13C-sialic acid labeling of glycans on glycoproteins using ST6Gal-I
13C-sialic acid labeling of glycans on glycoproteins using ST6Gal-I
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DOI：
10.1021/ja804614w
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发表时间：
2008-09-10
影响因子：
15
通讯作者：
Prestegard, James H.
中科院分区：
文献类型：
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作者：
Macnaughtan, Megan A.;Tian, Fang;Prestegard, James H.
N-连接到天冬酰胺或O-连接到丝氨酸或苏氨酸的聚糖是糖蛋白的标志，糖蛋白是一类在哺乳动物蛋白质组中占主导地位的蛋白质。这些聚糖在细胞中发挥重要作用，在某些情况下是蛋白质活性所必需的。核磁共振（NMR）光谱是研究聚糖结构和相互作用的有力工具，特别是在利用异核（如13 C）的形式中。本文介绍了一种利用α-2，6-唾液酸转移酶（ST 6 Gal-I）将13 C-N-乙酰神经氨酸（NeuAc或唾液酸）酶促添加到糖蛋白上的方法。ST 6 Gal-I本身是一种糖蛋白，并且在该初始应用中，说明了其自身聚糖的标记和通过NMR观察这些聚糖。在哺乳动物HEK 293细胞中表达来自大鼠ST 6 Gal-I的催化结构域。用质谱法分析来自两个糖基化位点的聚糖，发现其含有唾液酸化的双触角结构。同位素标记方法包括用神经氨酸酶从ST 6 Gal-I中去除天然NeuAc残基，用凝集素亲和柱分离神经氨酸酶，并将合成的13 C-CMP-NeuAc加入去唾液酸化的ST 6 Gal-I中。由于ST 6 Gal-I上各种13 C-NeuAc加合物的化学位移分散，在一个3D实验中观察到糖环的1H − 13 C3 − 13 C2原子。
Glycans that are eitherN-linked to asparagine orO-linked to serine or threonine are the hallmark of glycoproteins, a class of protein that dominates the mammalian proteome. These glycans perform important functions in cells and in some cases are required for protein activity. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying glycan structure and interactions, particularly in a form that exploits heteronuclei such as13C. Here an approach is presented that that uses α-2,6-sialyltransferase (ST6Gal-I) to enzymatically add13C-N-acetylneuraminic acid (NeuAc or sialic acid) to glycoproteins after their preparation using nonbacterial hosts. ST6Gal-I is itself a glycoprotein, and in this initial application, labeling of its own glycans and observation of these glycans by NMR are illustrated. The catalytic domain from rat ST6Gal-I was expressed in mammalian HEK293 cells. The glycans from the two glycosylation sites were analyzed with mass spectrometry and found to contain sialylated biantennary structures. The isotopic labeling approach involved removal of the native NeuAc residues from ST6Gal-I with neuraminidase, separation of the neuramindase with a lectin affinity column, and addition of synthesized13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating1H−13C3−13C2atoms of the sugar ring.