Plant cultured cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 26.8%], GalGNM4 18.4%], GalGNM3 19.6%], and GalGNM3X 35.2%]) is different from that found in intracellular glycoproteins (M7A 9.3%], M7B 15.9%], M6B 19.5%], M5 1.4%], M3X 6.6%], GalGNM5 35.5%], and GalGNM3 11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.