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Occurrence of complex type free N-glycans with a single GlcNAc residue at the reducing termini in the fresh-water plant, Egeria densa

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Abstract

In our previous study, we found unique free N-glycans (FNGs), which carry a single GlcNAc residue (GN1) at the reducing-end side and the Lewis-a epitope at the non-reducing-end side, in the culture broth of rice cells. Based on the FNG structural features and the substrate specificity of plant ENGase, we hypothesized that there might be a novel biosynthetic mechanism responsible for the production of these unique GN1-FNGs, in which high-mannose type (HMT)-GN1-FNGs produced in the cytosol from misfolded glycoproteins by ENGase are transported back into the endoplasmic reticulum and processed to plant complex type (PCT)-GN1-FNGs in the Golgi apparatus. Until now, however, PCT-GN1-FNGs had only been found in the culture broth of rice cultured cells and never in plants, suggesting that the formation of PCT-GN1-FNGs might be generated under special or artificial conditions. In this study, we confirm the presence of PCT-GN1-FNGs, HMT-GN1-FNGs and PCT-GN2-FNGs in the fresh-water plant Egeria densa. These results suggest that a mechanism responsible for the production of PCT-GN1-FNG is present in native plant tissues.

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Abbreviations

ER:

Endoplasmic reticulum

ERAD:

ER-associated degradation

ENGase:

endo-β-N-acetylglucosaminidase

PNGase:

peptide:N-glycanase

FNG:

Free N-glycan

GN1-FNG:

Free N-glycan bearing one GlcNAc residue at the reducing end side

GN2-FNG:

Free N-glycan bearing two GlcNAc residues at the reducing end side

HMT-FNG:

High-mannose type FNG

PCT-FNG:

Plant complex type FNG

PA-:

Pyridylamino

RP-HPLC:

Reversed-phase HPLC

SF-HPLC:

Size-fractionation HPLC

ESI-MS:

Electrospray ionization mass spectrometry

MS/MS:

Tandem mass

Hex:

Hexose

DeoxHex:

Deoxyhexose

HexNAc:

N-acetyl-D-hexosamine

Pen:

Pentose

Man:

D-mannose

Gal:

D-galactose

GlcNAc:

N-acetyl-D-glucosamine

M3XGN1:

Manα1-6(Manβ1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

GN2M3XGN1:

GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

Gal1GN2M3XGN1:

Galβ1-3GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA or GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

Gal1Fuc1GN2M3XGN1:

Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA or GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

Gal2Fuc1GN2M3XGN1:

Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA or Galβ1-3GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

Gal2Fuc2GN2M3XGN1:

Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAc-PA

GN2M3XGN2:

GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc-PA

Gal1GN2M3XGN2:

Galβ1-3(GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc-PA or GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc-PA

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Acknowledgements

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Basic Research C (no. 24580494) and Fostering Joint International Research (no. 15 K07841) to M.M.), Research Grants of Mizutani Foundation for Glycoscience (to Y.K.). We are grateful to the Department of Instrumental Analysis, Advanced Science Research a nonsensical phrase Center, Okayama University, for ESI-MS analysis.

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Authors and Affiliations

  1. Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-Naka, Okayama, 700-8530, Japan

    Megumi Maeda, Misato Tani, Christopher J. Vavricka & Yoshinobu Kimura

  2. Faculty of Agriculture, Division of Agricultural Science, Okayama University, 1-1-1 Tsushima-Naka, Okayama, 700-8530, Japan

    Megumi Maeda, Natsuki Ebara & Yoshinobu Kimura

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  1. Megumi Maeda
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Correspondence to Yoshinobu Kimura.

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Maeda, M., Ebara, N., Tani, M. et al. Occurrence of complex type free N-glycans with a single GlcNAc residue at the reducing termini in the fresh-water plant, Egeria densa . Glycoconj J 34, 229–240 (2017). https://doi.org/10.1007/s10719-016-9758-z

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