Free N-glycans (FNGs) are present at micromolar concentrations in plant cells during their differentiation, growth, and maturation stages. It has been postulated that these FNGs are signaling molecules involved in plant development or fruit ripening. However, the hypothetical biochemical and molecular function of FNGs has not been yet established. The structure of FNGs found ubiquitously in plant tissues such as hypocotyls, leaves, roots, developing seeds, or fruits can be classified into two types: high-mannose type and plant complex type; the former, in most cases, has only one GlcNAc residue at the reducing end (GN1 type), while the latter has the chitobiosyl unit at the reducing end (GN2 type). These findings suggest that endo-β-N-acetylglucosaminidase (ENGase) must be involved in the production of GN1 type FNGs, whereas only peptide:N-glycanase (PNGase) is involved in the production of GN2 type FNGs. It has been hypothesized that cytosolic PNGase (cPNGase) and ENGase in animal cells are involved in the production of high-mannose type FNGs in order to release N-glycans from the misfolded glycoproteins in the protein quality control systems. In the case of plants, it is well known that another type of PNGase, the acidic PNGase (aPNGase) is involved in the production of plant complex type FNGs in an acidic organelle, suggesting the de-N-glycosylation mechanism in plants is different from that in animal cells. To better understand the role of these FNGs in plants, the genes encoding these N-glycan releasing enzymes (ENGase and PNGase) were first identified, and then structure of FNGs in ENGase knocked-out plants were analyzed. These transgenic plants provide new insight into the plant-specific de-N-glycosylation mechanism and putative physiological functions of FNGs. In this review, we focus on the structural features of plant FNGs, as well as functional features of cPNGase/ENGase and plant specific PNGase, and putative functions of FNGs are also discussed.

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.

PGD2 is the major prostanoid produced during the acute phase of allergic reactions. Two PGD2 receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD2-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.

These results suggest that Cry j 1-related oligosaccharides are not major epitopes for IgE or T cells. However, these oligosaccharides have a novel potential to inhibit Cry j 1-specific T cell responses selectively.

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.