搜索

Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans
构巢曲霉中半乳甘露糖蛋白的D-呋喃半乳糖含量测定

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is known that furanoses, contained in oligosaccharides, are detected as pyranoses after hydrolysis, and that D-galactopyranose is not contained in the galactomannoproteins of Aspergillus spp. To determine the levels of D-galactofuranose in galactomannoproteins extracted from Aspergillus nidulans (A. nidulans), we measured the amount of D-galactopyranose production after galactomannoproteins hydrolysis. The method described in this manuscript allows determination of the D-galactofuranose content of galactomannoproteins in Aspergillus spp.

Keywords: Galactofuranose(呋喃), Aspergillus(曲霉), Cell wall(细胞壁), Galactomannan(半乳甘露聚糖)

Materials and Reagents

  1. Aspergillus nidulans conidia
  2. Trisodium citrate dihydrate (C6H5Na3O7.2H2O) (Wako Pure Chemical Industries, catalog number: 191-01785 )
  3. Citric acid (C6H8O7) (Wako Pure Chemical Industries, catalog number: 030-05525 )
  4. Ethyl-4-aminobenzoate (C9H11NO2) (ABEE) (Tokyo Chemical Industry, catalog number: A0271 )
  5. Sodium cyanoborohydride (NaBH3CN) (Tokyo Chemical Industry, catalog number: S0396 )
  6. Chloroform (Wako Pure Chemical Industries, catalog number: 038-02606 )
  7. Ethanol (Wako Pure Chemical Industries, catalog number: 050-00446 )
  8. Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, catalog number: 206-10731 )
  9. Glacial acetic acid (Wako Pure Chemical Industries, catalog number: 012-00245 )
  10. Methanol (Wako Pure Chemical Industries, catalog number: 136-09475 )
  11. D-galactose (Sigma-Aldrich, catalog number: G0750 )
  12. ABEE labeling mixture (see Recipes)
  13. HPLC solvent A (see Recipes)
  14. HPLC solvent B (see Recipes)
  15. Minimal medium (see Recipes)
  16. Hutner's trace elements (see Recipes)

Equipment

  1. 50-ml plastic centrifuge tube (e.g., Greiner Bio-One GmbH)
  2. 1.5-ml conical screw cap tube and cap
  3. Spreader
  4. 500-ml Sakaguchi flasks
  5. Centrifuge
  6. Rotator (e.g., TAITEC)
  7. High-performance liquid chromatography (HPLC) system equipped with a fluorescence detector (Hitachi, LaChrom Elite, model: L-2485) and software for HPLC peak analysis (e.g., Hitachi, model: D-2000 Elite HPLC)
  8. Centrifugal evaporator (e.g., SpeedVac®)
  9. Heat block or water bath
  10. GlycoScope Honenpak C18 column (4.6 mm x 75 mm) (COSMO BIO, catalog number: JOM-J715-1PC)
  11. Filter paper (Munktell & Filtrak GmbH, catalog number: 113053 )
  12. Dialysis membrane (BioDesign Inc. of New York, catalog number: D102 )

Procedure

  1. Streak Aspergillus nidulans conidia from frozen stock onto Minimal medium (MM) plate and cultivate for 3 days at 30 °C.
  2. Collect the formed conidia with a spreader.
  3. Spread Aspergillus nidulans conidia (1 x 105) onto MM plates and cultivate for 3 days at 30 °C.
  4. Inoculate the collected conidia (2 x 107) into 100 ml of MM in 500-ml Sakaguchi flasks.
  5. Shake the flasks at 126 rpm at 30 °C for 24 h.
  6. Collect the mycelial cells by paper filtration.
  7. Wash the cells twice with approximately 30 ml of distilled water (Video 1).

    Video 1. Collection and washing of the mycelial cells

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  8. Resuspend the cells (approx. 2 g of wet cells) in 10 ml of 100 mM citrate buffer (pH 7.0).
  9. Autoclave the sample at 121 °C for 120 min.
  10. Remove cell debris by paper filtration (Video 2).

