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Preparation of Arabinogalactan Glycoproteins from Plant Tissue
从植物组织中制备阿拉伯半乳聚糖蛋白

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Abstract

This supplements an earlier protocol (Popper, 2011) for the extraction and assay of cell surface arabinogalactan proteins (AGPs). These highly glycosylated glycoproteins (~95% carbohydrate) contain numerous glycomodules with paired glucuronic acid residues that bind Ca2+ in a pH dependent manner (Lamport and Varnai, 2013). Classical AGPs comprise the bulk of cell surface glycoproteins and are thus integral components of a Ca2+ oscillator involved in a signalling pathway where calcium is a “universal signalling currency” analogous to ATP as the universal energy currency. The central role of these peripheral glycoproteins is thus reason enough for their further study. However, problems arise due to the extensive glycosylation and its apparent microheterogeneity generally assumed to preclude a simple reductionist approach.
Here I describe a simple partial purification of classical AGPs based on their specific interaction with the β-D-glucosyl or galactosyl Yariv reagent, a synthetic diazo dye that precipitates AGPs as an insoluble complex in salt solutions at neutral pH. (The solubility of this complex in dilute alkali provides a rapid sensitive quantitative assay for AGPs.) Reduction of the Yariv diazo linkage releases soluble AGPs for further analysis. For example deglycosylation of AGPs in anhydrous hydrogen fluoride followed by column chromatography yields just a few major AGP polypeptides purified to homogeneity (Zhao et al., 2002). However, purification of individual AGP glycoproteins to homogeneity is rarely achieved (Darjania et al., 2002); not only do the closely related AGP glycosylation profiles vastly outweigh any contribution from the amino acid composition but the glycan polydispersity made isolation of a single molecular entity well-nigh impossible until AGPs genetically engineered with a hydrophobic green fluorescent protein tag allowed chromatographic purification (Zhao et al., 2002). New approaches to AGP fractionation into discrete classes is now also a distinct possibility based on their calcium content hitherto ignored!

[Principle] Disrupted plant tissues release soluble AGPs that can be precipitated as their Yariv complex. This procedure yields mainly classical AGPs; these comprise the bulk of cell surface AGPs. Extraction with CaCl2 rather than the more usual NaCl has two advantages:

  1. It results in Ca2+ tightly bound by the glucuronic acid residues (Lamport and Varnai, 2013) at > pH 4.5 thus enhancing AGP solubility after its release from the insoluble Yariv complex.
  2. It removes pectin as insoluble calcium pectate crosslinked by intermolecular Ca2+ bridges while AGPs with intramolecular Ca2+ remain soluble.

Materials and Reagents

  1. Tobacco BY-2 cells or other plant tissue
  2. Liquid nitrogen
  3. CaCl2 (2% w/v)
  4. Distilled water
  5. Na metabisulphite (Sigma-Aldrich, catalog number: S-1256 )
  6. Dialysis tubing 12 kDa MW cutoff (3.2 cm flat width) (Sigma-Aldrich, catalog number: D-0530 )
  7. Superose-6, 10/300 GL (GE Healthcare, catalog number: 17-5172-01 )
  8. Hydroxyproline
  9. Glucuronic acid
  10. Gum arabic (Sigma-Aldrich, catalog number: G-9752 )
  11. NaCl
  12. NaOH
  13. Yariv reagent (Biosupplies Australia Pty, catalog number: 100-2 ) (see Recipes)

Equipment

  1. Blender/coffee mill
  2. Minifuge centrifuge
  3. 2 ml Sarstedt tube (with screw cap)
  4. Spectrophotometer or microplate reader
  5. Mass spectroscope
  6. Microfuge
  7. Fine-tip pipette
  8. Fine tip sonic probe
  9. Block heater

