Dammers Christina, Gremer Lothar, Neudecker Philipp, Demuth Hans-Ulrich, Schwarten Melanie, Willbold Dieter
Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany.
Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
PLoS One. 2015 Oct 5;10(10):e0139710. doi: 10.1371/journal.pone.0139710. eCollection 2015.
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.
阿尔茨海默病（AD）是老年人痴呆的主要病因，其特征为记忆力丧失和认知能力下降。AD大脑的病理标志是细胞内神经原纤维缠结和细胞外淀粉样斑块。这些斑块的主要成分是高度异质的淀粉样β（Aβ）肽，其长度和修饰各不相同。近年来，焦谷氨酸修饰的淀粉样β（pEAβ）肽越来越受到关注，因为它们被认为是所有N端截短的Aβ中的主要种类。与未修饰的Aβ相比，已知pEAβ具有更高的疏水性、更高的毒性、更快的聚集速度和β-折叠稳定性，并且更耐降解。核磁共振（NMR）光谱是研究溶液中pEAβ异构体构象以及研究肽/配体相互作用以进行药物开发的一种特别强大的方法。然而，到目前为止，由于缺乏高度纯化的重组和同位素富集蛋白，pEAβ的生物物理特性及其与未修饰变体的比较受到严重阻碍。在此，据我们所知，首次提出了一种可重复的方案，用于从在大肠杆菌中表达的重组前体以天然同位素丰度以及均匀的[U-15N]或[U-13C, 15N]标记形式生产pEAβ，产量高达每升大肠杆菌培养液15毫克。通过反相高效液相色谱（RP-HPLC）评估纯化蛋白的化学状态，并通过质谱验证焦谷氨酸的形成。通过硫黄素-T测定监测，重组焦谷氨酸修饰的Aβ肽显示出特征性的S形聚集动力学。所生产的pEAβ40和pEAβ42的质量和数量使我们能够在溶液中进行异核多维NMR光谱，并在接近生理条件下对主链共振进行序列特异性归属。我们的结果表明所提出的方法将有助于获得具有成本效益的高质量重组pEAβ40和pEAβ42，用于进一步的生理和生化研究。
