Enzymatic properties of endo-β-N-acetylglucosaminidases from developing tomato fruits and soybean seeds: substrate specificity of plant origin endoglycosidase

Changes in Structural Features of FreeN-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

“…It is noteworthy that the in red mature fruits, Man 9 GlcNAc 1 and Man 8 GlcNAc 1 made up to 37.3% and 33.4% of total free N-glycans, indicating that these two structures were predominant species, and that the activity of cytosolic -mannosidase is suppressed at the late stage of fruit ripening. In a previous study, 13) we found that partially purified tomato ENGase (endo-LE) is highly active towards high-mannose type N-glycans, especially Man 9 GlcNAc 2 -PA and Man 8 GlcNAc 2 -PA. Hence the significant accumulation of Man 9 GlcNAc 1 and Man 8 GlcNAc 1 at high concentrations in red mature might indicate that endo-LE was vigorously involved in the de-N-glycosylation machinery for stimulating final ripening of the tomato fruits.…”

“…An enzyme solution, crude extract from tomato fruits, (50 ml) was mixed with M6B (120 pmol) and M3FX (74 pmol, as an internal standard) in 0.5 M MES buffer (25 ml), pH 6.0, containing 10 mM EDTA and 80 mM swainsonine. 13) After incubation at 37 C for 1 h, the reaction was stopped by heating at 100 C for 3 min. After centrifugation, an aliquot (20 ml) of the resulting supernatant was analyzed with a Jasco 880-PU HPLC apparatus with a Jasco Intelligent spectrofluorometer and the Cosmosil 5C18-AR column (0:6 Â 25 cm, Nacalai Tesque, Kyoto, Japan).…”

“…11,12) As a part of a study to elucidate the physiological functions of free N-glycans and the significance of the de-N-glycosylation mechanism working at the differentiating and developing stages of plant cells, Kimura et al analyzed the structural features of free N-glycans occurring in developing seeds, seedlings, and shoots, [5][6][7][8][9] and the functional futures of de-N-glycosylation enzymes, endo--N-acetylglucosaminidase (ENGase) and peptide: N-glycanase (PNGase). 9,[13][14][15] They found that two types of free N-glycan, a high-mannose type and the a plant complex type, are always found in developing seeds, hypocotyls undergoing elongation, and ripening fruits. [5][6][7][8][9][10] In such free N-glycans, most high-mannose type structures have a single GlcNAc residue at their reducing end moieties, while plant complex type free N-glycans with xylose/fucose residues have an Nacetylchitobiosyl unit, indicating that the former structures are produced by ENGase and the latter structures by PNGase.…”

“…[5][6][7][8][9][10] In such free N-glycans, most high-mannose type structures have a single GlcNAc residue at their reducing end moieties, while plant complex type free N-glycans with xylose/fucose residues have an Nacetylchitobiosyl unit, indicating that the former structures are produced by ENGase and the latter structures by PNGase. It has been found that plant ENGase is highly active against high-mannose type N-glycans bearing the Man 1-2Man 1-3Man 1-4GlcNAc 1-4GlcNAc unit 9,12,13) and that plant PNGase can release high-mannose type, hybrid type, and complex type N-glycans from the peptide backbone. 14,15) These facts suggest that plant ENGase plays a critical role in the accumulation of high-mannose type free N-glycans bearing one GlcNAc residue at their reducing end moieties, although the involvement of cytosolic PNGase in the turnover of misfolded nascent glycoproteins in plant cells remains to be elucidated.…”

Regulation of Substrate Specificity of Plant α-Mannosidase by Cobalt Ion:In VitroHydrolysis of High-Mannose TypeN-Glycans by Co2+-ActivatedGinkgoα-Mannosidase

“…9) As part of a study to elucidate the physiological function(s) of free N-glycans and deglycosylation mechanism in developing plant cells, we purified and characterized several endo--N-acetylglucosaminidases (endo--N-GlcNAcase) and peptide:N-glycanase (PNGase). 8,[10][11][12][13][14] On the basis of structural analysis of such free N-glycans in plant cells and functional features of plant endo--NGlcNAc-ase and PNGase, we suggested that the highmannose type should be generated by the endo--NGlcNAc-ase and plant complex type N-glycans by PNGase.…”