詳細

木下　充弘 （キノシタ　ミツヒロ）

  • 薬学部 創薬科学科　教授
Last Updated :2024/04/19

コミュニケーション情報 byコメンテータガイド

  • コメント

    生体内における糖鎖とタンパク質の網羅的解析により、疾患発症にかかわる分子あるいは分子群を特定し診断マーカーの開発を目指す研究。

研究者情報

学位

  • 博士（薬学）

ホームページURL

J-Global ID

研究キーワード

  • マイクロチップ電気泳動   バイオ医薬品   グリコサミノグリカン   糖タンパク質   レクチン   糖鎖生合成   高速液体クロマトグラフィー   質量分析   キャピラリー電気泳動   

現在の研究分野（キーワード）

    生体内における糖鎖とタンパク質の網羅的解析により、疾患発症にかかわる分子あるいは分子群を特定し診断マーカーの開発を目指す研究。

研究分野

  • ライフサイエンス / 薬系分析、物理化学 / 糖鎖構造解析
  • ライフサイエンス / 薬系衛生、生物化学 / 糖鎖生合成

経歴

  • 2022年04月 - 現在  近畿大学薬学部教授
  • 2014年04月 - 現在  近畿大学薬学部准教授
  • 2007年04月 - 2014年03月  近畿大学薬学部講師
  • 2000年04月 - 2007年03月  近畿大学薬学部助手
  • 1999年04月 - 2000年03月  近畿大学薬学部実験助手

学歴

  • 1997年04月 - 1999年03月   近畿大学   大学院薬学研究科博士前期課程   薬学専攻
  •         - 1999年   Kinki University   Faculty of Pharmacentical Sciences
  • 1993年04月 - 1997年03月   近畿大学   薬学部   薬学科
  •         - 1997年   Kinki University   Faculty of Pharmaceutical Science

所属学協会

  • 日本分析化学会   日本生化学会   日本糖質学会   日本薬学会   

研究活動情報

論文

  • Sachio Yamamoto; Naho Kato; Miki Wada; Mitsuhiro Kinoshita
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 39 7 1041 - 1046 2023年07月 
    Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30 min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.
  • 山本 佐知雄; 宮脇 直久; 川上 夏海; 木下 充弘; 鈴木 茂生
    分析化学 71 6 333 - 339 (公社)日本分析化学会 2022年06月 
    8-Aminopyrene-1,3,6-trisulfonic acid(APTS)は三つのスルホン酸基を有している蛍光試薬であり、その高い電気泳動移動度を利用することでAPTS標識化糖鎖がキャピラリー電気泳動(CE)で分析されている。しかしながらCE単独で糖鎖の構造解析を達成することは困難である。またAPTSは親水性が非常に高いため、一般的な逆相クロマトグラフィーや親水性相互作用クロマトグラフィー(HILIC)で分離が困難であった。そこで著者らは、官能基にテトラゾール基を有するポリマーが結合したDCpak PTZカラムの試作品を使用してHILICモードを利用したAPTS標識化糖鎖の分離方法を開発した。しかしながら開発した方法では再現性、構造の類似した糖鎖の分離能が低く、溶出時間も1時間程度を要した。特にシアル酸含有糖鎖においては、成分ごとの分離がほとんどできなかった。そこで本研究では市販のDCpak PTZカラムを利用し、糖鎖の分離方法を再検討した。また、DCpak PTZカラムで分離した糖鎖に関して分取、脱塩した試料をCEの試料に添加してがん細胞のピークの同定を行った。(著者抄録)
  • Yasuhiro Morikawa; Keiji Nishiwaki; Shigeo Suzuki; Mitsuhiro Kinoshita; Isao Nakanishi
    Analytical Sciences 2022年02月
  • Sachio Yamamoto; Shoko Yano; Mitsuhiro Kinoshita; Shigeo Suzuki
    Gels (Basel, Switzerland) 7 4 2021年12月 
    An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.
  • Recent advances and trends in sample preparation and chemical modification for glycan analysis
    Mitsuhiro Kinoshita; Keita Yamada
    Journal of Pharmaceutical and Biomedical Analysis 207 114424  2021年10月 [査読有り]
  • Mitsuhiro Kinoshita; Kazuki Nakajima; Sachio Yamamoto; Shigeo Suzuki
    Analytical and bioanalytical chemistry 413 19 4727 - 4738 2021年08月 [査読有り]
     
    N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.
  • Sachio Yamamoto; Kazuhito Maetani; Gai Tatsumi; Fuka Okada; Mitsuhiro Kinoshita; Shigeo Suzuki
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 2021年04月
  • Maho HIOKI; Hiroshi KOBAYASHI; Mitsuhiro KINOSHITA; Sachio YAMAMOTO; Shigeo SUZUKI
    CHROMATOGRAPHY 2021年04月
  • Sachio YAMAMOTO; Yume KAWAGUCHI; Mitsuhiro KINOSHITA; Shigeo SUZUKI
    CHROMATOGRAPHY 42 1 37 - 42 2021年02月
  • Mitsuhiro Kinoshita; Yumi Nakatani; Keita Yamada; Sachio Yamamoto; Shigeo Suzuki
    Journal of pharmaceutical and biomedical analysis 195 113875 - 113875 2021年02月 [査読有り]
     
    Glycoanalytical technology is required for a wide variety of scientific research, including basic glycobiological pharmaceutical, and biomarker research. Although several innovative analytical techniques have been developed for these purposes, quantitative glycan analysis based on electrophoretic separation, has often been impeded by the lack of cost-effective and facile sample preparation approaches. Here, we developed a rapid and facile sample preparation workflow for cost-effective glycan analysis and demonstrated its use with fully automated microchip electrophoresis (ME). Purification of 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans was based on the combination of ion-pair assisted extraction (IPAE) with hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE). Compared to commonly used sample preparation methods, the IPAE/HILIC-SPE method undergoes minimal nonspecific loss and undesirable degradation of N-glycans during the purification step. Furthermore, our method required only 10 min, and the entire workflow, including glycan release, labeling, and concentration processes was completed within 4 h. Although the present system should be improved to enable analysis of more complex mixtures, ME-based separation of APTS-labeled N-glycans offers a fully automated operation including conditioning, sample loading, separation, and can be analyzed with a sample-to-sample throughput of 120 s in parallel processes. The present workflow is easy to implement, does not require expensive reagents and instruments and may be useful for glycoscientists across disciplines.
  • Yuka Kishimoto; Fuka Okada; Tomohiro Maesako; Sachio Yamamoto; Mitsuhiro Kinoshita; Takao Hayakawa; Shigeo Suzuki
    Journal of chromatography. A 1625 461194 - 461194 2020年08月 [査読有り]
     
    Quantitative analysis of glycans released from glycoproteins using high-performance liquid chromatography (HPLC) requires fluorescent tag labeling to enhance sensitivity and selectivity. However, the methods required to remove large amounts of excess labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods, including solvent extraction and solid phase extraction (SPE), often impair quantitative analysis. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminopyridine (AP)-labeled glycans using a six-port/two-way valve and two small columns: one packed with a strong cation exchange resin (SCX) and the other comprising ODS silica gel. AP-labeled glycans delivered from an injection port were separated from excess AP by passing through an SCX column (4.6 mm i.d., 1 cm long) regulated to 40°C. The AP-labeled glycans were trapped on an ODS column (4.6 mm i.d., 1 cm long) to further separate them from inorganic contaminants. By changing the valve position after 2 min to connect the ODS column to an analysis column, AP-labeled glycans trapped in the ODS column were eluted with an acetonitrile-containing eluent followed by hydrophilic interaction liquid chromatography (HILIC) separation on an amide column or reversed-phase mode separation on a C30 column. This method was successfully used to analyze N-linked glycans released from several glycoprotein samples.
  • Keita Yamada; Koji Suzuki; Yoshihiko Hirohata; Mitsuhiro Kinoshita
    Journal of proteome research 19 8 3033 - 3043 2020年08月 [査読有り]
     
    Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column and were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples of the N-glycan analysis in human serum were reported, but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allow the analysis of minor acidic glycans that are typically difficult to detect.
  • Mitsuhiro Kinoshita; Sachio Yamamoto; Shigeo Suzuki
    ACS Omega 5 30 18608 - 18618 2020年08月 [査読有り]
  • Mitsuhiro Kinoshita; Ai Saito; Sachio Yamamoto; Shigeo Suzuki
    Journal of Pharmaceutical and Biomedical Analysis 186 113267 - 113267 2020年07月 [査読有り]
  • 光重合性ポリアクリルアミドゲルを用いるリン酸化化合物のオンライン濃縮・標識マイクロチップ電気泳動法の開発
    山本 佐知雄; 矢野 祥子; 増田 誠子; 木下 充弘; 鈴木 茂生
    日本薬学会年会要旨集 140年会 27F - pm03 (公社)日本薬学会 2020年03月
  • Sachio Yamamoto; Maki Ueda; Masataka Kasai; Yusuke Ueda; Mitsuhiro Kinoshita; Shigeo Suzuki
    Journal of pharmaceutical and biomedical analysis 179 112995 - 112995 2020年02月 [査読有り]
     
    An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200 μL volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure: (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30 mm from the tip was performed by irradiation with a 365 nm blue LED beam from a distance 10 mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8 min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.
  • Naonori UTAMURA; Sachio YAMAMOTO; Mitsuhiro KINOSHITA; Shigeo SUZUKI
    BUNSEKI KAGAKU 68 11 859 - 864 2019年11月 [査読有り]
  • ピンポイント重合Phos-tagアクリルアミドによるリン酸化化合物のオンライン特異的濃縮・標識マイクロチップ電気泳動法の開発
    山本 佐知雄; 矢野 祥子; 増田 誠子; 木下 充弘; 鈴木 茂生
    日本分析化学会講演要旨集 68年会 336 - 336 (公社)日本分析化学会 2019年08月
  • 非水系キャピラリー電気泳動方における過剰吸着を利用した新規分離モードの開発
    宇田村尚典; 山本佐知雄; 木下充弘; 鈴木茂生
    分析化学 69 2019年 [査読有り]
  • Simultaneous determination of inorganic anions and cations in water and biological samples by capillary electrophoresis with a capacitive coupled contactless conductivity detector using capillary filling method.
    Yamamoto S; Fujiwara H; Maruyama K; Tanaka Y; Kinoshita M; Suzuki S
    Anal. Sci. 35 295 - 300 2019年 [査読有り]
  • Analysis of 2-aminobenzoic acid-labeled monosaccharides and glycoprotein-derived oligosaccharides by online cleanup liquid chromatography in the reversed-phase and hydrophilic interaction liquid chromatography modes.
    Yamamoto S; Hayashi Y; Matsunaga H; Okada F; Kinoshita M; Suzuki S
    Chromatography 40 65 - 70 2019年 [査読有り]
  • Mitsuhiro Kinoshita; Sachio Yamamoto,; Shigeo Suzuki
    Electrophoresis Letters 63 2 47 - 54 2019年 [査読有り]
  • Yamamoto S; Okada F; Kinoshita M; Suzuki S
    The Analyst 143 18 4429 - 4435 2018年09月 [査読有り]
  • バイオ医薬品研究開発における電気泳動手法
    木下 充弘; 木下 英樹; 鈴木 茂生
    Journal of the Mass Spectrometry Society of Japan 66 4 154 - 160 2018年08月
  • Yamada K; Kayahara H; Kinoshita M; Suzuki S
    Anal.Chem. 90 14 8387 - 8395 2018年07月 [査読有り]
     
