Muchmore E A, Milewski M, Varki A, Diaz S
Department of Medicine, San Diego Veterans Administration Medical Center, California.
J Biol Chem. 1989 Dec 5;264(34):20216-23.
N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.
N-羟乙酰神经氨酸（Neu5Gc）是人类的一种癌胚抗原，在啮齿动物中受发育调控。我们探究了N-乙酰神经氨酸羟化酶的生物学特性，该酶负责将母体唾液酸N-乙酰神经氨酸（Neu5Ac）转化为Neu5Gc。我们发现，鼠骨髓瘤细胞系所有区室中的主要唾液酸是Neu5Gc。用唾液酸前体[6-³H]N-乙酰甘露糖胺对这些细胞进行脉冲追踪分析表明，大多数新合成的Neu5Gc最初出现在与CMP结合的胞质低分子量池中。在不同追踪时间，膜结合唾液酸上Neu5Gc的百分比与CMP结合池中Neu5Gc的百分比密切平行，而游离唾液酸池中Neu5Gc的百分比最初非常低，仅在追踪后期才升高。这意味着从Neu5Ac到Neu5Gc的转化主要发生在Neu5Ac处于其糖核苷酸形式时。支持这一点的是，来自各种组织和细胞的羟化酶将CMP-Neu5Ac转化为CMP-Neu5Gc，但对游离或α-糖苷键结合的Neu5Ac无活性。此外，大部分酶活性存在于胞质溶胶中。对分离的完整高尔基体囊泡的研究表明，CMP-Neu5Gc可以被转运并用于将Neu5Gc转移到糖缀合物上。我们还研究了该酶的一般特性。CMP-Neu5Ac的Km在0.6 - 2.5微摩尔范围内。对Neu5Ac的β-甲基糖苷无活性。另一方面，抑制研究表明该酶识别底物的5'-磷酸基团和嘧啶碱基。综上所述，这些数据使我们能够提出Neu5Gc的生物合成和再利用途径，最初从Neu5Ac的转化主要发生在糖核苷酸水平。随后Neu5Gc的释放和再利用可以解释在细胞所有唾液酸池中发现的较高稳态水平的Neu5Gc情况。
