ヒマ種子カルス化に伴うアスパラギン結合型糖鎖の構造変化

Using glycotechnology procedures, structural changes in asparagine-linked sugar chains (N-glycans) of glycoproteins in Ricinus communis seeds during dedifferentiation (callus induction) have been explored. N-Glycans were released from the glycoproteins in the 2,4-D derived callus tissues by hydrazinolysis, and the resulting oligosaccharides were N-acetylated and coupled with 2-aminopyridine. Structures of the purified pyridylaminated (PA-) N-glycans could be deduced by two-dimensional (2D) sugar chain mapping method. Structural analysis clearly showed that the relative amount of high-mannose type N-glycans of the endospermic glycoproteins decreased as the plant cells dedifferentiated, while that of complex type N-glycans increased. this observation suggested that enhancement of expression and/or activation of certain α-mannosidase(s) involved in N-glycan processing could occur during dedifferentiation of plant cells.植物細胞の脱分化と糖蛋白質糖鎖（アスパラギン結合型糖鎖）の発現機構との相関を明らかにすることを目的とし、ヒマ種子から誘導したカルス中に発現されるアスパラギン結合型糖鎖（N-グルカン）の構造解析を行った。2,4-D処理によるカルス化誘導を行い、経時的に採取した組織からTriton X-100を含むHEPES緩衝液（ph7.4)で全糖蛋白質を抽出した。得られた糖蛋白質からヒドラジン分解によりN-グリカンを遊離された後、N-アセチル化、ピリジルアミン(PA)化により蛍光標識糖鎖を調製した。逆相およびサイズフラクショネーションHPLCによりPA-糖鎖を単一に精製後、糖鎖2次元マップ法により構造の同定を行った。その結果、カルス化に伴い、ハイマンノース型糖鎖(Man7-4GlcNAc2)の相対量が顕著に減少するのに対して、キシロース/フコース含有型糖鎖( GlcNAclMan3FuclXyllGlcNAc2)の相対量が増加することが明らかになった。この現象は、植物細胞の脱分化に伴い、糖鎖プロセシングに関与するα-マンノシダーゼあるいはN-アセチルグリコサミン転移酵素の活性化が起こることを示唆するものと考えられる

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

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

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

タケノコ（ハチク）の中に存在する遊離N-グリカンの構造解析

Free N-glycans, the high-mannose-type and the plant complex-type, have been found in bamboo shoots. These free N-glycans were coupled with 2-aminopyridine and purified by gel filtration, Con A-Sepharose affinity chromatography, reversed-phase HPLC, and size-fractionation HPLC. The structures of these pyridylaminated free N-glycans were identified by two-dimensional sugar chain mapping, exomannosidase digestions, and ion-spray tandem mass spectrometry. The structual analyses showed that the various free high-mannose type sugar chains having one GlcNAc(Man8-5GlcNAc1) and free xylose/fucose containing sugar chains having the chitobiose segment occur in the developing bamboo shoots, suggesting that an endo-β-N-acetylglucosaminidase should produce the former structures, and a peptide: N-glycanase should produce the latter structures.植物細胞の分化・成長における遊離N-グリカンの植物ホルモン様活性が推定されている。そこで、分化・成長中の細胞における遊離N-グリカンの偏在性を立証する研究の一環として、成長速度の速いことで知られるタケノコから遊離N-グリカンを精製し、それらの製造解析を行った。タケノコ抽出物の透析外液から調整したオリゴ糖画分を2-アミノピリジンで蛍光標識した後、遊離N-グリカンを逆相HPLC、ConAアフィニティークロマト及びサイズフラッシュネーションHPLCにより精製した。得られた遊離N-グリカンの構造解析はα-マンノシダーゼ消化、糖鎖2次元マップ法、IS-MS及びMS/MS分析を組み合わせることにより行った。その結果、タケノコ中にはエンド-β-N-アセチルグリコサミニダーゼにより誘導されたと考えられるハイマンノース型糖鎖（Man8-5GlcNAc1、2.6nmol/gram fresh weight）が存在することが明らかになった。エンドウ胚軸同様、ハイマンノース型糖鎖の存在量が植物複合型糖鎖の存在量に比べかなり高いことから、前者構造の遊離糖鎖が植物細胞の分化・成長において何らかの重要な生理機能を担う可能性も考えられる