    Video 2. Removing cell debris by paper filtration

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  11. Collect the resultant sample (approx. 10 ml) in a 50-ml plastic centrifuge tube.
  12. Add 30 ml of cold 99.5% ethanol and keep the sample on ice for 30 min.
  13. Centrifuge at 14,000 x g at 4 °C for 10 min.
  14. Dry the pellet at room temperature using a centrifugal evaporator at 1,500 x g.
  15. Resuspend the pellet in 2 ml of distilled H2O (dH2O).
  16. Dialyze the sample with a dialysis membrane (15.5 mm x 300 mm) against 5 L of dH2O for 24 h at 4 °C. The resultant sample is designated as the extracted galactomannnoproteins.
  17. Incubate part (2.4 µg) of the extracted galactomannoproteins in 500 µl of 4 M trifluoroacetic acid (TFA) at 100 °C for 4 h in a 1.5-ml conical screw cap tube.
  18. Dry the resultant sample at room temperature using a centrifugal evaporator at 1,500 x g.
  19. Resuspend the sample in 10 µl of dH2O.
  20. Label the hydrolysates with fluorescent p-aminobenzoic acid ethyl ester (ABEE) using an ABEE labeling mixture.
    1. Add 40 µl of the ABEE labeling mixture (preheating at 70 °C for 5 min) to the sample.
    2. Incubate the sample for 1 h at 80 °C.
    3. Cool the sample to room temperature.
    4. Add 200 µl of dH2O and 200 µl of chloroform to the sample.
    5. Vigorously vortex the sample and then centrifuge it at 14,000 x g for 5 min at room temperature.
    6. Collect the upper aqueous layer.
  21. Analyze the ABEE-labeled D-galactopyranose using an HPLC system. Inject 20 µl of the sample into the column at a flow rate of 1.0 ml/min at 45 °C. ABEE-D-galactopyranose can be detected with a fluorescence detector at an excitation wavelength of 360 nm and an emission wavelength of 305 nm.
    Note: Use solvents containing acetonitrile and potassium borate buffer (see the Recipes section for solvents A and B). Set the gradient program at a flow rate of 1.0 ml/min (expressed as a percent of solvent A) and apply the following gradient to analyze the ABEE-D-galactopyranose: 0-45 min, isocratic 100%; 45-50 min, 100-0%; 50-55 min, 0-100%; 55-75 min, isocratic 100%. Use D-galactose as a standard for quantification purposes.

Recipes

  1. ABEE labeling mixture
    165 mg ABEE
    35 mg sodium cyanoborohydride
    41 µl glacial acetic acid
    350 µl methanol
    The mixture can be long-term stored at -20 °C
  2. HPLC solvent A [0.2 M potassium borate buffer (pH 9.0) containing 6% acetonitrile]
    12.4 g H3BO3
    60 ml acetonitrile
    Add water to bring the final solution to 1 L total volume while adjusting pH 9.0 with potassium hydroxide (KOH)
  3. HPLC solvent B [0.2 M potassium borate buffer (pH 9.0) containing 50% acetonitrile]
    12.4 g H3BO3
    500 ml acetonitrile
    Add water to bring the final solution to 1 L total volume while adjusting pH 9.0 with potassium hydroxide (KOH)
  4. Minimal medium (1 L)
    NaNO3
    6.0 g
    KCl
    0.52 g
    KH2PO4
    1.52 g
    Glucose
    10.0 g
    Hutner's trace elements
    2 ml
    Adjust the pH value to 6.8 using NaOH
    Add water to bring the final solution to 1 L total volume
    Autoclave for 20 min
  5. Hutner's trace elements
    H2O (60 ˚C)
    100 ml
    ZnSO4•7H2O
    2.2 g
    H3BO3
    1.1 g
    MnCl2•4H2O
    0.5 g
    FeSO4•7H2O
    0.5 g
    CoCl2•6H2O
    0.16 g
    CuSO4•5H2O
    0.16 g
    (NH4) 6Mo7O24•4H2O
    0.11 g
    EDTA
    5.0 g
    Adjust the pH value to 6.5-6.8 using KOH

Acknowledgments

This protocol was adapted from the previously published papers Komachi et al. (2013) and Yasuno et al. (1997). The work was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society of the Promotion of Science (JSPS) (21780313, 23780350 and 26450106) (to T.O.).

