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 mere 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 similar to 5)GlcNAc(1), 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 (Man(3)Xyl(1) GlcNAc(2), 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 similar to 5)GlcNAc(1)) in the soybean seedlings have a common core structural unit; Man alpha 1-6(Man1-3) Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. 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.