Biophysical Analyses of Synthetic Amyloid-β(1−42) Aggregates before and after Covalent Cross-Linking. Implications for Deducing the Structure of Endogenous Amyloid-β Oligomers

Abstract

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that soluble Abeta aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Abeta aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Abeta oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Abeta have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Abeta aggregates in vitro that resemble the endogenous soluble Abeta aggregates, we previously found that monomeric Abeta(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Abeta(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Abeta cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Abeta(1-42) with three cross-linking agents: transglutaminase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS.

Di-Tyrosine Cross-Link Decreases the Collisional Cross-Section of Aβ Peptide Dimers and Trimers in the Gas Phase: An Ion Mobility Study

“…This hypothesis has been challenged by the observation of Bitan et al [18] that SDS itself can artificially induce oligomerization. Other groups have confirmed this conclusion [19], [20], but it was also found that such artificial forms become disaggregated upon boiling in 1% SDS. Also, it is known that species resolved by SDS-PAGE differed markedly in terms of size and stability depending on the source of the oligomer: endogenous or synthetic [19], AD-derived or extracted material from transgenic mouse AD models [21].…”

Biochemistry of Amyloid -Protein and Amyloid Deposits in Alzheimer Disease

“…Depending on the stoichiometry, Cu 2þ -Ab interactions can cause synthetic Ab to aggregate in vitro principally via an oligomer-forming pathway or a fibrillogenic pathway (Brzyska et al 2009;Moore et al 2009;Olofsson et al 2009a;Tõugu et al 2009;Haeffner et al 2010). That is, at subequimolar Cu 2þ :Ab ratios, amyloid fibrils form; at supra-equimolar ratios, stable oligomers form first, then dityrosine cross-linkages occur (Smith et al 2007).…”

“…Cu 2þ as a redox-active entity can also induce oxidative modification to Ab, particularly at Tyr10 with consequent dityrosine covalent cross-links (Drew et al 2009a,b;Moore et al 2009;Jiang et al 2010). Other residues, such as Met35, may participate (Barman et al 2009;Butterfield et al 2010), but this is not proven (da Silva et al 2009).…”

APOE4-specific Changes in Aβ Accumulation in a New Transgenic Mouse Model of Alzheimer Disease

“…MOAB-2 was developed in the LaDu laboratory and is a high-affinity antibody specific for A␤ that does not detect APP. For the oA␤ ELISA, MOAB-2 was used as a capture antibody and biotinylated MOAB-2 as a detection antibody based on a previous protocol (46). This ELISA pairing detects synthetic oA␤ preparations but not monomeric A␤ using preparations of oA␤ optimized for the original protocol (data not shown) (46).…”

“…For the oA␤ ELISA, MOAB-2 was used as a capture antibody and biotinylated MOAB-2 as a detection antibody based on a previous protocol (46). This ELISA pairing detects synthetic oA␤ preparations but not monomeric A␤ using preparations of oA␤ optimized for the original protocol (data not shown) (46). These oA␤ preparations were used as a standard for measuring soluble oA␤ levels in EFAD mice.…”