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Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract
journal contribution
posted on 2018-04-16, 05:55 authored by Ryota Uemura, Mikako Ogura, Chihiro Matsumaru, Tsuyoshi Akiyama, Megumi Maeda, Yoshinobu KimuraAcidic 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.
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Acidic PNGasefree N-glycanstransgenic plantenzyme assayaffinity chromatographyFNG: free N-glycanFuc: L-fucoseGal: D-galactoseGlcNAc: N-acetyl-D-glucosamineHPLC: high-performance liquid chromatographyMan: D-mannoseNeuNAc: N-acetylneuraminic acidNeuNAc2Gal2GlcNAc2Man3GlcNAc2: NeuNAcα2–6Galβ1–4GlcNAcβ1–2Manα1–6(NeuNAcα2–6Galβ1–4GlcNAcβ1–2Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAcNeuNAc2Gal2GlcNAc2Man3GlcNAc1: NeuNAcα2–6Galβ1–4GlcNAcβ1–2Manα1–6(NeuNAcα2–6Galβ1–4GlcNAcβ1–2Manα1–3)Manβ1–4GlcNAcPA-: pyridylaminoPNGase: peptide:N-glycanasePNGase-A: aPNGase from almond seedPNGase-Le: aPNGase from tomato (Solanum lycopersium L.)PTC: plant complex typeRCA120: Ricinus communis agglutinin (120 kDa)RP-HPLC: reversed-phase HPLCSF-HPLC: size-fractionation HPLCXyl: D-xylose
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