Biosynthesis in vitro of core lacto-series glycosphingolipids by N-acetyl-D-glucosaminyltransferases from human colon carcinoma cells, Colo 205.

Two N-acetyl-D-glucosaminyltransferases have been detected in human colon carcinoma Colo 205 cells. These enzymes catalyze the biosynthesis in vitro of the core-glycolipid of Type 1 and Type 2 lacto-series antigens and of the polylactosamine-containing longer chain antigenic structures, respectively. The first enzyme, GlcNAcT-1, which catalyzes the formation of lactotriosylceramide [LcOse3Cer, beta-D-GlcpNAc-(1----3)-LcOse2Cer, the core for all lacto-series Type 1 and Type 2 chains] from lactosylceramide [beta-D-Galp-(1----4)-D-Glcp-Cer, LcOse2Cer] and UDP-GlcNAc shows optimum activity in the presence of nonionic detergent Triton CF-54. The other enzyme, GlcNAcT-2, which catalyzes the biosynthesis in vitro of iLcOse5Cer [beta-D-GlcpNAc-(1----3)-nLcOse4Cer, the core for polylactosamine-containing antigens] from nLcOse4Cer [beta-D-Galp-(1----4)-LcOse3Cer] and UDP-GlcNAc, is optimally active with the zwitterionic detergent, Zwittergent 3-14, when membrane-bound. Both of these activities, however, can be extracted from the membrane by use of a nonionic detergent. Triton X-114, with nearly the same efficiency. These two transferases showed different pH optima, different cation and anion effects, and differential heat-inactivation patterns at 55 degrees. Permethylation studies of the radioactive products isolated from both of the enzyme-catalyzed reactions using respective 3H-substrates and nonradioactive UDP-GlcNAc showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed products. This indicated the presence of a (1----3)-linked beta-D-GlcpNAc group at the nonreducing end in both cases. The linkage of the beta-D-GlcpNAc group to the subterminal D-Gal residue in the two products was confirmed by an almost 90% cleavage of the terminal [3H]GlcNAc group by purified clam and papaya beta-D-hexosaminidases.