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Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
核糖体灭活蛋白的核糖体RNA N-糖基化酶活性测定   

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Abstract

Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo’s fragment) is diagnostic of the action of RIPs.

Keywords: Ribosome-inactivating protein (RIP)(核糖体灭活蛋白(RIP)), rRNA N-glycosylase(rRNA N-糖基化酶), Protein synthesis (inhibition)(蛋白质合成(抑制)), Sarcin-ricin loop(八叠球菌素-篦麻毒素循环), Polynucleotide:adenosine glycosylase(多核苷酸:腺苷糖基化酶), Ricin(蓖麻毒素), Pokeweed Antiviral Protein (PAP)(商陆抗病毒蛋白(PAP)), Beetin 27(甜菜素27)

Background

N-glycosylase activity of RIPs on the eukaryotic 28S rRNA was first described by Endo and Tsurugi for ricin ( Endo and Tsurugi, 1988) in rat ribosomes; subsequently it was shown that some RIPs can also depurinate ribosomes from plants, bacteria and fungi. The result of this effect, upon treatment with aniline, is the release of an RNA fragment of between 240 and 500 nucleotides (depending on species) from the rRNA of the large subunit (Figure 1). Most RIPs depurinate ribosomes at one site (the adenine 4,324), whereas other RIPs such as saporins, PAP-R and trichokirin depurinate the rRNA at multiple sites. The protocol described here uses rabbit reticulocyte lysate, a eukaryotic cell-free model system very sensitive to the action of RIPs, which shows high rates of depurination for most RIPs. This allows obtaining a large amount of fragment which facilitates its detection by electrophoresis. In this procedure we use denaturing polyacrylamide minigels which require small quantities of sample and have higher resolution than the agarose gels when staining with fluorescent dyes.


Figure 1. Sarcin Ricin Loop of the large rRNA from rat, yeast and Escherichia coli. The sequences (accession numbers NR_046246, J01355 and AB035926) were downloaded from the NCBI sequence database (http://www.ncbi.nlm.nih.gov/nucleotide/). The adenine released by the RIP action (boldfaced), the site of splitting by either the aniline or α-sarcin (arrows) and the size of the generated fragment are also indicated. Partial sequence of rabbit 28S rRNA (AF460236) indicates that rat and rabbit share the same SRL sequence.

Materials and Reagents

Note: All the reagents used in preparing buffers should be of molecular biology grade purity (RNase, DNase-free). Water and solutions should be autoclaved at 120 °C for 15 min (except of aniline and ethanol).

