Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation
Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation
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DOI：
10.1074/jbc.m117.813097
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发表时间：
2017-12-29
影响因子：
4.8
通讯作者：
Goto, Yuji
中科院分区：
文献类型：
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作者：
Nitani, Ayame;Muta, Hiroya;Goto, Yuji
肝素是一种具有高负电荷密度的生物聚合物，已知会加速各种蛋白质的淀粉样蛋白纤维化。使用鸡蛋清溶菌酶，我们研究了肝素在低 pH、升高温度和超声波照射等促进淀粉样纤维颤动的条件下对蛋白质聚集的影响。肝素表现出复杂的双峰浓度依赖性效应，在 pH 2.0 和 60 摄氏度下加速或抑制纤维颤动。浓度低于 20 μg/ml 时，肝素通过瞬时形成异源寡聚体聚集体加速纤维颤动。在 0.1 至 10 mg/ml 之间，肝素迅速诱导无定形异质聚集，几乎没有伴随原纤维形成。高于 10 mg/ml，肝素在长时间滞后后再次诱发纤维颤动，随后形成寡聚体。与使用单价和二价阴离子进行的研究相比，结果表明肝素诱导的纤维性颤动有两种不同的机制。在低肝素浓度下，抗衡离子结合和排斥电荷的筛选促进了最初的鸡蛋清溶菌酶簇的形成和随后的原纤维化。在高肝素浓度下，纤维颤动是由盐析和大分子拥挤效应共同引起的，可能与蛋白质净电荷无关。两种纤维颤动机制都与无定形聚集竞争，产生复杂的肝素浓度依赖性相图。此外，结果表明，无定形寡聚聚集体在引发原纤维化方面发挥着积极作用，从而通过淀粉样蛋白在无定形聚集体上的异质成核来破坏过饱和度。
Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 degrees C. At concentrations lower than 20 g/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.