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

NeuGc通过N-乙酰基残基中的甲基部分羟基化衍生自NeuAc。这种羟基化通过将CMP-N-乙酰神经氨酸（CMP-NeuAc）转化为CMP-N-羟乙酰基CMP-NeuGc而发生，这是通过涉及NADH依赖性细胞色素b5还原酶、细胞色素b5和CMP-NeuAc羟化酶的电子传递系统进行的。CMP-NeuAc羟化酶是CMP-NeuAc羟化反应的关键酶，在电子传递系统的最后一步起作用。我们以前已经从小鼠肝脏中纯化了CMP-NeuAc羟化酶，并分离了该酶的cDNA，该酶含有编码577个氨基酸的蛋白质的ORF。为了提高NeuGc的表达量，将含有CMP-NeuAc羟基化酶cDNA的载体转染到3 T3细胞中。接受载体的细胞含有大量NeuGc（约占总唾液酸的85%），但对照细胞含有少量NeuGc（约占总唾液酸的3%）。研究了对PDGF、EGF、FGF和胰岛素的反应。在PDGF、EGF和胰岛素存在下，两种细胞的生长速度几乎相同。然而，在FGF存在下，含有高含量NeuGc的细胞比含有低含量NeuGc的细胞生长快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.