    Changes in the structures and quantities of sulfated glycans play important roles in inflammatory and neurological diseases and cancer. Therefore, sulfated glycans are expected to become diagnostic markers for a variety of diseases such as Alzheimer's disease and cancer. On the other hand, structural abnormalities in the phosphorylated glycans on lysosomal enzymes cause a number of lysosomal diseases, while novel phosphorylated glycans have been found in other proteins. As with sulfated glycans, structural and quantitative changes in these phosphorylated glycans and their associations with disease are also of interest. In this article, we introduce a new method for the simultaneous analysis of sulfated and phosphorylated glycans. We first employ a serotonin-immobilized column to enrich these glycans. Glycans obtained in this manner were sequentially subjected to other analytical techniques without desalting. We employed hydrophilic interaction chromatography to distinguish the sulfate and phosphate groups of the glycans and were able to analyze sulfated and phosphorylated N-glycans expressed on thyroglobulin, ovalbumin, and myozyme. We showed that our method not only analyzes sulfated and phosphorylated glycans, but also glycans containing the GlcNAc-HPO3 residue. We comparatively analyzed sulfated glycans, phosphorylated glycans, and GlcNAc-HPO3-residue-containing glycans expressed on MKN7 cells (well-differentiated gastric cancer cells) and MKN45 cells (poorly differentiated gastric cancer cells). To the best of our knowledge, this is the first report of a method for the simultaneous and quantitative analysis of sulfated and phosphorylated glycans.
  • 木下 英樹; 小杉 正幸; 矢部 公彦; 松永 貴輝; 木下 充弘
    電気泳動 62 1 13 - 18 日本電気泳動学会 2018年07月 [査読有り]
     
    抗体医薬は生体高分子であり，糖鎖修飾などの翻訳後修飾やジスルフィド結合による高次構造形成など，複雑な物理化学的特性が付加されるため，これらは本質的に不均一性を有する．近年，抗体医薬の機能性，製造プロセス，品質管理性を向上させるために，抗体医薬の構造を詳細に分析できる分析技術や評価方法の高度化が求められている．本研究開発では，2DE技術を用いた抗体医薬の高精度な評価方法の確立を目指して，2DEの自動化システムで使用できる分析用チップおよび分析方法を開発し，その有効性を検討した．
  • On-line derivatization and concentration of aspartic acid using in situ photopolymerized carboxylic acid type polyacrylamide gels as a permselective preconcentrator
    Yamamoto S; Nishida N; Kinoshita M; Suzuki S
    Chromatography 39 3 125 - 130 2018年07月 [査読有り]
  • 全自動マイクロチップ電気泳動装置MCE-202”MultiNA”を用いた糖鎖の迅速解析
    木下 充弘; 山本万莉; 御子柴柚子; 松本和樹; 山本佐知雄; 鈴木茂生; 曽我部有司
    74 3・4 193 - 204 2018年03月
  • Sachio Yamamoto; Mitsuhiro Kinoshita; Toru Ikegami; Shigeo Suzuki
    Journal of Chromatography A 1566 44 - 50 2018年 [査読有り]
     
    8-Aminopyrene-1,3,6-trisulfonate (APTS) is one of the most frequently used reagent in capillary electrophoresis. Three sulfonate groups in APTS generate fast electrophoretic mobilities of derivatized glycans, therefore very suitable for CE-LIF applications. However, these groups also make separation with partition chromatography difficult. A novel column for hydrophilic interaction liquid chromatography (HILIC) with an anionic tetrazole functionalized polymer-based silica was examined for the separation of APTS-labeled glycans derived from specific glycoproteins. This separation mode has enhanced capability for the size resolution of neutral and acidic oligosaccharides. In addition, specific glycan isomers, which are usually difficult to separate with HILIC methods, were also separated. IgG-derived complex-type glycans that have an isomeric pair of monogalactosylated glycans, as well as differences in the number of galactose residues, are separable using this mode. We also utilized this column for the fractionation of APTS-labeled glycans from porcine thyroglobulin and examined their migration times with CE by co-migration with a mixture of glycoprotein glycans. Combinational modes of HILIC and anionic repulsion show promise for the separation and preparation of glycoprotein-derived glycans labeled with APTS.
  • 西田 憲晃; 小鍛治 靖子; 山本 佐知雄; 木下 充弘; 鈴木 茂生
    分析化学 66 12 909 - 917 日本分析化学会 2017年12月 [査読有り]
     
    Compared to N-linked oligosaccharides, O-linked oligosaccharides were rarely reported for their analysis due to the absence of glycanases having wide substrate specificity to release all O-linked oligosaccharides. Therefore, O-linked oligosaccharides have often been released by chemical methods, such as hydrazinolysis and beta-elimination under alkaline conditions, but the liberated oligosaccharides released by these methods are susceptible to alkaline conditions which cause epimerization and degradation, called peeling reaction and the hydrolysis of N-acetyl groups. To compare the releasing efficiency and the degradation rate of previously reported releasing methods, we developed a novel HPLC method for the analysis of O-linked oligosaccharides: oligosaccharides released from glycoproteins are labeled quantitatively with 7-amino-4-methylcoumarin (AMC) and separated by reversed-phase HPLC, and then fluorimetrically detected with high sensitivity. In the releasing methods, we found that 25 % ammonia is most optimal, and can be applied to the release of N-linked oligosaccharides. We established a method that could be applied to the rapid and simple and nonspecific release of N-linked oligosaccharides. Various AMC-labeled N-linked oligosaccharides released from glycoproteins were used as samples, and were separated by reversed-phase HPLC and fluorimetrically detected with high sensitivity for comparing with the enzymatic releasing method using PNGase F.
  • Sachio Yamamoto; Miyuki Himeno; Masaya Kobayashi; Miki Akamatsu; Ryosuke Satoh; Mitsuhiro Kinoshita; Reiko Sugiura; Shigeo Suzuki
    ANALYST 142 18 3416 - 3423 2017年09月 [査読有り]
     
    A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N, N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis [2-methyl-N-(2-hydroxyethyl) propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 x 100 x 30 mu m) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.
  • Hideaki Yamada; Chiemi Matsumura; Keita Yamada; Koichiro Teshima; Takashi Hiroshima; Mitsuhiro Kinoshita; Shigeo Suzuki; Kazuaki Kakehi
    ELECTROPHORESIS 38 9-10 1344 - 1352 2017年05月 [査読有り]
     
    mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals? perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.
  • Maki Yamagami; Yurie Matsui; Takao Hayakawa; Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A 1496 157 - 162 2017年05月 [査読有り]
     
    An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5 w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined beta-galactosidase, alpha-mannosidase, beta-N-acetylhexosaminidase, alpha-neuraminidase, and alpha-fucosidase, and found only beta-galactosidase and alpha-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6 cm long plug of 200 and 50 mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5 U/mL. The beta-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10 min when saccharide derivatives were achieved in the enzyme plug. The reaction of alpha-fucosidase and alpha-mannosidase was completed by decreasing the electrophoretic voltage to -2 kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans. (C) 2017 Elsevier B.V. All rights reserved.
  • Yoshie NAGATOMO; Shinichi HASHIMOTO; Yuka KISHIMOTO; Takao HAYAKAWA; Sachio YAMAMOTO; Mitsuhiro KINOSHITA; Shigeo SUZUKI
    CHROMATOGRAPHY 38 1 23 - 30 2017年 [査読有り]
  • Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo Suzuki
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 130 130 273 - 300 2016年10月 [査読有り]
     
    This review covers the basics and some applications of methodologies for the analysis of glycoprotein glycans. Analytical techniques used for glycoprotein glycans, including liquid chromatography (LC), capillary electrophoresis (CE), mass spectrometry (MS), and high-throughput analytical methods based on microfluidics, were described to supply the essentials about biopharmaceutical and biomarker glycoproteins. We will also describe the MS analysis of glycoproteins and glycopeptides as well as the chemical and enzymatic releasing methods of glycans from glycoproteins and the chemical reactions used for the derivatization of glycans. We hope the techniques have accommodated most of the requests from glycoproteomics researchers. (C) 2016 Elsevier B.V. All rights reserved.
  • Hiromi Ito; Hiroyuki Kaji; Akira Togayachi; Parastoo Azadi; Mayumi Ishihara; Rudolf Geyer; Christina Galuska; Hildegard Geyer; Kazuaki Kakehi; Mitsuhiro Kinoshita; Niclas G Karlsson; Chunsheng Jin; Koichi Kato; Hirokazu Yagi; Sachiko Kondo; Nana Kawasaki; Noritaka Hashii; Daniel Kolarich; Kathrin Stavenhagen; Nicolle H Packer; Morten Thaysen-Andersen; Miyako Nakano; Naoyuki Taniguchi; Ayako Kurimoto; Yoshinao Wada; Michiko Tajiri; Pengyuan Yang; Weiqian Cao; Hong Li; Pauline M Rudd; Hisashi Narimatsu
    Glycoconjugate journal 33 3 405 - 415 2016年06月 
    The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.
  • リン酸化/硫酸化糖タンパク質糖鎖の網羅的解析
    山田 佳太; 栢原 春奈; 木下 充弘; 鈴木 茂生; 坂崎 文俊
    日本薬学会年会要旨集 136年会 3 105 - 105 (公社)日本薬学会 2016年03月
  • Yuto Takeda; Yuka Hayashi; Naonori Utamura; Chise Takamoto; Mitsuhiro Kinoshita; Sachio Yamamoto; Takao Hayakawa; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A 1427 170 - 176 2016年01月 [査読有り]
     
    Monoamine- and triamine-bonded silica nanoparticles were prepared using 3-aminopropyl-trimethoxysilane and N-1-(3-trimethoxysilylpropyl)diethylenetriamine, respectively, and used as pseudostationary phases for capillary electrochromatography. The amine-bonded silica nanoparticles were tightly adsorbed on the inner wall of a capillary and generated fast electro-osmotic flow (2.59 x 10(-4) cm(2) V-1 s(-1)) toward the anode in an electric field. The electro-osmotic velocities obtained with 20 nm triamine-bonded silica were three to five times larger than those generated by a fused silica capillary and two times faster than those for the commercial cationic polymer-modified capillary. Fast electro-osmotic flow enables rapid analysis. This method was applied to hydrophilic interaction chromatography (HILIC) mode separation of various samples including the size separation of glucose oligomer derivatives and the resolution of four nucleic acid bases. (C) 2015 Elsevier B.V. All rights reserved.
  • Sachio Yamamoto; Yoko Tamata; Kaori Sejima; Mitsuhiro Kinoshita; Shigeo Suzuki
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 407 20 6201 - 6206 2015年08月 [査読有り]
     
    A novel method was developed for d/l-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of d/l isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of d/l- galactose in red seaweed.
  • 糖鎖のリン酸化及び硫酸化にフォーカスしたグライコミクス技術の開発
    山田 佳太; 栢原 春奈; 木下 充弘; 鈴木 茂生; 坂崎 文俊; 閔 庚善
    日本薬学会年会要旨集 135年会 3 104 - 104 (公社)日本薬学会 2015年03月
  • Sachio Yamamoto; Eriko Nagai; Yuki Asada; Mitsuhiro Kinoshita; Shigeo Suzuki
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 407 5 1499 - 1503 2015年02月 [査読有り]
     
    A selective separation method using a poly(methylmethacrylate) microchip was developed for 7-amino-4-methylcoumarin-labeled saccharides in a crude reaction mixture. In an alkaline borate buffer, saccharide derivatives formed strong anionic borate complexes. These complexes moved from the cathode to the anode in an electric field and were detected near the anode. Excess labeling reagents and other foreign substances remained at the inlet reservoir. A confocal fluorimetric detection system enabled the determination of monosaccharide derivatives with good linearity between at least 5 and 100 nM, corresponding to 50 fmol to 1 pmol per injection. The lower limit of detection (signal-to-noise=5) was 2 nM. The sensitivity and linear quantitation range were comparable to reported values using fluorometric detection, capillary electrophoresis, or liquid chromatography. Application of the method showed excellent resolution in the analysis of O-linked glycans chemically released from glycoproteins.
  • Mitsuhiro Kinoshita; Kazuaki Kakehi
    Glycoscience: Biology and Medicine 111 - 118 2015年01月 [査読有り]
     
    The N-glycosylation on therapeutic monoclonal antibodies (mAbs) plays important biological roles such as antibody-dependent cellular cytotoxicity (ADCC) or circulatory half-time in vivo. Although the attached N-glycans are mostly core α1-6-fucosylated biantennary glycans, minor nonhuman N-glycans having N-glycolylneuraminic acid (NeuGc) and Galα1-3Gal epitopes (αGal) have been reported to be present in therapeutic mAbs. Determination of nonhuman N-glycans is important in the evaluation of the mAb characteristics, because the presence of nonhuman N-glycans may affect the safety of mAb products leading to immunogenicity or adverse incident. In this chapter, we present the method for validation of mAbs based on glycan profiles using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The presented method using CE-LIF allows the routine testing to ensure safety and efficacy of the mAb products.
  • Sachio YAMAMOTO Tomoyuki IWATA; Keiji NISHIWAKI; Mitsuhiro KINOSHITA; Shigeo SUZUKI
    Chromatography 36 93 - 98 The Society for Chromatographic Sciences 2015年 [査読有り]
     