Procedure

  1. Freeze ~10 to 100 g plant tissue in liquid nitrogen.
  2. Pulverise frozen fresh tissue to a fine powder in a cold blender/coffee mill.
  3. Stir tissue in 2% w/v CaCl2 for 2-3 h at RT (2 ml for each gram of tissue).
  4. Centrifuge 30 minutes at ~10,000 x g (e.g. minifuge at RT)。
  5. Assay 10, 50 and 100 μl aliquots to estimate total AGP in extract (see Notes).
  6. Add a slight excess (see Notes) of Yariv reagent to the remaining extract.
  7. Allow to precipitate at least 1 h or overnight at RT.
  8. Collect precipitate by low speed centrifugation (10 min at 2,000 x g).
  9. Resuspend precipitate in 1.5 ml distilled water.
  10. Transfer to 2 ml Sarstedt tube (with screw cap).
  11. Add ~25 mg Na metabisulphite (final conc. 70 mM) to reduce the diazo linkage (top up with H2O to exlude oxygen which otherwise results in the formation of elemental sulfur).
  12. Cap tube tightly and heat at ~50 °C until decolourised (5-20 min).
  13. Transfer to small (~5 ml) dialysis bag; stir overnight at RT in 500 ml distilled H2O and change H2O three times.
  14. Freeze dialysate in liquid nitrogen, lyophilise, then and weigh the product. (AGP yields vary from 30-300 μg AGP/g fresh weight depending on tissue source) (see Notes).
  15. Validate classical AGPs by size and composition:
    1. Gel filtration on Superose-6 (Lamport, et al., 2006).
    2. Hydroxyproline content (Kivirikko and Liesmaa, 1959).
    3. Uronic acid content (Blumenkrantz and Asboe-Hansen, 1973).
    4. Bound calcium via colourimetry (Gindler and King, 1972) or ICPMS (inductively coupled plasma mass spectroscopy) (Lamport and Varnai, 2013).
    5. Amino acid and sugar analyses.

Notes

  1. Arabinogalactan protein assay via Yariv reagent.
    All steps at RT.
    1. Add test samples to Eppendorf microfuge tubes.
    2. Make up to ~500 μl in 1% CaCl2.
    3. Use 10 and 20 μg gum arabic as AGP "standards" as follows:
      Add 10 μl gum arabic (1 mg/ml) to 500 μl 1% CaCl2
      Add 20 μl gum arabic (1 mg/ml) to 500 μl 1% CaCl2
    4. Use 500 μl 1% CaCl2 as a reagent blank.
    5. Then to each tube:
      Add 200 μl β-D-Galactosyl-Yariv reagent (1 mg/ml in 2% CaCl2)
      Or use β-D-Glucosyl-Yariv reagent – choice depends on availability.
    6. Mix well and leave for at least 30 min at room temperature.
    7. Spin 10 min at ~15,000 x g in microfuge.
    8. With CARE use fine-tip pipette to remove & discard supernate.
    9. Wash pellet twice with 1 ml 2% CaCl2.
    10. Add 1 ml 20 mM NaOH.
    11. Shake vigorously to dissolve pellet or sonicate 10-20 sec with fine tip probe or in a sonic bath. If solution is turbid, spin at ~15,000 x g to clarify.
    12. Read A457 nm against reagent blank within an hour or so. A457 avoids phenolic interference when eluting Yariv from intact BY-2 cells; for general assays read at 440 nm.
    13. Plot "standard curve" and calculate unknowns as μg AGP/tube.
      Note: Gum arabic quantification is only an approximation as each AGP binds different amounts of Yariv.
      As a general guide, however, a given weight of Yariv reagent will precipitate the same weight of AGP. So for AGP isolation a 10% excess of Yariv reagent generally suffices to precipitate all the AGP.
  2. AGP cellular distribution (background information):
    Classical AGPs are essential glycoproteins distributed in three cell surface compartments: bound to the outer surface of the plasma membrane by a GPI anchor; soluble in the periplasm; and "bound" or trapped in the wall matrix.
    Thus AGP cellular distribution T = M + S + W (Lamport et al., 2006)
    M = AGPs bound to plasma membrane
    S = Soluble AGPs released by cell disruption
    W = AGPs bound to cell wall

    Table 1. Total of AGPs in tobacco BY-2 cells. In tobacco BY-2 cells, T = 600 μg AGPs g fresh weight.
    BY-2 cells
    (data in 6)
    T Total
     M Membrane bound
     S Soluble periplasmic
    W Wall bound
    Salt-adapted*
    600
    60
    354
    186
    Control
    600
    210
    282
    108
    * AGPs upregulated by high salt appear in the growth medium.

Recipes

  1. Preparation of Yariv reagent (1,3,5-tri-(p-β-D-galactosyloxyphenylazo)-2,4,6-trihydroxybenzene) or the β-D-glucosyl derivative
    Dissolve 100 mg β-D-galactosyl Yariv in 100 ml 2% w/v CaCl2

Acknowledgments

This protocol is adapted from Popper (2011), Lamport and Varnai (2013) and Lamport et al. (2006).

