Mammalian sialyltransferase ST3Gal-II: Its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin type sialylated glycoproteins and specific gangliosides

摘要

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some members 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 NeuAcα2,3Galβ1,3GalNAc- unit [Chandrasekaran, E V et al. (2008) Biochemistry 47, 320–330]. The present study shows by using [9-³H] or [¹⁴C] sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and NeuAcα2,3Galβ1,3GalNAc- unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease as evident from a) radiolabeleling of fetuin was ~2 fold that of asialo fetuin when CMP- [9-³H] NeuAc was generated in situ from 5′-CMP and [9-³H] NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II; b) ST3Gal-II exchanged radiolabels between [¹⁴C] sialyl fetuin and [9-³H] NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[¹⁴C] and -[9-³H] NeuAc through 5′-CMP; only 20.3% [¹⁴C] and 28.0%[³H] remained with the parent compounds after the sialyl exchange. The [9-³H] sialyl tagged MN Glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins and haptoglobin were analysed 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. The present 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.