Namba Norihiro, Danjo Tokiko, Kitagawa Yuichiro, Naito Yoshito, Ohgita Takashi, Shimanouchi Toshinori, Saito Hiroyuki
Laboratory of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Misasagi-Nakauchi-cho, Yamashina-ku, Kyoto, 607-8414, Japan.
Center for Instrumental Analysis, Kyoto Pharmaceutical University, 1 Misasagi-Shichono-cho, Yamashina-ku, Kyoto, 607-8412, Japan.
Sci Rep. 2025 Apr 16;15(1):13203. doi: 10.1038/s41598-025-97992-3.
Apolipoprotein A-IV (apoA-IV), the largest member of the exchangeable apolipoprotein family, is a common constituent of amyloid deposits in renal and cardiac amyloidosis. In this study, we characterized the aggregation propensity of the apoA-IV N-terminal fragment to form amyloid fibrils using a variety of biophysical techniques. Thioflavin T fluorescence assay, circular dichroism measurement, and microscopic observations revealed that the N-terminal 1-70 amino acid fragment of apoA-IV readily forms amyloid fibrils by a transition from a random coil to a β-sheet-rich structure. Sequence-based analysis indicated that residues 7-16 and 38-42 are the major aggregation-prone segments within the N-terminal 1-70 residues of apoA-IV. Consistent with this, deletion of these residues strongly inhibited the β-transition and fibril formation of apoA-IV 1-70. Kinetic and thermodynamic analyses of fibril formation by the apoA-IV 1-70 fragment demonstrated that primary nucleation is the dominant step in fibril formation, for which the activation energy barrier is entirely entropic. In addition, we found that the presence of heparin, a representative glycosaminoglycan, accelerated fibril formation kinetics and enhanced the yield of apoA-IV 1-70 fibrils, and the positively charged residues K58-K59 play a critical role in heparin interaction. Overall, our results suggest that the strong amyloid-forming propensity of the N-terminal fragment of apoA-IV may play a key role in amyloid deposition associated with apoA-IV amyloidosis.
载脂蛋白A-IV（apoA-IV）是可交换载脂蛋白家族中最大的成员，是肾和心脏淀粉样变性中淀粉样沉积物的常见成分。在本研究中，我们使用多种生物物理技术对apoA-IV N端片段形成淀粉样纤维的聚集倾向进行了表征。硫黄素T荧光测定、圆二色性测量和显微镜观察表明，apoA-IV的N端1-70个氨基酸片段通过从无规卷曲转变为富含β-折叠的结构，很容易形成淀粉样纤维。基于序列的分析表明，第7-16位和38-42位残基是apoA-IV N端1-70个残基内主要的易于聚集的片段。与此一致的是，缺失这些残基强烈抑制了apoA-IV 1-7至0的β转变和纤维形成。对apoA-IV 1-70片段形成纤维的动力学和热力学分析表明，初级成核是纤维形成的主导步骤，其活化能垒完全是熵性的。此外，我们发现，代表性糖胺聚糖肝素的存在加速了纤维形成动力学并提高了apoA-IV 1-70纤维的产量，并且带正电荷的残基K58-K59在肝素相互作用中起关键作用。总体而言，我们的结果表明，apoA-IV N端片段强烈的淀粉样形成倾向可能在与apoA-IV淀粉样变性相关的淀粉样沉积中起关键作用。
