|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
The purpose of this research project is to elucidate physiological function(s) of free N-glycans and N-glycan releasing enzymes (endo-beta-N-acetylglucosaminidase (endo-beta-GIcNAc-ase) and peptide : N-glycanase (PNGase)) for growth and differentiation of plant cells. We have already purified some endo-beta-GlcNAc-ases and PNGase from various plant cells and revealed their detail substrate specificity. The analysis of substrate specificity of these plant endo-beta-GlcNAc-ase led us to propose that the plant endoglycosidase have a specific subsite for alpha 1-2 mannosyl residue(s) to facilitate the hydrolytic reaction of chitobiose linkages in high-mannose type N-glycans. However, it is still obscure the physiological significance of the plant endo-beta-GlcNAc-ase. For elucidation of physiological function of the enzyme in plant cells, it seems to be very important to clone a gene encoded the endoglycosidase as first step. At this moment, we have not succeed to identify N-terminal or in
ternal amino acid sequences of the enzyme, since the amount of the purified plant endoglycosidase was not enough for such analysis. Therefore, in this fiscal year, to recover the plant endoglycosidase in reasonable amount for amino acid sequence analysis, we have developed a new affinity chromatographic procedure for purification of the plant endoglycosidase using the alkylated yeast invertase (cm-YI) -Sepharose 4B, in which the various high-mannose type N-glycans linked to the denatured glycoprotein would serve as ligands for the plant endo-beta-GlcNAc-ases. Several plant endoglycosidase could bind to cm-YI -Sepharose 4B column and the enzyme activities were recovered by increasing the concentration of NaCl, Using this affinity chromatography, two endo-beta-GlcNAc-ases from Ginkgo seeds and tomato fruits could be purified to homogeneity.
We have revealed that free N-glycans occur in hypocotyls of soybean seedling, bamboo shoot, and developing Ginkgo seed and determined the detailed structures of such free N-glycans. The revealed structures of high-mannose type free N-glycans showed that these N-glycans should be derived by the plant endoglycosidase, since these high-mannose type N-glycans had only one GIcNAc residue at the reducing end. On the contrary, plant complex type free N-glycans should be derived by PNGase, since these oligosaccharides had the intact chitobiose segment. Comparing the amount of high-mannose type structures and plant complex type structures, the relative amount of the former structure overwhelmed that of the latter structure. Less