Stanley P, Atkinson P H
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1988 Aug 15;263(23):11374-81.
Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Lex determinant (Gal beta 1,4(Fuc alpha 1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Lex determinant (NeuAc alpha 2,3Gal beta 1,4(Fuc alpha 1,3)GlcNAc), and both mutants have been shown to possess a distinct alpha(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Lex to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Lex determinant and that G from LEC11/VSV also possesses sialylated Lex. Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of alpha(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking alpha(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.
中国仓鼠卵巢细胞突变体LEC11和LEC12合成的糖蛋白带有Lex决定簇（Galβ1,4(Fucα1,3)GlcNAc），而LEC11细胞合成的糖蛋白还带有唾液酸化Lex决定簇（NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc），并且已证明这两种突变体都拥有一种具有适当特异性的独特α(1,3)-岩藻糖基转移酶来合成这些决定簇（坎贝尔，C.，和斯坦利，P.（1983年）《细胞》35卷，303 - 309页；坎贝尔，C.，和斯坦利，P.（1984年），《生物化学杂志》259卷，11208 - 11214页；霍华德，D.R.，福田，M.，福田，M.N.，和斯坦利，P.（1987年）《生物化学杂志》262卷，16830 - 16837页）。因此LEC11细胞提供了一种以唾液酸化、岩藻糖基化乳糖胺结尾的碳水化合物来源，这是一种相对罕见的结构，当与N - 连接的碳水化合物结合时，以前尚未通过1H核磁共振光谱进行表征。在本文中，我们使用一种对Lex特异的单克隆抗体来表明，在LEC11和LEC12细胞中生长的水疱性口炎病毒（VSV）的G糖蛋白带有Lex决定簇，并且来自LEC11/VSV的G还带有唾液酸化Lex。因此，从LEC11/VSV和LEC12/VSV中纯化的双天线碳水化合物被用于研究唾液酸化和未唾液酸化乳糖胺单元上存在α(1,3)-岩藻糖残基对1H核磁共振光谱的影响。将在500 MHz下从LEC11/VSV和LEC12/VSV糖肽在神经氨酸酶处理前后获得的一维光谱与缺乏α(1,3)-岩藻糖的双天线碳水化合物的光谱进行比较，从而确定了几个新的共振峰。通过在400 MHz下的二维相关光谱（COSY）实现了某些信号的分辨率和耦合常数的测定，并在一维光谱中确定了更多的共振峰。
