Mammalian Sialyltransferase ST3Gal-II: Its Exchange Sialylation Catalytic Properties Allow Labeling of Sialyl Residues in Mucin-Type Sialylated Glycoproteins and Specific Gangliosides

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAc alpha 2,3Gal beta 1,3Ga1NAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-H-3]- or [C-14]sialy1 mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAca2,3-Gal beta 1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was similar to 2-fold stronger than that of asialo fetuin when CMP- [9-H-3]NeuAc was generated in situ from 5'-CMP and [9-(3)]NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,3Gal alpha-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [C-14]sialyl fetuin and [9-H-3]NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,3Gal alpha-O-Me by generating CMP-[C-14]- and [9-H-3]NeuAc through 5'-CMP; only 20.3% C-14 and 28.0% H-3 remained with the parent compounds after the sialyl exchange. The [9-H-3]sialyl-tagged MN glycophorin A, human chorionic gonadotropin beta subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.