Macnaughtan Megan A, Tian Fang, Liu Shan, Meng Lu, Park Seongha, Azadi Parastoo, Moremen Kelley W, Prestegard James H
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, USA.
J Am Chem Soc. 2008 Sep 10;130(36):11864-5. doi: 10.1021/ja804614w. Epub 2008 Aug 14.
Glycans that are either N-linked to asparagine or O-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 as 13C. Here an approach is presented that that uses alpha-2,6-sialyltransferase (ST6Gal-I) to enzymatically add 13C-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 synthesized 13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various 13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating 1H-13C3-13C2 atoms of the sugar ring.
与天冬酰胺N连接或与丝氨酸或苏氨酸O连接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功能，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，特别是以利用13C等异核的形式。本文介绍了一种方法，该方法使用α-2,6-唾液酸转移酶（ST6Gal-I）在使用非细菌宿主制备糖蛋白后，将13C-N-乙酰神经氨酸（NeuAc或唾液酸）酶促添加到糖蛋白上。ST6Gal-I本身就是一种糖蛋白，在这个初步应用中，展示了其自身聚糖的标记以及通过NMR对这些聚糖的观察。大鼠ST6Gal-I的催化结构域在哺乳动物HEK293细胞中表达。用质谱分析了两个糖基化位点的聚糖，发现其含有唾液酸化的双天线结构。同位素标记方法包括用神经氨酸酶去除ST6Gal-I上的天然NeuAc残基，用凝集素亲和柱分离神经氨酸酶，以及将合成的13C-CMP-NeuAc添加到去唾液酸化的ST6Gal-I中。在一个将糖环的1H-13C3-13C2原子相关联的三维实验中，观察到了由于ST6Gal-I上各种13C-NeuAc加合物导致的化学位移分散。
