Title:In vitro Aggregation Ability of Five Commercially Available Aβ42 peptide
Volume: 18
Issue: 9
关键词:
阿尔茨海默病,Aβ42,聚集,细胞毒性,测量,差异。
摘要:
Background: As the most basic material, synthetic human Amyloid-β (1-42) (Aβ42) peptide
from different manufacturers have been widely used. Their aggregation ability is vital to the reliability,
repeatability and comparability of studies on Aβ42 physiology and pathology. However, it
has not been evaluated and compared.
Objective: To analyze the consistency of the aggregation ability of 5 commercially available Aβ42
peptide.
Methods: 5 Aβ42 peptide represented as A, B, C, D and E were pretreated by HFIP. The pretreated
Aβ42 peptide were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was
monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptide were aggregated
in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of
ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission
electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to
SH-SY5Y cells was further evaluated using Cell Counting Kit-8.
Results: For aggregation kinetics, peptide A, C and E remained low level curves, while peptide B
and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of peptide
B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37
mdeg, while the height of peptide A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence
intensity of the aggregates of peptide B and D were 7.79 and 8.82, higher than 1.19,
1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained
monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from
dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers remained
in all aggregates. Furthermore, peptide B and D aggregated to dimers and trimers, peptide
A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils
were observed only in peptide B, while substantial spherical aggregates were formed in
other peptide. Additionally, peptide B, D and E exhibited higher cytotoxicity after aggregated for 8
h, whereas peptide A, B and D presented relatively high cytotoxicity after 12-hour aggregation.
Conclusion: Commercially available Aβ42 peptide showed obvious differences in aggregation ability,
which should arouse enough attention in the field of basic study related to Aβ42. The aggregation
ability evaluation with the various assay methods has some discrepancies, and it is highly urgent to
establish a reasonable and uniform measurement strategy.