Single chain variable fragment against aβ expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.
Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" Aβ aggregation and will assist in the development of therapeutic strategies for AD.
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Ying
Y
Zhang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Hai-Qiang
HQ
Yang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Fang
F
Fang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Lin-Lin
LL
Song
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Yue-Ying
YY
Jiao
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
He
H
Wang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Xiang-Lei
XL
Peng
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Yan-Peng
YP
Zheng
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Jun
J
Wang
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China.
Jin-Sheng
JS
He
College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, China; Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
Tao
T
Hung
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D066263:Protein Aggregation, Pathological / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification
D016229:Amyloid beta-Peptides; D057127:Single-Chain Antibodies