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Glycophosphosphingolipid (GSPL) Purification Protocol
糖磷酸神经鞘脂类(GSPL)的纯化方法   

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Abstract

Glycosylated ceramide phosphorylinositol are present in many species of fungi and mushrooms and bacteria and parasitic organisms like leishmania. These are usually membrane raft associated and are not easily extracted by conventional methodologies. This extraction method gives higher yield of the glycolipid. Glycosphingolipids are usually associated with the detergent resistance membrane rafts. They are difficult to be extracted by neutral solvents from rigid promastigote cell wall with neutral or less polar solvents. For more polar phospholipids, more polar solvents are essential. Ammonia is used to extract phosphatidylinositols.

Materials and Reagents

  1. Promastigotes
  2. Tris-HCl
  3. PMSF
  4. Fetal bovine serum (FBS)
  5. Sucrose
  6. Cholorform
  7. Methanol
  8. Ethyl acetate
  9. Pyridine
  10. Ammonia
  11. Ammonium Hydroxide
  12. Sodium Taurodeoxycholic Acid
  13. KCl
  14. Acetone
  15. Silicic Acid
  16. Medium 199
  17. Phosphate buffered saline (PBS) (see Recipes)
  18. Sucrose density gradient (see Recipes)
  19. PBS-AT (see Recipes)

Equipment

  1. Vortexer
  2. Centrifuges
  3. DEAE-Sephadex A-25
  4. HPTLC plate
  5. Iodine chamber
  6. TLC
  7. Erythina cristagalli-agarose column
  8. Galactose
  9. Dialysis device
  10. Lyophilizor
  11. Swinging bucket rotor

Procedure

  1. Promastigote culture
    1. Promastigotes of L. donovani, strain AG83, are grown at 22 °C in medium 199 (pH 7.4) supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U of penicillin G sodium, and 100 μg of streptomycin sulfate per ml and subcultured in the same medium at an average density of 2 × 106 cells/ml. Cells are harvested after 3 days of growth by centrifugation at 1,500 x g for 10 min. Harvested cells are washed twice with PBS (10 ml, 1,500 x g, 10 min).

  2. Preparation of promastigote membrane ghost
    1. Take up promastigotes (1 g wet weight) in 50 ml of chilled 50 mM Tris-HCl (pH 7.4), containing 2 mM PMSF (Phenylmethanesulfonyl fluoride) in glass tubes and keep on ice. Tube is mixed by mildly vortexing six times on a cyclomixer and kept on ice for 10 min in between each vortexing. Time of vortexing is 2 min each.
    2. Collect cells by centrifugation at 3,300 x g for 20 min at 40 °C and wash extensively (3-5 times) with PBS (20 ml each).
    3. The pellet is re-suspended in 10 ml of a 5% sucrose solution in 50 mM Tris-HCl (pH 7.4). 5 ml of this solution was layered onto a 10 ml solution of 25% sucrose in the same buffer in a corex tube. This is centrifuged at 1,500 x g for 30 min. The pellet formed was again taken up in 2.5 ml of 5% sucrose in 50 mM Tris-HCl (pH 7.4). And layered onto a 5 ml 25% sucrose solution in the same buffer. This is centrifuged at 1500x g for 45 min and the pellet (membrane ghost) was processed further.

