Abstract
Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEAβ from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-15N]- or [U-13C, 15N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEAβ40 and pEAβ42 for further physiological and biochemical studies.
Highlights
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive functions and has become the main cause for dementia in the elderly [1, 2]
Besides Aβ isoforms starting with the amino acid D at position 1 (D1), a significant amount of N-terminally truncated Aβ variants is deposited in the brains of AD patients [10, 11], whereby pyroglutamate-modified Aβ species were described as the major isoforms [12,13,14,15]
To show the applicability and advantage of this mutation for non-enzymatic pyroglutamate-modified amyloid-β (pEAβ) conversion, we produced Aβ(3–42) starting at the N-terminal position with the original E instead of Q, which in vivo is the primary substrate for QC and is catalytically converted to pEAβ but can be modified non-enzymatically [28]
Summary
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive functions and has become the main cause for dementia in the elderly [1, 2]. The conversion results in altered biophysical and biochemical properties since: (1) pEAβ shows higher hydrophobicity due to the formation of the N-terminal pE lactam ring and the loss of three charges resulting in increased aggregation propensity [12, 19]. We report a method for reproducible expression and purification of recombinant pEAβ(3–40) and pEAβ(3–42) with natural isotope abundance, as well as uniformly [U-15N] or [U-13C, 15N]-labeled protein with yields up to 15 mg/l culture based on a previously published protocol for Aβ by Finder, Glockshuber and coworkers [37].
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