Smith Ian W, d'Aquino Anne E, Coyle Christopher W, Fedanov Andrew, Parker Ernest T, Denning Gabriela, Spencer Harold Trent, Lollar Pete, Doering Christopher B, Spiegel Paul Clint
Department of Chemistry, Western Washington University, Bellingham, Washington.
Graduate Program in Molecular and Systems Pharmacology, Graduate Division of Biological and Biomedical Sciences, Laney Graduate School, Emory University, Atlanta, Georgia.
J Thromb Haemost. 2020 Jan;18(1):57-69. doi: 10.1111/jth.14621. Epub 2019 Sep 8.
Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies.
To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin.
The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics.
Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics.
These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.
凝血因子VIII是最古老的基于蛋白质的治疗药物之一，作为一种有效的血友病A治疗药物已有半个世纪。最佳治疗方案是终身每周重复静脉输注凝血因子VIII（FVIII）。尽管总体治疗取得成功，但仍存在重大局限性，包括治疗的侵入性、持续时间、免疫原性和成本。这些问题激发了对生物工程FVIII产品和基因疗法开发的研究。
对一种称为ET3i的FVIII生物工程构建体进行结构表征，它是一种人/猪嵌合的缺失B结构域的异二聚体，具有改善的表达和凝血酶蛋白水解激活后较慢的A2结构域解离。
用X射线晶体学和基于串联质谱的糖蛋白质组学对ET3i的结构进行表征。
在此，我们报告了ET3i的3.2 Å晶体结构，并用液相色谱-串联质谱糖蛋白质组学表征了N-连接聚糖的分布。该结构与人类FVIII蛋白具有显著的保守性，并提供了A2结构域与其余FVIII结构之间界面的详细视图。在晶体中有两个FVIII分子，我们观察到C2结构域相对于其余FVIII结构的两种构象。ET3i改进的模型和立体化学作为支架，生成了改进的、精细化的人类FVIII结构。利用分辨率为3.7 Å和4.0 Å的原始数据集，这个新结构产生了改进的精细化统计数据。
这些改进的结构为下一代工程努力提供了更可靠的模型，以开发具有更长半衰期、更高表达水平和更低免疫原性的FVIII治疗药物。
