Elucidation of physiological function of free N-glycans and expression control of N-glycan releasing enzymes in plant cells.

本研究旨在阐明游离N-糖链和N-糖链释放酶(内切-β-N-乙酰氨基葡萄糖苷酶(Endo-β-GIcNAc-ase，Endo-beta-GIcNAc-ase)和多肽：N-葡聚糖酶(PNGase))对植物细胞生长和分化的生理功能(S)。我们已经从各种植物细胞中纯化了一些内切-β-GlcNAc酶和PNGase，并揭示了它们的底物特异性。通过对这些植物内切酶底物专一性的分析，我们提出植物内切糖苷酶具有α1-2甘露糖基的专一性亚基(S)，以促进高甘露糖型N-糖链中的壳二糖键的水解反应。然而，植物内切β-GlcNAc-ase的生理意义仍不清楚。为了阐明该酶在植物细胞中的生理功能，克隆内切糖苷酶基因似乎是非常重要的一步。目前，我们还没有成功地鉴定出N-端或…中的更多的内部氨基酸序列，因为纯化的植物内切糖苷酶的量不足以进行这样的分析。因此，在本财政年度，为了回收合理数量的植物内切糖苷酶用于氨基酸序列分析，我们开发了一种新的亲和层析方法，使用烷基化酵母转化酶(Cm-YI)-Sepharose4B来纯化植物内切糖苷酶，其中与变性糖蛋白相连的各种高甘露糖型N-糖链将作为植物内切-β-GlcNAc-酶的配体。几种植物内切糖苷酶可以结合到Cm-yi-Sepharose4B柱上，并能通过增加NaC l浓度恢复酶的活性。利用这种亲和层析，银杏种子和番茄果实中的两种内切β-GlcNAc酶可以纯化为均一。我们揭示了大豆幼苗、竹笋和发育中的银杏种子的下胚轴中存在游离N-糖链，并确定了这种游离N-糖链的详细结构。揭示的高甘露糖型游离N-糖链的结构表明，这些N-糖链应该是由植物内切糖苷酶产生的，因为这些高甘露糖型N-链的还原末端只有一个GIcNAc残基。相反，植物复合型游离N-糖链应由PNGase产生，因为这些寡糖含有完整的壳二糖片段。比较高甘露糖型结构和植物复合型结构的数量，前者的相对数量超过后者。较少

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 … More 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