  3. Purification of GSPL
    1. Promastigote membrane ghost (1 g wet weight) is mixed with 19 volumes of chloroform:methanol:ethyl acetate:pyridine:4.5 N ammonia:water (15:15:5:0.5:0.5:0.5, v/v) and homogenized by 18-20 strokes in tight fitting Dounce-type homogenizer (on ice). The tube is kept on ice for 10 min and then sonicated in a bath sonicator (30 x 20 sec). Tube was kept on ice for 30 sec in between each sonication.
    2. Concentrate (to dryness) the extracted material by rotary evaporation, dissolve in 1 ml PBS-AT by sonication in a water bath (5 x 20 sec, with 30 sec intervals inbetween) and load onto a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated in 0.01 M phosphate buffer (pH 6.4), containing 0.05 N ammonium hydroxide and 0.1% sodium salt of taurodeoxycholic acid (PBS-AT). Wash the column with 5 ml of PBS-AT and elute anionic glycolipids with a linear gradient of KCl (0-0.1 M in PBS-AT). The gradient is produced by mixing Anion-exchange buffer PBS-AT (no KCl) with PBS-AT containing 0.1 M KCl.
      Note: 10 times the column volume is used to prepare the gradient. For example: For a 2 ml column, a 20 ml gradient is used. To prepare this gradient, 10 ml of PBS-AT without salt is placed in the container that pumps into the column and 10 ml PBS-AT (containing 0.1 M KCl) is placed in the other container.
    3. Line the developing chamber on the inside with Whatman 3 mm paper. Equilibrate the developing chamber with ~100 ml solvent for ~ 1 h. Spot 20 μl fractions on a HPTLC plate and develop in chloroform:methanol:0.25 N ammonium hydroxide in 0.25% KCl (65:45:9, v/v). Identify glycolipid containing fractions in an iodine chamber. To prepare the iodine chamber, add a piece of filter paper and a few crystals of iodine in a dry TLC jar. The jar soon becomes saturated with iodine vapors. Insert the TLC plate inside the jar. Glycolipids appear as dark brown spots.
    4. Pool fractions containing glycolipids together and precipitate glycolipids with 1 volume of acetone.
    5. After 3 days at 40 °C to allow the precipitate to settle down, decant the supernatant carefully, wash the precipitate with acetone and air dry.
    6. Take up the dried precipitate in 1 ml of Chloroform:Methanol (9:1, v/v) and load onto a silicic acid column pre-equilibrated in chloroform (10 g silicic acid/g lipid). Sequentially elute the column with 10 bed volume each of chloroform and then Chloroform:Methanol (8:2; 7:3; 6:4; 1:1; 4:6; 3:7; 2:8; v/v) and finally with methanol (10 bed volumes).
    7. Monitor the fractions by TLC and further purify the fraction containing the desired glycolipid (GSPL) by affinity chromatography on an erythina cristagalli-agarose column (1 ml) pre-equlibrated in PBS-AT (In our case the presence of the desired glycolpid was monitored by its antileishmanial efficacy in the in vitro infected macrophage model).
    8. Wash the column with 10 bed volumes of PBS-AT and elute the glycolipid with 5 ml of 0.1 M galactose in PBS-AT.
    9. Dialyze the galactose eluent against PBS at 40 °C for 8 h with frequent changes, and finally lyophilize.
    10. Take up the lyophilized material in C:M (100 μl, 2:1, v/v) and precipitate with acetone as before.

Recipes

  1. Phosphate buffered saline (PBS)
    0.02 M phosphate buffer (pH 7.2)
    Containing 0.15 M NaCl
  2. Sucrose density gradient
    Cells collected by centrifugation from step A-2, was taken up in 5 ml of 5% sucrose in 50 mM Tris-HCl buffer (pH 7.4). 5 ml each of this suspension was layered on to a 10-ml cushion of 20% sucrose in the same buffer in a corex tube. These were centrifuged in a swinging bucket rotor at 1,500 x g for 30 min. The band at the interface consisted of the flagella, and the pellet formed consisted of membrane ghosts.
  3. PBS-AT
    To a 0.01 M potassium phosphate buffer containing 0.15 M NaCl (pH 6.4), ammonium hydroxide and sodium salt of taurodeoxycholic acid was added to a final concentration of 0.05 N ammonium hydroxide and 0.1% sodium salt of taurodeoxycholic acid.

Acknowledgments

This protocol is adapted from Karmakar et al. (2012).

References

  1. Karmakar, S., Bhaumik, S. K., Paul, J. and De, T. (2012). TLR4 and NKT cell synergy in immunotherapy against visceral leishmaniasis. PLoS Pathog 8(4): e1002646.

