Kosloski Matthew P, Miclea Razvan D, Balu-Iyer Sathy V
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260, USA.
AAPS J. 2009 Sep;11(3):424-31. doi: 10.1208/s12248-009-9119-y. Epub 2009 Jun 5.
Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.
凝血因子 VIII（FVIII）是一种多结构域糖蛋白，是血液凝固级联反应中必不可少的辅助因子。其缺乏或功能障碍会导致甲型血友病，这是一种出血性疾病。使用外源性重组人凝血因子 VIII（rFVIII）进行替代是治疗甲型血友病的一线疗法。糖基化对 FVIII 的活性、稳定性、蛋白质 - 脂质相互作用和免疫原性的作用尚不清楚。为了研究糖基化的作用，通过酶切碳水化合物链生成了去糖基化形式的 FVIII。使用酶联免疫吸附测定、尺寸排阻色谱和凝血活性研究比较了完全糖基化和完全去糖基化形式的 rFVIII（去糖 rFVIII）的生化特性。与完全糖基化的蛋白质相比，去糖基化 FVIII 的生物活性降低。去糖 rFVIII 与含磷脂酰丝氨酸膜相互作用的能力部分受损。数据表明，糖基化显著影响 FVIII 的稳定性和生物学相关的大分子相互作用。在甲型血友病小鼠模型中研究了糖基化对免疫原性的影响。研究表明，糖基化的缺失不会增加免疫原性。
