Chen Helen Y, Challa Anil K, Varki Ajit
Glycobiology Research and Training Center, Department of Medicine, University of California San Diego, La Jolla, California 92093-0687, USA.
J Biol Chem. 2006 Mar 24;281(12):7825-33. doi: 10.1074/jbc.M512379200. Epub 2006 Jan 24.
Sialic acids are sometimes 9-O-acetylated in a developmentally regulated and cell-type-specific manner. Cells naturally expressing the disialoganglioside GD3 often O-acetylate the terminal sialic acid residue, giving 9-O-acetyl-GD3 (9AcGD3), a marker of neural differentiation and malignant transformation. We also reported that Chinese hamster ovary cells transfected with GD3 synthase can spontaneously O-acetylate some of the newly synthesized GD3. It is unclear whether such phenomena result from induction of the 9-O-acetylation machinery and whether induction is caused by the GD3 synthase protein or by the GD3 molecule itself. We now show that exogenously added GD3 rapidly incorporates into the plasma membrane of Chinese hamster ovary cells, and 9AcGD3 is detected after approximately 6 h. The incorporated GD3 and newly synthesized 9AcGD3 have a half-life of approximately 24 h. This phenomenon is also seen in other cell types, such as human diploid fibroblasts. Inhibitors of gene transcription, protein translation, or endoplasmic reticulum-to-Golgi transport each prevent induction of 9-O-acetylation, without affecting GD3 incorporation. Inhibition of the initial clathrin-independent internalization of incorporated GD3 also blocks induction of 9-O-acetylation. Thus, new synthesis of one or more components of the 9-O-acetylation machinery is induced by incorporation and internalization of GD3. Prepriming with structurally related gangliosides fails to accelerate the onset of 9-O-acetylation of subsequently added GD3, indicating a requirement for specific recognition of GD3. To our knowledge, this is the first example wherein a newly expressed or exogenously introduced ganglioside induces de novo synthesis of an enzymatic machinery to modify itself, and the first evidence for a mechanism of induction of sialic acid O-acetylation.
唾液酸有时会以发育调控和细胞类型特异性的方式进行9-O-乙酰化修饰。天然表达双唾液酸神经节苷脂GD3的细胞通常会将末端唾液酸残基进行O-乙酰化修饰，生成9-O-乙酰-GD3（9AcGD3），它是神经分化和恶性转化的标志物。我们还报道过，转染了GD3合酶的中国仓鼠卵巢细胞能够自发地将一些新合成的GD3进行O-乙酰化修饰。目前尚不清楚这种现象是否源于9-O-乙酰化修饰机制的诱导，以及这种诱导是由GD3合酶蛋白还是由GD3分子本身引起的。我们现在发现，外源添加的GD3能迅速整合到中国仓鼠卵巢细胞的质膜中，大约6小时后可检测到9AcGD3。整合的GD3和新合成的9AcGD3的半衰期约为24小时。这种现象在其他细胞类型中也能观察到，比如人二倍体成纤维细胞。基因转录抑制剂、蛋白质翻译抑制剂或内质网到高尔基体运输抑制剂均可阻止9-O-乙酰化修饰的诱导，且不影响GD3的整合。抑制整合的GD3最初的非网格蛋白依赖性内化也会阻断9-O-乙酰化修饰的诱导。因此，9-O-乙酰化修饰机制中一种或多种组分的新合成是由GD3的整合和内化所诱导的。用结构相关的神经节苷脂进行预引发并不能加速随后添加的GD3的9-O-乙酰化修饰的起始，这表明需要对GD3进行特异性识别。据我们所知，这是新表达或外源引入的神经节苷脂诱导酶促机制从头合成以修饰自身的首个例子，也是唾液酸O-乙酰化修饰诱导机制的首个证据。
