搜索

Lectin Binding Analysis of Streptococcus mutans Glycoproteins
变异链球菌糖蛋白的凝集素结合分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various techniques, this is often limited by the specific characteristics of individual proteins such as type and level of glycosylation. Lectins are sugar-binding proteins that recognize specific glycoconjugates in a manner similar to antigen-antibody interactions. Here, we describe a simple method for the detection of glycoproteins using lectin-based Western blot analysis, which can be applied to different organisms and coupled with various other strategies for complementary analysis.

Materials and Reagents

  1. Bacterial whole cell lysates
  2. Brain heart infusion medium (BHI) (BD Biosciences, catalog number: 237500 )
  3. Biotinylated lectin [Vector Laboratories, for Wheat Germ Agglutinin (WGA), catalog number: B-1025]
  4. HRP-conjugated streptavidin (Cell Signaling Technology, catalog number: 3999 )
  5. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
  6. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  7. Enhanced chemiluminiscent detection kit (GE Healthcare, catalog number: RPN2108 )
  8. Autoradiography films (Carestream Health, catalog number: 864-6770 )
  9. NaCl (J.T.Baker®, catalog number: 3628-01 )
  10. KCl (J.T.Baker®, catalog number: 3045-01 )
  11. Na2HPO4 (J.T.Baker®, catalog number: 3827-01 )
  12. KH2PO4 (J.T.Baker®, catalog number: 3246-01 )
  13. Tris base (J.T.Baker®, catalog number: 4109-02 )
  14. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  15. Glycine (Thermo Fisher Scientific, catalog number: BP381-1 )
  16. 30% Acrylamide/Bis solution (Bio-Rad Laboratories, catalog number: 161-0158 )
  17. Tetramethylethylenediamine (TEMED) (Life Technologies, catalog number: 15524-010 )
  18. Ammonium persulfate (Bio-Rad Laboratories, catalog number: BP179-25 )
  19. Glycerol (Alfa Aesar, catalog number: A16205 )
  20. Methanol (BDH Chemicals, catalog number: BDH1135-4LP )
  21. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
  22. 1x phosphate buffer solution (PBS) (see Recipes)
  23. Resolving gel (see Recipes)
  24. Stacking gel (see Recipes)
  25. 2x sample buffer (see Recipes)
  26. Running buffer (see Recipes)
  27. Transfer buffer (see Recipes)
  28. Blocking solution (see Recipes)
  29. Primary solution (see Recipes)
  30. Secondary solution (see Recipes)
  31. Washing solution (see Recipes)

Equipment

  1. 0.1 mm glass beads (Research Products International)
  2. Polyvinylidene fluoride (PVDF) membranes (EMD Millipore, catalog number: P2563-10EA )
  3. 3 mm Whatman paper (GE Healthcare)
  4. CO2 Incubator
  5. Sterile culture tubes (15 ml)
  6. Screw cap tubes (1.7 ml)
  7. Bead beater (BioSpec Products)
  8. Protein electrophoresis running apparatus
  9. Protein transfer apparatus
  10. Power supply
  11. Rocker
  12. Film developer
  13. pH meter

