Posttranslational modifications of recombinant myotube-synthesized human factor IX
Posttranslational modifications of recombinant myotube-synthesized human factor IX
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DOI：
10.1182/blood.v97.1.130
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发表时间：
2001-01-01
期刊：
影响因子：
20.3
通讯作者：
High, KA
中科院分区：
文献类型：
--
作者：
Arruda, VR;Hagstrom, JN;High, KA
最近的数据表明，将表达凝血因子IX(F.IX)的腺相关病毒(AAV)载体导入骨骼肌可以导致转基因产物的长期表达，并改善血友病B动物的出血质量。这些数据表明，骨骼肌中可以合成具有生物活性的F.IX。因子IX在肝脏中经历了广泛的翻译后修饰，肝脏是正常的合成部位。除了影响特定的活性，这些翻译后修饰还可以影响蛋白质的回收率、循环中的半衰期和免疫原性。在启动AAV介导的肌肉导向治疗血友病B的人体试验之前，我们对骨骼肌合成的F.IX进行了详细的生化分析，作为模型系统，使用了表达F.IX的AAV载体转导的人肌管。用一种新的策略从条件培养液中提纯F.IX，该方法旨在纯化代表培养液中所有rF.IX种类的物质。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)、N-末端序列分析、化学γ-羧基谷氨酰基分析、糖类分析、酪氨酸硫化和丝氨酸磷酸化等方法对纯化的F.IX进行分析，并测定其比活性。结果表明，肌管合成的F.IX具有与肝脏合成的F.IX相似的比活性。对比活性至关重要的翻译后修饰，包括信号序列和前肽的去除，以及N端谷氨酸残基的伽马羧化，也是相似的，但碳水化合物分析和酪氨酸硫化和丝氨酸磷酸化的评估揭示了差异。在老鼠身上的实验表明，这些差异影响美国血液学学会肌肉合成的F.IX(C)2001的恢复，但不影响半衰期。
Recent data demonstrate that the introduction into skeletal muscle of an adenoassociated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. in addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out, As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma -carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma -carboxylation of the N-terminal glutamic acid residues,are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX. (C) 2001 by The American Society of Hematology.