From the Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Institute of Advanced Technology, Kindai University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan.
J Biol Chem. 2017 Dec 29;292(52):21219-21230. doi: 10.1074/jbc.M117.813097. Epub 2017 Nov 3.
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 °C. At concentrations lower than 20 μg/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.
肝素是一种带有高负电荷密度的生物聚合物，已知它可以加速各种蛋白质的淀粉样纤维形成。我们使用鸡蛋白溶菌酶研究了肝素在低 pH 值、升高的温度和应用超声波辐射条件下对蛋白质聚集的影响，这些条件促进了淀粉样纤维形成。肝素表现出复杂的双模态浓度依赖性效应，即在 pH 值为 2.0 和 60°C 时，要么加速，要么抑制纤维形成。在低于 20μg/ml 的浓度下，肝素通过瞬态形成杂寡聚体聚集来加速纤维形成。在 0.1 到 10mg/ml 之间，肝素迅速诱导无定形杂聚集，几乎没有伴随的纤维形成。在 10mg/ml 以上时，在寡聚体聚集形成之前，经过长时间的滞后期，肝素再次诱导纤维形成。与使用单价和二价阴离子进行的研究相比，这些结果表明肝素诱导纤维形成存在两种不同的机制。在低肝素浓度下，通过抗衡离子结合和排斥电荷的屏蔽，最初的鸡蛋白溶菌酶簇形成和随后的纤维形成得到促进。在高肝素浓度下，纤维形成是由盐析和大分子拥挤效应的组合引起的，可能与蛋白质净电荷无关。这两种纤维形成机制与无定形聚集竞争，产生了复杂的肝素浓度依赖性相图。此外，结果表明无定形寡聚体聚集物在触发纤维形成中起着积极的作用，其中通过在无定形聚集物上异质成核使过饱和度发生崩塌。