Procedure

  1. Preparation of whole cell protein lysates
    1. Grow bacterial cells [Streptococcus mutans (S. mutans) OMZ175] in 10 ml BHI broth at 37 °C in a humidified 5% CO2 atmosphere.
    2. After 18 h, stationary phase cultures (OD600~0.9) are washed twice (2x) by centrifuging at 4,000 rpm for 10 min, discarding supernatants and resuspending pelleted cells with 1x PBS in original culture volume. After the second wash, cells are concentrated 5x by adding five times less the original culture volume of 1x PBS. The concentrated suspension is then transferred to a 1.7 ml screw cap tube containing approximately 500 μl of glass beads.
    3. Whole cell protein lysates are obtained using a bead-beater at maximum setting (3,450 oscillations per min) and the suspensions are homogenized for three cycles of 30 sec with 2 min of incubation on ice between cycles.
    4. Alternative mechanical methods to obtain whole cell protein lysates such as sonication or French-press, with or without freeze-thawing cycles, are also acceptable.
  2. Protein lysates are separated on a 10% SDS-PAGE and subsequently transferred to a PVDF membrane.
    1. For gel preparation, the cassette is assembled with glass and silica plates with 0.75-1.0 mm spacers and placed on a caster.
    2. Resolving gel is prepared, poured and let to polymerize for approximately 30 min.
    3. Stacking gel is prepared, poured and a comb with desired number of wells inserted. Allow gel to polymerize (~ 30 min).
    4. Remove cassette from caster and place on running apparatus.
    5. Prepare samples by mixing equal volume of sample and 2x sample buffer and boil for 10 min. Incubate on ice for 1 min, quick spin tube to settle any sediment and load sample on gel.
    6. Run SDS-PAGE at 100 V for 2 h.
    7. Transfer “sandwich” is assembled (Figure 1) in the presence of transfer buffer. Gel is removed from plates and placed in assembling “sandwich”.


      Figure 1. Schematic of transfer “sandwich” assembling for Western blotting. In the presence of transfer buffer, place two (2) pieces of Whatman paper A soaked in transfer buffer followed by the gel with separated proteins B, the PVDF membrane C soaked in methanol then transfer buffer, and two (2) more pieces of soaked Whatman papers. Arrow shows the direction of protein transfer.

    8. An oversized piece of PVDF membrane, to cover the entire gel, is soaked in methanol first followed by transfer buffer and then placed on top of the gel.
    9. The rest of the transfer “sandwich” is completed and proteins are transferred to the membrane for 1 h at 100 V in the presence of transfer buffer and an ice unit block to absorb heat produced during transfer.
    10. Sandwich is disassembled, membrane is removed and any extra piece of membrane that is bigger than the gel is cut out.
  3. Block membrane with 5% BSA solution for 18-20 h at 4 °C with slow rocking. Wash membrane 3x by adding 20 ml of washing solution and slow rocking for 5 min to remove excess BSA. Then, incubate the membrane with the primary solution (biotinylated lectin in 0.5% BSA) at room temperature for 1 h with slow rocking.
  4. Wash membrane 3x followed by the addition of the secondary solution (HRP-streptavidin in 0.5% BSA) and incubate at room temperature for 1 h with slow rocking.
  5. After incubation with secondary solution, wash the membrane 4x to visualize biotinylated lectins bound to glycoproteins using enhanced chemiluminescent detection kit, mix equal volumes of detection solution 1 and detection solution 2 (for a final volume of 0.125 ml per cm2 of membrane) and incubate for 1 min at room temperature.
  6. Drain excess detection solution and place membrane in an autoradiography cassette for film exposure. Due to the variability of carbohydrate content on glycoproteins, different exposure of the membrane to the film should be performed.

Representative data

A representative image of Cnm analysis for different strains of S. mutans with anti-rCnmA and biotinylated-WGA is shown below.


Figure 2. Example of lectin binding analysis of S. mutans. Cnm is a surface protein of invasive S. mutans that undergoes PgfS-dependent glycosylation (Aviles-Reyes et al., 2014). A. Size difference in Cnm due to PgfS modification can be observed using anti-rCnmA. B. The presence of glycoconjugates attached to Cnm in OMZ175 was determined using biotinylated wheat germ agglutinin.

Notes

  1. Due to the variability of the type and the level of glycosylation for each protein, protocol optimization may be required for successful identification of glycoproteins by lectin analysis.
  2. Control glycoproteins with known reactivity to specific lectins should be included in each experiment.

