Structure of the disaccharide chain of galactosyl-N-acetylgalactosaminyl-protein synthesized in vitro.
In order to obtain a [14C]galactosyl-N-acetylgalactosaminyl-protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N-acetylgalactosaminyl-protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [14C]galactosyl-N-acetylgalactosaminyl-protein product and as glycopeptides by proteolytic digestion of the glycoprotein. The reduced disaccharide consisted of equimolar amounts of galactose and N-acetylgalactosaminitol and was characterized by thin-layer chromatography, high-voltage electrophoresis and gas-liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [14C]galactose was linked to position C-3 on the N-acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [14C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was beta. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: alpha-D-N-acetylgalactosaminyl-protein beta 1 leads to 3 transferase activity.
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000032:analysis; D000818:Animals; D004187:Disaccharides / Q000032:analysis*; D005690:Galactose / Q000031:analogs & derivatives / Q000032:analysis; D006023:Glycoproteins / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000378:metabolism; D009077:Mucins / Q000096:biosynthesis; D013363:Submandibular Gland / Q000378:metabolism*; D013552:Swine; D000519:alpha-Galactosidase; D001616:beta-Galactosidase
D004187:Disaccharides; D006023:Glycoproteins; D009077:Mucins; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; D000116:Acetylgalactosamine; D005690:Galactose