    In this work, quaternary ammonium group-modified celluloses (QCs) were homogeneously synthesized by reacting cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride in NaOH/urea solutions. The structure and solution properties of the QCs were characterized by elemental analysis, FT/IR, 1H-NMR, and size exclusion chromatography. The results show that water-soluble QCs, with degree of substitution values, defined as the substitution of free hydroxyl groups of cellulose, 0.49– 0.72 and molecular weight 21–66 kDa could be obtained by optimizing the reaction time. The synthesized QCs were tested for protein separation as physically adsorbed coatings in capillary electrophoresis. Among the derivatives studied, quaternary ammonium cellulose showed rapid electroosmotic mobility and effective suppression of protein adsorption. The EOF can be manipulated for various applications by using QCs with different molecular weight. Because the reversed EOF can be obtained over a broad pH range, it is possible to separate basic, neutral, and acidic proteins under physiological conditions. Eight proteins; lysozyme, ribonuclease A, cytochrome C, α-chymotrypsinogen, α-lactalbumin, ovalbumin, transferrin, and myoglobin were baseline separated within 25 min by 25 mM sodium phosphate buffers (pH 7.0) containing 100 µg/mL QC.
  • Keita Yamada; Mitsuhiro Kinoshita; Yoshio Jo; Toshiki Inoue; Motonori Aoshima; Kiyotaka Hasegawa; Kazuo Sei; Soichiro Kita; Kazuaki Kakehi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 134 11 1209 - 1217 2014年11月 [査読有り]
     
    Carboxymethyl cellulose (CMC) is one of the most important cellulose derivatives and used in the fields of food, pharmaceuticals, cosmetics, and paint. Fibrous CMC is used an antiadhesive material to prevent postoperative wound adhesions. The degree of substitution and distribution of the substituent (i.e., the carboxymethyl group) are the most important parameters for the function of CMC. Thus, CMC used for antiadhesive material must be carefully evaluated, because the CMC product is retained in patients' bodies over the long term. Although identification tests of CMC are defined in the Japanese Pharmacopoeia, it is difficult to evaluate its structure using those tests. In the present study, we propose improved methods for evaluating CMC products by. analyzing monosaccharides after hydrolysis.
  • Higashi K; Asano K; Yagi M; Yamada K; Arakawa T; Ehashi T; Mori T; Sumida K; Kushida M; Ando S; Kinoshita M; Kakehi K; Tachibana T; Saito K
    The Journal of biological chemistry 289 37 25833 - 25843 2014年09月 [査読有り]
     
    Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of > 239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAc alpha 2-3Gal beta O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.
  • Kinya Iwatsuka; Hiroki Iwamoto; Mitsuhiro Kinoshita; Katsuhiro Inada; Shin-ichi Yasueda; Kazuaki Kakehi
    CURRENT EYE RESEARCH 39 7 686 - 694 2014年07月 [査読有り]
     
    Purpose: We compared cultured Statens Seruminstitut rabbit cornea (SIRC) cells and corneal epithelial cells from rabbit eyes by analyzing their N-glycans and glycosaminoglycans (GAGs). This work is a fundamental study on the efficacy of using cultured cells instead of animals for drug development. Materials and methods: N-Glycans and GAGs from SIRC cell monolayers and corneal epithelial cells of rabbit eyes were analyzed by capillary electrophoresis (CE) and a combination of high-performance liquid chromatography (HPLC) and mass spectrometry. Results: High mannose-type glycans and a hybrid-type glycan were the common N-glycans in SIRC cells and corneal epithelial cells of rabbit eyes. Mono-fucosylated biantennary glycans with or without one N-acetylneuraminic acid residue were observed only in SIRC cells. Hyaluronic acid was the only measurable GAG in the corneal epithelial cells of rabbit eyes. In contrast, hyaluronic acid and chondroitin sulfates were abundantly present in SIRC cells. Conclusions: Profiles of both N-glycans and GAGs were conspicuously different between SIRC cells and corneal epithelial cells of rabbit eyes. This report will be useful for the evaluation of pharmaceutical candidates when animals or cultured cells are employed in drug development studies.
  • 癒着防止材中カルボキシメチルセルロースの構造評価技術
    山田 佳太; 木下 充弘; 徐 吉夫; 井上 利樹; 青島 元法; 長谷川 清孝; 清 一雄; 北 荘一郎; 掛樋 一晃
    日本薬学会年会要旨集 134年会 2 276 - 276 (公社)日本薬学会 2014年03月
  • ロタウイルスエントリー機構の解明に向けたグライコミクス
    山田 佳太; 栢原 春奈; 稲垣 瑞穂; 木下 充弘; 金丸 義敬; 鈴木 徹; 矢部 富雄; 中込 とよ子; 中込 治; 掛樋 一晃
    日本薬学会年会要旨集 134年会 3 100 - 100 (公社)日本薬学会 2014年03月
  • Mitsuhiro Kinoshita; Yosuke Mitsui; Naotaka Kakoi; Keita Yamada; Takao Hayakawa; Kazuaki Kakehi
    JOURNAL OF PROTEOME RESEARCH 13 2 1021 - 1033 2014年02月 [査読有り]
     
    Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [Mitsui et al., J. Pharm. Biomed. Anal. 2012, 70, 718-726]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.
  • Mitsuhiro Kinoshita; Kazuaki Kakehi
    Methods in Molecular Biology 1200 131 - 146 2014年 [査読有り]
     
    Capillary affinity electrophoresis (CAE) is a powerful technique for glycan analysis, and one of the analytical approaches for analyzing the interaction between lectins and glycans. The method is based on the high-resolution separation of fluorescently labeled glycans by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) in the presence of lectins (or glycan binding proteins). CAE allows simultaneous determination of glycan structures in a complex mixture of glycans. In addition, we can calculate the binding kinetics on a specific glycan in the complex mixture of glycans with a lectin. Here, we show detailed procedures for capillary affinity electrophoresis of fluorescently labeled glycans with lectins using CE-LIF apparatus. Its application to screening a sialic acid binding protein in plant barks is also shown. © 2014 Springer Science+Business Media New York.
  • Mitsuhiro Kinoshita; Yuki Nakatsuji; Shigeo Suzuki; Takao Hayakawa; Kazuaki Kakehi
    JOURNAL OF CHROMATOGRAPHY A 1309 76 - 83 2013年09月 [査読有り]
     
    Monoclonal antibody (mAb) pharmaceuticals are much more complex than small-molecule drugs. Such complex characteristics raise challenging questions for regulatory evaluation. Although heterogeneity in mAbs based on their charge variants has been mainly evaluated using gel-based isoelectric focusing (IEF) method, recent development in capillary electrophoresis and microchip electrophoresis has made it possible to assure their heterogeneities in more easy and rapid manner. In the present paper, we customized the imaged microchip isoelectric focusing (mIEF) for the analysis of mAbs, and compared the customized version with the conventional capillary isoelectric focusing (cIEF) method, and found that mIEF has much higher performance in operations, and its resolving powers are comparable with those obtained by cIEF. (C) 2013 Elsevier B.V. All rights reserved.
  • Kinya Iwatsuka; Sakie Watanabe; Mitsuhiro Kinoshita; Kazuya Kamisue; Keita Yamada; Takao Hayakawa; Tadashi Suzuki; Kazuaki Kakehi
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 928 16 - 21 2013年06月 [査読有り]
     
    During the course of studies on the analysis of O-glycans in biological samples, we found that significant amount of free glycans are present in normal human serum samples. The most abundant free glycan was disialo-biantennary glycan typically observed in transferrin which is one of the abundant glycoproteins found in sera. Minor glycans were also considered to be mainly due to transferrin, but some glycans were derived from mucin-type O-glycans, although the amount was quite minute. However, high mannose-type glycans could not be detected at all. Although there have been many reports on the presence of intracellular "free" N-glycans (mainly derived from high mannose-type glycans) generated either from lipid-linked oligosaccharides or from misfolded glycoproteins through endoplasmic-reticulum associated protein degradation pathway, there is little information on the presence of free glycans in extracellular matrix and biological fluids such as serum. This report is the first one which demonstrates the presence of free glycans due to glycoproteins in sera. (C) 2013 Elsevier B.V. All rights reserved.
  • ヒト胃腺癌細胞による細胞内糖タンパク質の分解と遊離糖鎖の細胞外排出
    神末 和哉; 大河原 周平; 山田 佳太; 岩塚 欣也; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 133年会 3 123 - 123 (公社)日本薬学会 2013年03月
  • ヒトロタウイルスが認識する糖鎖の解析とその臨床化学的応用の可能性
    山田 佳太; 栢原 春奈; 稲垣 瑞穂; 矢部 富雄; 金丸 義敬; 鈴木 徹; 中込 とよ子; 中込 治; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 133年会 3 123 - 123 (公社)日本薬学会 2013年03月
  • 多様なムチン型糖鎖アレイの作製を可能にする糖鎖ライブラリー構築技術
    山田 佳太; 平林 淳; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 133年会 3 124 - 124 (公社)日本薬学会 2013年03月
  • Yosuke Mitsui; Keita Yamada; Sayaka Hara; Mitsuhiro Kinoshita; Takao Hayakawa; Kazuaki Kakehi
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 70 718 - 726 2012年11月 [査読有り]
     
    In the series of our previous reports, we showed that some cancer cell lines specifically express polylactosamine-type N-glycans and such glycans were often modified with fucose and sulfate residues. To confirm the proteins expressing these glycans, glycopeptide mixture obtained by digestion of whole protein fractions with trypsin was captured by a polylactosamine-specific lectin, Datura strasmonium agglutinin (DSA). And the peptides and glycans of the captured glycopeptides after digestion with N-glycoamidase F were extensively analyzed by HPLC and MS techniques. We found that some glycoproteins such as CD107a and CD107b commonly contained polylactosamine-type glycans in all the examined cancer cells. But integrin-alpha 5 (CD49e) and carcinoembryonic antigen-related cell adhesion molecule 5 (CD66e) having these glycans were specifically found in U937 (human T-lymphoma) and MKN45 (human gastric cancer) cells, respectively. These data clearly indicate that specific glycans attached to specific proteins will be promising markers for specific tumors with high accuracy. (C) 2012 Elsevier B.V. All rights reserved.
  • 消化器系癌細胞に発現するCEA上の高フコシル化糖鎖の比較解析
    原 沙弥香; 三ツ井 洋輔; 山田 佳太; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 132年会 3 184 - 184 (公社)日本薬学会 2012年03月
  • Eiki Maeda; Soichiro Kita; Mitsuhiro Kinoshita; Koji Urakami; Takao Hayakawa; Kazuaki Kakehi
    ANALYTICAL CHEMISTRY 84 5 2373 - 2379 2012年03月 [査読有り]
     
    Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or alpha-Gal epitopes (i.e., galactose-alpha 1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.
  • Keita Yamada; Yosuke Mitsui; Naotaka Kakoi; Mitsuhiro Kinoshita; Takao Hayakawa; Kazuaki Kakehi
    ANALYTICAL BIOCHEMISTRY 421 2 595 - 606 2012年02月 [査読有り]
     
    We developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins [Anal. Biochem. 371 (2007) 52-61; Anal. Chem. 82 (2010) 7436-7443] and applied the device to analyze them in some cancer cell lines [J. Proteome Res. 8 (2009) 521-537]. We also found that the device is useful to release glycosaminoglycans from proteoglycans [Anal. Biochem. 362 (2007) 245-251]. Based on these studies, we developed a method for one-pot analysis of mucin-type glycans and glycosaminoglycans after releasing them from total protein pool obtained from some cancer cell lines. Mucin-type glycans were analyzed by a combination of high-performance liquid chromatography and mass spectrometry techniques, and glycosaminoglycans were analyzed by capillary electrophoresis as fluorescent-labeled unsaturated disaccharides after digestion with specific eliminases followed by fluorescent labeling. Ten cancer cell lines, including blood cancer cells as well as epithelial cancer cells, were used to assess the method. The results clearly revealed that both mucin-type glycans and glycosaminoglycans showed quite interesting profiles. Thus, the current technique will be a powerful tool for discovery of glycan markers of diseases. (C) 2011 Elsevier Inc. All rights reserved.
  • Kinya Iwatsuka; Shin-ichi Yasueda; Eiji Bando; Hiroyuki Fujii; Takashi Terada; Hiroya Okubo; Hiroki Iwamoto; Mitsuhiro Kinoshita; Kazuaki Kakehi
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 879 27 2866 - 2870 2011年10月 [査読有り]
     
    Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples. (C) 2011 Elsevier B.V. All rights reserved.
  • Akira Harazono; Tetsu Kobayashi; Nana Kawasaki; Satsuki Itoh; Minoru Tada; Noritaka Hashii; Akiko Ishii; Teruyo Arato; Shigehiro Yanagihara; Yuki Yagi; Akiko Koga; Yuriko Tsuda; Mikiko Kimura; Masashi Sakita; Satoshi Kitamura; Hideto Yamaguchi; Hisashi Mimura; Yoshimi Murata; Yasuki Hamazume; Takayuki Sato; Shunji Natsuka; Kazuaki Kakehi; Mitsuhiro Kinoshita; Sakie Watanabe; Teruhide Yamaguchi
    BIOLOGICALS 39 3 171 - 180 2011年05月 [査読有り]
     
    The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAECPAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study. (C) 2011 The International Alliance for Biologicals. Published by Elsevier Ltd. All rights reserved.
  • Mitsuhiro Kinoshita; Naotaka Kakoi; Yu-ki Matsuno; Takao Hayakawa; Kazuaki Kakehi
    BIOMEDICAL CHROMATOGRAPHY 25 5 588 - 593 2011年05月 
    Carbohydrates having sulfate groups such as glycosaminoglycans and chemically synthesized sucrose sulfate show interesting and important biological activities. We adapted CE with indirect UV detection technique to the determination of sulfate ester in sulfated carbohydrates, which were previously hydrolyzed with HCl. The liberated sulfate ion was analyzed using a background electrolyte consisting of triethanolamine-buffered chromate with hexamethonium bromide. Sulfate contents of glucose 3-sulfate and sucrose octasulfate used as a model were in good agreement with theoretical values (accuracy, 95.9-96.7 and 97.4-101.9%, respectively), and relative standard deviation values run-to-run were 0.977 and 1.90%, respectively. We applied the method to the determination of the sulfate contents of some glycosaminoglycan samples and showed that the contents were in good agreement with those calculated from sulfur content. Copyright (C) 2010 JohnWiley & Sons, Ltd.
  • 癌特異的糖タンパク質のグライコプロテオーム解析
    三ツ井 洋輔; 山田 佳太; 田中 佑樹; 栫 直孝; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 131年会 3 109 - 109 (公社)日本薬学会 2011年03月
  • ヒト胃癌細胞中の高フコシル化糖タンパク質の探索
    三ツ井 洋輔; 原 沙弥香; 山田 佳太; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 131年会 3 109 - 109 (公社)日本薬学会 2011年03月
  • Mitsuhiro Kinoshita; Naotaka Kakoi; Yu-Ki Matsuno; Takao Hayakawa; Kazuaki Kakehi
    Biomedical Chromatography 25 5 588 - 593 2011年03月 [査読有り]
     
    Carbohydrates having sulfate groups such as glycosaminoglycans and chemically synthesized sucrose sulfate show interesting and important biological activities. We adapted CE with indirect UV detection technique to the determination of sulfate ester in sulfated carbohydrates, which were previously hydrolyzed with HCl. The liberated sulfate ion was analyzed using a background electrolyte consisting of triethanolamine-buffered chromate with hexamethonium bromide. Sulfate contents of glucose 3-sulfate and sucrose octasulfate used as a model were in good agreement with theoretical values (accuracy, 95.9-96.7 and 97.4-101.9%, respectively), and relative standard deviation values run-to-run were 0.977 and 1.90%, respectively. We applied the method to the determination of the sulfate contents of some glycosaminoglycan samples and showed that the contents were in good agreement with those calculated from sulfur content. © 2010 John Wiley & Sons, Ltd.
  • グライコプロテオミクスによるポリラクトサミン型糖鎖キャリアータンパク質の解析
    三ツ井 洋輔; 山田 佳太; 栫 直孝; 田中 佑樹; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 4P - 0216 (公社)日本生化学会 2010年12月
  • Eiki Maeda; Koji Urakami; Kiyohito Shimura; Mitsuhiro Kinoshita; Kazuaki Kakehi
    JOURNAL OF CHROMATOGRAPHY A 1217 45 7164 - 7171 2010年11月 [査読有り]
     
    A robust and highly reproducible capillary isoelectnc focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte In order to optimize major analytical parameters for robust mobilization experimental responses from three pI markers were selected The optimized method gave excellent repeatability and intermediate precision in estimated pi values of charge variants with relative standard deviations (RSDs) of not more than 0 06% and 0 95% respectively when using IgG(4) as a model Furthermore RSDs of charge variant compositions were less than 5 0% These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution and It is applicable to quality testing and detailed characterization in the pharmaceutical industry In addition it should be noticed that the method provided non-linear pH gradient within the tested ranges from pI 9 50 to 3 78 and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values (C) 2010 Elsevier B V All rights reserved
  • Eiki Maeda; Koji Urakami; Kiyohito Shimura; Mitsuhiro Kinoshita; Kazuaki Kakehi
    JOURNAL OF CHROMATOGRAPHY A 1217 45 7164 - 7171 2010年11月 
    A robust and highly reproducible capillary isoelectnc focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte In order to optimize major analytical parameters for robust mobilization experimental responses from three pI markers were selected The optimized method gave excellent repeatability and intermediate precision in estimated pi values of charge variants with relative standard deviations (RSDs) of not more than 0 06% and 0 95% respectively when using IgG(4) as a model Furthermore RSDs of charge variant compositions were less than 5 0% These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution and It is applicable to quality testing and detailed characterization in the pharmaceutical industry In addition it should be noticed that the method provided non-linear pH gradient within the tested ranges from pI 9 50 to 3 78 and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values (C) 2010 Elsevier B V All rights reserved
  • Toru Hiratsuka; Shinsuke Matsuzaki; Shingo Miyata; Mitsuhiro Kinoshita; Kazuaki Kakehi; Shinji Nishida; Taiichi Katayama; Masaya Tohyama
    PLOS ONE 5 10 e1328  2010年10月 [査読有り]
     
    Background: Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death. Methods: We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein. Results: Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs. Conclusions: Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.
  • Keita Yamada; Satomi Hyodo; Mitsuhiro Kinoshita; Takao Hayakawa; Kazuaki Kakehi
    Analytical Chemistry 82 17 7436 - 7443 2010年09月 [査読有り]
     
    We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k. Yamada, K. Tanabe, A. Kinoshita, M. Maruyama, S.-z. Osaka, Y.-s. Masuko, T. Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K. Hyodo, S. Matsuno, Y. K. Kinoshita, M. Maruyama, S. Z. Osaka, Y. S. Casal, E. Lee, Y. C. Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within 10 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K. Kinoshita, M. Hayakawa, T. Nakaya, S. Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works. © 2010 American Chemical Society.
  • Keita Yamada; Satomi Hyodo; Mitsuhiro Kinoshita; Takao Hayakawa; Kazuaki Kakehi
    ANALYTICAL CHEMISTRY 82 17 7436 - 7443 2010年09月 
    We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k.; Yamada, K.; Tanabe, A.; Kinoshita, M.; Maruyama, S.-z.; Osaka, Y.-s.; Masuko, T.; Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within 10 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K.; Kinoshita, M.; Hayakawa, T.; Nakaya, S.; Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works.
  • 松野祐樹; 掛樋 一晃; 木下 充弘; 山田佳太; 尾坂裕輔; 丸山
    Anal Biochem. 362 2 245 - 257 2010年05月 
    An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions.
  • 培養癌細胞中のムチン型およびプロテオグリカン型糖鎖の一斉解析
    栫 直孝; 山田 佳太; 木下 充弘; 早川 堯夫; 掛樋 一晃
    日本薬学会年会要旨集 130年会 3 115 - 115 (公社)日本薬学会 2010年03月
  • Hirofumi Takada; Aiko Nishida; Mitsuhiro Domae; Ayako Kita; Yuki Yamano; Atsushi Uchida; Shunji Ishiwata; Yue Fang; Xin Zhou; Takashi Masuko; Mitsuhiro Kinoshita; Kazuaki Kakehi; Reiko Sugiura
    MOLECULAR BIOLOGY OF THE CELL 21 4 674 - 685 2010年02月 [査読有り]
     
    The highly conserved fission yeast Pmk1 MAPK pathway plays a key role in cell integrity by regulating Atf1, which belongs to the ATF/cAMP-responsive element-binding (CREB) protein family. We identified and characterized ecm33(+), which encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. We demonstrated that the gene expression of Ecm33 is regulated by two transcription factors Atf1 and a MADS-box-type transcription factor Mbx1. We identified a putative ATF/CREB-binding site and an RLM1-binding site in the ecm33(+) promoter region and monitored the transcriptional activity of Atf1 or Mbx1 in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of the CRE and six tandem repeats of the Rlm1-binding sequence, respectively. These reporter genes reflect the activation of the Pmk1 pathway by various stimuli, thereby enabling the real-time monitoring of the Pmk1 cell integrity pathway. Notably, the Delta ecm33 cells displayed hyperactivation of the Pmk1 signaling together with hypersensitivity to Ca2+ and an abnormal morphology, which were almost abolished by simultaneous deletion of the components of the Rho2/Pck2/Pmk1 pathway. Our results suggest that Ecm33 is involved in the negative feedback regulation of Pmk1 cell integrity signaling and is linked to cellular Ca2+ signaling.
  • Keita Yamada; Sakie Watanabe; Soichiro Kita; Mitsuhiro Kinoshita; Takao Hayakawa; Kazuaki Kakehi
    ANALYTICAL BIOCHEMISTRY 396 1 161 - 163 2010年01月 [査読有り]
     
    An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate Structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. in the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells. (C) 2009 Elsevier Inc. All rights reserved.
  • Naoki Kamei; Rie Fukui; Yoshiyuki Suzuki; Yasuhiro Kajihara; Mitsuhiro Kinoshita; Kazuaki Kakehi; Hironobu Hojo; Katsunari Tezuka; Takashi Tsuji
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 391 1 557 - 563 2010年01月 [査読有り]
     
    Glycosylation is a widespread post-translational modification Found in glycoproteins Glycans play key roles in protein folding. quality control in the endoplasmic reticulum (ER) and protein trafficking within cells However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present oil inducible costimulator (ICOS), a T-cell costimulatory molecule. in proper protein folding and intracellular trafficking to the cell surface membrane We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89). but not proteins lacking either N23 or N110. were retained within the cell and were not detected oil the cell surface membrane Additional evidence Suggested that N89 glycosylation was indirectly involved in ICOS ligand binding These data suggest that amongst the three putative ICOS glycosylation sites, N89 is requried for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins. (C) 2009 Elsevier Inc All rights reserved
  • Naotaka Kakoi; Mitsuhiro Kinoshita; Nana Kawasaki; Teruhide Yamaguchi; Takao Hayakawa; Kazuaki Kakehi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 129 10 1255 - 1264 2009年10月 [査読有り]
     
