Glycoproteins secreted from suspension-cultured tobacco BY2 cells have distinct glycan structures from intracellular glycoproteins

Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular glycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35-39]. Extracellular glycoproteins have six distinct N-glycans (marked by *) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40% (Man₅GlcNAc₂, 28.8%; Man₆GlcNAc₂*, 6.4%; and Man₇GlcNAc₂*, 3.8%), while the plant-complex-type structures account for nearly 60% (GlcNAc₂Man₃ Xyl₁GlcNAc₂*, 32.1%; GlcNAc₁Man₃Xyl₁GlcNAc₂ (containing two isomers)*, 6.2%; GlcNAc₂Man₃ GlcNAc₂*, 4.9%; Man₃Xyl₁Fuc₁GlcNAc₂, 8.3%; and Man₃Xyl₁GlcNAc₂, 3.7%).