Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G, Conradt H S
Department of Gene Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
Eur J Biochem. 1995 Sep 15;232(3):718-25. doi: 10.1111/j.1432-1033.1995.718zz.x.
The human beta-trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of beta-trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2,3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.
人β-微球蛋白已被克隆，并首次在哺乳动物宿主细胞系中表达。通过将细胞与质粒pMT-βTP或pAB3-1（分别含有编码人分泌糖蛋白β-微球蛋白或抗凝血酶III的cDNA）以及含有人类高尔基体酶CMP-神经氨酸：Gal(β1-4)GlcNAc-Rα2,6-唾液酸转移酶（ST6N）基因的pABSial共转染，构建了具有改变的末端唾液酸化特性的稳定BHK-21细胞系。通过免疫亲和色谱法纯化β-微球蛋白，并对N-连接寡糖进行碳水化合物结构分析。发现酶解释放的寡糖中90%是二天线型链，这与来自人脑脊液的天然β-微球蛋白情况相同。总寡糖的约90%以1:5的比例回收在单唾液酸和双唾液酸部分中。发现与人类ST6N共表达的β-微球蛋白的单唾液酸化寡糖中，NeuAc以α2,6-或α2,3-连接的比例相同。通过1H-NMR分析以及HPLC获得的峰面积计算，发现双唾液酸部分60%的分子中NeuAc以α2,3-和α2,6-连接到Galβ(1-4)GlcNAc-R，而该部分40%的分子中NeuAc仅以α2,3-连接到Gal(β1-4)GlcNAc-R。α2,6-连接的NeuAc显示优先连接到二天线型结构的Gal(β1-4)GlcNAc(β1-2)Man(α1-3)分支上。因此，本研究中观察到的新引入的重组人ST6N的体内特异性支持了先前报道的牛初乳ST6N活性的体外分支特异性。此外，这些研究证明了具有新型糖基化特性的基因工程哺乳动物宿主细胞系适合用于生产人源化糖基化分泌重组多肽。