  1. Disposable gloves
  2. Pipettes and tips (either RNase, DNase-free or autoclaved)
  3. Eppendorf tubes (1.5 ml polypropylene microcentrifuge tubes , either RNase, DNase-free or autoclaved)
  4. Paper towels
  5. Corning 50 ml PP centrifuge tubes (Corning, catalog number: 430291 )
  6. Pasteur pipettes
  7. Cotton swabs
  8. Rabbit reticulocyte lysate, untreated with micrococcal nuclease, either obtained as indicated by Pelham and Jackson (1976) or purchased from a biochemical supplier (for example: Promega, catalog number: L4151 )
  9. RIP either obtained as indicated by Barbieri et al. (2001) or purchased from a biochemical supplier (for example Ricin A chain: Sigma-Aldrich, catalog number: L9514 )
  10. Crushed ice
  11. Phenol/TRIS saturated sol., for molecular biology, stabilized, DNAse, RNAase and Protease free (ACROS Organics, catalog number: 327125000 )
  12. Ethanol absolute (EMD Millipore, catalog number: 100983 )
  13. Deionized, RNase and DNase-free water
    Note: For example, Millipore Elix 5 (UV) water autoclaved 120 °C 15 min.
  14. Aniline (Sigma-Aldrich, catalog number: 242284 )
  15. Urea (Thermo Fisher Scientific, Affymetrix, catalog number: 75826 )
  16. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  17. N,N,N’,N’-tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  18. Acrylamide bis-acrylamide 19:1, 40% (w/v) solution (Thermo Fisher Scientific, Affymetrix, catalog number: 75848 )
  19. Either ethidium bromide (Sigma-Aldrich, catalog number: E7637 ) or GelRed (Biotium, catalog number: 41003 )
  20. EDTA·2H2O
  21. NaOH pellets
  22. Hydrochloric acid (HCl) (EMD Millipore, catalog number: 100317 )
  23. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, Affymetrix, catalog number: 75819 )
  24. Sodium acetate trihydrate (EMD Millipore, catalog number: 106267 )
  25. Glacial acetic acid (EMD Millipore, catalog number: 100063 )
  26. Diethyl pyrocarbonate (Sigma-Aldrich, catalog number: D5758 )
  27. Diethyl ether (EMD Millipore, catalog number: 100921 )
  28. Trimethylol aminomethane (Tris base) (Fisher Scientific, catalog number: BP154-1 )
  29. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  30. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  31. RNA markers (Roche Diagnostics, catalog number: 1062 638 )
    Note: This product has been discontinued. Can be replaced by Low Range ssRNA Ladder (New England Biolabs, catalog number: N0364S )
  32. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
  33. 0.5 M EDTA (pH 8.0) (see Recipes)
  34. 50 mM Tris/0.5% SDS (pH 7.8) (see Recipes)
  35. 3 M sodium acetate (pH 5.2) (see Recipes)
  36. 70% ethanol (see Recipes)
  37. 2 M aniline (pH 4.5) (see Recipes)
  38. Water saturated ether (see Recipes)
  39. 10x TBE buffer (see Recipes)
  40. 2x gel loading buffer (see Recipes)

Equipment

  1. Deep freezer (-80 °C freezer) (Thermo Fisher Scientific, Thermo ScientificTM, model: TSE Series , catalog number: TSE400SSV)
  2. Fume hood (BURDINOLA, model: V21-Space BAJA ST 1500 )
  3. Two microcentrifuges (DJB Labcare, model: Heraeus Biofuge Pico , catalog number: 75003235), one of them kept at 4 °C
  4. 30 °C water bath (JP SELECTA, catalog number: 6000140 )
  5. Vortex mixer for test tubes (IKA, catalog number: 0003617000 )
  6. BECKMAN DU-640 spectrophotometer (Beckman Coulter, model: DU-640 )
  7. Quartz cuvette (Hellma Analytics, model: 104-QS , catalog number: 104-10-40)
  8. Polyacrylamide gel electrophoresis system (GE Healthcare, catalog number: 80-6418-77 )
  9. Power supply (GE Healthcare, catalog number: 18-1130-01 )
  10. Molecular Imager® Gel DocTM XR+ System with Image LabTM Software (Bio-Rad Laboratories, catalog number: 1708195 )
  11. Gel staining tray
  12. Magnetic stirrer
  13. Autoclave (JP SELECTA, catalog number: 4002516 )

Procedure

The general procedure to assay N-glycosylase activity is illustrated in a flowchart in Figure 2.


Figure 2. Flowchart illustrating protocol Procedure parts A-H. The flowchart shows the key steps describing the eight main parts of the protocol procedure. Capital letters (A-H) refer to the subsections in the Procedure section.

  1. Treatment of ribosomes with RIPs
    1. Thaw frozen aliquots of both rabbit reticulocyte lysate (stored at -80 °C) and RIP (stored at -20 °C) at room temperature and place them on crushed ice.
    2. Reactions are prepared on crushed ice into four Eppendorf tubes as indicated in the following:
      Tube
      1 (control)
      2 (control)
      3 (+RIP)
      4 (+RIP)
      lysate
      40 µl
      40 µl
      40 µl
      40 µl
      RIP (1 mg/ml)
      -
      -
      1 µl
      1 µl

    3. Pipette 40 µl of rabbit reticulocyte lysate into the four tubes.
    4. Add 1 µl of 1 mg/ml RIP into tubes 3 and 4.
    5. Mix by gently vortexing.
    6. Incubate at 30 °C for 1 h in a water bath.
    7. Stop the reaction by adding 2 µl of 0.5 M EDTA (pH 8.0).
    8. Mix by gently vortexing.
    9. Add 500 µl of 50 mM Tris/0.5% SDS.
    10. Vortex hard for 30 sec (with the purpose of denaturing proteins with the aid of SDS).

