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

Abstract

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 [LcOse 3Cer, β- d-Glc pNAc-(1→3)-LcOse 2Cer, the core for all lacto-series Type 1 and Type 2 chains] from lactosylceramide [β- d-Gal p-(1→4)- d-Glc p-Cer, LcOse 2Cer] 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 iLcOse 5Cer [β- d-Glc pNAc-(1→3)-nLcOse 4Cer, the core for polylactosamine-containing antigens] from nLcOse 4Cer [β- d-Gal p-(1→4)-LcOse 3Cer] 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°. 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 β- d-Glc pNAc group at the nonreducing end in both cases. The linkage of the β- d-Glc pNAc 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 β- d-hexosaminidases.