简介

糖基化的神经酰胺磷酰肌醇存在于许多真菌和蘑菇以及细菌和寄生生物如利什曼原虫中。 这些通常是膜筏相关的并且不容易通过常规方法提取。 该提取方法提供更高的糖脂产量。 糖鞘脂通常与洗涤剂抗性膜筏相关。 它们难以用中性溶剂从具有中性或较低极性溶剂的刚性前鞭毛细胞壁中提取。 对于极性较强的磷脂,极性较大的溶剂是必需的。 氨用于提取磷脂酰肌醇。

材料和试剂

  1. promastigotes
  2. Tris-HCl
  3. PMSF
  4. 胎牛血清(FBS)
  5. 蔗糖
  6. 氯仿
  7. 甲醇
  8. 乙酸乙酯
  9. 吡啶
  10. li>氨>
  11. 氢氧化铵
  12. 牛磺脱氧胆酸钠
  13. KCl
  14. 丙酮
  15. 硅酸
  16. 中等199
  17. 磷酸盐缓冲盐水(PBS)(见Recipes)
  18. 蔗糖密度梯度(参见配方)
  19. PBS-AT(参见配方)

设备

  1. Vortexer
  2. 离心机
  3. DEAE-Sephadex A-25
  4. HPTLC板
  5. 碘室
  6. TLC
  7. Erythina cristagalli琼脂糖柱
  8. 半乳糖
  9. 透析装置
  10. 冻干机
  11. 摆动轮转子

程序

  1. Promastigote文化
    1. Promastigotes of em。 在22℃下在补充有10%热灭活的FBS,2mM l-谷氨酰胺,100U青霉素G钠和100μg硫酸链霉素的培养基199(pH7.4)中培养菌株AG83 并在相同培养基中以2×10 6个细胞/ml的平均密度传代培养。 通过在1,500×g离心10分钟来生长3天后收获细胞。 收获的细胞用PBS(10ml,1,500×g,10分钟)洗涤两次。

  2. 前鞭毛膜幽灵的制备
    1. 在玻璃管中含有2mM PMSF(苯基甲磺酰氟)的50ml冷却的50mM Tris-HCl(pH7.4)中吸收前鞭毛体(1g湿重),并保持在冰上。通过在环形混合器上轻轻涡旋6次来混合管,并在每次涡旋之间在冰上保持10分钟。涡旋时间为2分钟。
    2. 通过在40℃下以3,300×g离心20分钟收集细胞,并用PBS(各20ml)充分洗涤(3-5次)。
    3. 将沉淀重悬于10ml 50mM Tris-HCl(pH7.4)中的5%蔗糖溶液中。将5ml该溶液在Corex管中铺在10ml的25%蔗糖的相同缓冲液中的溶液上。将其在1,500×g离心30分钟。形成的沉淀再次溶于2.5ml 50%Tris-HCl(pH7.4)中的5%蔗糖中。并分层到相同缓冲液中的5ml 25%蔗糖溶液上。将其在1500×g离心45分钟,并进一步处理沉淀(膜重影)。