References

  1. Blumenkrantz, N. and Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Anal Biochem 54(2): 484-489.
  2. Darjania, L., Ichise, N., Ichikawa, S., Okamoto, T., Okuyama, H. and Thompson Jr, G. A. (2002). Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells. Plant Physiol Biochem 40(1): 69-79.
  3. Gindler, E. and King, J. (1972). Rapid colorimetric determination of calcium in biologic fluids with methylthymol blue. Am J Clin Pathol 58(4):376-82.
  4. Kivirikko, K. I. and Liesmaa, M. A. (1959). A Colorimetric method for determination of hydroxyproline in tissue hydrolysates. Scandinavian J Clin Lab 11(2):128-133.
  5. Lamport, D. T. and Varnai, P. (2013). Periplasmic arabinogalactan glycoproteins act as a calcium capacitor that regulates plant growth and development. New Phytol 197(1): 58-64.
  6. Lamport, D. T., Kieliszewski, M. J. and Showalter, A. M. (2006). Salt stress upregulates periplasmic arabinogalactan proteins: using salt stress to analyse AGP function. New Phytol 169(3): 479-492.
  7. Popper, Z. A. (2011). Extraction and Detection of Arabinogalactan Proteins in The Plant Cell Wall - Methods and Protocols, edited by John M. Walker. Humana Press, New York, pp.245-254.
  8. Qi, W., Fong, C. and Lamport, D. T. (1991). Gum arabic glycoprotein is a twisted hairy rope: a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site. Plant Physiol 96(3): 848-855.
  9. Zhao, Z. D., Tan, L., Showalter, A. M., Lamport, D. T. and Kieliszewski, M. J. (2002). Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis. Plant J 31(4): 431-444.

简介

这补充了用于细胞表面阿拉伯半乳聚糖蛋白(AGP)的提取和测定的早期方案(Popper,2011)。这些高度糖基化的糖蛋白(〜95%碳水化合物)含有许多具有以pH依赖性方式结合Ca 2+ 2+的成对葡萄糖醛酸残基的糖模块(Lamport和Varnai,2013)。经典AGP包含大量细胞表面糖蛋白,因此是参与信号传导途径的Ca 2+ 2+振荡器的整体组分,其中钙是类似于作为通用能量货币的ATP的"通用信号传导货币"。因此,这些外周糖蛋白的中心作用是足以进行进一步研究的理由。然而,由于广泛的糖基化及其明显的微不均一性,通常假定排除简单的还原剂方法而出现问题。在这里,我描述了基于其与β-D-葡糖基或半乳糖基的特异性相互作用的经典AGP的简单部分纯化Yariv试剂,一种合成的重氮染料,其在中性pH的盐溶液中沉淀AGP作为不溶性复合物。 (该络合物在稀碱中的溶解度为AGPs提供快速灵敏的定量测定。)Yariv重氮键的还原释放可溶性AGP用于进一步分析。例如,AGP在无水氟化氢中的去糖基化,随后通过柱色谱法仅产生纯化至同质的几种主要AGP多肽(Zhao等人,2002)。然而,很少实现将单个AGP糖蛋白纯化至同质性(Darjania等人,2002);不仅紧密相关的AGP糖基化谱远远超过来自氨基酸组成的任何贡献,而且聚糖多分散性使得几乎不可能分离单个分子实体,直到用疏水性绿色荧光蛋白标签遗传工程化的AGP允许色谱纯化(Zhao& et al。,,2002)。 AGP分离成离散类的新方法现在也是基于其迄今忽略的钙含量的不同可能性。

[Principle] 中断的植物组织释放可溶性AGP,可以沉淀为其Yariv复合物。 该程序主要产生经典AGP; 这些包括大量的细胞表面AGP。 用CaCl 2提取而不是更常用的NaCl具有两个优点:

  1. 其导致由葡萄糖醛酸残基紧密结合的Ca 2+ 2+(Lamport和Varnai,2013),在> pH 4.5,从而在其从不溶性Yariv复合物释放后增强AGP溶解度
  2. 它将果胶作为通过分子间Ca 2+ 2+桥键交联的不溶性钙果胶,而具有分子内Ca 2+ 2 +的AGP保持溶解。

