Identification of the primary peptide contaminant that inhibits fibrillation and toxicity in synthetic amyloid-β42.
Since introduction of the amyloid hypothesis of Alzheimer's Disease (AD) over 20 years ago, an significant amount of literature has reinforced the central importance of Aβ42 in the mechanism of the disease. The Aβ42 peptide, is a strongly favored molecule for elucidating disease mechanisms and phenotypes in relevant models including in animal models and cell culture systems. Its extreme propensity to aggregate in solution among other handling issues has largely impacted its performance in functional assays. While synthetic Aβ42 is favorably used for several years, studies relating to identifying possible contaminants/impurities that in turn inhibit toxicity of the peptide, calls for the use of recombinant Aβ42 emerging as interesting alternatives.
A recent study by Adams D, J et al (2017) has shown that the ability of Aβ42 to aggregate correlates with its toxicity validated by examining the mechanism of toxicity-driven fibrillation. The authors used G37L mutant of the Aβ42 peptide and demonstrated that this mutation can act as a dominant negative in an aggregation assay by exhibiting a protective effect towards toxicity. Further, they observed a disparity in aggregation dynamics and toxicity between recombinant and synthetic Aβ42, leading to the possibility of contaminants present in the recombinant peptide as causal for increased toxicity and aggregation patterns.
In addition to performing doping experiments, the authors used RP-HPLC and Mass Spectrometry analyses to identify and characterize contaminants and found that synthetic Aβ42 peptide (from AnaSpec) contains by-products that inhibit Aβ42 aggregation. Further characterization revealed the presence of a peak of 4417.25 Da consistent to the identity of Aβ42Δ39 as an inhibitor of Aβ42 aggregation. They confirmed that a synthesized Aβ42Δ39 (custom synthesized by AnaSpec) was in fact a potent inhibiting agent likely formed due to the relative inefficiency of β-branched amino acid coupling during solid phase peptide synthesis. Another suggested contaminant, Aβ42-HIS, also was shown to exhibit some inhibition of aggregation.
The authors conclude, given that Aβ42 is particularly prone to synthetic contaminants arising from coupling inefficiencies, the presence of potentially minor impurities that may alter both the biophysical and biological properties of synthetically prepared peptides should be taken into consideration. The observed correlation between Aβ42 toxicity and aggregation kinetics, from their study also suggests that Aβ42Δ39 and perhaps Aβ42-HIS may exhibit neuroprotective activity in AD models. Future studies are recommended to analyze various analogs of Aβ42Δ39 or Aβ42-HIS which can serve to attenuate Aβ42 toxicity, possibly yielding an important new class of compounds for therapeutic intervention in Alzheimer's disease.
Citation: Adams DJ., et al. (2017)Identification of the primary peptide contaminant that inhibits fibrillation and toxicity in synthetic amyloid-β42. PLoS One. 9;12(8):e0182804. doi:10.1371/journal.pone.0182804.