    Heparin is widely used as an anticoagulant for the treatment and prevention of thrombotic disorders. Recently, hundreds of cases of anaphylactic reaction as adverse effects were reported by the presence of contaminating oversulfated chondroitin sulfate (OSCS) in some heparin preparations. In addition, these heparin preparations often contaminated dermatan sulfate (DS). Unfortunately, the Japanese Pharmacopoeia (JP) does not include appropriate purity tests. In the present paper, we show that capillary electrophoresis (CE) is a powerful tool for the analysis of OSCS and DS ill heparin preparations. CE method shows high resolution and good quantification of OSCS in heparin preparations. This method (OSCS method) was evaluated for accuracy (93.7%), repeatability (R.S.D. = 2.11), linearity (R-2 = 0.9996), detection limit (0.1% OSCS) and specificity. In contrast, DS was not able to be detected in high sensitivity by OSCS method. However, a modified CE method (DS method) using the buffer at lower pHs showed good parameters for accuracy (88.1%,), repeatability (R.S.D. = 1.99), linearity (R-2 = 0.9998), detection limit (0.25% DS) and specificity. In conclusion, CE will be an alternative to the NMR method which is being adopted for purification test of heparin sodium in the present version of JP.
  • フコシル化を回復させたHCT116細胞上に観察される糖タンパク質糖鎖
    栫 直孝; 山田 佳太; 田中 佑樹; 岩本 竜昇; 木下 充弘; 三善 英知; 森脇 健太; 早川 堯夫; 掛樋 一晃
    日本生化学会大会プログラム・講演要旨集 82回 4P - 010 (公社)日本生化学会 2009年09月
  • Mitsuhiro Kinoshita; Hiroko Ohta; Kanata Higaki; Yoko Kojima; Tadasu Urashima; Kazuki Nakajima; Minoru Suzuki; Kit M. Kovacs; Christian Lydersen; Takao Hayakawa; Kazuaki Kakehi
    Analytical Biochemistry 388 2 242 - 253 2009年05月 
    A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position. © 2009 Elsevier Inc. All rights reserved.
  • Mitsuhiro Kinoshita; Hiroko Ohta; Kanata Higaki; Yoko Kojima; Tadasu Urashima; Kazuki Nakajima; Minoru Suzuki; Kit M. Kovacs; Christian Lydersen; Takao Hayakawa; Kazuaki Kakehi
    ANALYTICAL BIOCHEMISTRY 388 2 242 - 253 2009年05月 [査読有り]
     
    A complex mixture of diverse oligosaccharides related to the carbohydrates in glycocojugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and Structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW similar to 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk Was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Gal beta 1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Ga beta 1-4GlcNAc beta 1-6[Gal beta 1-4Glc-NAc beta 1-3]Gal beta 1-4Glc) as the common core structure, and most of them contained Fuc alpha 1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Gal beta 1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Gal beta 1-4Glc-NAc beta 1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also Substituted with all N-acetyllactosamine at the 6-OH position. (c) 2009 Elsevier Inc. All rights reserved.
  • Keita Yamada; Mitsuhiro Kinoshita; Takao Hayakawa; Shuuichi Nakaya; Kazuaki Kakehi
    JOURNAL OF PROTEOME RESEARCH 8 2 521 - 537 2009年02月 [査読有り]
     
    Recently, we developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins and proteoglycans (Anal. Biochem. 2007, 362, 245-251; 2007, 371, 52-61). In the present paper, we released O-glycans from some leukemia and epithelial cells using the apparatus, and compared the profiles of O-glycans among these cells after fluorescent labeling of the released glycans with 2-aminobenzoic acid. The fluorescent labeled glycans were analyzed using a combination of HPLC and off-line MALDI-(QIT)TOF mass spectrometry We found that leukemia cells generally showed simple glycan profiles and commonly contained sialyl-T (NeuAc alpha 2-3Gal beta 1-3GalNAc) and disialyl-T (NeuAc alpha 2-3Gal beta 1-3 (NeuAc alpha 2-6)GalNAc) antigens as major O-glycans. In contrast, epithelial cancer cell lines usually showed extremely complex profiles. We found that polylactosamine-type O-glycans were abundantly present in MKN45 cells. Especially, we found characteristic glycans, of which Gal beta 1-3 residue of core1 structure is modified with biantennary polylactosamine units. In contrast, this cell line did not contain polylactosamine-type N-glycans (J. Proteome Res. 2006, 6, 88-97). These results suggest that the different biosynthetic pathways for N- and O-glycans are proposed, The method presented here will accelerate the speed for comprehensive analysis of O-glycans in biological samples and will be a powerful tool for clinical/biochemical analysis in cancer biology.
  • Kazuaki Kakehi; Mitsuhiro Kinoshita
    Methods in Molecular Biology 534 93 - 105 2009年 [査読有り]
     
    Glycosylation is one of the most important post-translational events for proteins, affecting their functions in healthdisease,plays significant roles in various information trafficking for intercellularintracellular biological events. The glycans which show such important effects are generally present as quite complex mixtures in minute amounts. The approach described here makes it possible to profile glycans for the analysis of post-translational modification of proteins with carbohydrates. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The technique affords simultaneous determination of glycans having similar structures even in complex mixtures. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.
  • Yu-Ki Matsuno; Naotaka Kakoi; Mitsuhiro Kinoshita; Yuji Matsuzaki; Jun-Ichi Kumada; Kazuaki Kakehi
    ELECTROPHORESIS 29 17 3628 - 3635 2008年09月 [査読有り]
     
    Hyaluronic acid (HA) samples showing inhibition effect on digestion with testicular hyaluronidase (HAase) were found from 16 commercially available HA products, which were supplied from 11 different manufacturers. Most of these HA samples (six samples) were derived from the rooster comb, and one sample was derived from the human umbilical cord. HA oligosaccharides produced by exhaustive digestion of these HA samples with testicular HAases were monitored by capillary electrophoresis, and we found that a few HA samples gave no oligosaccharide products. Detailed analysis of HA samples by cellulose acetate membrane electrophoresis revealed that the HA samples were not digested with HAase because of the presence of a small amount of dermatan sulfate (DS). Analysis of disaccharide units of these HA samples produced by digestion with chondroitinase ABC supported the observations. And the content of DS in the sample was estimated to be ca. 8%. In contrast, these HA samples were easily digested with bacterial hyaluronate lyases from Streptomyces hyalurolyticus and Streptococcus dysgalactiae and gave endproducts of unsaturated disaccharide or unsaturated tetra- or hexasaccharides. The results suggested that the inhibitory effect of DS on HAase is specific to endo-type hydrolase (i.e. testicular HAase). In addition, pharmaceutical preparations of HA derived from rooster comb were easily digested with testicular HAase. These findings will be useful information for clinical or cosmetic use of HA preparations in terms of their half-life.
  • Aya Ishizuka; Yuki Hashimto; Ryosuke Naka; Mitsuhiro Kinoshita; Kazuaki Kakehi; Junichi Seino; Yoko Funakoshi; Tadashi Suzuki; Akihiko Kameyama; Hisashi Narimatsu
    BIOCHEMICAL JOURNAL 413 2 227 - 237 2008年07月 [査読有り]
     
    During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 8897], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancerderived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3Man beta 1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.
  • 質量分析法によるセネガとニンジン中のグリコシドエステルを有するサポニン類の分析
    王 峰; 木下 充弘; 松田 秀秋; 森川 敏生; 掛樋 一晃
    日本薬学会年会要旨集 128年会 3 47 - 47 2008年03月
  • 多賀 淳; 掛樋 一晃; 木下 充弘; 北 荘一郎; 西浦 香織; 林 智則; 佐藤 睦; 鈴木 健太郎; 小玉 修嗣
    J. Sep. Sci. 31 5 853 - 858 2008年03月 [査読有り]
     
    Antibody pharmaceuticals are becoming more and more prevalent due to their excellent effectiveness in clinical medications, and are expected to allow tailor-made medical treatment for rheumatic diseases, immunosuppression in cardiac transplantation, and cancer. Antibody-type pharmaceuticals of immunoglobulin G (IgG) commonly have N-glycosylated carbohydrate chains attached to heavy chains. The carbohydrate chains play important roles in the effectiveness of antibodies. Therefore evaluation of a glycosylated species is important in the first step of quality control of antibody pharmaceuticals. In the present work, we examined capillary electrophoresis with a newly developed, chemically modified capillary, the inner surface of which is modified with carboxyl groups, for evaluation of IgG molecular species which have carbohydrate chains; tocilizumab was used as a model. The analytical system developed in the present study is useful for determining the content of non-glycosylated peptides. In the analysis of tocilizumab, the ratio of non-glycosylated peptide was estimated to be 1.23% with a relative standard deviation of 3.05%. The method affords high reproducibility with simple operation, and analysis can be completed within 6 min.
  • Keita Yamada; Satomi Hyodo; Yu-ki Matsuno; Mitsuhiro Kinoshita; Shu-zou Maruyama; Yu-suke Osaka; Enriqueta Casal; Yuan C. Lee; Kazuaki Kakehi
    ANALYTICAL BIOCHEMISTRY 371 1 52 - 61 2007年12月 [査読有り]
     
    Rapid and sensitive analysis of glycans is essential for glycomics. We previously reported an apparatus, the AutoGlycoCutter (AGC), for rapid release of O-linked glycans under alkaline conditions and its application to rapid analysis of glycans in proteoglycans. We now report an application of the AGC to obtain mucin-type glycans with reducing end (i.e., hemiacetal group) within only 3 min. The released oligosaccharides could be labeled with fluorescent 2-aminobenzoic acid for analysis by normal-phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We could detect O-glycans from as low as 5 pmol of bovine caseino glycomacropeptide (CGMP) by the proposed procedures. The validity of the current method was shown by the analyses of the released O-glycans from some standard glycoproteins: bovine submaxillary mucin, bovine fetuin, porcine stomach mucin, and human colostrum immunoglobulin A. The advantage of the current method was also demonstrated in comparative analysis of mucin-type glycans in CGMP derived from three different animal species. (c) 2007 Elsevier Inc. All rights reserved.
  • Yu-ki Matsuno; Keita Yamada; Ayumi Tanabe; Mitsuhiro Kinoshita; Shu-zou Maruyama; Yu-suke Osaka; Takashi Masuko; Kazuaki Kakehi
    ANALYTICAL BIOCHEMISTRY 362 2 245 - 257 2007年03月 [査読有り]
     
