Solid‐Phase Synthesis and Characterization of N‐Terminally Elongated Aβ_−3–x‐Peptides

Abstract

In addition to the prototypic amyloid‐β (Aβ) peptides Aβ1–40 and Aβ1–42, several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid‐phase peptide synthesis of the N‐terminally elongated Aβ‐peptides Aβ−3–38, Aβ−3–40, and Aβ−3–42. Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ−3–38 and Aβ−3–40 are generated by transfected cells even in the presence of a tripartite β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(−3) can be separated from N‐terminally‐truncated Aβ forms by high‐resolution isoelectric‐focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N‐terminally elongated Aβ variants in Alzheimer's disease.

Reorientational Dynamics of Amyloid-β from NMR Spin Relaxation and Molecular Simulation

“…34 Using HYCUD, we calculated the rotational correlation time of Aβ40 segments for an ensemble of 5000 random Aβ40 structures (Supporting Information). The HYCUD-predicted translational diffusion coefficient for Aβ40 at 278 K was 7.54 × 10 –7 cm 2 ·.s –1 , which corresponds to a hydrodynamic radius of 1.8 ± 0.1 nm, in excellent agreement with experiment 20,35 and the predicted value for disordered peptides of the same size. 36 The HYCUD-predicted correlation time for Aβ40 at 300 K was 3.0 ± 0.1 ns.…”

Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

“…The functionality of the peptides in forming aggregates can be assessed using various spectroscopic methods 39,48,49 , and also by incubating the peptides in PBS at room temperature for more than 1 h, which will yield aggregates. In this case, precipitates and large aggregates should be seen in solution.…”

Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples

“…Recombinant sAPPα was obtained from Sigma-Aldrich (Munich, Germany). Aβ −3–40 , Aβ −3–38 , and Aβ −3–42 were synthesized as described in detail previously [ 25 ]. The synthetic peptides Aβ 1–40 , Aβ 2–40 , Aβ 3–40 , Aβ N3pE–40 , Aβ 4–40 , and Aβ 5–40 were obtained from AnaSpec (Fremont, CA, USA).…”

“…The synthetic calibrator peptide Aβ −3–40 was synthesized as described before [ 25 ]. The lyophilized peptide was initially solubilized in DMSO (dimethyl sulfoxide) at a concentration of 1 mg/mL and stored in aliquots at −80 °C.…”

“…The APP 669–711 peptide starts three amino acids N-terminal to the canonical Aβ amino-terminus (Asp1) and encompasses the amino acid sequence of an N-terminally elongated Aβ −3–40 peptide. Neuronal BE(2)-C cells and SH-SY5Y cells overexpressing APP were shown to be able to generate Aβ −3–40 in cell culture [ 23 , 25 ], and it appears that its cellular production occurs independently of BACE1 [ 25 ]. The self-assembly tendency of Aβ −3–40 was found to be lower than that of Aβ 1–42 [ 23 ].…”