Edmunds T, Van Patten S M, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J M, Cole E S
Cell and Protein Therapeutics Department, Genzyme Corp, and Genzyme Transgenics Corp, Framingham, MA 01701-9322, USA.
Blood. 1998 Jun 15;91(12):4561-71.
Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
从转基因山羊奶中生产的重组人抗凝血酶（rhAT）纯度达到了99%以上。在体外凝血酶抑制试验中，rhAT的比活性与源自人血浆的抗凝血酶（phAT）相同。然而，rhAT对肝素的亲和力比phAT高四倍。通过反相高效液相色谱、圆二色性、荧光辅助碳水化合物电泳（FACE）、氨基酸序列以及液相色谱/质谱肽图谱分析对rhAT和phAT进行了分析和比较。基于这些分析，除了糖基化差异外，rhAT在结构上被确定与phAT相同。在转基因蛋白的Asn 155位点发现了寡甘露糖结构，而在血浆蛋白上仅观察到复杂结构。rhAT在一些N-连接寡糖上含有用于半乳糖替代的N-乙酰半乳糖胺，以及高度的岩藻糖基化。rhAT的唾液酸化程度低于phAT，并且同时含有N-乙酰神经氨酸和N-羟乙酰神经氨酸。我们推测，rhAT对肝素亲和力的增加是由于Asn 155糖基化位点上存在寡甘露糖型结构以及唾液酸化差异所致。
