Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts.

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.

Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer.

Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or ß1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.

Plant complex type free N-glycans occur in tomato xylem sap.

Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant. Abbreviations: RP-HPLC: reversed-phase HPLC; SF-HPLC: size-fractionation HPLC; PA-: pyridylamino; PCT: plant complex type; Hex: hexose; HexNAc: N-acetylhexosamine; Pen: pentose; Deoxyhex: deoxyhexose; Man: D-mannose; GlcNAc: N-acetyl-D-glucosamine; Xyl: D-xylose; Fuc: L-fucose; Lea: Lewis a (Galß1-3(Fucα1-4)GlcNAc); PCT: plant complex type; M3FX: Manα1-6(Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; GN2M3FX: GlcNAcß1-2Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; (Lea)1GN1M3FX: Galß1-3(Fucα1-4)GlcNAc1-2 Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA or GlcNAc1-2Manα1-6(Galß1-3(Fucα1-4)GlcNAc1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

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.