Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.
Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.
复制标题
果蝇 CMP-唾液酸合成酶活性的表征揭示了不寻常的酶特性。
DOI：
10.1042/bcj20160347
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Panin,VladislavM
中科院分区：
文献类型：
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作者：
Mertsalov,IlyaB;Novikov,BorisN;Scott,Hilary;Dangott,Lawrence;Panin,VladislavM
CMP-唾液酸合成酶（CSAS）是唾液酸化途径的关键酶。CSAS产生活化的糖供体CMP-唾液酸，其作为唾液酸转移酶的底物以用唾液酸修饰聚糖末端。与其他动物CSAS通常定位于细胞核，果蝇CSAS（DmCSAS）定位于细胞分泌室，主要是在高尔基体，这表明这种酶具有不同于其脊椎动物对应物的特性。为了验证这一假设，我们纯化了重组DmCSAS并表征了其体外活性。我们的实验揭示了这种酶的几个独特特征。DmCSAS显示特异性forN-乙酰神经氨酸作为底物，显示偏好较低的pH值，并可以与广泛的金属辅因子的功能。当在对应于高尔基体隔室的pH下测试时，该酶与几种金属阳离子（包括Zn 2+、Fe 2+、Co 2+和Mn 2+）显示出显著的活性，而与Mg 2+的活性被发现是低的。蛋白质序列分析和位点特异性诱变鉴定了酶活性所必需的天冬氨酸残基，并预测其参与协调金属辅因子。发现DmCSAS酶活性对于挽救DmCSAS突变体的表型是必需的。最后，我们的实验揭示了酶活性对温度的急剧依赖性。两者合计，我们的研究结果表明，DmCSAS经历了进化适应的pH值和离子环境不同的对应合成酶在脊椎动物。我们的数据还表明，环境温度可以调节果蝇唾液酸化，从而调节神经传递。
CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus,Drosophila melanogasterCSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activityin vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity forN-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+and Mn2+, whereas the activity with Mg2+was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essentialin vivofor rescuing the phenotype ofDmCSASmutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulateDrosophilasialylation, thus modulating neural transmission.