Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.与非唾液酸化形式相比，高糖基化蛋白质（hyperglycosylated proteins）稳定性更强、血清半衰期（serum half-life）更长，且对蛋白水解作用（proteolysis）的敏感性更低。这一特性在重组人促红细胞生成素（rhEPO）上体现得尤为显著。近年来，针对非哺乳动物表达宿主（non-mammalian expression hosts）的N糖基工程（N-glycoengineering）技术取得进展，实现了均一性极佳的体内蛋白质唾液酸化（in vivo protein sialylation）。但迄今为止，多唾液酸化N聚糖（multi-sialylated N-glycans）的合成仍仅限于哺乳动物细胞。本研究采用植物基表达系统（plant based expression system），实现了多天线型蛋白质唾液酸化（multi-antennary protein sialylation）。我们将人促红细胞生成素融合蛋白（EPOFc）在本氏烟草（Nicotiana benthamiana）ΔXTFT——一种缺失植物特异性N聚糖残基（plant specific N-glycan residues）的糖基化突变体（glycosylation mutant）——中进行了瞬时表达。将该激素的互补DNA（cDNA）与三类必要基因共同递送至植物中：(i) 糖链分支相关基因、(ii) β1,4-半乳糖基化（β1,4-galactosylation）相关基因，以及(iii) 唾液酸（sialic acid）的合成、转运与转移相关基因。最终获得了携带二、三、四唾液酸化复合N聚糖（bi-, tri- and tetra-sialylated complex N-glycans）的重组EPOFc。这一高度复杂寡糖结构（oligosaccharide structure）的形成，需要11种人类蛋白质在糖基化通路（glycosylation pathway）的不同阶段、不同亚细胞区室（subcellular compartments）内协同表达（coordinated expression）。体外受体结合实验（in vitro receptor binding assays）证实，我们获得了具有生物活性的分子（biologically active molecules）。本研究证明了可在植物体内合成其中一种最为复杂的哺乳动物糖型（mammalian glycoforms），这表明植物对糖基化通路的改变具有极高的耐受度。