Development of plant cell growth control technology using oligosaccharide of plantglycoprotein as signaling molecule.

1999年，从银杏种子、番茄果实、南瓜幼苗和烟草培养细胞中纯化并鉴定了一些endo-β- n -乙酰氨基葡萄糖苷酶。这些分子量为63 kDa的植物内糖苷酶的最适pH值在6 ~ 7之间，对含有Manα1-2Manα1-3Man单元的高甘露糖型n -聚糖具有较强的活性。这一结果表明，植物内糖苷酶可能具有一个特定于结构单元的共同亚位点，由此产生的游离n -聚糖可能对植物的发育或分化具有关键作用。2000年对植物内糖苷酶的亚细胞分布进行了研究。亚细胞分离分析表明，植物内糖苷酶和高甘露糖型游离n -聚糖定位在细胞质中，而不在内质网或高尔基体中。从2000年到2001年，我们发现至少在每一种分析的植物材料中，在微摩尔（μM）浓度下，存在更多只含有一个GlcNAc残基（Man9 ~ 4GlcNAc1）的n -聚糖。另一方面，虽然也检测到含有n -乙酰基壳生物基片段的植物复合物型游离聚糖（GlcNAc2 ~ 0Man3Xyl1 Fuc1 ~ 0GlcNAc2），但这些类型结构的数量约为高甘露糖型结构的几十分钟之一。植物细胞中的高甘露糖型游离n -聚糖（Man9 ~ 5GlcNAc1）具有共同的核心结构(Manα1-6（Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc）。我们还从银杏种子中纯化并鉴定了α-甘露糖苷酶。由于该酶对含1个GlcNAc残基的高甘露糖型n -聚糖的活性较强，而对含2个GlcNAc残基的n -聚糖的活性较弱，提示该α-甘露糖苷酶可能参与了胞浆中游离n -聚糖的降解。该α-mamosidase的分子克隆及基因在大肠杆菌中的表达正在进行中。从2001年到2002年，我们从水稻细胞中纯化出了内糖苷酶的部分氨基酸序列，并成功克隆了内糖苷酶基因。DNA序列与拟南芥中功能未知蛋白的DNA序列高度同源，提示该蛋白可能是内切-β- n-乙酰氨基葡萄糖苷酶。目前，我们正在构建一种抑制内糖苷酶基因的转基因植物（水稻植物），以证实n -聚糖在植物生长和分化中的作用。此外，从1999年到2001年，我们以豆角α-甘露糖苷酶为模型糖蛋白，揭示了游离n -聚糖可能在低聚蛋白的四级结构形成中起关键作用。游离n -聚糖参与植物细胞生长的这种类似伴侣的功能似乎是关于寡糖功能的一个新概念。少

In 1999, some endo-β-N-acetylglucosaminidases were purified and characterized from Ginkgo seeds, tomato fruits, pumpkin seedlings, and tobacco cultured cells. The optimum pH of these plant endoglycosidases having the molecular weight of 63 kDa was between 6 to 7 and the enzyme showed strong activity toward the high-mannose type N-glycans bearing the Manα1-2Manα1-3Man unit. This result suggested that the plant endoglycosidase would have a common subsite specific for the structural unit and the resulting free N-glycans might have a critical function for the plant development or differentiation.In 2000, the subcellular distribution of the plant endoglycosidase was investigated. Subcellular fractionation analysis using ultracentrifugation method showed that the plant endoglycosidase and the high-mannose type free N-glycans are localized in the cytosol but not in the ER or Golgi apparatus.From 2000 to 2001, we have revealed that at least in every plant material analyzed the high-mannose typ … More e N-glycans with only one GlcNAc residue (Man9〜4GlcNAc1) always existed at the micromolar (μM) concentration. On the other hand, although the plant complex type free glycans with the N-acetylchitobiosyl segment (GlcNAc2〜0Man3Xyl1 Fuc1〜0GlcNAc2) were also detected, the amount of these type structures was about one of the several decade minutes of that of high-mannose type structures. The high-mannose type free N-glycans (Man9〜5GlcNAc1) in plant cells have a common core structure (Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc). We also purified and characterized an α-mannosidase from Ginkgo biloba seed. Since this enzyme showed a strong activity against the high-mannos type N-glycan bearing one GlcNAc residue rather than the N-glycan bearing two GlcNAc residues, suggesting this α-mannosidase could be involved in the free N-glycan degradation produced in the cytosol. The molecular cloning of this α-mamosidase and expression of the gene in E. Coli is in progress.From 2001 to 2002, we have determined the partial amino acid sequences of the endoglycosidase purified from rice cells and succeeded the molecular cloning of the endoglycosidase gene. The DNA sequence determined showed a high homology with a DNA sequence of function-unknown protein in Arabidopsis thaliana, suggesting this Arabidopsis protein may be endo-β-N-acetylglucosaminidase.Now, we are constructing a transgenic plant (rice plant) in which the endoglycosidase gene is suppressed to confirm the N-glycan function involved in plant growth and differentiation.Moreover, from 1999 to 2001, using jack bean α-mannosidase as a model glycoprotein, we have revealed that free N-glycans could play a critical role in the formation of quaternary structure of oligomeric proteins. This chaperone-like function of free N-glycans involved in plant cell growth seems to be a new concept on the oligosaccharide's function. Less