  2. Deproteinization
    Note: This process should be carried out under a fume hood.
    1. Add 500 µl of phenol.
    2. Vortex hard for 30 sec.
    3. Centrifuge at 16,060 x g (13,000 rpm) for 5 min at room temperature.
    4. Recover the aqueous (upper) phase (400 µl) to a new Eppendorf tube without disturbing the protein interphase.

  3. RNA precipitation
    1. To carry out RNA precipitation with 3 volumes of ethanol in 1.5 ml tubes, the aqueous phase is split into two Eppendorf tubes (A and B), with 200 µl in each. Perform ethanol precipitation by mixing the following:
      Tube
      1A (control)
      1B (control)
      2A (control)
      2B (control)
      Aqueous phase
      200 µl
      200 µl
      200 µl
      200 µl
      3 M sodium acetate, pH 5.2
      20 µl
      20 µl
      20 µl
      20 µl
      Absolute ethanol
      600 µl
      600 µl
      600 µl
      600 µl

      Tube
      3A (+RIP)
      3B (+RIP)
      4A (+RIP)
      4B (+RIP)
      Aqueous phase
      200 µl
      200 µl
      200 µl
      200 µl
      3 M sodium acetate, pH 5.2
      20 µl
      20 µl
      20 µl
      20 µl
      Absolute ethanol
      600 µl
      600 µl
      600 µl
      600 µl

    2. Mix by vortexing.
    3. Keep overnight at -80 °C (or at least 3 h).
    4. Centrifuge at 16,060 x g (13,000 rpm) for 15 min at 4 °C. Orient each tube in the rotor with the cap hinge pointing outward, this will indicate the position of the pelleted RNA since pellets will be scarcely visible.
    5. Take the tubes out. Carefully decant the supernatant liquid and discard it into paper towel without losing sight of the off-white pellet avoiding its displacement.
    6. Add 250 µl of 70% ethanol.
    7. Centrifuge at 16,060 x g (13,000 rpm) for 15 min at 4 °C. Orient each tube in the rotor with the cap hinge pointing outward.
    8. Carefully decant the supernatant liquid and discard it into paper towel avoiding pellet displacement. Remove any droplet on the tube wall with the aid of a cotton swab.
    9. Place the tubes up-side-down onto a clean paper towel and allow the pellet to air-dry for 15 min.
    10. Resuspend the pellet in 10 µl of RNase-free deionized water. Mix the contents of tubes 1A and 1B, and 3A and 3B, obtaining now the tubes 1 and 3 with 20 µl each. Store these samples at -80 °C. Tubes 2A, 2B, 4A and 4B still remain separated.

  4. Aniline treatment of RNA
    1. Add one volume (10 µl) of 2 M aniline (pH 4.5) to tubes 2A, 2B, 4A and 4B.
      Note: Reaction is carried out on crushed ice.
    2. Mix by vortexing.
    3. Incubate for 10 min on ice.
    4. Stop the reaction by adding 200 µl of RNase-free deionized water and mix by vortexing.
    5. Add 200 µl of water saturated ether.
    6. Vortex hard for 20 sec.
    7. Let the sample stand for 20 sec to allow phase separation.
    8. Discard the ether (upper) phase.
    9. Repeat steps D5 to D8.
    10. Add 20 µl of 3 M sodium acetate (pH 5.2) and 600 µl of absolute ethanol.
    11. Mix by vortexing.
    12. Keep overnight at -80 °C (or at least 3 h).
    13. Centrifuge at 16,060 x g (13,000 rpm) for 15 min at 4 °C. Orient each tube in the rotor with the cap hinge pointing outward.
    14. Take the tubes out. Carefully decant the supernatant liquid and discard it into paper towel without losing sight of the off-white pellet avoiding its displacement.
    15. Add 250 µl of 70% ethanol.
    16. Centrifuge at 16,060 x g (13,000 rpm) for 15 min at 4 °C. Orient each tube in the rotor with the cap hinge pointing outward.
    17. Carefully decant the supernatant liquid and discard it into paper towel avoiding pellet displacement. Remove any droplet on the tube wall with the aid of a cotton swab.
    18. Place the tubes up-side-down onto a clean paper towel and allow the pellet to air-dry for 15 min.
    19. Resuspend the pellet in 10 µl of RNase-free, deionized water. Mix the contents of tubes 2A and 2B, and 4A and 4B, obtaining now the tubes 2 and 4 with 20 µl each. Store these samples at -80 °C.

