• Journal of Plant Biotechnology
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• v.41 no.3
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• pp.159-167
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• 2014

In eukaryotes, proteins that are secreted into ER are post-translationally modified by N-glycosylation, the patterns of which are significantly different between plant and animal cells. Biotechnology industry has already produced a number of therapeutic glycoproteins in plant cells. However, the aberrant glycosylation of therapeutic recombinant proteins in plant systems can cause immune problems in humans. Therefore, it is important to develop strategies for producing non-glycosylated forms to preserve biological activity and native conformation by a peptide: N-glycanase (PNGase). In this study, we try to isolate PNGase T gene from tomato, which can use as a platform plant for biotechnology industry. We isolated a cDNA (GenBank Accession number KM401550) from tomato leaves with 1,767 bp, which encoded a polypeptide of 588 amino acids with a predicted molecular mass of 65.8 kDa. We also investigated the expression patterns of PNGase T during fruit ripening of tomato. The transcripts of PNGase T, which were constitutively induced in tomato fruit from green stage, were significantly increased and reached a peak at orange stage. After which, those transcripts were continuously reduced. The expression pattern of PNGase T was coincided well with transcripts profiles of metacaspase gene, LeMCA, and senescence-related gene members of ACC synthase, LeACS2, LeACS4, and LeACS6, for ethylene biosynthesis during fruit ripening. These results suggest that PNGase T is involved in a de-glycosylation process associated with senescence and fruit ripening.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.191-195
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• 2003

The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diretyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N'-diacetyl chitobiose was about $60\%$. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylaton, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of tie sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.

• Mass Spectrometry Letters
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• v.4 no.2
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• pp.38-40
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• 2013

Glycans released from ovalbumin by PNGase F were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using three different dihydroxybenzoic acid (DHB) matrix systems: 2,5-DHB, 2,6-DHB, and a 2,5-DHB/2,6-DHB binary matrix. Relative to the results obtained with the single-component matrices (2,5-DHB or 2,6-DHB), the 2,5-DHB/2,6-DHB binary matrix boasted lower background noise and higher sensitivity. A total of 16 glycan peaks were observed using the 2,5-DHB/2,6-DHB binary matrix, while only 10 and 9 glycan peaks were observed using the 2,5-DHB and 2,6-DHB matrices, respectively.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.495-495
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• 2003

• Korean Journal of Food Science and Technology
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• v.29 no.4
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• pp.630-634
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• 1997

A fluorescence tagging agent, FMOC-Cl (9-fluorenylmethyl chloroformate) was used for the determination of 1-amino-oligosaccharide intermediates generated from glycoproteins by peptide-N $(N-acetyl-{\beta}-D-glucosaminyl)$ asparagine amidase (N-Glycanase, PNGase F). The derivatives were separated on an Amido 80 column by HPLC using a gradient system with 25 to 51% aqueous acetonitrile and monitored by a fluorometric detector. The detection limit of FMOC-amino-oligosaccharides was $0.05{\sim}1.5$ pmol with fluorometric detection at 278 nm.

• Mass Spectrometry Letters
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• v.8 no.1
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• pp.14-17
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• 2017

The effect of cationization agent concentration on glycan detection via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was investigated using $Na^+$ ions in the form of NaCl as the cationization agent. NaCl solution concentrations ranging from 1 mM to 1 M were investigated. Glycans from ovalbumin were mixed with the cationization agent solution and the 2,5-dihydroxybenzoic acid (2,5-DHB) matrix solution in a volume ratio of 1:1:1. The resulting mixture was loaded onto the MALDI plate. Two MALDI-TOF MS instruments (Voyager DE-STR MALDI-TOF MS and Tinkerbell RT MALDI-TOF MS) were used for detection of glycans. The best detection, in terms of the number of identified glycans, the peak intensity, and the signal-to-noise (S/N) ratio, was obtained with NaCl concentrations of 0.01-0.1 M for both MALDI-TOF MS instruments.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.29-32
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• 2004

Inhibition of the early N-glycosylation process in the endoplasmic reticulum prevents the activation of tyrosinase, a key enzyme for melanin biosynthesis. This work aims at evaluating the increased activity of N-glycosylation inhibitors in vitro b, employing a nano-sized pH-sensitive liposome as a delivery carrier. Melexsome, a pH-sensitive nano carrier loaded with glycosylation inhibitos, was prepared by the hydration method with phospholipids and cholresterol-based amphiphiles. Inhibitory effects of Melexsome on the N-glycosylation process were evaluated by EndoH ＆ PNGaseF digestion and the western blotting. Melanin synthesis was also monitored after treatment with Melexsome Interestingly, Melexsome effectively increased the efficacy of N-glycosylation inhibitors. Melexsome was also much more efficiently translocated into the cytoplasm as observed in CLSM. These results demonstrated that the amphiphilic lipid-based pH-sensitive nano-carriers could be, used as an efficient delivery system for N-glycosylation inhibitor to enhance the effects of skin whitening cosmetics.