Nakano Y, Noda K, Endo T, Kobata A, Tomita M
Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Arch Biochem Biophys. 1994 May 15;311(1):117-26. doi: 10.1006/abbi.1994.1216.
CD59 is an 18-kDa glycoprotein widely expressed on human cells. An important structural feature of CD59 is its attachment to the cell surface via a glycosyl-phosphatidylinositol (GPI) anchor. CD59, like many GPI-anchored proteins, has been found in urine, serum, and other body fluids. The structures of the GPI anchor and the asparagine-linked sugar chain of a soluble form of CD59 in urine, U-CD59, were determined. Purified U-CD59 released 1 mol of inositol per mole of protein by nitrous acid deamination, which cleaved between glucosamine and inositol present commonly in the GPI anchor. This indicates that a GPI anchor, which ended with inositol, is linked at the carboxy terminus of U-CD59. The peptide containing an asparagine-linked sugar chain and the peptide containing a glycan portion of the GPI anchor were isolated after trypsin digestion of U-CD59. The asparagine-linked sugar chains and the glycan portion of the GPI anchor were isolated from these peptides following hydrazinolysis or deamination and dephosphorylation, respectively. Their structures were analyzed by sequential exoglycosidase digestion and methylation analyses. The structures of the asparagine-linked sugar chains of U-CD59 were biantennary complex type, only 4.2% of which are monosialylated. The backbone structure of the GPI anchor was similar to that of Try-panosoma brucei variant surface glycoprotein, but showed significant variations in its side-chain moieties. This is the first detailed structural analysis of the human GPI anchor and the first detailed analysis of the carboxyl-terminal structure of the soluble-form GPI-anchored protein. The results indicate that the backbone structure of the GPI anchor is conserved from parasites to human and that at least a part of the soluble-form GPI-anchored protein has the structure produced by the action of glycan-phosphatidylinositol-specific phospholipase D.
CD59是一种18 kDa的糖蛋白，在人类细胞上广泛表达。CD59的一个重要结构特征是它通过糖基磷脂酰肌醇（GPI）锚定在细胞表面。与许多GPI锚定蛋白一样，CD59已在尿液、血清和其他体液中被发现。已确定尿液中可溶性形式的CD59（U-CD59）的GPI锚和天冬酰胺连接糖链的结构。纯化的U-CD59通过亚硝酸脱氨作用，每摩尔蛋白质释放1摩尔肌醇，该反应在GPI锚中常见的葡糖胺和肌醇之间裂解。这表明以肌醇结尾的GPI锚连接在U-CD59的羧基末端。在对U-CD59进行胰蛋白酶消化后，分离出含有天冬酰胺连接糖链的肽段和含有GPI锚聚糖部分的肽段。分别通过肼解或脱氨及去磷酸化作用，从这些肽段中分离出天冬酰胺连接糖链和GPI锚的聚糖部分。通过顺序外切糖苷酶消化和甲基化分析对它们的结构进行了分析。U-CD59的天冬酰胺连接糖链结构为双天线复合型，其中只有4.2%是单唾液酸化的。GPI锚的主干结构与布氏锥虫可变表面糖蛋白的主干结构相似，但在其侧链部分显示出显著差异。这是对人类GPI锚的首次详细结构分析，也是对可溶性形式GPI锚定蛋白羧基末端结构的首次详细分析。结果表明，GPI锚的主干结构从寄生虫到人类是保守的，并且至少部分可溶性形式的GPI锚定蛋白具有由聚糖磷脂酰肌醇特异性磷脂酶D作用产生的结构。
