Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds

Yoshinobu Kimura, Erika Kitahara

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9-5GlcNAC1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1 GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9-5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1-6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.

Original languageEnglish
Pages (from-to)1847-1855
Number of pages9
JournalBioscience, Biotechnology and Biochemistry
Volume64
Issue number9
Publication statusPublished - 2000

Fingerprint

Seedlings
Soybeans
Structural analysis
Polysaccharides
Seed
Seeds
polysaccharides
soybeans
seedlings
seeds
Sugars
stems
Gels
Mannosidases
mannosidases
Affinity chromatography
Salt removal
Xylose
Jacks
Fractionation

Keywords

  • End-β-n-acetylglucosaminidase
  • Free n-glycan
  • Glycine max
  • N-glycan metabolism
  • Pepetid: N-glycanase

ASJC Scopus subject areas

  • Food Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Chemistry (miscellaneous)
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds. / Kimura, Yoshinobu; Kitahara, Erika.

In: Bioscience, Biotechnology and Biochemistry, Vol. 64, No. 9, 2000, p. 1847-1855.

Research output: Contribution to journalArticle

@article{a36653acd9b743ee9185b45a5713616a,
title = "Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds",
abstract = "The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9-5GlcNAC1, 93{\%}), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1 GlcNAc2, 7{\%}), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9-5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1-6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.",
keywords = "End-β-n-acetylglucosaminidase, Free n-glycan, Glycine max, N-glycan metabolism, Pepetid: N-glycanase",
author = "Yoshinobu Kimura and Erika Kitahara",
year = "2000",
language = "English",
volume = "64",
pages = "1847--1855",
journal = "Bioscience, Biotechnology and Biochemistry",
issn = "0916-8451",
publisher = "Japan Society for Bioscience Biotechnology and Agrochemistry",
number = "9",

}

TY - JOUR

T1 - Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds

AU - Kimura, Yoshinobu

AU - Kitahara, Erika

PY - 2000

Y1 - 2000

N2 - The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9-5GlcNAC1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1 GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9-5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1-6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.

AB - The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9-5GlcNAC1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1 GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9-5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1-6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.

KW - End-β-n-acetylglucosaminidase

KW - Free n-glycan

KW - Glycine max

KW - N-glycan metabolism

KW - Pepetid: N-glycanase

UR - http://www.scopus.com/inward/record.url?scp=0034279448&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034279448&partnerID=8YFLogxK

M3 - Article

C2 - 11055387

AN - SCOPUS:0034279448

VL - 64

SP - 1847

EP - 1855

JO - Bioscience, Biotechnology and Biochemistry

JF - Bioscience, Biotechnology and Biochemistry

SN - 0916-8451

IS - 9

ER -