  5. Spectrophotometric quantification of RNA
    1. Thaw frozen samples 1-4 with 20 µl each and place them on ice.
    2. Determine the RNA concentration of each sample in the spectrophotometer at 260 nm (in quartz cuvette of 10 mm path length, using water as blank). If A260 = 1, then the RNA concentration is 40 µg RNA/ml. Usually the yield of the process is 45-50 µg RNA for samples treated with aniline and 50-60 µg RNA for untreated samples.

  6. Preparation of 7 M urea/5% (w/v) polyacrylamide gel
    Prepare 25 ml of mix for two gels (glass plates of 10 x 10.5 cm, spacers of 0.1 cm) in a RNase-free Corning tube: 2.5 ml 10x TBE; 3.1 ml acrylamide/bisacrylamide (40%); 10.5 g urea; 12.3 ml RNase-free, deionized water; 0.2 ml 10% ammonium persulfate; 0.05 ml TEMED.

  7. Preparation of samples
    Samples are prepared into Eppendorf tubes placed on crushed ice.
    1. Add 3 µg of RNA.
    2. Complete the volume to 5 µl with water.
    3. Add 5 µl of 2x gel loading buffer.
    4. Mix by vortexing.
    5. Boil 30 sec (in a 100 °C water bath) and place the tubes on crushed ice immediately.

  8. Electrophoresis
    1. Attach the gel plates to the gel apparatus.
    2. Add TBE electrophoresis buffer (900 ml water and 100 ml 10x TBE) to the top and bottom chambers.
    3. Remove the comb and rinse the wells with buffer to remove residual urea (using a Pasteur pipette).
    4. Using a micropipettor, load the samples.
    5. Run the gel at 15 mA for 1 h 50 min.
    6. Disassemble the gel.
    7. Stain the gel with either 0.5 µg/ml ethidium bromide or GelRed solution at room temperature for 40 min with gentle shaking (destaining is not needed after staining).
    8. Place the gel on an ultraviolet transilluminator. Visualize and photograph RNA bands.
      Note: Be aware that RNA will diffuse within the gel over time, therefore examination and photography should take place shortly after the end of electrophoresis.
      A representative data is shown in Figure 3.


      Figure 3. rRNA N-glycosylase activity of BE27 in rabbit reticulocyte lysate. Each lane contained 3 µg of RNA isolated from either untreated (control) or RIP (+BE27) treated ribosomes from rabbit. The arrows indicate the 28S, 18S and 5.8S rRNAs, and the RNA fragment released as a result of RIP action after aniline treatment (+). The number of the corresponding tube in section A is indicated. Samples from lanes 5 and 6 were processed as samples from lanes 3 and 4, respectively, but the ribosomes were treated with 3 µg instead of 1 µg of BE27. The size of RNA markers in nucleotides are also indicated (M). The gel to the left was stained with ethidium bromide and the gel to the right with GelRed.

Data analysis

This procedure is very specific for identifying rRNA N-glycosylase activity and the results are very reliable. Controls are included to rule out nonspecific RNA breaks produced at random by aniline treatment. On the other hand, a comparative analysis of RIP treated-rRNA in the absence and presence of aniline is carried out in each experiment to exclude unspecific effects due to possible contaminants that could fragment or degrade the RNA. Besides, the result can be confirmed by treating the RNA with different RIP concentrations. RNA molecular weight markers can be used to determine the size of the RNA fragment released (which would depend on the type of ribosomes used in the assay). It is also recommended to include in the experiment a previously published RIP (e.g., ricin) to unequivocally localize the fragment.

