Purification, Characterization, and Subunit Structure of Rat

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Gal 133GalNAc 13Ser/ Thr, which is also the precursor for many extended Oglycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-Ser/Thr is UDP-Gal:GalNAc-Ser/Thr 3-galactosyltransferase (core1 3-Gal-T). Core 1 3-Gal-T activity, which requires Mn , was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of 84and 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of 43 kDa. Reducing SDS-PAGE of these proteins gave 42and 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 M and an apparent Vmax of 206 mol/mg/h protein using GalNAc 1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Gal 133GalNAc. These studies demonstrate that activity of the core 1 1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.