材料和试剂

  1. 烟草BY-2细胞或其他植物组织
  2. 液氮
  3. CaCl 2(2%w/v)
  4. 蒸馏水
  5. 焦亚硫酸钠(Sigma-Aldrich,目录号:S-1256)
  6. 透析管12kDa MW截留(3.2cm平宽)(Sigma-Aldrich,目录号:D-0530)
  7. Superose-6,10/300GL(GE Healthcare,目录号:17-5172-01)
  8. 羟脯氨酸
  9. 葡萄糖醛酸
  10. 阿拉伯胶(Sigma-Aldrich,目录号:G-9752)
  11. NaCl
  12. NaOH
  13. Yariv试剂(Biosupplies Australia Pty,目录号:100-2)(参见Recipes)

设备

  1. 搅拌机/咖啡磨机
  2. Minifuge离心机
  3. 2 ml Sarstedt管(带螺旋盖)
  4. 分光光度计或酶标仪
  5. 质谱仪
  6. Microfuge
  7. 精细移液器
  8. 细尖音波探头
  9. 阻止加热器

程序

  1. 在液氮中冷冻〜10至100g植物组织
  2. 在冷混合器/咖啡磨中将冷冻的新鲜组织粉碎成细粉
  3. 在室温下将组织在2%w/v CaCl 2中搅拌2-3小时(每克组织2ml)。
  4. 以〜10,000英寸×g (例如例如离心机在RT)离心30分钟。
  5. 测定10,50和100μl等分试样以估计提取物中的总AGP(参见注释)
  6. 在剩余的提取物中加入稍微过量的Yariv试剂(见注释)。
  7. 在室温下至少沉淀1小时或过夜。
  8. 通过低速离心(在2,000×g下10分钟)收集沉淀物
  9. 在1.5ml蒸馏水中重悬沉淀
  10. 转移到2ml Sarstedt管(带螺旋盖)
  11. 加入〜25mg焦亚硫酸钠(最终浓度为70mM)以减少重氮连接(补充H 2 O以排出氧,否则会导致形成元素硫)。
  12. 盖管密封并在〜50℃加热直至脱色(5-20分钟)
  13. 转移至小(〜5ml)透析袋; 在室温下在500ml蒸馏H 2 O中搅拌过夜,并更换H 2 O三次。
  14. 在液氮中冷冻透析液,冻干,然后称重产品。 (AGP产量从30-300μgAGP/g鲜重取决于组织来源)(见注释)
  15. 通过大小和组成验证经典AGP:
    1. 在Superose-6上凝胶过滤(Lamport,et al。,2006)。
    2. 羟脯氨酸含量(Kivirikko和Liesmaa,1959)
    3. 糖酸含量(Blumenkrantz和Asboe-Hansen,1973)
    4. 通过色度法结合钙(Gindler和King,1972)或ICPMS(电感耦合等离子体质谱)(Lamport和Varnai,2013)。
    5. 氨基酸和糖分析。

笔记

  1. 通过Yariv试剂进行的阿拉伯半乳聚糖蛋白测定。
    所有步骤在RT。
    1. 将试样加入Eppendorf微量离心管中
    2. 补充至〜500μl在1%CaCl 2中。
    3. 使用10和20μg阿拉伯胶作为AGP"标准品",如下:
      将10μl阿拉伯胶(1mg/ml)加入到500μl1%CaCl 2中。
      将20μl阿拉伯树胶(1mg/ml)加入500μl1%CaCl 2
    4. 使用500μl1%CaCl 2 2作为试剂空白。
    5. 然后对每个管:
      加入200μlβ-D-半乳糖基 - Yariv试剂(1mg/ml,在2%CaCl 2中)
      或使用β-D-葡萄糖基 - Yariv试剂 - 根据可用性选择
    6. 混合均匀,在室温下放置至少30分钟
    7. 在微型离心机中以约15,000英尺/分钟的速度旋转10分钟。
    8. 用CARE使用细尖移液管去除& 丢弃上清液
    9. 用1ml 2%CaCl 2洗涤沉淀两次。
    10. 加入1ml 20mM NaOH
    11. 剧烈摇动以溶解沉淀或用细探针或在声波浴中声处理10-20秒。 如果溶液是混浊的,在〜15,000×g下旋转以澄清
    12. 在一小时左右内,相对于试剂空白读取A 457 nm。 当从完整BY-2细胞中洗脱Yariv时,a <457 避免酚类干扰; 用于在440nm读取的一般测定。
    13. 绘制"标准曲线"并计算未知数,如μgAGP /管 注意:阿拉伯胶定量仅是近似值,因为每个AGP结合不同量的Yariv。
      然而,作为一般指导,给定重量的Yariv试剂将沉淀相同重量的AGP。 因此,对于AGP分离,10%过量的Yariv试剂通常足以沉淀所有AGP
  2. AGP细胞分布(背景信息):
    经典AGP是分布在三个细胞表面区室中的必需糖蛋白:通过GPI锚与细胞膜的外表面结合; 可溶于周质; 并"结合"或捕获在壁基质中。
    因此AGP细胞分布T = M + S + W(Lamport等人,2006)
    M =与质膜结合的AGP S =细胞破坏所释放的可溶性AGP
    W =与细胞壁结合的AGP

