Firrman Jenni, Wang Qizhao, Wu Wenman, Dong Biao, Cao Wenjing, Moore Andrea Rossi, Roberts Sean, Konkle Barbara A, Miao Carol, Liu LinShu, Li Dong, Xiao Weidong
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA, USA.
Dairy and Functional Foods Research Unit, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA.
Mol Ther Methods Clin Dev. 2020 Jan 15;17:328-336. doi: 10.1016/j.omtm.2019.12.019. eCollection 2020 Jun 12.
It is well known that canine factor VIII (cFVIII) has a higher specific activity than does human FVIII (hFVIII), and it has been previously demonstrated that cFVIII light chain is able to enhance hFVIII activity. The goal of this study was to first determine which amino acids in cFVIII light chain were responsible for enhancing hFVIII activity, and second to use these amino acids to develop a hFVIII variant with enhanced functional activity. We systemically screened segments of cFVIII light chain by testing an array of human-canine light chain hybrids and found that canine amino acids 1857-2147 were key to this enhancement. Each canine amino acid within this span was screened individually using a negative selection method, which led to the identification of 12 aa (JF12) in the FVIII light chain that could enhance activity. Substitution of the corresponding 12 aa into hFVIII (hFVIIIJF12BDD) elevated the specific activity profile . Furthermore, hFVIIIJF12BDD expressed an -displayed increased coagulation activity compared to wild-type, while maintaining normal secretion efficiency. In conclusion, we identified the amino acids in cFVIII that are the key determinants for higher specific activity and may be the basis for future development of therapeutic treatments for hemophilia A.
众所周知，犬因子VIII（cFVIII）比人因子VIII（hFVIII）具有更高的比活性，并且先前已经证明cFVIII轻链能够增强hFVIII活性。本研究的目的，一是确定cFVIII轻链中的哪些氨基酸负责增强hFVIII活性，二是利用这些氨基酸开发一种具有增强功能活性的hFVIII变体。我们通过测试一系列人 - 犬轻链杂交体，系统地筛选了cFVIII轻链的片段，发现犬氨基酸1857 - 2147是这种增强作用的关键。使用阴性选择方法对该范围内的每个犬氨基酸进行单独筛选，从而在FVIII轻链中鉴定出12个能够增强活性的氨基酸（JF12）。将相应的12个氨基酸替换到hFVIII中（hFVIIIJF12BDD）提高了比活性。此外，与野生型相比，hFVIIIJF12BDD表达并显示出凝血活性增加，同时保持正常的分泌效率。总之，我们鉴定出了cFVIII中具有更高比活性的关键决定氨基酸，这可能是未来开发A型血友病治疗方法的基础。