植物複合型遊離Ｎ－グリカンが細胞壁に局在する可能性

In this report, we bring up the possibility that complex type free N-glycans may localize in cell wall fraction of developing seeds (Ginkgo biloba seeds). Several free N-glycans extracted by mild acid hydrolysis of cell wall fraction were coupled with 2-aminopyridine and purified by gel filtration, size-fractionation HPLC, and reversed-phase HPLC. The structures of the pyridylaminated free N-glycans were identified by two-dimensional sugar chain mapping, α-1,2-mannosidase digestions, and ionspray tandem mass spectrometry. The structural analyses showed that highmannose type free N-glycans having one GlcNAc residue (Man8-5GlcNAc1) and plant complex type free N-glycans having the N-acetyl chitobiose unit also occur in the call wall fraction of the developing Ginkgo seeds. However, quantitative analysis of such free N-glycans showed that the plant complex type free glycans found in small amounts(～3%) in the cytosolic fraction accounted for nearly 40% of total free N-glycans, This observation suggested that the complex type free N-glycans might occur and localized in the cell wall fraction.登熟期銀杏種子の細胞壁画分に局在する遊離型-グリカンの構造解析を行った．登熟期銀杏種子抽出物を徹底的に透析して細胞質中に存在する遊離-グリカンを除去した後，細胞壁画分を遠心分離により回収した．得られた細胞壁画分を希酸処理（0.01NHCl/20メタノール中で100℃，30分間処理）した後，透析によりオリゴ糖鎖を透析外液に回収した．オリゴ糖鎖画分を２-アミノピリジンで蛍光標識した後，遊離-グリカンをゲルろ過およびサイズフラクショネーションHPLCにより精製した。遊離-グリカンの構造解析は，IS-MS分析，α-1，2-マンノシダーゼ消化，および糖鎖２次元マップ法を組み合わせることにより行った．その結果，細胞壁画分からは，ハイマンノース型および植物複合型構造を有する遊離-グリカンが検出されたが，細胞質画分には74pmol/freshweightofseed（全遊離グリカンの３構相当）程度の存在量であった植物複合型遊離-グリカンが，細胞壁画分では310pmol/freshweightofseed（全遊離グリカンの40相当）程度の濃度を占めることが明らかとなった。この結果は，植物複合型し造の遊離-グリカンは主に細胞壁画分に局在し，水素結合あるいは疎水結合により細胞壁マトリックスに強固に結合とていることを示唆するものと考えられ

登熟期ヒマ種子ミクロゾーム画分からのアリル-α-マンノシダーゼの精製と酵素学的諸性質

An α-mannosidase, which would be involved in N-linked glycoprotein metabolism, was purified and characterized from microsomal fraction of developing Ricinus communis endosperms. The purified enzyme with 43 kDa on SDS-PAGE showed maximal activity at pH5.0 and 50℃, when p-nitrophenyl-α-mannopyranoside was used as a substrate. α-Mannosidase activity was inhibited by EDTA and the reduced activity was rescued by addition of Zn2+ or Ca2+, suggesting this α-mannosidase should be a metal enzyme. Ricinus aryl-α-mannosidase was able to convert the Man6GlcNAc2-PA and Man5GlcNAc2-PA to Man4GlcNAc2-PA but was completely inactive toward Man4GlcNAc2-PA, Man4Xy11GlcNAc2-PA and GlcNAc1Man5GlcNAc2-PA.登熟期ヒマ種子のミクロゾーム画分からp-nitrophenyl-α-D-mannopyranoside(PNP-α-Man)及び蛍光標識N-グリカンに対して活性を示すα-マンノシダーゼを精製後、酵素学的諸性質を解析した。精製酵素は還元条件下のSDS-PAGEでは43kDaの相対分子量を示し、PNP-α-Manを基質とした場合の至適反応条件は50-60℃、pH4.5-5.0であった。本酵素はEDTAにより阻害を受けたが、Zn2+及びCa2+添加により活性が回復することから金属イオン要求性の酵素であると思われる。また、PNP-α-Manに対するKm値は1.3mM、Man5GLcNAc2-PAに対するKm値は0.4mMであった。本酵素はMan5GlcNAc2-PAに作用しMan4GlcNAc2-PAを誘導するものの、Man6GlcNAc2-PA対してむしろ強い活性を示し、Man5GlcNAc2-PA(88%)及びMan4GlcNAc2-PA(12%)を誘導した。しかしながら、Man4GlcNAc2-PA,GlcNAc1Man5GlcNAc2-PA、Man4Xy11GlcNAc2-PAは本酵素の有効な基質とはなり得なかった。これらの結果から、本酵素はα-1,2-マンノース残基を優先的に加水分解し、その後、β-結合マンノースに結合するα-1,3マンノース残基を遊離することが示唆された

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

Evaluation of Myelotoxicity in Dietary Restricted Rats

The purpose of this study was to clarify the effect of decreased food consumption on evaluation of myelotoxicity in routine general toxicity studies. Male rats were divided into the following 7 groups: 12, 15, and 18 mg/kg 5-fluorouracil (5-FU) treatment groups (FU12, FU15 and FU18); dietary restriction groups (R12, R15 and R18 receiving the same amount of food as the rats in the FU12, FU15 and FU18 groups, respectively); and a nontreated control group (NT). We compared the changes in body weight, hematology and the results of cytological analyses of bone marrow and histopathology among the groups after administration and recovery periods of 14 and 7 days, respectively. At the end of the administration period, the FU15 and FU18 groups showed decreases in many hematologic and bone marrow parameters that were all similar to those in the corresponding dietary restriction groups (R15 and R18). A granulocyte abnormality (polyploidy: frequency of 1% or less) was also observed in all 5-FU treated groups. At the end of the recovery period, increases in the reticulocyte and platelet counts and extramedullary hematopoiesis of the spleen were observed in the 5-FU treated groups. These results indicate that the results of general toxicity studies in rats should be evaluated in consideration of dietary restriction effects when food consumption is decreased at about 30-40% or more. Careful morphological observation of hemocytes would be helpful in distinguishing the effect of a drug from that of dietary restriction in relation to hematological and bone marrow parameters. Performance of a recovery test to determine the reactive response of hematopoiesis is also recommended