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J. Biol. Chem., Vol. 281, Issue 23, 15929-15940, June 9, 2006

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Expression of a Functional Drosophila melanogaster CMP-sialic Acid Synthetase

DIFFERENTIAL LOCALIZATION OF THE DROSOPHILA AND HUMAN ENZYMES^*

Karthik Viswanathan, Noboru Tomiya, Jung Park^¶, Sundeep Singh^¶, Yuan C. Lee, Karen Palter^¶, and Michael J. Betenbaugh¹

From the Departments of Chemical and Biomolecular Engineering and Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and the ^¶Department of Biology, Temple University, Philadelphia, Pennsylvania 19122

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.

Received for publication, November 14, 2005 , and in revised form, February 21, 2006.

^* This work was supported by National Science Foundation Grant BES9814100 and National Institutes of Health Grant 5R01GM067935-03 of the Metabolic Engineering Program and from the Howard Hughes Medical Institute through the undergraduate Biological Science education Program (to J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Chemical and Biomolecular Engineering, The Johns Hopkins University, MD221, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-5461; Fax: 410-516-5510; E-mail: beten{at}jhu.edu.

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