Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes
Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes
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DOI：
10.1093/glycob/cwr034
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发表时间：
2011-08-01
期刊：
影响因子：
4.3
通讯作者：
Kim, Jung Hoe
中科院分区：
文献类型：
--
作者：
Son, Young-Dok;Jeong, Yeon Tae;Kim, Jung Hoe
具有暴露的半乳糖 (Gal) 残基的治疗性糖蛋白会被肝细胞中的脱唾液酸糖蛋白受体迅速从血流中清除。已使用各种方法来增加唾液酸的含量，唾液酸占据 N-或 O-连接聚糖的末端位点，从而延长治疗性糖蛋白的半衰期。我们通过中国仓鼠卵巢（CHO）细胞唾液酸化途径的基因工程增强了人促红细胞生成素（EPO）的唾液酸化。 GNE（尿苷二磷酸-N-乙酰氨基葡萄糖2-差向异构酶）/MNK（N-乙酰甘露糖胺激酶）在唾液酸生物合成的前两个步骤中起关键作用，通过反馈机制受到胞苷单磷酸（CMP）-唾液酸的调节。由于唾液尿症患者细胞无法通过反馈机制调节唾液酸生物合成，因此建立了各种唾液尿症样突变大鼠GNE并进行体外活性测定。与野生型相比，GNE/MNK-R263L-R266Q 突变体显示出 93.6% 的相对活性，并且不表现出反馈抑制。将唾液酸突变的大鼠 GNE/MNK、中国仓鼠 CMP-唾液酸转运蛋白和人 α2,3-唾液酸转移酶 (α2,3-ST) 的基因同时转染到重组人 (rh) 产生 EPO 的 CHO 细胞中。唾液酸突变的 GNE/MNK (R263L-R266Q) 表达使工程细胞的 CMP-唾液酸浓度显着（> 10 倍）增加。工程细胞产生的 rhEPO 的唾液酸含量比对照细胞高 43%。与对照相比，工程细胞产生的rhEPO的四唾液酸化聚糖的比例增加了约32%，但脱唾液酸和单唾液酸化聚糖的比例降低了约50%。这些发现表明，唾液酸突变的大鼠 GNE/MNK 有效增加了细胞内 CMP-唾液酸水平。新构建的宿主 CHO 细胞系通过过度表达唾液酸突变的 GNE/MNK、CMP-SAT 和 α 2,3-ST，产生更高唾液酸化的治疗性糖蛋白。
Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human alpha 2,3-sialyltransferase (alpha 2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (> 10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased similar to 32%, but ratios of asialo- and mono-sialylated glycans were decreased similar to 50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and alpha 2,3-ST.