    An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method. (c) 2006 Elsevier Inc. All rights reserved.
  • S Kamoda; Y Nakanishi; M Kinoshita; R Ishikawa; K Kakehi
    JOURNAL OF CHROMATOGRAPHY A 1106 1-2 67 - 74 2006年02月 
    Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved. 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of oil-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antermary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform, detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides. (c) 2005 Elsevier B.V. All rights reserved.
  • R Naka; S Kamoda; A Ishizuka; M Kinoshita; K Kakehi
    JOURNAL OF PROTEOME RESEARCH 5 1 88 - 97 2006年01月 
    Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.
  • K Nakajima; M Kinoshita; N Matsushita; T Urashima; M Suzuki; A Suzuki; K Kakehi
    ANALYTICAL BIOCHEMISTRY 348 1 105 - 114 2006年01月 
    Animal colostrum and milk contain complex mixtures of oligosaccharides, which have species-specific profiles. Milk oligosaccharides have various types of structure related to the core Structures of glycolipids and N- and O-glycans of glycoproteins and provide a good library to examine the binding of oligosaccharides to various lectins. Recently, we reported a capillary affinity electrophoresis (CAE) method for analyzing the interactions between lectins and complex mixtures of N-linked oligosaccharides prepared from serum glycoproteins [K. Nakajima, Y. Oda, M. Kinoshita, K. Kakehi, J. Proteome Res. 2 (2003) 81-88]. The present paper reports the interactions between 24 milk oligosaccharides and six lectins (PA-1, RCA(120), SBA, WGA, UEA-I, and AAL) analyzed using CAE. Based on the resulting data, we constructed a library that enables LIS to determine nonreducing terminal monosaccharides, such as Gal, GalNAc, GlcNAc, and Fuc, and to differentiate Gal- or Fuc-linked isomers, such as lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose 11 and III. In addition, using the library, we show that a combination of the lectins can characterize the neutral oligosaccharides derived from bovine colostrum. (c) 2005 Elsevier Inc. All rights reserved.
  • Shin-Ichi Yasueda; Kazuhiro Yamakawa; Yasuharu Nakanishi; Mitsuhiro Kinoshita; Kazuaki Kakehi
    Journal of Pharmaceutical and Biomedical Analysis 39 1-2 187 - 195 2005年09月 
    Characteristics of tear-film may be influenced by contact lens wear, because contact lenses present the habitual, direct rubbing action of the lids upon the covered ocular surface and may cause changes of tear-film. In the present paper, influence of contact lens on proteins in tear samples was studied using carbohydrates attached to the protein as a marker. We found that N-acetylneuraminic acid (Neu5Ac) was significantly decreased in tear samples of volunteers wearing contact lens (wearing, 86.1 ± 57.7 nmol/ml normal, 190.2 ± 121.9 nmol/ml). Analysis by polyacrylamide gel electrophoresis demonstrated that the amounts of major proteins in tear fluids, such as lactoferrin and secretory immunoglobulin A were not changed upon wearing contact lenses. In contrast, cellulose acetate membrane electrophoresis revealed that mucin band in tear samples from contact lens wearers showed significant decrease as examined by lectin staining. © 2005 Elsevier B.V. All rights reserved.
  • S Kamoda; M Nakano; R Ishikawa; S Suzuki; K Kakehi
    JOURNAL OF PROTEOME RESEARCH 4 1 146 - 152 2005年01月 
    There are a large number of labeling methods for asparagine-type oligosaccharides with fluorogenic and chromophoric reagents. We have to choose the most appropriate labeling method based on the purposes such as mass spectrometry, high-performance liquid chromatography and capillary electrophoresis. Asparagine-type glycans are released from core proteins as N-glycosylamine at the initial step of the releasing reaction when glycoamidase F is employed as the enzyme. The N-glycosylamine-type oligosaccharicles thus released by the enzyme are subjected to hydrolysis or mutarotation to form free-form oligosaccharides. In the detailed studies on the enzyme reaction, we found a condition in which the released N-glycosylamine-type oligosaccha rides were exclusively present at least during the course of enzyme reaction, and developed a method for in situ derivatization of the glycosylamine-type oligosaccharides with 9-fluorenylmethyl chloroformate (Fmoc-CI). The Fmoc labeled sialo- and asialo- (or high-mannose and hybrid) oligosaccha rides were successfully analyzed on an amine-bonded polymer column and amide-silica column, respectively. The present method showed approximately 5 times higher sensitivities than that using 2-aminobenzoic acid (2-AA). The separation profile was similar to that observed using 2-AA method as examined by the analyses of carbohydrate chains derived from several glycoproteins including complex-type, high-mannose type and hybrid type of N-linked oligosaccha rides. The labeled oligosaccharicles were stable at least for several months when stored at -20 degreesC. Furthermore, it should be emphasized that the Fmoc-derivatized oligosaccha rides could be easily recovered as free reducing oligosaccharides simply by incubation with morpholine in dimethylformamide solution. We obtained a pure triantennary oligosaccharide with 3 sialic acid residues as a free reducing form from fetuin in good yield after isolation of the corresponding Fmoc oligosaccharide followed by removing reaction of the Fmoc group. The proposed method will be useful for preparation of free oligosaccha rides as standard samples at pmol-nmol scale from commercially available glycoproteins.
  • 機能性高分子ゲルを用いる生体組織中の目的成分の選択的捕捉
    林 友典; 木下 充弘; 安枝 真一; 三木 康義; 掛樋 一晃
    日本薬学会年会要旨集 122年会 3 79 - 79 (公社)日本薬学会 2002年03月
  • Y Oda; M Kinoshita; K Nakayama; K Kakehi
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 21 11 1215 - 1217 1998年11月 [査読有り]
     
    The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.
  • Y Oda; M Kinoshita; K Kakehi
    ANALYTICAL BIOCHEMISTRY 254 1 41 - 48 1997年12月 [査読有り]
     
    A fluorometric assay of lectin binding to yeast cells is reported. The relative amount of biotinylated lectins bound to the yeast cells was estimated by enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate for the lectin-bound beta-galactosidase through biotin-avidin interaction. Binding properties of 4 mannose specific and 3 glucose/mannose-specific lec tins to 22 different species of yeast cells were studied. The binding reaction of biotinylated lectins to the yeast cells was rapid and became constant within 10 min. Each lectin showed its characteristic binding specificity to each yeast species. The relative fluorescent intensities observed for 4-methylumbelliferone released by the action of bound beta-galactosidase were good indicators for the classification of yeast cells in quantitative base. We found that the yeast cells of the Saccharomyces genus can be classified into three groups, and those of Pichia were grouped into two groups. The present method can examine many samples simultaneously and be completed within 3 h. (C) 1997 Academic Press.

書籍

  • ナノテク・バイオMEMS時代の分離・計測技術, グライコーム解析
    掛樋 一晃; 木下 充弘 (担当:共著範囲:)シーエムシー出版 2006年02月 
    本稿ではグライコーム解析の現状について紹介するとともに、キャピラリー電気泳動法を用いるグライコーム解析へのアプローチとその高速化を目指したマイクロチップ電気泳動による糖鎖の分析法について紹介する。
  • 糖鎖科学の新展開-機能解明・次世代型材料・医薬品開発に向けて-, キャピラリー電気泳動による構造グライコミクスへのアプローチ
    掛樋 一晃; 木下 充弘 (担当:共著範囲:)株式会社エヌ・ティー・エス 2005年08月 
    構造グライコミクスは糖鎖機能を解明するために糖鎖構造情報を包括的に取得するための方法論として不可欠な要素であり、これを達成するためには高網羅性・高スループット・高感度という技術要素が要求される。キャピラリー電気泳動法とレーザー励起蛍光検出法を組合せる方法は、糖鎖分析において最も高感度が期待できる方法であり、構造グライコミクスを達成できる手法の一つである。本稿ではキャピラリー電気泳動法を用いる構造グライコミクスへのアプローチについて、現状と将来の展望について述べる。
  • Lectin from bulbs of Crocus sativus recognizing N-linked core glycan: isolation and binding studies using fluorescence polarization
    Methods Enzymol 2003年

講演・口頭発表等

  • 多分岐流路マイクロチップと光重合性ポリアクリルアミドゲルを用いるリン酸化化合物のオンライン濃縮・標識マイクロチップ電気泳動法の開発  [通常講演]
    矢野祥子; 増田誠子; 山本佐知雄; 木下充弘; 鈴木茂生
    第39回キャピラリー電気泳動シンポジウム 2019年11月 口頭発表（一般）
  • 構造グライコミクス技術の現状と高スループット解析プラットホームとしての電気泳動の可能性  [招待講演]
    木下充弘; 山本佐知雄; 鈴木茂生
    第39回キャピラリー電気泳動シンポジウム 2019年11月 口頭発表（招待・特別）
  • マイクロチップ電気泳動装置を利用する全自動糖鎖解析システムの開発  [通常講演]
    中谷祐美; 寺井佑奈; 山本佐知雄; 鈴木茂生; 木下充弘
    第69回日本薬学会関西支部総会・大会 2019年10月 ポスター発表
  • リキッドバイオプシーによる糖鎖バイオマーカーの探索  [通常講演]
    池内紘子; 西畑星奈; 山本佐知雄; 鈴木茂生; 木下充弘
    第69回日本薬学会関西支部総会・大会 2019年10月 ポスター発表
  • 大腸がん細胞の細胞外環境とアスパラギン結合型糖鎖の高分岐化  [通常講演]
    谷ノ上顕大; 寺口瑠果; 山本佐知雄; 鈴木茂生; 木下充弘
    第69回日本薬学会関西支部総会・大会 2019年10月 ポスター発表
  • ラット肝癌細胞のヘキソサミン合成経路の活性化によるアスパラギン結合型糖鎖の高分岐化  [通常講演]
    寺口瑠果; 栗須里彩; 山本佐知雄; 鈴木茂生; 木下充弘
    第69回日本薬学会関西支部総会・大会 2019年10月 ポスター発表
  • ヘキソサミン経路活性化によるアスパラギン結合型糖鎖の高分岐化  [通常講演]
    木下充弘; 松本和樹; 山本佐知雄; 鈴木茂生
    第92回日本生化学会大会 2019年09月 ポスター発表
  • 全自動マイクロチップ電気泳動装置を利用した高スループット糖鎖解析プラットフォーム  [通常講演]
    木下充弘; 山本万莉; 中谷裕美; 山本佐知雄; 鈴木茂生
    日本分析化学会第68年会 2019年09月 ポスター発表
  • 部分導入アフィニティーキャピラリー電気泳動とHPLC を組み合わせた8-Aminopyrene-1,3,6-trisulfonic acid 標識化ガン細胞由来糖鎖の二次元解析  [通常講演]
    山本佐知雄; 中住智典; 宮脇直久; 川上夏海; 田中冬馬; 木下充弘; 鈴木茂生
    第32回 バイオメディカル分析科学シンポジウム 2019年08月 口頭発表（一般）
  • 蛍光標識糖タンパク質糖鎖分析のためのオンライン固相抽出HPLC 分析法の開発  [通常講演]
    鈴木茂生; 前迫知宏; 岡田風花; 長友淑恵; 岸本有加; 山本佐知雄; 木下充弘
    第32回 バイオメディカル分析科学シンポジウム 2019年08月 ポスター発表
  • 細胞外環境および増殖シグナルがN-結合型糖鎖の分岐に及ぼす影響  [通常講演]
    木下充弘; 松本和樹; 山本佐知雄; 鈴木茂生
    第38回日本糖質学会年会 2019年08月 ポスター発表
  • ヘキソサミン経路とその周辺代謝環境が糖鎖生合成に与える影響  [通常講演]
    寺口瑠果; 山本佐知雄; 鈴木茂生; 木下充弘
    第68回 日本薬学会近畿支部総会・大会 2018年10月 ポスター発表
  • 抗体医薬の定常領域配列に着目した迅速糖鎖プロファイリング  [通常講演]
    河崎拓也; 山本佐知雄; 鈴木茂生; 木下充弘
    第68回 日本薬学会近畿支部総会・大会 2018年10月 ポスター発表
  • ヒト肝臓がん細胞タンパク質および分泌タンパク質の発現糖鎖解析  [通常講演]
    松本和樹; 山本佐知雄; 木下充弘; 鈴木茂生
    第68回 日本薬学会近畿支部総会・大会 2018年10月 ポスター発表
  • マイクロチップアフィニティ電気泳動による糖タンパク質糖鎖の高速定量/定性解析  [通常講演]
    木下 充弘; 山本佐知雄; 鈴木茂生
    第91回 日本生化学会大会 2018年09月 ポスター発表
  • 抗体医薬の定常領域配列に着目した迅速糖鎖プロファイリング  [通常講演]
    河崎 拓也; 山本 佐知雄; 鈴木 茂生; 木下充弘
    第31回 バイオメディカル分析科学シンポジウム 2018年08月 ポスター発表
  • 高密度アミノ化スライドガラスにアレイ化したタンパク質の翻訳後修飾解析  [通常講演]
    木下 充弘; 田中晴佳; 松本和樹; 山本佐知雄; 鈴木茂生
    第37回 日本糖質学会年会 2018年08月 ポスター発表
  • グライコーム/グライコプロテオーム解析技術としての電気泳動の可能性  [通常講演]
    木下 充弘; 山本佐知雄; 鈴木茂生; 木下英樹
    第69回 日本電気泳動学会総会 2018年08月 シンポジウム・ワークショップパネル（指名）
  • 電気泳動法を組合せた糖タンパク質のトップダウン解析手法の合理的設計  [通常講演]
    木下 充弘; 御子柴柚子; 山本万莉; 山本佐知雄; 鈴木茂生
    第37回 キャピラリー電気泳動シンポジウム 2017年11月 口頭発表（一般）
  • 糖鎖解析において示されるマイクロチップ電気泳動装置”MultiNA”の実力と可能性  [通常講演]
    木下 充弘
    第16回 糖質科学コンソーシアムシンポジウム 2017年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • 糖タンパク質性バイオ医薬品のPAT指向型分析技術基盤の開発に向けた取り組み  [通常講演]
    木下 充弘; 御子柴柚子; 山本佐知雄; 鈴木茂生
    第37回 日本糖質学会年会 2017年07月 ポスター発表
  • ゲル電気泳動からの抽出とインタクト質量分析法による抗体医薬品の高選択的特性解析手法の構築  [通常講演]
    山田英明; 松村千恵美; 山田佳太; 手島浩一郎; 廣島高志; 木下充弘; 鈴木茂生; 掛樋一晃
    第65回 質量分析総合討論会 2017年05月 口頭発表（一般）
  • バイオ医薬品開発における糖鎖解析技術  [招待講演]
    木下 充弘; 山本佐知雄; 鈴木茂生
    第29回 バイオメディカル分析科学シンポジウム 2016年09月 シンポジウム・ワークショップパネル（指名）
  • 大学生の肥満と朝食欠食およびやせ志向との関連性に関する研究  [通常講演]
    川﨑 直人; 緒方 文彦; 冨永壽人; 木下 充弘; 掛樋 一晃; 佐川和則
    2011年03月
  • 大学生の朝食欠食と体格誤認に関する実態調査  [通常講演]
    冨永壽人; 緒方 文彦; 掛樋 一晃; 木下 充弘; 川﨑 直人; 佐川和則
    2011年03月
  • 調剤薬局における薬剤師需要の動向予測に関する研究  [通常講演]
    長井 紀章; 川瀬 篤史; 木下 充弘; 関口 富美子; 桑島 博; 鈴木 茂生; 西田 升三; 松尾 圭造; 掛樋 一晃
    医療薬学フォーラム2010 第18回クリニカルファーマシーシンポジウム 2010年07月 広島 医療薬学フォーラム2010 第18回クリニカルファーマシーシンポジウム
  • キャピラリー電気泳動による抗体医薬品の品質管理  [通常講演]
    多賀 淳; 木下 充弘; 掛樋 一晃; 北 荘一郎; 佐藤 睦; 鈴木 健太郎; 小玉 修嗣
    第18回クロマトグラフィー科学会議 2007年11月 函館 第18回クロマトグラフィー科学会議
  • 化学修飾シリカキャピラリーを利用する抗体医薬品のキャピラリー電気泳動  [通常講演]
    多賀 淳; 木下 充弘; 掛樋 一晃; 北荘一郎; 佐藤睦; 鈴木健太郎; 小玉修嗣; 鴨田聡
    第27回日本糖質学会年会 2007年08月 福岡 第27回日本糖質学会年会
  • プロテオグリカン中のグリコサミノグリカン結合領域糖鎖の高速高感度解析法  [通常講演]
    掛樋 一晃; 木下 充弘; 田辺亜弓; 松野
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    我々はムチン型糖鎖を高速かつ高感度で分析するために糖鎖自動切断装置“AutoGlycoCutter”（AGC）をNEDO糖鎖エンジアリングプロジェクトの一環として開発中である。これまでにAGCがムチン型糖鎖の高速切り離し法として有効であることを報告しており、現在プロテオグリカン（PG）中のグリコサミノグリカン（GAGs）の解析への適用についても検討を進めている。本研究では開発中のAGCを用いてPGの結合領域糖鎖を切り離し構造解析を行った結果について報告する。
  • 2次元クロマトグラフィーシステムを利用するグライコプロテオミクスへの試み  [通常講演]
    掛樋 一晃; 木下 充弘; 田中寿和
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    生体内タンパク質の50％以上が糖鎖修飾を受けていると言われているが、現在のプロテオミクス研究は、タンパク質のプロファイリングと同定に重点が置かれ、糖鎖による翻訳後修飾を含めた形でグライコプロテオミクスと呼べる方法論は確立していないと思われる。本研究では、2次元クロマトグラフィーシステム（2D-HPLC）によりタンパク質を等電点と疎水性の違いに基づき2次元分画し、タンパク質プロファイルを比較する方法ならびに分離された各タンパク質の糖鎖解析を行う方法を検討したので報告する
  • 低分子量ヘパリンの品質評価法に関する研究  [通常講演]
    掛樋 一晃; 木下 充弘; 中村仁美; 松野
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    生物由来医薬品中の成分が複合糖質である場合、糖鎖構造の違いに基づく不均一性を生じる。この不均一性は原材料、製造条件等の違いによっても変動することから、成分の同等性を評価できる分析手法はこれらの医薬品の品質確保において重要である。本研究では低分子量ヘパリン製剤であるダルテパリンナトリウムの同等性を各種分離分析法により評価した。
  • アザラシミルク中の高分子量オリゴ糖の構造解析  [通常講演]
    掛樋 一晃; 木下 充弘; 太田弘子; 檜垣かなた; 中嶋和紀; 浦島 匡
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    哺乳類のミルクオリゴ糖は、80％以上のラクトースとラクトース以外のオリゴ糖からなるが、個々の糖鎖の含量と構造は動物種によって著しく異なることが知られている。真獣類であるアザラシミルクの場合、H抗原を有する高分子量のオリゴ糖が多く含まれ、未知の糖鎖を含む糖鎖ライブラリーとしても興味深い。本研究では、ズキンおよびアゴヒゲアザラシミルク中の高分子量オリゴ糖画分に含まれる糖鎖について順相HPLCと質量分析法により構造解析を行った。
  • κカゼイン由来のムチン型糖ペプチドの精製と構造解析  [通常講演]
    掛樋 一晃; 木下 充弘; 山田佳太; 兵頭里美; Yuan C. Lee
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    我々はムチン型糖鎖を高速かつ高感度で分析するために、インフロー方式による高速糖鎖切断法を提案し、コアタンパク質より還元末端を持つムチン型糖鎖を数分以内で遊離することができる糖鎖自動切断装置“AutoGlycoCutter”（AGC）を開発中である。本研究ではAGCの詳細な性能評価を実施するため、ウシκカゼインからムチン型糖ペプチドの精製を行い、その構造解析を行った結果について報告する。
  • 癌細胞に特異的に観察される遊離糖鎖の解析  [通常講演]
    掛樋 一晃; 木下 充弘; 橋本有樹; 石塚; 文; 仲 亮輔; 中家修一; 亀山昭彦; 成松 久
    第126年会日本薬学会 2006年03月 仙台 第126年会日本薬学会
     
