Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.
A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.
Wang
Y
Y
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Abernethy
J L
JL
Eckhardt
A E
AE
Hill
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases; D012756:Sheep; D013363:Submandibular Gland / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine; D013912:Threonine / Q000378:metabolism*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D005700:Galactosyltransferases; D008670:Metals; D017350:N-Acetylgalactosaminyltransferases; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase