Intracellular and extracellular free N-glycans produced by plant cells: occurrence of unusual plant complex-type free N-glycans in extracellular spaces.

As a part of the study to reveal the biological significance of de-N-glycosylation in plants, we analysed the structural features of free N-glycans (FNGs) accumulated inside cells and secreted to the extracellular space using a rice cell culture system. The structural analysis of FNGs obtained from the intracellular fraction revealed that the high-mannose type N-glycans with one GlcNAc residue (GN1-type) occurred at a concentration of ∼10 nmol/g, while the truncated complex type N-glycans with a N, N'-diacetylchitobiosyl unit (GN2-type) occurred at a concentration of ∼1 nmol/g. This result suggested that two kinds of glycoenzymes, cytosolic endo-β-N-acetylglucosaminidase (ENGase) and intracellular acidic peptide:N-glycanse (PNGase), are involved in the production of FNGs in rice cell as well as in other plant cells. On the other hand, in the culture medium, Lewis a epitope-containing complex and high-mannose type FNGs with the N, N'-diacetylchitobiosyl unit were found, suggesting extracellular acidic PNGase to be involved in the release of N-glycans from folded/processed glycoproteins in extracellular space. Furthermore, in the culture medium, we found unusual GN1-FNGs that have a biantennary complex type structure harbouring the Lewis a epitope, suggesting cytosolic ENGase and golgi N-glycan-processing enzymes to be involved in the production of these plant complex type FNGs.