Notes

Due to its sensitivity and simplicity, rabbit reticulocyte lysate is the best system to check rRNA N-glycosylase activity. This method can also be used to test specific RNase activity (alpha sarcin-like activity) but without aniline treatment. Other sensitive ribosomes can also be used with minor modifications but because many RNA bands can be seen after gel electrophoresis, optimization is required before the Endo’s fragment can be detected. Such modified protocols have been used successfully for yeast, Penicillium digitatum, Escherichia coli, Agrobacterium tumefaciens, Streptomyces lividans, Brevibacterium lactofermentum, Vicia sativa, and Colo 320 and HeLa cell cultures.

Recipes

  1. 0.5 M EDTA (pH 8.0)
    1. Add 186.1 g of disodium EDTA·2H2O to 800 ml of ddH2O
    2. Stir vigorously on a magnetic stirrer
    3. Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets)
    4. Dispense into aliquots and sterilize by autoclaving
    Note: The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to ~8.0 by the addition of NaOH. Autoclave at 120 °C for 15 min.
  2. 50 mM Tris/0.5% SDS
    Mix 45 ml of ddH2O, 2.5 ml of 1 M Tris-HCl (adjusted to pH 7.8 with HCl) and 2.5 ml of 10% SDS. Autoclave 120 °C 15 min
  3. 3 M sodium acetate (pH 5.2)
    20.412 g sodium acetate trihydrate
    Add approximately 6 ml glacial acetic acid to pH 5.2 and up to 50 ml with ddH2O
    Autoclave at 120 °C for 15 min
  4. 70% ethanol
    70% ethanol absolute and 30% deionized, RNase and DNase-free water
  5. 2 M aniline (pH 4.5)
    1. Add 18.25 ml of aniline to RNase and DNase-free water
    2. Adjust to pH 4.5 with glacial acetic acid using an RNase-free electrode washed with either RNase and DNase-free water or with 1% diethyl pyrocarbonate in water
    3. Add double distilled water up to 100 ml
    4. Aliquot in Eppendorf tubes and store at -20 °C
  6. Water saturated ether
    Add one volume of deionized, RNase and DNase-free water to one volume of diethyl ether and mix. The upper phase is the water saturated ether
    Note: It is stored in small quantities (10 ml) at room temperature under a fume hood.
  7. 10x TBE buffer
    1. Dissolve (use magnetic stirrer) 108 g Tris base and 55 g boric acid in 800 ml deionized, RNase and DNase-free water
    2. Add 40 ml 0.5 M EDTA (pH 8.0)
    3. Adjust volume to 1 L
    4. Autoclave at 120 °C for 15 min
    5. Store at room temperature
  8. 2x gel loading buffer
    TBE buffer
    100 mg/ml sucrose
    7 M urea
    0.4 µg/ml bromophenol blue
    Store at 4 °C until use

Acknowledgments

This protocol was modified from previous publications (Endo and Tsurugi, 1988; Sallustio and Stanley, 1990). This work was supported by grants BIO39/VA39/14 and BIO/VA17/15 to L.C.

References

  1. Barbieri, L., Bolognesi, A. and Stirpe, F. (2001). Purification and conjugation of type 1 ribosome-inactivating proteins. In: Hall, W. A. (Ed.). Immunotoxin Methods and Protocols-Series Methods in Molecular Biology. Humana Press, pp: 71-85.
  2. Citores, L., Iglesias, R., Gay, C. and Ferreras, J. M. (2016). Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mould Penicillium digitatum. Mol Plant Pathol 17(2): 261-271.
  3. Endo, Y. and Tsurugi, K. (1988). The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA. J Biol Chem 263(18): 8735-8739.
  4. Iglesias, R., Citores, L., Di Maro, A. and Ferreras, J. M. (2015). Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.). Planta 241(2): 421-433.
  5. Iglesias, R., Citores, L., Ragucci, S., Russo, R., Di Maro, A. and Ferreras, J. M. (2016). Biological and antipathogenic activities of ribosome-inactivating proteins from Phytolacca dioica L. Biochim Biophys Acta 1860(6): 1256-1264.
  6. Pelham, H. R. and Jackson, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 67(1): 247-256.
  7. Sallustio, S. and Stanley, P. (1990). Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin. J Biol Chem 265(1): 582-588.