    細胞内で生合成された糖タンパク質は、高次構造不全の場合プロテアソームによる分解に先立ちタンパク質から糖鎖が除去されることが知られている。通常、遊離した糖鎖はリソソームでさらに分解を受けるが、何らかの原因で糖鎖分解機構に異常が起こると、細胞内に遊離糖鎖が蓄積する。この遊離糖鎖の蓄積は糖鎖の生合成および分解・代謝における異常とも密接に関係し、種々の疾患との関係についても興味が持たれる。本研究では癌細胞中の遊離糖鎖を順相HPLCおよびMALDI-TOF-MSを組み合わせ構造解析を行った結果を報告する。
  • 糖鎖自動切断装置“Auto Glyco Cutter”のムチン型糖鎖構造解析への応用  [通常講演]
    掛樋 一晃; 木下 充弘
    日本薬学会 2005年03月 東京 日本薬学会
     
    我々はムチン型糖鎖の分析において律速段階であった糖鎖の遊離反応を高速化するため、インフロー方式によるムチン型糖鎖高速遊離法について検討し、糖鎖自動切断装置“Auto Glyco Cutter（AGC）”を開発中である。本研究ではAGCの基本性能の評価を行うとともに、AGCを用いて入手可能なムチン型糖タンパク質中の糖鎖分析を行った結果を報告する。
  • 二次元電気泳動ゲル上で検出される糖タンパク質糖鎖の解析  [通常講演]
    掛樋 一晃; 木下 充弘; 久保 兼信; 鴨田聡
    日本薬学会 2005年03月 東京 日本薬学会
     
    2次元ゲル電気泳動（2DE）はタンパク質の網羅的解析技術で重要な役目を果たしている。また、ゲル上で観察された各たんぱく質について、プロテオーム解析技術と連動したタンパク質翻訳後修飾解析技術の開発が望まれる。本研究では2次元ゲル電気泳動により分離された微量糖タンパク質中の糖鎖を蛍光標識し、高感度に分析するために試料イオンの濃縮法について検討した。さらに、多発性骨髄腫患者より得られたIgG糖鎖の癌性変化の解析に利用した結果を報告する。
  • ウリナスタチンの品質評価に関する検討  [通常講演]
    掛樋 一晃; 木下 充弘
    日本薬学会 2005年03月 日本薬学会
     
    糖タンパク質は糖鎖組成の違いにより、グライコフォームと呼ばれる不均一性を生じる。したがって、糖タンパク質性医薬品の品質評価の指標として糖鎖の評価は極めて重要である。本研究では急性膵炎および急性循環不全治療薬として局方に収載されている糖タンパク質性医薬品ウリナスタチン(UTI)について、タンパク質分子を指標とする同等性評価法ならびに糖鎖を指標とする評価法について検討した結果について報告する。
  • 癌細胞糖タンパク質糖鎖の網羅解析  [通常講演]
    掛樋 一晃; 木下 充弘
    日本薬学会 2005年03月 東京 日本薬学会
     
    複合糖質中の糖鎖は生体内においてクロストークを行いながら、調和を保ち生理機能の発現や維持に関与している。糖鎖の意義を明らかにするためには、生体中の糖鎖を網羅的に精密に解析できる方法の確立が求められる。本研究では、細胞糖鎖、特に糖タンパク質中のアスパラギン結合型糖鎖に的を絞り、総糖鎖を一斉に解析する方法を開発した結果を報告する。
  • グリコサミノグリカン由来オリゴ糖の大量調製  [通常講演]
    掛樋 一晃; 木下 充弘
    日本薬学会 2005年03月 東京 日本薬学会
     
    グリコサミノグリカン類（GAGs）は結合組織の構造維持のための成分として重要であるが、最近、正常組織ではほとんど観察されないGAG由来オリゴ糖が示す様々な生理活性に興味が持たれている。本研究ではGAG由来オリゴ糖の生理活性の探索や誘導体合成のための材料となるオリゴ糖を効率良く高スケールで調製する技術の開発を目指し、バイオリアクターとしての酵素固定化カラムと生成オリゴ糖の分離を目的とする透析装置を組み合わせたオリゴ糖合成装置と選択的エタノール沈殿によるヒアルロン酸及びコンドロイチン由来オリゴ糖の大量調製法について検討した。
  • キャピラリーアフィニティー電気泳動による糖鎖クラスターとレクチンとの相互作用解析  [通常講演]
    掛樋 一晃; 木下 充弘; Yougjum Gao; Yuan; C. Lee
    日本薬学会 2005年03月 東京 日本薬学会
     
    細胞間認識等に関与する糖鎖-タンパク質間相互作用には、適切な空間的距離と配向を保って集積した糖鎖クラスターによるクラスター効果が重要である。本研究ではPolyhedral oligosilsesquioxane（POSS）骨格を母体とする糖鎖クラスターや高分枝複合型糖鎖をミミックした糖鎖を用いて、2、3のレクチン及び糖鎖認識抗体との相互作用におけるクラスター効果をキャピラリーアフィニティー電気泳動法（CAE）を用いて解析した。
  • 2次元電気泳動上で検出される糖タンパク質  [通常講演]
    掛樋 一晃; 木下 充弘; 久保 兼信
    第24回キャピラリー電気泳動シンポジウム 2004年12月 仙台 第24回キャピラリー電気泳動シンポジウム
     
    本研究では代表的なタンパク質翻訳後修飾の一つである糖鎖によるタンパク質修飾を網羅的に解析するために、SDS-PAGEまたは2次元電気泳動により分離された微量の糖タンパク質から糖鎖を遊離後、蛍光標識しレーザー励起蛍光検出キャピラリー電気泳動を用いて糖鎖を高感度で分析する技術を開発した。開発した手法を用いて多発性骨髄腫患者より得られた血清糖タンパク質糖鎖の癌性変化を解析した結果について報告する。
  • キャピラリー電気泳動を利用する細胞内糖転移酵素活性の比較解析法  [通常講演]
    掛樋 一晃; 木下 充弘
    第24回キャピラリー電気泳動シンポジウム 2004年12月 仙台 第24回キャピラリー電気泳動シンポジウム
     
