Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.
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.
Ju
Tongzhong
T
W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Cummings
Richard D
RD
Canfield
William M
WM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography; D005700:Galactosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D021122:Protein Subunits; D051381:Rats
D021122:Protein Subunits; D005700:Galactosyltransferases; C076385:glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase