Runkel L, Meier W, Pepinsky R B, Karpusas M, Whitty A, Kimball K, Brickelmaier M, Muldowney C, Jones W, Goelz S E
Biogen, Inc., Cambridge, Massachusetts 02142, USA.
Pharm Res. 1998 Apr;15(4):641-9. doi: 10.1023/a:1011974512425.
Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined.
In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences.
IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure.
Together these results suggest that the greater biological activity of IFN-beta-1a is due to a stabilizing effect of the carbohydrate on structure.
两种重组干扰素-β产品已被批准用于治疗多发性硬化症，一种是具有预测天然氨基酸序列的糖基化形式（干扰素-β-1a），另一种是非糖基化形式，其具有Met-1缺失和Cys-17至Ser突变（干扰素-β-1b）。确定了干扰素-β-1a和干扰素-β-1b之间活性差异的结构基础。
采用体外抗病毒、抗增殖和免疫调节试验直接比较这两种干扰素-β产品。使用尺寸排阻色谱法（SEC）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、热变性和X射线晶体学来检查结构差异。
在标准抗病毒试验中，干扰素-β-1a的比活性为20×10⁷IU/mg，干扰素-β-1b为2×10⁷IU/mg，干扰素-β-1a的活性比干扰素-β-1b高10倍。在干扰素-β-1a和干扰素-β-1b之间已知的结构差异中，只有糖基化影响体外活性。干扰素-β-1a的去糖基化导致总活性降低，这主要是由不溶性二硫键连接的干扰素沉淀物的形成引起的。去糖基化还导致对热变性的敏感性增加。干扰素-β-1b的SEC数据显示出具有降低抗病毒活性（约为0.7×10⁷IU/mg）的大的可溶性聚集体。干扰素-β-1a的晶体学数据显示，聚糖与肽主链形成氢键，并使一个不带电荷的表面免受溶剂暴露。
这些结果共同表明，干扰素-β-1a更大的生物学活性归因于碳水化合物对结构的稳定作用。
