Pallister Edward G, Choo Matthew S F, Tai Jien-Nee, Leong Dawn S Z, Tang Wen-Qin, Ng Say-Kong, Huang Kun, Marchesi Andrea, Both Peter, Gray Christopher, Rudd Pauline M, Flitsch Sabine L, Nguyen-Khuong Terry
Bioprocessing Technology Institute, Agency for Science Technology and Research, Singapore 138668.
School of Chemistry and Manchester Institute of Biotechnology (MIB), The University of Manchester, Manchester M1 7DN, United Kingdom.
Biochemistry. 2020 Sep 1;59(34):3123-3128. doi: 10.1021/acs.biochem.9b00563. Epub 2019 Oct 11.
Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.
唾液酸是存在于许多动物糖蛋白中的糖类，在生物制药领域具有特殊意义，因为唾液酸化的缺失会降低生物活性。在此，我们描述了如何利用来自[具体来源未提及]的α-2,6-唾液酸转移酶显著提高中国仓鼠卵巢细胞（CHO）产生的α-1-抗胰蛋白酶的唾液酸化水平。对唾液酸化产物的详细分析表明，除了预期的半乳糖α-2,6-唾液酸化外，还产生了第二个双唾液酸半乳糖基序Neu5Ac-α2,3(Neu5Ac-α2,6)Gal，据我们所知，这种基序从未在哺乳动物糖蛋白上被检测到。我们在多酶反应中利用[具体来源未提及]的这种双唾液酸半乳糖活性来生产高度唾液酸化的α-1-抗胰蛋白酶。研究了这种独特的双唾液酸化对α-1-抗胰蛋白酶活性的影响，并开发了一套用于在复杂混合物中鉴定这种新的双唾液酸半乳糖基序的质谱方法工具包。