    表1.烟草BY-2细胞中AGP的总量。 在烟草BY-2细胞中,T = 600μgAGPs g鲜重
    BY-2单元格
    ( 6 中的数据)
    T总计
      M膜结合
      S可溶性周质
    W有界限
    盐适应*
    600
    60
    354
    186
    控制
    600
    210
    282
    108
    *由高盐上调的AGP出现在生长培养基中

食谱

  1. Yariv试剂(1,3,5-三 - (对-β-D-半乳糖基氧基苯基偶氮)-2,4,6-三羟基苯)或β-D-葡糖基衍生物的制备
    将100mgβ-D-半乳糖基Yariv溶解在100ml 2%w/v CaCl 2中

致谢

该协议改编自Popper(2011),Lamport和Varnai(2013)和Lamport等人(2006)。

参考文献

  1. Blumenkrantz,N。和Asboe-Hansen,G。(1973)。 定量测定糖醛酸的新方法 Anal Biochem 54(2):484-489。
  2. Darjania,L.,Ichise,N.,Ichikawa,S.,Okamoto,T.,Okuyama,H。和Thompson Jr,G.A。(2002)。 在培养的拟南芥细胞中动态更新阿拉伯半乳聚糖蛋白。 Plant Physiol Biochem 40(1):69-79
  3. Gindler,E。和King,J。(1972)。 用甲基百里酚蓝快速比色测定生物液中的钙。美国Clin Pathol 58(4):376-82。
  4. Kivirikko,K.I.和Liesmaa,M.A。(1959)。 用于测定组织水解产物中羟脯氨酸的比色法。斯堪的纳维亚J Clin Lab 11(2):128-133
  5. Lamport,D.T.和Varnai,P.(2013)。 周质阿拉伯半乳聚糖糖蛋白作为钙电容器调节植物生长, New Phytol 197(1):58-64。
  6. Lamport,D.T.,Kieliszewski,M.J.and Showalter,A.M。(2006)。 盐胁迫上调周质阿拉伯半乳聚糖蛋白:使用盐胁迫分析AGP功能。 New Phytol 169(3):479-492。
  7. Popper,Z.A。(2011)。提取和检测植物细胞壁中的阿拉伯半乳聚糖蛋白 - 方法和方案,由John M. Walker编辑。 Humana Press ,纽约,第245-254页。
  8. Qi,W.,Fong,C。和Lamport,D.T。(1991)。 阿拉伯胶糖蛋白是一种扭曲的毛状绳:基于o-半乳糖基羟基脯氨酸作为多糖附着的新模型site。 Plant Physiol 96(3):848-855。
  9. Zhao,Z.D.,Tan,L.,Showalter,A.M.,Lamport,D.T.and Kieliszewski,M.J。(2002)。 从转基因烟草中纯化的番茄LeAGP-1阿拉伯半乳聚糖蛋白证实了Hyp毗连假说。 Plant J 31(4):431-444。
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引用:Lamport, D. T. (2013). Preparation of Arabinogalactan Glycoproteins from Plant Tissue. Bio-protocol 3(19): e918. DOI: 10.21769/BioProtoc.918.
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asoke banerji
school of biotechnology
pl provide the method of preparation of yariv reagent
A BANERJI
4/24/2014 3:52:12 AM Reply
Bio-protocol Editorial Team
bio-protocol.org

Please see the detail in Recipes section and let us know with any questions/comments on that.

Thanks,
Bio-protocol Editorial Team

4/27/2014 3:54:12 PM