Characterization of a beta 1----3-N-acetylglucosaminyltransferase associated with synthesis of type 1 and type 2 lacto-series tumor-associated antigens from the human colonic adenocarcinoma cell line SW403.
Previous studies have indicated that activation of a normally unexpressed beta 1----3-N-acetylglucosaminyltransferase is responsible for the accumulation of a wide diversity of both type 1 and 2 lacto-series antigens in human colonic adenocarcinomas. A beta 1----3-N-acetylglucosaminyltransferase has been solubilized from the human colonic adenocarcinoma cell line SW403 by 0.2% Triton X-100 and some of its properties have been studied. The enzyme was active over a broad pH range from 5.8 to 7.5 and had a strict requirement for Mn2+ as a divalent metal ion. Transfer of N-acetylglucosamine (GlcNAc) to lactosylceramide was optimal when assayed in the presence of a final concentration of Triton CF-54 of 0.3%. Inclusion of CDPcholine in the reaction mixture stimulated the activity by protecting the UDP[14C]GlcNAc from hydrolysis by endogenous enzymes. The kinetic parameters of the enzyme were studied. Km values for acceptors nLc4 and nLc6 were determined to be 0.19 mM for each. However, the Vmax values calculated for these acceptors were 150 and 110 pmol/h/mg protein for nLc4 and nLc6, respectively, suggesting reduced potential for further elongation as the chain length increases. The Km for UDPGlcNAc was determined to be 0.17 mM. Studies of the acceptor specificity have indicated transfer of GlcNAc occurs mainly to type 2 chain nonfucosylated structures. However, elongation of the type 1 chain structure Lc4 was also detected.
D000230:Adenocarcinoma / Q000201:enzymology* / Q000276:immunology; D015703:Antigens, CD; D000951:Antigens, Neoplasm / Q000096:biosynthesis*; D015295:Antigens, Tumor-Associated, Carbohydrate; D002460:Cell Line; D002855:Chromatography, Thin Layer; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000276:immunology; D003902:Detergents; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007790:Lactosylceramides; D017351:N-Acetylglucosaminyltransferases; D012995:Solubility
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D003902:Detergents; D006028:Glycosphingolipids; D007790:Lactosylceramides; C009744:CDw17 antigen; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase