Study for the biological role of N-glycolylneuraminic acid-containing glycoconjugates

NeuGc是由NeuAc通过N-乙酰残基中的甲基部分羟基化而得。这种羟化反应是通过cMP-N-乙酰神经氨酸(cMP-N-NeuAc)转化为cMP-N-乙醇基cMP-NeuGc进行的，该过程是由一个电子传递系统完成的，该系统包括依赖于NADH的细胞色素b5还原酶、细胞色素b5和cMP-NeuAc羟基酶。CMP-NeuAc羟基酶是cMP-NeuAc羟基化的关键酶，在电子传递系统的最后一步发挥作用。我们之前已经从小鼠肝脏中纯化了cMP-NeuAc羟基酶，并分离出该酶的一个cDNA，它包含一个编码577个氨基酸的蛋白质的ORF。在本研究中，我们研究了细胞内NeuGc含量增加的影响。为了增加NeuGc的量，将肌动蛋白启动子启动子下的含cMP-NeuAc羟基化基因的载体导入3T3细胞。接受载体的细胞具有较高的NeuGc含量(约占总唾液酸的85%)，而对照细胞的NeuGc含量较少(约占总唾液酸的3%)。观察PDGF、EGF、成纤维细胞生长因子和胰岛素的治疗效果。在PDGF、EGF或胰岛素存在下，两种细胞的生长速度几乎相似。然而，在有成纤维细胞生长因子的情况下，高含量NeuGc的细胞生长速度是低NeuGc的1.8倍。

NeuGc is derived from NeuAc by hydroxylation of a methyl moiety in the N-acetyl residue. This hydroxylation occurs by the conversion of CMP-N-acetylneuraminic acid (CMP-NeuAc) to CMP-N-glycolylCMP-NeuGc, which is carried out by an electron transport system involving NADH-dependent cytochrome b5 reductase, cytochrome b5, and CMP-NeuAc hydroxylase. CMP-NeuAc hydroxylase is the key enzyme in the hydroxylation of CMP-NeuAc, and functions at the final step of the electron transport system. We have previously purified CMP-NeuAc hydroxylase from the mouse liver, and isolated a cDNA for this enzyme which contains an ORF encoding a protein of 577 amino acids.In this study, we investigated the effect of the increasing amount of NeuGc in cells. In order to increase the amount of NeuGc, a vector containig CMP-NeuAc hydroxylade cDNA under the actin promoter was transfected into 3T3 cells. The cells receiving the vector had the high amount of NeuGc (about 85% of the total sialic acid), but the control cells had the small amount (about 3% of the total sialic acid). The responses for PDGF,EGF,FGF and insulin were investigated. The growth rate betweem the two cells was almost simillar in the presence of PDGF,EGF or insulin. However, the cells containing the high amount of NeuGc grew 1.8 folds faster than those with the low amount of NeuGc in the presence of FGF.