Expression of a Functional Drosophila melanogaster CMP-sialic Acid Synthetase

Abstract

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.

Dynamic Developmental Elaboration of N-Linked Glycan Complexity in the Drosophila melanogaster Embryo

“…The identification and characterization of key synthetic and processing enzymes in Drosophila (GlcNAcT-I, Fdl, dSiaT, N-acetylneuraminic acid phosphate synthase, and CMP-sialic acid synthase), coupled with the expanding genomic annotation of relevant Drosophila genes, have built an increasingly convincing argument that complex glycans should be present in this organism (72,73). Also, extensive efforts to bioengineer mammalian-like glycosylation into insect cell lines have demonstrated that dipteran and lepidopteran cells can accommodate the components necessary for complex glycan synthesis (74,75).…”

Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal)

“…The humans cannot synthesize CMP-Neu5Gc from CMP-Neu5Ac because the human CMAH gene was inactivated 2 million years ago [32,33], an activity that was independently lost in the ferrets [34], birds and reptiles [35] (Figure 2B) . Interestingly, a cmas gene was identified and characterized in the D. melanogaster genome [36,37] and moreover, 1 gne , 2 st and 2 neu genes were identified in the porifera Oscarella carmella [28,29,38,39], and a SLC35A1 -related gene was identified in the tunicate Ciona intestinalis and C. elegans genomes (personal data) suggesting the ancient occurrence and subsequent divergent evolution of the sialylation machinery.…”

Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

“…Recent studies identified CSAS in Drosophila [7] and found that its function is important for the control of neural transmission [8]. CSAS mutations in Drosophila cause defects in neural excitability and locomotion, while also resulting in temperature-sensitive paralysis and significantly shortened life span [8].…”