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Protein Expr Purif. 2019 Jun;158:81-88. doi: 10.1016/j.pep.2019.02.014. Epub 2019 Feb 27.

Production of recombinant human acid β-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Author information

1
Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do, 54896, Republic of Korea.
2
Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon, 22212, Republic of Korea.
3
Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do, 54896, Republic of Korea; Biology Department, University of Education, Hue University, 34 Le Loi, Hue, Viet Nam.
4
Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06944, Republic of Korea.
5
Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon, 22212, Republic of Korea. Electronic address: kimdi@inha.ac.kr.
6
Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do, 54896, Republic of Korea; National Institute of Horticultural & Herbal Science (NIHHS), Rural Development Administration (RDA), Wanju, Jeollabuk-do, 55365, Republic of Korea. Electronic address: nskims@jbnu.ac.kr.

Abstract

Gaucher disease is an inherited metabolic disease caused by genetic acid β -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

KEYWORDS:

Acid β-glucosidase (GBA); Gaucher disease; N-acetylglucosaminyltransferase-I (GnT1); N-glycosylation; Rice cell suspension culture; Rice gnt1 mutant

PMID:
30822514
DOI:
10.1016/j.pep.2019.02.014

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