earticle

영어

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 a gene encoding sialyltransferase (ST) from marine invertebrate organism, Ciona savignyi (Csav), and analyzed its phylogenetic relationship with other animal sialyltransferases. Csav-ST was clustered with ST3Gal subfamily. 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.