Here, we present a detailed immunochemical and mass spectrometric characterization of primary structures of Aβ peptides secreted by 7PA2 cells. Immunoprecipitation and western blot of 7PA2 cell culture media revealed abundant anti-Aβ immunoreactive bands in the molecular weight range of 4-20 kDa. Mass spectrometric analysis showed that these bands contain several AβPP/Aβ peptides, starting at the N-terminal of the Aβ sequence and extending across the BACE1 cleavage site. Treatment of cells with a BACE1 inhibitor decreased the abundance of the Aβ monomer band by western blot and resulted in lower levels of Aβ1-40, Aβ1-42, and sAβPPβ as measured by ELISA. However, western blot bands thought to represent oligomers of Aβ increased in response to BACE1 inhibition. This increase was paralleled by the emergence of N-terminally truncated Aβ species (Aβ5-40 in particular) and Aβ species that spanned the β-secretase site in AβPP according to mass spectrometric analyses. The formation of these AβPP/Aβ peptides may have implications for the use of the 7PA2 cell line as a model for Aβ pathology. The enzyme(s) responsible for this particular BACE1-independent AβPP-processing remains to be identified.
authors.affiliation: Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden. erik.portelius@neuro.gu.se
authors.forename: Erik
authors.initials: E
authors.lastname: Portelius
authors.forename: Maria
authors.initials: M
authors.lastname: Olsson
authors.forename: Gunnar
authors.initials: G
authors.lastname: Brinkmalm
authors.forename: Ulla
authors.initials: U
authors.lastname: Rüetschi
authors.forename: Niklas
authors.initials: N
authors.lastname: Mattsson
authors.forename: Ulf
authors.initials: U
authors.lastname: Andreasson
authors.forename: Johan
authors.initials: J
authors.lastname: Gobom
authors.forename: Ann
authors.initials: A
authors.lastname: Brinkmalm
authors.forename: Mikko
authors.initials: M
authors.lastname: Hölttä
authors.forename: Kaj
authors.initials: K
authors.lastname: Blennow
authors.forename: Henrik
authors.initials: H
authors.lastname: Zetterberg
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments