Mucin biosynthesis: purification and characterization of a mucin beta 6N-acetylglucosaminyltransferase.
We have purified, to apparent homogeneity, a mucin beta 6N-acetylglucosaminyltransferase (beta 6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Gal beta 1-3GalNAc alpha benzyl (Bzl) to stabilize the beta 6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Superflow affinity column equilibrated with 1 mM Gal beta 1-3GalNAc alpha Bzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 mumol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the beta 6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The Km values for UDP-GlcNAc and Gal beta 1-3GalNAc alpha Bzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified beta 6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAc beta 1-3Gal beta R as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a beta 1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all beta 6GlcNAc structures found in mucin-type oligosaccharides.
Ropp
P A
PA
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Little
M R
MR
Cheng
P W
PW
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004848:Epithelium / Q000201:enzymology; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis*; D017351:N-Acetylglucosaminyltransferases; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014132:Trachea / Q000201:enzymology*
D009077:Mucins; D009844:Oligosaccharides; D008345:Manganese; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C039548:UDP-N-acetylglucosamine-galactose(1-6)-N-acetylglucosaminyltransferase