Recipes

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
  2. Resolving gel (10 % Tris glycine SDS-PAGE; 10 ml)
    4.0 ml of H2O
    3.3 ml of 30% acrylamide
    2.5 ml of 1.5 M Tris (pH 8.8)
    0.1 ml of 10% SDS
    0.1 ml of 10% APS
    8 μl of TEMED
  3. Stacking gel (3 ml)
    2.1 ml of H2O
    0.5 ml of 30% acrylamide
    0.38 ml of 0.5 M Tris (pH 6.8)
    0.03 ml of 10% SDS
    0.03 ml of 10% APS
    6 μl of TEMED
  4. 2x sample buffer (200 ml)
    50 ml of 0.5 M Tris (pH 6.8)
    8 g of SDS
    40 ml of glycerol
    H2O to 200 ml
    Add bromophenol blue until dark
  5. Running buffer
    25 mM Tris
    192 mM glycine
    0.1% SDS
    pH 8.3
  6. Transfer buffer (1 L)
    200 ml of methanol
    3.0 g of Tris
    14.4 g of glycine
    H2O to 1 L
  7. Blocking solution
    5% BSA (w/v) in 1x PBS
  8. Primary solution
    0.5% BSA (w/v) in 1x PBS
    0.1% Tween 20
    20 μg/ml biotinylated lectin
  9. Secondary solution
    0.5% BSA (w/v) in 1x PBS
    0.1% Tween 20
  10. Washing solution
    1x PBS + 0.1% Tween 20

Acknowledgements

We would like to thank Fred Hagen for technical support. This work has been supported by grants from the American Heart Association (10GRNT420049) and NIH-NIDCR (R01 DE022559). A.A.-R. was supported by NIH-NHLBI (F31 HL124951).

References

  1. Avilés-Reyes, A., Miller, J. H., Simpson-Haidaris, P. J., Hagen, F. K., Abranches, J. and Lemos, J. A. (2014). Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence. J Bacteriol 196(15): 2789-2797.
  2. Nothaft, H. and Szymanski, C. M. (2010). Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8(11): 765-778.

简介

细菌糖蛋白由于其在自然界中的丰富性和在健康和传染病中的重要性而日益受到关注。 然而,只有非常小部分的细菌糖蛋白已被表征和其翻译后修饰机制鉴定。 尽管通过各种技术可以实现糖蛋白的分析,但是这常常受到单个蛋白质的特定特征的限制,例如糖基化的类型和水平。 凝集素是以类似于抗原 - 抗体相互作用的方式识别特异性糖缀合物的糖结合蛋白。 在这里,我们描述了一种简单的方法使用基于凝集素的蛋白质印迹分析检测糖蛋白,这可以应用于不同的生物体,并与各种其他策略的补充分析。

材料和试剂

  1. 细菌全细胞裂解物
  2. 脑心浸液介质(BHI)(BD Biosciences,目录号:237500)
  3. 生物素化凝集素[Vector Laboratories,for Wheat Germ Agglutinin(WGA),目录号:B-1025]
  4. HRP-缀合的链霉亲和素(Cell Signaling Technology,目录号:3999)
  5. 吐温20(Sigma-Aldrich,目录号:P2287)
  6. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  7. 增强化学发光检测试剂盒(GE Healthcare,目录号:RPN2108)
  8. 放射自显影胶片(Carestream Health,目录号:864-6770)
  9. NaCl(J.T.Baker ,目录号:3628-01)
  10. KCl(J.T.Baker ,目录号:3045-01)

  11. ,目录号:3827-01)

  12. (J.T.Baker ®,目录号:3246-01)
  13. Tris碱(J.T.Baker ,目录号:4109-02)
  14. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  15. 甘氨酸(Thermo Fisher Scientific,目录号:BP381-1)
  16. 30%丙烯酰胺/双溶液(Bio-Rad Laboratories,目录号:161-0158)
  17. 四甲基乙二胺(TEMED)(Life Technologies,目录号:15524-010)
  18. 过硫酸铵(Bio-Rad Laboratories,目录号:BP179-25)
  19. 甘油(Alfa Aesar,目录号:A16205)
  20. 甲醇(BDH Chemicals,目录号:BDH1135-4LP)
  21. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  22. 1×磷酸盐缓冲溶液(PBS)(参见配方)
  23. 解决凝胶(见配方)
  24. 堆叠胶(见配方)
  25. 2x样品缓冲液(见配方)
  26. 运行缓冲区(参见配方)
  27. 传输缓冲区(请参阅配方)
  28. 阻止解决方案(参见配方)
  29. 主要解决方案(参见配方)
  30. 次要解决方案(参见配方)
  31. 洗涤液(见配方)

