Unidentified N-glycans by N-glycosidase A were Identified by N-glycosidase F under Denaturing Conditions in Plant Glycoprotein
- Authors
- Kim, Jeongeun; Kim, Jihye; Ryu, Changsoo; Lee, Jaeryong; Park, Chi Soo; Jin, Mijung; Kang, Minju; Kim, Ahyon; Mun, Chulmin; Kim, Ha Hyung
- Issue Date
- 2022
- Publisher
- Bentham Science Publishers
- Keywords
- Allergenicity; mammalian cell cultures; N-glycan; N-glycosidase; plant biotechnology; plant glycoprotein
- Citation
- Protein and Peptide Letters, v.29, no.5, pp 440 - 447
- Pages
- 8
- Journal Title
- Protein and Peptide Letters
- Volume
- 29
- Number
- 5
- Start Page
- 440
- End Page
- 447
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/60762
- DOI
- 10.2174/0929866529666220328152941
- ISSN
- 0929-8665
1875-5305
- Abstract
- Background: The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the N-glycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of N-glycosidase A activity in plant glycoproteins remains unclear. Objective: The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by N-glycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). Methods: Liquid chromatography–tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. Results: N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional core-fucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 core-fucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by N-glycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only N-glycansodase F. Conclusion: These results demonstrate that N-glycosidase A alone is insufficient to release the N-glycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required. © 2022 Bentham Science Publishers.
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