Abstract
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 Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, 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 beta3-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 approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 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 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-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 Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.
Highlights
The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures
Rat liver was chosen as a tissue source for the purification of core 1 3-Gal-T, because our preliminary experiments demonstrated that rat liver homogenates contain relatively high core 1 3-Gal-T activity compared with other organs
4-GP-1 was an excellent acceptor and perhaps slightly better than GalNAc␣1-O-phenyl, sufficient quantities of the synthetic glycopeptide were not available for use to determine precisely its Km value. These results demonstrate that the rat liver core 1 UDP-Gal: GalNAc-␣-R 3-galactosyltransferase has been purified 71,034-fold
Summary
GalNAc␣1-O-phenyl, Gal1,3GalNAc-␣-phenyl UDP-Gal, DTT, ATP, UDP, PMSF, benzamidine, leupeptin, pepstatin A, Triton X-100, bovine submaxillary mucin (type I-S), and the disaccharides Gal1–3GalNAc, Gal1– 4GlcNAc, Gal1– 6GalNAc, Gal1– 6Gal, and Gal1–3GlcNAc were obtained from Sigma. The concentrated sample was loaded onto a column of asialo-BSM-UltralinkTM (1.2 ϫ 5 cm) equilibrated with 25 mM Tris-HCl (pH 7.0), 0.01% Triton X-100, 20 mM MnCl2, 150 mM NaCl, containing 1 mM PMSF, 2 g/ml leupeptin, 1 mM benzamidine, and 0.7 g/ml pepstatin A. Triprep 30 and Centricon 30 (Omicon), and loaded on Superose 12 (1.0 ϫ 35 cm; Amersham Biosciences), which was pre-equilibrated with 25 mM Tris-HCl (pH 7.2), 0.005% Triton X-100, and 150 mM NaCl. Core 1 3-Gal-T was eluted with the same buffer, and fractions (0.25 ml) were collected and assayed for activity. Bovine submaxillary mucin (BSM) (5 mg) was dissolved in 0.80 ml of 1 N HCl and incubated at 100 °C for 90 s and cooled in ice. The reaction mixture was neutralized with NaOH and desalted on a G-25 Sephadex, PD-10 column (Amersham Biosciences) equilibrated with water. Fractions (ϳ100 l) were collected and pooled, and the first radioactive peak was concentrated to 50 l
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