Sialyltransferase is a glycosyltransferase transferring sialic acid from CMP-Neu5Ac to the non-reducing terminal position of glycoconjugates. Twenty different sialyltransferases containing so-called sialylmotifs are highly conserved from invertebrates to vertebrates. These well conserved sialylmotifs suggested that all sialyltransferases had been evolved from a common ancestor. Many sialyltransferases have been known in vertebrate linage, however, only a few have been characterized in non-vertebrates. In this study, we cloned genes encoding sialyltransferase (ST) from marine invertebrate organisms, Ciona savignyi (Csav), Halocynthia roretzi (Hror), and Strongylocentroutus intermedius (Sint), and analyzed their phylogenetic relationship with other animal sialyltransferases. Csav-ST and Hror-ST were clustered with ST3Gal and ST6GalNAc subfamilies, but Sint-ST was not grouped with other sialyltransferases. Among these sialyltransferases, Csav-ST has two putative transcript isoforms identified in of the sea squirt C. savignyi genome database. To indentify a sialyltransferase activity, Csav-ST3Gal was functionally characterized by using recombinant enzyme expressed in Saccharomyces cerevisiae. Csav-ST3Gal is localized to Golgi membrane when expressed in yeast. Enzymatic assays indicate that Csav-ST3Gal is capable of transferring sialic acids to the glycans attached to asialofetuin. Substrate specificities and kinetic properties indicated that Csav-ST3Gal could prefer O-glycans rather than N-glycan of asialoglycoproteins as substrates. Further, it has been displayed that Csav-ST3Gal catalyzes the formation of α(2,3)-linkage by a lectin blot analysis with Maackia amurensis lectin and by the linkage-specific sialidase treatments. In addition, Csav-ST3Gal has three putative N-glycosylation sites in its stem region, Interestingly, only one site for N-glycosylation was occupied with N-glycan when expressed in yeast. By N-glycan trimming with glycosidases treatments, we determined that this glycan of Csav-ST could not be required for in vitro enzyme activity, because of no difference in non-glycosylated and glycosylated sialyltransferase activities. These results indicate that Csav-ST3Gal could be an common ancestral glycosyltransferase belonged to ST3Gal I/II subfamily, transferring sialic acid to Gal-β1,3-GalNAc moiety.
