Viswanathan Karthik, Tomiya Noboru, Park Jung, Singh Sundeep, Lee Yuan C, Palter Karen, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
J Biol Chem. 2006 Jun 9;281(23):15929-40. doi: 10.1074/jbc.M512186200. Epub 2006 Mar 14.
CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12-24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.
CMP-N-乙酰神经氨酸是糖缀合物生成过程中的一种关键代谢物，这些糖缀合物在发育及其他生理过程中发挥作用。虽然其生成途径在脊椎动物中已得到明确确立，但昆虫和其他原口动物中相关合成酶的存在及功能尚不清楚。在本研究中，我们鉴定了首个来自原口动物谱系的功能性CMP-唾液酸合成酶（DmCSAS），它由黑腹果蝇的一个cDNA克隆表达。随后在其他昆虫物种中鉴定出了同源基因。该基因受发育调控，在胚胎发育12至24小时时首次出现表达，在幼虫和蛹期表达水平较低，而在成虫头部表达大幅富集，这表明其可能在中枢神经系统中发挥作用。当在异源宿主中表达时体外和体内CMP-N-乙酰神经氨酸水平的升高证实了该酶的活性。与所有已知定位于细胞核的脊椎动物CMP-唾液酸合成酶（CSAS）蛋白不同，当在异源哺乳动物和昆虫细胞系中表达时，黑腹果蝇CSAS蛋白靶向高尔基体区室。用人类CSAS的N端序列替换DmCSAS的N端前导序列导致嵌合CSAS蛋白重新定位于细胞核，但同时酶活性丧失。据我们所知，CSAS直系同源物定位于不同细胞内细胞器代表了真核生物中直系同源物蛋白质靶向差异的首个例子，并揭示了原口动物和后口动物进化过程中唾液酸化途径是如何分化的。
