The formation of amyloid- (A) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited A is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-A). These forms show enhanced neurotoxicity compared to full-length A. Although the truncation may occur by aminopeptidases after formation of A, recently discovered processing pathways of amyloid- protein precursor (APP) by proteases such as meprin  may also be involved. Here, we assessed a role of meprin  in forming A 3-40/42 , which is the precursor of pGlu-A 3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin  mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin  in neurons and astrocytes in the vicinity of pGlu-A containing deposits. Cleavage of APP-derived peptides by meprin  in vitro results in peptides A 1-x , A 2-x , and A 3-x . The formation of N-truncated A by meprin  was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-A 3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of A 3-40 . Further analysis of the meprin  cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of A 1-x . Thus, our data suggest that meprin  contributes to the formation of N-truncated A by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-A formation.
Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity.
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