We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.

It is known that oligosaccharyltransferase (OST) has hydrolytic activity toward dolichol-linked oligosaccharides (DLO), which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation. Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of DLO, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the ER in yeast. The possible role of FNGs on protein folding is discussed.

High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-β-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Δeng mutants (T0 and T1 generations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Δeng1-2, -22, and -26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Δeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Δeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Δeng mutants (T2 generation) also showed loss of ENGase activity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Δeng mutants and the wild type. The Δeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.

Deletion of α-1,3/4-fucosidase activity in Arabidopsis thaliana resulted in the accumulation of GN1-type free N-glycans with the Lewis a epitope (GN1-FNG). This suggests that the release of α-fucose residue(s) may trigger rapid degradation of the plant complex-type (PCT) GN1-FNG. The fact that PCT-GN1-FNG has rarely been detected to date is probably due to its easier degradation compared with PCT-GN2-FNG.

Parkinson\'s disease is characterized by the accumulation of amyloid, which consists of α-synuclein (α-Syn). To screen compounds with amyloid aggregation inhibitory activity, an effective method for the preparation of α-Syn is a prerequisite. We established a simpler method for α-Syn preparation using freeze-thaw treatment of transformed Escherichia coli. Furthermore, we found that the high-mannose type free N-glycans could prevent α-Syn aggregation.

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Complex N-glycan modification of secretory glycoproteins in plants is still not well understood. Essential in animals, where a lack of complex N-glycans is embryo-lethal, their presence in plants seemed less relevant for a long time mostly because Arabidopsis thaliana cgl1 mutants lacking N-acetyl-glucosaminyltransferase I (GNTI, the enzyme initiating complex N-glycan maturation in the Golgi apparatus) are viable and showed only minor impairments regarding stress tolerance or development. A different picture emerged when a rice (Oryza sativa) gntI T-DNA mutant was found to be unable to reach the reproductive stage. Here, we report on tomato (Solanum lycopersicum) lines that showed severe impairments upon two RNA interference (RNAi) approaches. Originally created to shed light on the role of core α1,3-fucose and β1,2-xylose residues in food allergy, plants with strongly reduced GNTI activity developed necrotic fruit-attached stalks and early fruit drop combined with patchy incomplete ripening. Correspondingly, semiquantitative RT-PCR of the abscission zone (az) revealed an increase of abscission markers. Also, GNTI-RNA interference (RNAi) plants were more susceptible to sporadic infection. To obtain vital tomatoes with comparable low allergenic potential, Golgi α-mannosidase II (MANII) was chosen as the second target. The resulting phenotypes were oppositional: MANII-reduced plants carried normal-looking fruits that remained attached for extended time without signs of necrosis. Fruits contained no or only few, but enlarged, seeds. Furthermore, leaves developed rolled-up rims simultaneously during the reproductive stage. Trials to cross MANII-reduced plants failed, while GNTI-reduced plants could be (back-)crossed, retaining their characteristic phenotype. This phenotype could not be overcome by ethephon or indole-3-acetic acid (IAA) application, but the latter was able to mimic patchy fruit ripening in wild-type. Phytohormones measured in leaves and 1-aminocyclopropane-1-carboxylic acid (ACC) contents in fruits showed no significant differences. Together, the findings hint at altered liberation/perception of protein-bound N-glycans, known to trigger auxin-like effects. Concomitantly, semiquantitative RT-PCR analysis revealed differences in auxin-responsive genes, indicating the importance of complex N-glycan modification for hormone signaling/crosstalk. Another possible role of altered glycoprotein life span seems subordinate, as concluded from transient expression of Arabidopsis KORRIGAN KOR1-GFP fusion proteins in RNAi plants of Nicotiana benthamiana. In summary, our analyses stress the importance of complex N-glycan maturation for normal plant responses, especially in fruit-bearing crops like tomato.

During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited β-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewisa epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry β1-2xylosyl and/or α1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man9-7GlcNAc1 and Man9-8GlcNAc2) could be found in microsomes, which almost consist of the ER compartments.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.