    本研究ではキャピラリー電気泳動の高分解能分離を利用し、蛍光性糖鎖混合物を酵素基質として用い、ヒト癌細胞中に存在する糖転移酵素活性を一斉解析し、個々の糖鎖に対する特異性ならびに細胞間における酵素活性を比較解析する手法について報告する。
  • キャピラリーアフィニティー電気泳動法によるミルクオリゴ糖－レクチン間相互作用解析  [通常講演]
    掛樋 一晃; 木下 充弘; 鈴木明身; 鈴; 浦島匡
    第77回日本生化学会大会 2004年10月 横浜 第77回日本生化学会大会
  • 癌患者血清由来α1酸性糖タンパク質中の新規フコシル化N結合型糖鎖  [通常講演]
    掛樋 一晃; 木下 充弘; 矢澤伸; 西村東洋; 巨智部直久
    第77回日本生化学会大会 2004年10月 横浜 第77回日本生化学会大会
  • グリコサミノグリカン由来オリゴ糖と大量調製法  [通常講演]
    掛樋 一晃; 木下 充弘
    第77回日本生化学会大会 2004年10月 横浜 第77回日本生化学会大会
  • 糖タンパク質性医薬品中の糖鎖プロファイル分析へのキャピラリー電気泳動法の適用  [通常講演]
    掛樋 一晃; 木下 充弘; 鴨田聡 石川リカ; 川崎ナナ 早川尭夫
    第17回バイオメディカル分析科学シンポジウム 2004年06月 西宮 第17回バイオメディカル分析科学シンポジウム
  • マイクロチップ電気泳動を用いるグリコサミノグリカン製剤の高速簡易分析  [通常講演]
    掛樋 一晃; 木下 充弘
    第17回バイオメディカル分析科学シンポジウム 2004年06月 西宮 第17回バイオメディカル分析科学シンポジウム
  • Binding analysis of alpha-galactose-binding proteins by capillary affinity electrophoresis  [通常講演]
    掛樋 一晃; 木下 充弘; 今井康之
    InterLec21 2004年05月 湘南 InterLec21
     
    In this study, following oligosaccharides were prepared enzymatically or from bird eggs to examine the binding characteristics of alpha-galactose-binding proteins: N-glycans with one to three a-Gal epitope at the nonreducing end for anti-Gal antibody; and those with three Gala1-4Gal residues at nonreducing end for verotoxin. We studied the binding characteristics of anti-Gal antibody and verotoxin using capillary affinity electrophoresis (CAE), and found the following results.
  • Size-requirement of hyaluronan for the recognition by hyaluronan-binding protein  [通常講演]
    掛樋 一晃; 木下 充弘
    InterLec21 2004年05月 湘南 InterLec21
     
    Previously, we reported high-resolution separation of HA using capillary electrophoresis and a buffer containing neutral polymer as sieving matrix, and found that smaller oligosaccharides than octasaccharide migrated in the reverse order of their molecular sizes and larger oligosaccharides migrated in the order of molecular sizes. The reversal of migration order of these oligomers exhibited good correlation with the expression of biological functions. These results suggested that formation of a three-dimensional structure of HA oligomer played an important role in molecular recognition in the interaction between HA and their binding protein. The studies above prompted us to investigate the binding between HA oligomers having different molecular sizes and HABP. We used capillary affinity electrophoresis for the determination of the binding between HA oligomers and HABP from bovine nasal cartilage.
  • マイクロチップ電気泳動を用いるグリコサミノグリカン類の高速分析  [通常講演]
    掛樋 一晃; 松野裕樹; 木下 充弘
    第23 回キャピラリー電気泳動シンポジウム（岡山） 2003年12月 第23 回キャピラリー電気泳動シンポジウム（岡山）
     
    本研究では核酸の高速分析技術として利用されているマイクロチップ電気泳動装置を用い、ヒアルロン酸やコンドロチン硫酸などのグリコサミノグリカン類オリゴ糖の高速分析法ならびに蛍光性第四級アンモニウム塩を利用しグリコサミノグリカン類をラベル化や染色操作なしに直接分析する方法について検討した結果を報告した。
  • ヒアルロン酸オリゴマーとヒアルロン酸結合性タンパク質との相互作用解析 分子サイズと相互作用の関係  [通常講演]
    掛樋 一晃; 木下 充弘; 林友典; 益子 高
    日本薬学会第123年会（長崎） 2003年03月 日本薬学会第123年会（長崎）
     
    我々はヒアルロン酸（HA）を中性ポリマーを含む緩衝液を用いて表面修飾キャピラリー中で電気泳動を行うことによりあるサイズで泳動順序の逆転現象が起こることを見出し、この現象が生理活性と密接な関係があることを既に明らかにしている。本研究ではHAオリゴ糖とヒアルロン酸結合性タンパク質（HABP）との相互作用に着目し、HAオリゴ糖の重合度とHABP結合性の関係についてキャピラリー電気泳動を用い詳細な解析を行った結果について報告した。

作品等

  • 細胞内エネルギー代謝と糖鎖生合成
    木下 充弘
  • グライコフォームフォーカストプロテオミクス手法よる疾患マーカー探索
    木下 充弘
  • キャピラリー電気泳動を用いるバイオ医薬品評価技術開発
    木下 充弘
  • 糖タンパク質糖鎖の網羅的解析と細胞個性解析
    木下 充弘
  • 糖鎖-タンパク質間相互作用解析
    木下 充弘
  • キャピラリー電気泳動を用いた酸性多糖類の重合度解析
    木下 充弘

MISC

産業財産権

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 木下 充弘
     
    本年度においては、研究期間開始前より着手していた全自動マイクロチップ電気泳動装置を用いる、糖タンパク質糖鎖分析法の各種条件について詳細に検討し、リニアーポリアクリルアミドケルを含む緩衝液系にて、8-アミノピレン-1,3,6-トリスルフォネート標識された糖鎖を、高い分離能を発揮ししつ100秒以内に分離可能な条件を開発することに成功した。また、確立した方法を開発済みの迅速簡便な糖鎖試料調製法と組み合わせて用いる一連の糖鎖解析ワークフローの実行可能性について検証した。その結果、精製糖タンパク質試料およびヒト血清糖タンパク質からの糖鎖試料の調製から糖鎖プロファイリングまでを同一日中に完了できることを実証できた。さらに、レクチンを用いた糖鎖ピークの自動アノテーション法についても検討を開始した結果、レクチンとしてConA、DSA、WFAを組合せて用いることで、糖鎖のコア構造を明瞭に識別できることを明らかにした。
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2009年 -2010年 
    代表者 : 木下 充弘
     
    生体内の糖鎖は複数の遺伝子産物の協調作業により生合成されるため、細胞の生理的環境の変化を受け容易に変化する。細胞レベルにおけるこれらの微細な変化の蓄積は、老化の過程においても重要な一面を担うと考えられ、種々のバイオマーカー開発の標的としても興味深い。本研究では、抗加齢医学の実践に欠くことのできない加齢マーカーとしての糖鎖の可能性を検証するため、キャピラリー電気泳動(CE)を用いてラット血清糖タンパク質糖鎖の加齢に伴う変化を追跡した。また、メタボリックシンドロームモデル作製用に高脂肪食で飼育したラットの加齢及び食環境の違いが血清糖鎖プロファイルに及ぼす変化を明らかにした。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2005年 -2007年 
    代表者 : 掛樋 一晃; 木下 充弘
     
    本研究課題では、「細胞表面糖鎖を指標とする進化論的ならびに分化論的細胞分類」を目的とし、種々の分離分析手段を組み合わせた、細胞表面糖鎖の大規模解析技術を開発するとともに、培養癌細胞表面に分布する糖タンパク質糖鎖の定量的比較解析を行った。平成17年度はアフィニティークロマトグラフィーと順相分配型アミノカラムを用いるHPLCを組み合わせて、細胞表面の発現糖鎖プロファイリングを行う方法を開発し、糖鎖を指標とする細胞の系統的な分類法を検討した。その結果、細胞の発現糖鎖プロファイルを糖鎖の基本骨格構造とシアル酸ならびにフコース残基数に基づいて比較すると、細胞種によらず普遍的に存在する糖鎖と、細胞の個性解析に有用な特徴的糖鎖が存在することがわかった。また、ポリラクトサミン型の糖鎖や高フコシル化糖鎖など癌マーカーとなりうる糖鎖も発見できた。平成18年度はヒト由来低分化型胃癌細胞MKN45中に大量に蓄積する遊離糖鎖の構造解析と蓄積機構の解析を行った。解析の結果、MKN45中に蓄積する遊離糖鎖はNeuAcα2-6Galβ1-4GlcNAcβ1-3Manβ1-4GlcNAcの配列を持つ遊離糖鎖であり、その原因がリソソームにおけるシアリダーゼの活性異常あるいはリソソームの機能不全によると考えられた。平成19年度は平成18年度研究で明かにした、MKN45中の遊離糖鎖の蓄積機構の解析を行い、MKN45での遊離糖鎖の蓄積はリソソームシアリダーゼあるいはリソソームの機能異常が原因であることがわかった。開発した技術は、糖鎖を網羅的に定量解析できるという優れた特性を持ち、細胞・組織間における比較グライコミクスを実現する技術として、診断マーカーの開発や癌や難治性疾患の治療法開発に向けた新たな展開も期待できる。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2004年 
    代表者 : 掛樋 一晃; 木下 充弘; 小田 泰雄
     
    本研究プロジェクトでは「銀染色レベルに対応できる糖タンパク質糖鎖の網羅的解析法の開発」の目的で銀染色により検出された糖タンパク質をゲルから抽出し、酵素あるいは化学的な方法により糖鎖を切り離したのち、レーザー励起蛍光検出法によりアトモルレベルで糖鎖を分析するため、生体成分中からの複合糖質の分離精製法および糖タンパク質糖鎖の高速糖鎖構造解析について基盤的な研究を行った。以下に開発した技術とその概要を列挙する。(1)生体成分中の複合糖質の選択的捕捉:複合糖質を認識する分子を結合させた温度応答性ポリマーを用いて、血液や組織抽出液などから標的複合糖質を簡便かつ迅速に捕捉する技術を開発した。本技術を利用して、ポリリジンを結合した温度応答性ポリマーによるガン細胞由来プロテオグリカンの捕捉に成功した。(2)糖鎖-タンパク質間相互作用解析を利用する高速糖鎖解析法:細胞表面や糖タンパク質などから切り離された糖鎖の混合物は通常極めて複雑な混合物であり、混合物中の個々の糖鎖の構造を解析することは現在の技術をもってしても困難である。我々は糖鎖と糖鎖結合性タンパク質の特異性の高い相互作用を利用して、溶液中分離しながら糖鎖の構造を解析するという一石二鳥の方法を開発した。この方法は糖鎖アレーの基礎的データを得る方法としても非常に有用である。(3)糖タンパク質からの超高速糖鎖切り離し:糖タンパク質から糖鎖を切り離す方法として、化学的な方法や酵素を用いる方法などの多くの方法が開発されているが、我々は従来の方法の革新的な改良に取り組み、糖鎖の切り離しから解析までをほぼ1日で完了する方法の開発に目処をつけた。(4)複合糖質由来糖鎖の超高速分析:最近、キャピラリー電気泳動法のミクロ化ならびに高速化を目指したチップ電気泳動法の技術が進展し、高い実用性が期待できる装置も市販されるようになった。従来、生体中のグリコサミノグリカン類をセルロースアセテート膜電気泳動法により2〜3時間をかけて分析する方法が臨床化学の分野で適用されていたが、チップ電気泳動法とインターカレーター試薬を使用するin-situ蛍光標識法を利用して、秒単位の分析を可能とした。本プロジェクトにより開発した技術は各技術それぞれでも有効であるが、各技術をシステムとして統合すれば、従来少なくとも「週」〜「月」単位の作業を必要とした複合糖質中の糖鎖構造解析を「3時間」〜「4時間」程度にまで短縮可能であり、糖鎖解析技術に習熟していない技術者でも対応できるレベルにまで自動化するための基盤技術の開発に成功した。

委員歴

  • 2022年04月 - 現在   日本薬学会物理系薬学部会   委員
  • 2016年04月 - 現在   日本分析化学会電気泳動分析研究懇談会   委員
  • 2014年04月 - 2018年03月   次世代バイオ医薬品製造技術開発組合   委員

担当経験のある科目

  • 創薬科学演習近畿大学薬学部
  • キャリアデザイン近畿大学
  • 情報科学実習近畿大学
  • 薬学概論近畿大学
  • 医薬品開発論近畿大学
  • 基礎化学英語近畿大学
  • 基礎生物学英語近畿大学
  • 応用生物学演習近畿大学
  • 機器分析学近畿大学
  • 情報科学近畿大学

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