Structural analysis of free N-glycans occurring in soybean seedlings and dry seeds.
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 alpha-mannosidase digestion, alpha-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 (Man 9 approximately 5 GlcNAc1, 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 (Man3Xyl1GlcNAc2, 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 (Man 9 approximately 5 GlcNAc1) in the soybean seedlings have a common core structural unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. 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.
Kimura
Y
Y
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan. yosh8mar@cc.okayama-u.ac.jp
Kitahara
E
E
D000117:Acetylglucosamine / Q000032:analysis; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D008361:Mannosidases; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D013025:Glycine max / Q000737:chemistry*
D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011134:Polysaccharides; D008361:Mannosidases; D000117:Acetylglucosamine