Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans

An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers

Purification, Characterization, and Gene Expression of Rice Endo-beta-N-Acetylglucosaminidase, Endo-Os

In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-beta-N-acetylglucosaminidase (endo-beta-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-beta-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-beta-GlcNAc-ases and preliminarily reported the gene information of rice endo-beta-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-beta-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-beta-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Man alpha 1-2Man alpha 1-3Man beta 1 unit; this substrate specificity was almost the same as that of other plant endo-beta-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os

Cytosolic Free N-Glycans Are Retro-Transported Into the Endoplasmic Reticulum in Plant Cells

During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-beta-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited beta-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewis(a) epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry beta 1-2xylosyl and/or alpha 1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man(9-7)GlcNAc(1) and Man(9-8)GlcNAc(2)) could be found in microsomes, which almost consist of the ER compartments

大豆発芽時期におけるグリシニン分解酵素の活性変動

　Changes in glycinin-digesting protease activity during soybean germination have been investigated. The glycinin-digesting protease activities of imbibed or germinated soybean seed were assayed by RP‒HPLC using a tryptic peptide from CM‒glycinin or by SDS‒PAGE using CM‒glycinin as the endogenous substrate. Proteolytic activities of the germinated soybean seeds were found through the whole period of germination, the activities were maintained significantly unchanged during germination for 4 days, and then those specific activities declined slowly. AE‒HPLC analysis of the glycinin-digesting protease in the imbibed or germinated soybean seeds showed unchanged peaks corresponding to glycinin- digesting activity, suggesting that the glycinin-digesting protease was not induced during germination but had already been synthesized during seed maturation.　大豆発芽期におけるグリシニン分解酵素 (98 kDa SBP) の活性変動を解析した．大豆種子を４時間水で膨潤後， 25 ℃ 暗黒下で発芽させた．経時的にサンプリングを行い，2M NaCl を含むトリス緩衝液 (㏗ 7.0) により粗酵素を抽出 後，グリシニン由来のトリプシン分解ペプチドを基質としてグリシニン分解酵素の活性変動を逆相 HPLC により追 跡した．その結果，種子膨潤後４日間比活性はほぼ一定の値を保ち，以後徐々に低下することが分かった．次いで， 粗酵素溶液からイオン交換 HPLC により98 kDa SBP を部分精製するとともに，発芽期における 98 kDa SBP の消長 を解析したところ，98 kDa SBP は乾燥種子及び各発芽段階の種子中全てに認められ，かつグリシン分解活性もグリ シニン由来のトリプシン分解ペプチド基質に対する活性と同様に認められた．以上の結果から，98 kDa SBP は種子 発芽に伴い誘導されるプロテアーゼではなく，種子貯蔵型のプロテアーゼであることが明らかになった

Examining the assembly pathways and active microtubule mechanics underlying spindle self-organization

The bipolar organization of the microtubule-based mitotic spindle is essential for the faithful segregation of chromosomes in cell division. Despite our extensive knowledge of genes and proteins, the physical mechanism of how the ensemble of microtubules can assemble into a proper bipolar shape remains elusive. Here, we study the pathways of spindle self-organization using cell-free Xenopus egg extracts and computer-based automated shape analysis. Our microscopy assay allows us to simultaneously record the growth of hundreds of spindles in the bulk cytoplasm and systematically analyze the shape of each structure over the course of self-organization. We find that spindles that are maturing into a bipolar shape take a route that is distinct from those ending up with faulty structures, such as those of a tripolar shape. Moreover, matured structures are highly stable with little occasions of transformation between different shape phenotypes. Visualizing the movement of microtubules further reveals a fraction of microtubules that assemble between extra poles and push the poles apart, suggesting the presence of active extensile force that prevents pole coalescence. Together, we propose that a proper control over the magnitude and location of the extensile, pole-pushing force is crucial for establishing spindle bipolarity while preventing multipolarity.Comment: 22 pages, 5 + 2 figure

Plant complex type free N-glycans occur in tomato xylem sap

Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant

Ginkgo biloba α-fucosidase with activity towards plant complex type N-glycans containing the Lewis a epitope: Purification and characterization

　We have identified, and purified to homogeneity, a high molecular weight Ginkgo biloba α-fucosidase (α-fucosidase Gb, 120 kDa estimated by SDS‒PAGE) with activity against α-fucosylated oligosaccharides. When a Lewis a epitope-containing N-glycan was used as a substrate, α-fucosidase Gb showed optimum activity at approximately pH 5.5, suggesting that it functions in acidic environments such as the vacuole. It remains uncertain, however, whether this Ginkgo α-fucosidase belongs to the GH29 family, since its N-terminal sequence could not be determined, probably due to a chemical modification. α-Fucosidase Gb showed substantial activity towards the α1,3-fucosyl linkage in Lacto-N-fucopentaose III and an α1,4-fucosyl linkage in the Lewis a epitope found in plant complex type N-glycans, indicating an involvement in the degradation process of α-fucosylated oligosaccharides or N-glycoproteins.　銀杏種子から高分子量 （SDS-PAGE で120 kDa） を有し，α-フコース含有オリゴ糖に活性を示すα-フコシダーゼ（α-fucosidase Gb）を均一に精製した．ルイス a エピトープ含有 N-グリカンを基質とした場合，α-fucosidase Gb の至適 pH は 5.5 付近であることから，本酵素は液胞のような酸性環境で機能していることが示唆された．N‒末端アミノ酸配列が化学修飾のため同定できなかったため，本酵素が GH29 ファミリーに属するかどうかは不明である．α-Fucosidase Gb は，Lacto-N-fucopentaose IIIの α1,3-フコース残基やルイス a エピトープ含有の植物複合型N-グリカンのα1,4-フコース残基を加水分解することから，α-フコース含有オリゴ糖やN 型糖タンパク質の分解プロセスに関与することが示唆された

Biological Effects of Negatively Charged Particle-Dominant Indoor Air Conditions

To identify health-promoting indoor air conditions, we developed negatively charged particle-dominant indoor air conditions (NCPDIAC). Experiments assessing the biological effects of NCPDIAC comprised (1) 2.5-h stays in NCPDIAC or control rooms, (2) 2-week nightly stays in control followed by NCPDIAC rooms, (3) 3-month OFF to ON and ON to OFF trials in individual living homes equipped with NPCDIAC in their sleeping or living rooms, and (4) in vitro assays comparing the immune effects between negatively charged particle-dominant and control cell culture incubators. The most significant difference examined between NCPDIAC and control rooms in the 2.5-h stays was an increase in interleukin (IL)-2 with occupancy of the NCPDIAC room. For the 2-week nightly stay experiments, natural killer (NK) cell activity increased with occupancy of the NCPDIAC room. The 3-month OFF to ON trial showed an increase in NK cell activity, while the ON to OFF trial yielded a decrease in NK cell activity. Additionally, the in vitro assays also showed an increase in NK cell activity. The use of NCPDIAC resulted in increased NK cell activity, which has the effect of enhancing immune surveillance for the occurrence of cancer and improving symptoms associated with viral infections