References

  1. Komachi, Y., Hatakeyama, S., Motomatsu, H., Futagami, T., Kizjakina, K., Sobrado, P., Ekino, K., Takegawa, K., Goto, M., Nomura, Y. and Oka, T. (2013). GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol Microbiol 90(5): 1054-1073.
  2. Yasuno, S., Murata, T., Kokubo, K., Yamaguchi, T. and Kamei, M. (1997). Two-mode analysis by high-performance liquid chromatography of ρ-aminobenzoic ethyl ester-derivatized monosaccharides. Biosci Biotec Biochem 61(11): 1944-1946.

简介

半乳糖呋喃糖(Galactofuranose)(Gal ff)是某些种类的丝状真菌中几种多糖和糖缀合物的组分。 Gal< em>残基经常在曲霉属糖蛋白中发现,包括修饰许多蛋白质的聚糖和 细胞壁蛋白和细胞外酶。 已知包含在寡糖中的呋喃糖在水解后被检测为吡喃糖,并且D-吡喃半乳糖不包含在曲霉属的半乳糖蛋白中。 为了测定从构巢曲霉(构巢曲霉)提取的半乳糖蛋白中的D-半乳糖呋喃糖的水平,我们测量了半乳糖蛋白水解后D-吡喃半乳糖产生的量。 该手稿中描述的方法允许确定曲霉属中半乳糖甘露糖蛋白的D-半乳糖呋喃糖含量。

关键字:呋喃, 曲霉, 细胞壁, 半乳甘露聚糖

材料和试剂

  1. 构巢曲霉分生孢子
  2. 柠檬酸三钠二水合物(C 6 H 5 H 5 Na 3 O 7) 2> O)(Wako Pure Chemical Industries,目录号:191-01785)
  3. 柠檬酸(C 6 H 8 O 7)(Wako Pure Chemical Industries,目录号:030-05525)
  4. 乙基-4-氨基苯甲酸酯(C 9 H 11 NO 2)(ABEE)(Tokyo Chemical Industry,目录号:A0271)
  5. 氰基硼氢化钠(NaBH 3 CN)(Tokyo Chemical Industry,目录号:S0396)
  6. 氯仿(Wako Pure Chemical Industries,目录号:038-02606)
  7. 乙醇(Wako Pure Chemical Industries,目录号:050-00446)
  8. 三氟乙酸(TFA)(Wako Pure Chemical Industries,目录号:206-10731)
  9. 冰乙酸(Wako Pure Chemical Industries,目录号:012-00245)
  10. 甲醇(Wako Pure Chemical Industries,目录号:136-09475)
  11. D-半乳糖(Sigma-Aldrich,目录号:G0750)
  12. ABEE标签混合物(参见配方)
  13. HPLC溶剂A(参见配方)
  14. HPLC溶剂B(参见配方)
  15. 最小培养基(见配方)
  16. Hutner的微量元素(参见配方)

设备

  1. 50-ml塑料离心管(例如Greiner Bio-One GmbH)
  2. 1.5毫升锥形螺帽管和帽
  3. 撒布机
  4. 500 ml Sakaguchi烧瓶
  5. 离心机
  6. 旋转器(例如,TAITEC)
  7. 配备有荧光检测器(Hitachi,LaChrom Elite,型号:L-2485)和用于HPLC峰分析的软件(例如,Hitachi,型号:D-2000 Elite)的高效液相色谱 HPLC)
  8. 离心蒸发器(例如,SpeedVac ®
  9. 热块或水浴
  10. GlycoScope Honenpak C18柱(4.6mm×75mm)(COSMO BIO,目录号:JOM-J715-1PC)
  11. 滤纸(Munktell& Filtrak GmbH,目录号:113053)
  12. 透析膜(New York的BioDesign Inc.,目录号:D102)

程序

  1. 将来自冷冻原种的构巢曲霉分生孢子分生孢子在最小培养基(MM)平板上培养,并在30℃下培养3天。
  2. 使用撒布机收集形成的分生孢子。
  3. 将分生孢子(1×10 5)铺在MM平板上,在30℃培养3天。
  4. 将收集的分生孢子(2×10 7个)接种到500ml坂口烧瓶中的100ml MM中。
  5. 将烧瓶以126rpm在30℃摇动24小时
  6. 通过纸过滤收集菌丝体细胞
  7. 用约30ml蒸馏水洗涤细胞两次(视频1)。

