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

Abstract

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 CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (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 characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-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+, while 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 coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. 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 regulate Drosophila sialylation, thus modulating neural transmission.

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

“…Taken together, our results suggested that the CSAS activity is a regulatory “bottleneck” of the pathway. Notably, previous in vitro analyses revealed a uniquely steep dependence of CSAS enzymatic activity on temperature, demonstrating that the activity increases with temperature about an order of magnitude between 20 ° and 40°C, a range of temperature of Drosophila natural habitats (Mertsalov et al, 2016a). Drosophila is a poikilotherm, with its body temperature being regulated by ambient conditions, thus the changes in environmental temperature are predicted to modulate neural functions via the effect on CSAS activity.…”

“…Genetic and phenotypic analyses previously demonstrated that CSAS and DSiaT genes work in the same functional pathway affecting neural transmission (Repnikova et al, 2010;; Islam et al, 2013). Although the biochemical activities of their protein products were characterized in vitro (Koles et al, 2004;; Mertsalov et al, 2016a), the roles of these genes in sialylation were not examined in vivo . Considering the unusual separation of CSAS and DSiaT expression patterns at the cellular level, we decided to test their requirements for the biosynthesis of sialylated glycans in vivo .…”

“…The two penultimate steps in the biosynthetic pathway of sialylation are mediated by sialic acid synthase (also known as N- acetylneuraminic acid synthase, or NANS) and CMP-sialic acid synthetase (CSAS, also known as CMAS), the enzymes that synthesize sialic acid and carry out its activation, respectively (Varki et al, 2015). These enzymes have been characterized in Drosophila and found to be closely related to their mammalian counterparts (Kim et al, 2002;; Viswanathan et al, 2006;; Mertsalov et al, 2016b). In vivo analyses of DSiaT and CSAS demonstrated that Drosophila sialylation is a tightly regulated process limited to the nervous system and required for normal neural transmission.…”

Structural and functional characterization of CMP-N-acetylneuraminate synthetase fromVibrio cholerae

“…All CMAS enzymes require divalent cations (Mg 2+ or Mn 2+ ) for optimal activity (Horsfall et al, 2010;Sellmeier et al, 2015;Mertsalov et al, 2016). Biochemical and structural data on the closely related lipopolysaccharide-specific (L) and capsule-specific (K) CMP-Kdo synthetases (L-CKS and K-CKS, respectively) from E. coli (Heyes et al, 2009), H. ducreyi (Samuels et al, 1999) and Aquifex aeolicus (AA-LCKS; Schmidt et al, 2011) suggest the involvement of two Mg 2+ ions in the catalytic reaction.…”

Diverse subcellular localizations of the insect CMP-sialic acid synthetases

“…In stark contrast to vertebrate CSSs, Drosophila CSS (DmCSS) is localized predominantly in the Golgi (Viswanathan et al 2006;Islam et al 2013). This is the most prominent property of DmCSS, although DmCSS has recently been shown to have distinct pH-and metal ions-dependency than human CSS (Mertsalov et al 2016). Expression of DmCSS is tightly regulated in CNS, and in vivo inactivation studies of the CSS gene showed the same neuron-related abnormal phenotypes as that of DSiaT (Islam et al 2013).…”