  3. 纯化GSPL
    1. 将Promastigote膜重影(1g湿重)与19体积的氯仿:甲醇:乙酸乙酯:吡啶:4.5N氨:水(15:15:5:0.5:0.5:0.5,v/v)混合,并通过18 -20冲程在紧密型Dounce型均质器(在冰上)。将管保持在冰上10分钟,然后在浴超声器(30×20秒)中超声处理。在每次超声处理之间将管保持在冰上30秒
    2. 通过旋转蒸发浓缩(干燥)萃取的物质,通过在水浴中超声处理(5×20秒,中间间隔30秒)溶解在1ml PBS-AT中,并装载到DEAE-Sephadex A-25 0.5×5cm),其在含有0.05N氢氧化铵和0.1%牛磺脱氧胆酸钠盐(PBS-AT)的0.01M磷酸盐缓冲液(pH6.4)中平衡。用5ml PBS-AT洗涤柱,并用线性梯度的KCl(PBS-AT中的0-0.1M)洗脱阴离子糖脂。通过将阴离子交换缓冲液PBS-AT(无KCl)与含有0.1M KCl的PBS-AT混合来产生梯度。
      注意:10倍柱体积用于准备梯度。例如:对于2ml柱,使用20ml梯度。为了制备此梯度,将10ml无盐的PBS-AT置于泵入柱中的容器中,并将10ml PBS-AT(含有0.1M KCl)置于另一个容器中。
    3. 将内部的显影室与Whatman 3 mm纸张对齐。平衡显影室用〜100 ml溶剂〜1 h。在HPTLC板上点样20μl级分,并在氯仿:甲醇:在0.25%KCl(65:45:9,v/v)中的0.25N氢氧化铵中展开。在碘室中鉴定含糖脂的级分。为了制备碘室,在干燥的TLC罐中加入一张滤纸和几个碘晶体。瓶子很快被碘蒸气饱和。将TLC板插入罐内。糖脂显示为深棕色斑点。
    4. 将糖脂汇集在一起​​,并用1倍体积的丙酮沉淀糖脂
    5. 在40℃下3天后,使沉淀物沉降,小心地倾析上清液,用丙酮洗涤沉淀物并空气干燥。
    6. 在1ml氯仿:甲醇(9:1,v/v)中吸收干燥的沉淀,并上样到在氯仿(10g硅酸/g脂质)中预平衡的硅酸柱上。依次用10倍柱床体积的氯仿,然后氯仿:甲醇(8:2; 7:3; 6:4; 1:1; 4:6; 3:7; 2:8; v/v)洗脱该柱,最后用甲醇(10床体积)
    7. 通过TLC监测级分,并通过在PBS-AT预平衡的红藻cristagalli琼脂糖柱(1ml)上的亲和层析进一步纯化含有所需糖脂(GSPL)的级分(在我们的情况下,监测所需的糖脂的存在通过其在体外感染的巨噬细胞模型中的抗利什曼病效力)
    8. 用10倍柱床体积的PBS-AT洗柱,并用PBS-AT中的5ml 0.1M半乳糖洗脱糖脂。
    9. 在40℃下用PBS透析半乳糖洗脱液8小时,频繁更换,最后冷冻干燥。
    10. 用C:M(100μl,2:1,v/v)吸收冻干物质,并用丙酮沉淀,如前所述。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    0.02 M磷酸盐缓冲液(pH 7.2) 含有0.15M NaCl
  2. 蔗糖密度梯度
    通过从步骤A-2离心收集的细胞被吸收在5ml的50mM Tris-HCl缓冲液(pH7.4)中的5%蔗糖中。 将每种5ml悬浮液在corex管中的相同缓冲液中层叠到10-ml 20%蔗糖垫上。 将它们在1500×g的摇摆转子中离心30分钟。 界面上的带由鞭毛组成,形成的团块由膜重影组成
  3. PBS-AT
    向含有0.15M NaCl(pH6.4)的0.01M磷酸钾缓冲液中加入氢氧化铵和牛磺脱氧胆酸钠盐,使最终浓度为0.05N氢氧化铵和0.1%牛磺脱氧胆酸钠盐。

致谢

该协议改编自Karmakar等人(2012)。

参考文献

  1. Karmakar,S.,Bhaumik,S.K.,Paul,J.and De,T。(2012)。 TLR4和NKT细胞协同作用在针对内脏利什曼病的免疫治疗中。 PLoS Pathog 8(4):e1002646。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. De, T. (2012). Glycophosphosphingolipid (GSPL) Purification Protocol. Bio-protocol 2(23): e299. DOI: 10.21769/BioProtoc.299.
  2. Karmakar, S., Bhaumik, S. K., Paul, J. and De, T. (2012). TLR4 and NKT cell synergy in immunotherapy against visceral leishmaniasis. PLoS Pathog 8(4): e1002646.
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