Abstract：
Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We employed phage display technology to identify novel single-chain variable fragments (scFvs) that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size exclusion chromatography combined with multi-angle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.
摘要：
血浆凝溶胶蛋白异常片段的聚集，AGelD187N,是芬兰凝溶胶蛋白淀粉样变性病理生理学的关键事件，全身性淀粉样变性的遗传形式。淀粉样蛋白片段AGelD187N在体内不发挥任何生理作用，与其他蛋白质错误折叠疾病相关的大多数聚集蛋白不同。然而,不存在特异性和有效靶向和中和AGelD187N的治疗剂。我们采用噬菌体展示技术来鉴定与单体AGelD187N中的不同表位结合的新型单链可变片段（scFvs），这些表位通过可变域改组进一步成熟并转化为抗原结合片段（Fab）抗体。产生的抗体片段对全长AGelD187N具有纳摩尔结合亲和力，通过生物层干涉法评估。重要的是,通过硫黄素荧光测定法和透射电子显微镜检查，所有选择用于功能研究的四个Fab均有效抑制全长AGelD187N的淀粉样蛋白形成。两个Fabs,两者都不与先前提出的AGelD187N的原纤维形成区结合，完全阻断AGelD187N的淀粉样蛋白形成。此外，没有小的可溶性聚集体，它们被认为是蛋白质错误折叠疾病的致病物种，是由最有前途的聚集抑制剂成功抑制淀粉样蛋白形成后形成的，如通过尺寸排阻色谱法结合多角度光散射所研究的。我们得出的结论是，全长AGelD187N的所有区域在调节其组装成原纤维方面都很重要，并且发现的表位特异性抗AGelD187N抗体片段为gelsolin淀粉样变性的疾病修饰疗法提供了有希望的起点，这是目前所缺乏的。