设备

  1. 0.1mm玻璃珠(Research Products International)
  2. 聚偏二氟乙烯(PVDF)膜(EMD Millipore,目录号:P2563-10EA)
  3. 3mm Whatman纸(GE Healthcare)
  4. CO 2 孵化器
  5. 无菌培养管(15ml)
  6. 螺旋盖管(1.7ml)
  7. Bead beater(BioSpec产品)
  8. 蛋白电泳运行装置
  9. 蛋白转移装置
  10. 电源
  11. 摇杆
  12. 电影开发商
  13. pH计

程序

  1. 全细胞蛋白裂解液的制备
    1. 在37℃,在潮湿的5%CO 2中,在10ml BHI肉汤中培养细菌细胞[变异链球菌(变形链霉菌)OMZ175] 大气层。
    2. 18小时后,将固定相培养物(OD 600〜0.9)洗涤两次 (2x)通过在4,000rpm离心10分钟,弃去上清液 并在原始培养体积中用1×PBS重悬浮沉淀的细胞。 第二次洗涤后,通过加入5次将细胞浓缩5倍 减少1×PBS的原始培养体积。 浓缩悬浮液 然后转移到含有约1.5ml螺旋盖管的1.7ml螺旋盖管中 500μl玻璃珠。
    3. 获得全细胞蛋白裂解物 使用珠磨机在最大设置(每分钟3,450次振荡)和 将悬浮液匀化3个循环的30秒,2分钟   在循环之间在冰上孵育
    4. 替代机械方法   以获得全细胞蛋白裂解物,例如超声处理或 法式压榨,有或没有冻融循环,也是 可接受。
  2. 蛋白裂解物在10%SDS-PAGE上分离,随后转移到PVDF膜
    1. 对于凝胶制备,盒与具有0.75-1.0mm间隔物的玻璃和二氧化硅板组装并放置在脚轮上
    2. 制备溶解凝胶,倾倒并让其聚合约30分钟
    3. 制备堆叠凝胶,倒入并插入具有所需数量的孔的梳子。 允许凝胶聚合(〜30分钟)。
    4. 从脚轮上取下纸盒,并放置在正在运行的设备上。
    5. 通过混合等体积的样品和2x样品制备样品 缓冲液并煮沸10分钟。 在冰上孵育1分钟,快速旋转管 以沉淀任何沉积物并在凝胶上装载样品
    6. 运行SDS-PAGE在100 V 2小时。
    7. 在存在的情况下组装转移"三明治"(图1) 传送缓冲器。 从板上除去凝胶并放置在组装中 "三明治"。


      图1.转移"三明治" 装配用于Western印迹。在转移缓冲液存在下, 将两(2)片Whatman纸A浸泡在转移缓冲液中 其次是凝胶与分离的蛋白质B,PVDF膜C. 浸泡在甲醇中然后转移缓冲液,再加两(2)片 浸泡的Whatman论文。 箭头显示蛋白质转移的方向
    8. 一个超大的PVDF膜片,覆盖整个凝胶 先浸泡在甲醇中,然后加入转移缓冲液,然后放置 凝胶顶部。
    9. 其余的"三明治"是 完成并将蛋白质在100V下转移至膜1小时 在转移缓冲液和冰单元块的存在下吸收热量 转移期间产生
    10. 三明治被拆卸,膜被去除,并且切除大于凝胶的任何额外的膜片。
  3. 用5%BSA溶液封闭膜在4℃下缓慢摇动18-20小时。通过加入20ml洗涤溶液并缓慢摇动5分钟以洗涤过滤BSA来洗涤膜3次。然后,将膜与初级溶液(生物素化凝集素在0.5%BSA中)在室温下孵育1小时,同时缓慢摇动。
  4. 洗涤膜3x,然后加入第二溶液(HRP-链霉亲和素在0.5%BSA中),并在室温下缓慢摇动孵育1小时。
  5. 在与第二溶液温育后,洗涤膜4x以使用增强的化学发光检测试剂盒显现与糖蛋白结合的生物素化的凝集素,混合等体积的检测溶液1和检测溶液2(对于终体积为0.125ml/cm 2)膜)并在室温下孵育1分钟。
  6. 排出过量的检测溶液,将膜置于放射自显影盒中进行胶片曝光。由于糖蛋白上碳水化合物含量的可变性,应当进行膜对膜的不同暴露。

