Roux L, Holojda S, Sundblad G, Freeze H H, Varki A
Department of Medicine, University of California at San Diego, La Jolla 92093.
J Biol Chem. 1988 Jun 25;263(18):8879-89.
The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).
已有报道几种特定蛋白质的硫酸化N - 连接寡糖的结构。我们最近证明，此类寡糖存在于许多不同类型的组织培养细胞系中（弗里兹，H. H.，和瓦尔基，A.（1986年）《生物化学与生物物理学研究通讯》140，967 - 973）。在此我们报告用³⁵SO₄代谢标记细胞系以及用肽：N - 糖苷酶F释放硫酸化N - 连接寡糖的改进方法，以及其中一些新型寡糖的部分结构。从中国仓鼠卵巢（CHO）细胞和牛肺动脉内皮细胞（CPAE）释放的³⁵SO₄标记链通过凝胶过滤、阴离子交换和凝集素亲和色谱以及各种酶促和化学处理进行表征。每个细胞系都含有一类硫酸化寡糖链，其带有两到六个负电荷，以不同组合的O - 硫酸酯和唾液酸形式存在。这些分子在总³⁵SO₄标记物和总阴离子N - 连接寡糖中都占相当大的比例。它们在缺乏糖胺聚糖链合成的CHO突变体中也相对富集。对来自CPAE细胞的此类分子进行凝集素亲和色谱分析表明，大多数是唾液酸化的多天线复杂型链。硫酸酯完全是初级类型。包括在低pH下用β - 己糖胺酶A处理的顺序外切糖苷酶消化表明，这些硫酸酯中至少三分之一具有以下结构，（分子式；见正文）其中R是基础寡糖的其余部分，SA是唾液酸。除了这些分子外，在CPAE细胞中发现了一组带电量更高的硫酸化N - 连接寡糖，它们与糖胺聚糖具有共同的结构特征，但在CHO细胞中未发现。这些将在以下论文中描述（桑德布拉德，G.，霍洛伊达，S.，鲁克斯，L.，瓦尔基，A.，和弗里兹，H. H.（1988年）《生物化学杂志》263，8890 - 8896）。
