COMPARATIVE STUDY
JOURNAL ARTICLE

Biophysical analyses of synthetic amyloid-beta(1-42) aggregates before and after covalent cross-linking. Implications for deducing the structure of endogenous amyloid-beta oligomers

Brenda D Moore, Vijayaraghavan Rangachari, William M Tay, Nicole M Milkovic, Terrone L Rosenberry
Biochemistry 2009 December 15, 48 (49): 11796-806
19916493
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.

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