简介

核糖体失活蛋白(RIP)是由于其N-糖基化酶(EC 3.2.2.22)活性而不可逆地灭活核糖体的酶。酶从28S rRNA的腺嘌呤编号4324和大鼠核糖体中的核糖(或来自其他生物体的敏感核糖体中的等同腺嘌呤)切割N-糖苷键。该腺嘌呤位于α-原丝素 - 蓖麻毒素环(SRL)中,这对于在原核生物和真核生物中的mRNA-tRNA易位期间分别在核糖体上锚定延伸因子(EF)G和EF2至关重要。 RIP主要从植物中分离出来,这些蛋白质的实例是蓖麻毒蛋白或者口服抗病毒蛋白(PAP)。这些蛋白质,单独或作为免疫毒素的一部分,是癌症治疗的有用工具。以下方案描述了当通过在聚丙烯酰胺凝胶上电泳在来自兔网织红细胞裂解物的RIP处理的脱嘌呤RNA在酸性苯胺存在下孵育时检测释放的RNA片段的方法。发布的片段(Endo的片段)是RIP的动作的诊断。

Endo和Tsurugi首先在大鼠核糖体中描述了RIP对真核28S rRNA的N-糖基化酶活性,蓖麻毒素(Endo and Tsurugi,1988)随后显示出一些RIP也可以从植物,细菌和真菌中去除核糖体。在用苯胺处理时,这种效应的结果是从大亚基的rRNA释放240至500个核苷酸(取决于物种)的RNA片段(图1)。大多数RIPs在一个位点(腺嘌呤4,324)上消除核糖体,而其他RIP,如通丝蛋白,PAP-R和trichokirin可以在多个位点上去除rRNA。这里描述的方案使用兔网织红细胞裂解物,一种对RIP的作用非常敏感的真核细胞免疫模型系统,其对于大多数RIP显示高的脱嘌呤率。这样可以获得大量有助于通过电泳检测的片段。在这个过程中,我们使用变性聚丙烯酰胺微型凝胶,当用荧光染料染色时,需要少量样品并且具有比琼脂糖凝胶更高的分辨率。


图1.来自大鼠,酵母和大肠杆菌的大rRNA的Sarcin蓖麻毒素环。从NCBI序列数据库中下载序列(登录号NR_046246,J01355和AB035926) ( http://www.ncbi.nlm.nih.gov/核苷酸/ )。还通过RIP作用(粗体)释放的腺嘌呤,苯胺或α-丝氨酸(箭头)分裂的位点和产生的片段的大小。兔28S rRNA(AF460236)的部分序列表明大鼠和兔共享相同的SRL序列。

关键字:核糖体灭活蛋白(RIP), rRNA N-糖基化酶, 蛋白质合成(抑制), 八叠球菌素-篦麻毒素循环, 多核苷酸:腺苷糖基化酶, 蓖麻毒素, 商陆抗病毒蛋白(PAP), 甜菜素27

材料和试剂

注意:用于制备缓冲液的所有试剂应具有分子生物学级纯度(RNase,不含DNA酶)。水和溶液应在120℃高压灭菌15分钟(苯胺和乙醇除外)