代表数据

不同菌株的Cnm分析的代表性图像。突变体与抗rCnmA和生物素化-WGA的结合如下所示。


图2. S的凝集素结合分析的实施例。 mutans 。 Cnm是侵入性的表面蛋白。变异体,其经历PgfS依赖性糖基化(Aviles-Reyes等人,2014)。可以使用抗rCnmA观察到由于PgfS修饰引起的Cnm的尺寸差异。 B.使用生物素化的小麦胚芽凝集素测定OMZ175中与Cnm连接的糖缀合物的存在。

笔记

  1. 由于每种蛋白质的类型和糖基化水平的可变性,可能需要方案优化以通过凝集素分析成功鉴定糖蛋白。
  2. 应该在每个实验中包括对特定凝集素具有已知反应性的对照糖蛋白。

食谱

  1. 1x PBS
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
  2. 拆分凝胶(10%Tris甘氨酸SDS-PAGE; 10ml) 4.0ml H 2 O x/v 3.3ml 30%丙烯酰胺 2.5ml 1.5M Tris(pH8.8)
    0.1ml 10%SDS 0.1ml 10%APS
    8μlTEMED
  3. 堆叠凝胶(3ml)
    2.1ml H 2 O 2 / 0.5ml 30%丙烯酰胺 0.38ml 0.5M Tris(pH 6.8)
    0.03ml 10%SDS 0.03ml 10%APS
    6μlTEMED
  4. 2×样品缓冲液(200ml) 50ml 0.5M Tris(pH6.8)
    8克SDS
    40ml甘油 H 2 O至200ml
    将溴酚蓝加到黑色
  5. 运行缓冲区
    25 mM Tris
    192 mM甘氨酸 0.1%SDS
    pH 8.3
  6. 转移缓冲液(1 L)
    200ml甲醇
    3.0克Tris
    14.4g甘氨酸 H sub 2 O到1L
  7. 封锁解决方案
    5%BSA(w/v)在1x PBS中
  8. 主要解决方案
    0.5%BSA(w/v)在1x PBS中 0.1%Tween 20
    20μg/ml生物素化凝集素
  9. 次要解决方案
    0.5%BSA(w/v)在1x PBS中 0.1%Tween 20
  10. 洗涤溶液
    1×PBS + 0.1%Tween 20

致谢

我们要感谢Fred Hagen的技术支持。 这项工作得到了美国心脏协会(10GRNT420049)和NIH-NIDCR(R01 DE022559)的资助。 A.A.-R. 由NIH-NHLBI(F31 HL124951)支持。

参考文献

  1. Avilés-Reyes,A.,Miller,J.H.,Simpson-Haidaris,P.J.,Hagen,F.K.,Abranches,J.and Lemos,J.A。(2014)。 PgfS对变形链球菌 Cnm的修饰有助于粘附,内皮细胞侵袭 ,和毒力。细菌 196(15):2789-2797。
  2. Nothaft,H.and Szymanski,C.M。(2010)。 细菌中的蛋白质糖基化:比以往任何时候都更甜。 Nat Rev Microbiol < em> 8(11):765-778。
  • 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。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Avilés-Reyes, A., Lemos, J. A. and Abranches, J. (2015). Lectin Binding Analysis of Streptococcus mutans Glycoproteins. Bio-protocol 5(7): e1431. DOI: 10.21769/BioProtoc.1431.
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。