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

阐明植物细胞中游离 N-聚糖的生理功能和 N-聚糖释放酶的表达控制。

• 批准号: 09660358
• 负责人: KIMURA Yoshinobu
• 金额: $ 2.11万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660358/
• 关键词: plant glycoprotein; free N-glycan; endoglycosidase; glycoamidase; N-glycan structure; N-glycan function; glycobioilogy; 糖鎖生物学; plant glycoprotein; N-glycan; free N-glycan; endoglycosidase; glycoamidase; glycobiology; N-glycan structure; N-g

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

本研究项目的目的是阐明游离N-聚糖和N-聚糖释放酶（内切-β-N-乙酰氨基葡萄糖苷酶（内切-β-GlcNAc-酶）和肽：N-聚糖酶（PNGase））在植物细胞生长和分化中的生理功能。我们已经从各种植物细胞中纯化了一些内切-β-GlcNAc-酶和PNGase，并揭示了它们的详细底物特异性。对这些植物内切-β-GlcNAc-酶的底物特异性的分析使我们提出植物内切糖苷酶具有α 1-2甘露糖残基的特异性亚位点，以促进高甘露糖型N-聚糖中的壳二糖键的水解反应。然而，植物内切β-GlcNAc酶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，首先克隆该内切糖苷酶的编码基因是非常重要的。目前，我们还没有成功地鉴定出N-末端或 ...更多信息 由于纯化的植物糖苷内切酶的量不足以进行这样的分析，因此不能对酶的内部氨基酸序列进行分析。因此，在本财政年度，为了回收合理量的植物糖苷内切酶用于氨基酸序列分析，我们开发了一种新的亲和色谱法，用于使用烷基化酵母转化酶（cm-YI）-Sepharose 4 B纯化植物糖苷内切酶，其中与变性糖蛋白连接的各种高甘露糖型N-聚糖将作为植物内切-β-GlcNAc-酶的配体。几种植物内切糖苷酶都能与CM-YI -Sepharose 4 B柱结合，且随着NaCl浓度的增加，酶活性恢复。利用这种亲和层析法，从银杏种子和番茄果实中纯化了两种内切-β-GlcNAc-酶。并确定了这些游离N-聚糖的详细结构。高甘露糖型游离N-聚糖的结构表明，这些N-聚糖应该是由植物糖苷内切酶衍生的，因为这些高甘露糖型N-聚糖在还原末端只有一个GlcNAc残基。相反，植物复合型游离N-聚糖应该是由PNGase衍生的，因为这些寡糖具有完整的壳二糖片段。高甘露糖型结构与植物复合型结构的相对含量比较，前者的相对含量大于后者。少

项目成果

Y.Kimura.et al.: "Enzymatic properties of Ginkgo biloba endo-β-N-acetylglucosaminidase and N-glycan Structures of storage glycoproteins in the seeds" Biosci.Biotechnol.Biochem.62(2). 251-261 (1998)

Y.Kimura.等人：“银杏内切-β-N-乙酰氨基葡萄糖苷酶的酶特性和种子中储存糖蛋白的N-聚糖结构”Biosci.Biotechnol.Biochem.62(2) (1998)。

Palacpac.N., Y.Kimura et al.: "Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells" Biosci.Biotechnol.Biochem.63(1). 31-34 (1999)

Palacpac.N.、Y.Kimura 等人：“来自烟草 BY2 悬浮培养细胞的糖蛋白的 N-连接寡糖的结构”Biosci.Biotechnol.Biochem.63(1)。

Y.Kimura et al.: "Enzymatic properties of endo-β-N-acetylglucosaminidases from tomato fruits and dcycloping soybean seed:Substrate specificity of plant endoglycosidase" Biochime.Biophys.Acta. (in press). (1998)

Y.Kimura 等人：“番茄果实和 dcycloping 大豆种子的内切 β-N-乙酰氨基葡萄糖苷酶的酶学特性：植物内切糖苷酶的底物特异性”Biochime.Biophys.Acta（出版中）。

木村吉伸: "植物糖蛋白質糖鎖の構造と機能および植物細胞由来のN-グリカン遊離酵素" 日本農芸化学会誌. 71(61). 612-616 (1997)

Yoshinobu Kimura：“植物糖蛋白糖链和植物细胞中的 N-聚糖释放酶的结构和功能”，日本农业化学学会杂志 71(61) (1997)。

Palacpac, N.Q., Yoshida, S., Sakai, H., Kimura, Y., Fujiyama, K., Yoshida, T., and Seki, T.: "Expression of human beta1,4-galactosyl transferase gene modifies N-glycosylation in tobacco BY2 suspension cells" Proc.Natl.Acad.Sci.USA. (in press).

Palacpac, N.Q.、Yoshida, S.、Sakai, H.、Kimura, Y.、Fujiyama, K.、Yoshida, T. 和 Seki, T.：“人 β1,4-半乳糖基转移酶基因的表达修饰 N-糖基化