    视频1.收集和洗涤菌丝体细胞
  8. 将细胞(约2g湿细胞)重悬在10ml的100mM柠檬酸盐缓冲液(pH7.0)中。
  9. 将样品在121℃高压灭菌120分钟
  10. 通过纸过滤除去细胞碎片(视频2)。

    视频2.通过纸张过滤除去细胞碎片
  11. 在50ml塑料离心管中收集所得样品(约10ml)
  12. 加入30ml冷的99.5%乙醇,并将样品在冰上保持30分钟
  13. 在4℃下以14,000×g离心10分钟
  14. 在室温下使用离心蒸发器在1500×g下干燥沉淀。
  15. 将沉淀重悬在2ml蒸馏H 2 O(dH 2 O)中。
  16. 用透析膜(15.5mm×300mm)对5L dH 2 O在4℃透析样品24小时。 将所得样品称为提取的半乳糖甘露糖蛋白
  17. 将部分(2.4μg)提取的半乳糖蛋白在500μl4M三氟乙酸(TFA)中在100℃下在1.5-ml锥形螺旋盖管中孵育4小时。
  18. 在室温下使用离心蒸发器在1500×g下干燥所得样品。
  19. 将样品重悬于10μldH 2 O中。
  20. 使用ABEE标记混合物用荧光的p-氨基苯甲酸乙酯(ABEE)标记水解产物。
    1. 向样品中加入40μlABEE标记混合物(在70℃预热5分钟)
    2. 在80℃下孵育样品1小时。
    3. 将样品冷却至室温。
    4. 向样品中加入200μldH 2 O和200μl氯仿
    5. 剧烈涡旋样品,然后在室温下以14,000×g离心5分钟。
    6. 收集上层水层。
  21. 使用HPLC系统分析ABEE标记的D-吡喃半乳糖。以45℃下1.0ml/min的流速将20μl样品注入柱中。 ABEE-D-吡喃半乳糖可以用荧光检测器在360nm的激发波长和305nm的发射波长下检测。
    注意:使用含有乙腈和硼酸钾缓冲液的溶剂(参见溶剂A和B的配方部分)。以1.0ml/min(表示为溶剂A的百分比)的流速设置梯度程序并应用以下梯度以分析ABEE-D-吡喃半乳糖:0-45分钟,等度100%; 45-50分钟,100-0%; 50-55分钟,0-100%; 55-75分钟,等度100%。使用D-半乳糖作为定量标准。

食谱

  1. ABEE标签混合物
    165 mg ABEE
    35mg氰基硼氢化钠 41μl冰乙酸
    350μl甲醇
    该混合物可以长期储存在-20℃下
  2. HPLC溶剂A [含有6%乙腈的0.2M硼酸钾缓冲液(pH9.0)] 12.4g H sub 3 BO Sub 3
    60 ml乙腈 加入水,使最终溶液达到1L总体积,同时用氢氧化钾(KOH)调节pH9.0
  3. HPLC溶剂B [含有50%乙腈的0.2M硼酸钾缓冲液(pH9.0)] 12.4g H sub 3 BO Sub 3
    500ml乙腈 加入水,使最终溶液达到1L总体积,同时用氢氧化钾(KOH)调节pH9.0
  4. 最小培养基(1L)
    用NaOH
    调节pH值至6.8 加水,使最终溶液的体积为1 L 高压灭菌20分钟
  5. Hutner的微量元素

    致谢

    该协议改编自先前发表的论文Komachi等人(2013)和Yasuno等人(1997)。 这项工作部分得到日本科学促进会(JSPS)(21780313,23780350和26450106)(到T.O.)的青年科学家助学金(B)的支持。

    参考文献

    1. Komachi,Y.,Hatakeyama,S.,Motomatsu,H.,Futagami,T.,Kizjakina,K.,Sobrado,P.,Ekino,K.,Takegawa,K.,Goto,M.,Nomura, Oka,T。(2013)。 GfsA编码参与构巢曲霉中O-聚糖的半乳糖呋喃糖抗原生物合成的新型半乳糖呋喃糖基转移酶 和 Aspergillus fumigatus 。 Mol Microbiol 90(5):1054-1073。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Oka, T., Katafuchi, Y., Fukuda, K., Ekino, K., Goto, M. and Nomura, Y. (2014). Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans. Bio-protocol 4(17): e1223. DOI: 10.21769/BioProtoc.1223.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。