Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates
Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates
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DOI：
10.1093/glycob/11.8.685
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发表时间：
2001-08-01
期刊：
影响因子：
4.3
通讯作者：
Kitajima, K
中科院分区：
文献类型：
--
作者：
Nakata, D;Münster, AK;Kitajima, K
2-酮基-3-脱氧-D-甘油基-D-半乳糖基-壬糖酸（KDN）是一种唾液酸（Sia），在脊椎动物的正常发育和肿瘤发生过程中广泛表达。它的表达被认为是由几种酶催化的多个生物合成步骤调节的，包括CMP-Sia合成酶。使用粗酶制剂，显示哺乳动物CMP-Sia合成酶具有非常低的从KDN和CTP合成CMP-KDN的活性，并且来自虹鳟鱼睾丸的相应酶具有高的合成CMP-KDN和CMP-N-乙酰神经氨酸（Neu 5Ac）的活性（Terada等人[1993] J. Biol Chem.，268，2640-2648）。为了证明在粗鳟鱼酶中发现的独特底物特异性是否由单一酶传递，通过基于PCR的策略进行鳟鱼CMP-Sia合成酶的cDNA克隆。鳟鱼酶被证明是由432个氨基酸与两个潜在的核定位信号，和cDNA序列显示53.8%的同一性，小鼠酶。根据Vmax/K-m值，重组鳟鱼酶对KDN和Neu 5Ac都具有高活性（1.1对0.68 min（-1））。相反，重组鼠酶对KDN的活性比Neu 5Ac低15倍（0.23对3.5 min（-1））。北方印迹分析表明，几种大小的mRNA表达在睾丸，卵巢和肝脏中的组织特异性的方式。这些结果表明，至少一种克隆的酶具有有效利用KDN和Neu 5Ac作为底物的能力，并且可用于生产CMP-KDN。
2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al [1993] J. Biol Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the V-max/K-m values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.