  1. 一次性手套
  2. 移液器和提示(RNase,无DNA酶或高压灭菌)
  3. Eppendorf管(1.5ml聚丙烯微量离心管,RNase,不含DNA酶或高压灭菌)
  4. 纸巾
  5. Corning 50 ml PP离心管(Corning,目录号:430291)
  6. 巴斯德移液器
  7. 棉签
  8. 根据Pelham和Jackson(1976)所述获得或从生物化学供应商处购买的兔网织红细胞裂解物(未经微球菌核酸酶处理)或购自生物化学供应商(例如:Promega,目录号:L4151)
  9. RIP如Barbieri等人所述获得。 (2001)或从生物化学供应商购买(例如蓖麻毒素A链:Sigma-Aldrich,目录号:L9514)
  10. 碎冰
  11. 苯酚/TRIS饱和溶胶,用于分子生物学,稳定化,DNAse,RNAse和无蛋白酶(ACROS Organics,目录号:327125000)
  12. 绝对乙醇(EMD Millipore,目录号:100983)
  13. 去离子,RNase和无DNA酶的水
    注意:例如,Millipore Elix 5(UV)水高压灭菌120°C 15分钟。
  14. 苯胺(Sigma-Aldrich,目录号:242284)
  15. 尿素(Thermo Fisher Scientifc,Affymetrix,目录号:75826)
  16. 过硫酸铵(Sigma-Aldrich,目录号:A3678)
  17. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  18. 丙烯酰胺双丙烯酰胺19:1,40%(w/v)溶液(Thermo Fisher Scientifc,Affymetrix,目录号:75848)
  19. 溴化乙锭(Sigma-Aldrich,目录号:E7637)或GelRed(Biotium,目录号:41003)
  20. EDTA·2H 2 O
  21. NaOH颗粒
  22. 盐酸(HCl)(EMD Millipore,目录号:100317)
  23. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientifc,Affymetrix,目录号:75819)
  24. 乙酸钠三水合物(EMD Millipore,目录号:106267)
  25. 冰醋酸(EMD Millipore,目录号:100063)
  26. 碳酸二乙酯(Sigma-Aldrich,目录号:D5758)
  27. 二乙醚(EMD Millipore,目录号:100921)
  28. 三羟甲基氨基甲烷(Tris碱)(Fisher Scientific,目录号:BP154-1)
  29. 硼酸(Sigma-Aldrich,目录号:B6768)
  30. 蔗糖(Sigma-Aldrich,目录号:84097)
  31. RNA标记(Roche Diagnostics,目录号:1062 638)
    注意:本产品已停产。可以由低范围ssRNA梯子替代(New England Biolabs,目录号:N0364S)
  32. 溴酚蓝(Bio-Rad Laboratories,目录号:161-0404)
  33. 0.5 M EDTA(pH 8.0)(参见食谱)
  34. 50mM Tris/0.5%SDS(pH 7.8)(参见食谱)
  35. 3 M醋酸钠(pH 5.2)(见配方)
  36. 70%乙醇(见食谱)
  37. 2 M苯胺(pH 4.5)(见配方)
  38. 水饱和醚(参见食谱)
  39. 10x TBE缓冲区(见配方)
  40. 2x凝胶加载缓冲液(见配方)

设备

  1. (-80°C冰箱)(Thermo Fisher Scientific,Thermo Scientific TM,型号:TSE系列,目录号:TSE400SSV)
  2. 通风柜(BURDINOLA,型号:V21-Space BAJA ST 1500)
  3. 两个微量离心机(DJB Labcare,型号:Heraeus Biofuge Pico,目录号:75003235),其中一个保存在4°C
  4. 30℃水浴(JP SELECTA,目录号:6000140)
  5. 用于试管的涡街搅拌机(IKA,目录号:0003617000)
  6. BECKMAN DU-640分光光度计(Beckman Coulter,型号:DU-640)
  7. 石英比色皿(Hellma Analytics,型号:104-QS,目录号:104-10-40)
  8. 聚丙烯酰胺凝胶电泳系统(GE Healthcare,目录号:80-6418-77)
  9. 电源(GE Healthcare,目录号:18-1130-01)
  10. 分子成像仪 Gel Doc TM XR + System with Image Lab TM软件(Bio-Rad Laboratories,目录号:1708195)
  11. 凝胶染色托盘
  12. 磁力搅拌器
  13. 高压灭菌器(JP SELECTA,目录号:4002516)

程序

测定N-糖基化酶活性的一般程序如图2中的流程图所示

图2.说明协议的流程图过程部分A-H。流程图显示了描述协议过程的八个主要部分的关键步骤。大写字母(A-H)是指程序部分中的小节。

  1. 用RIP处理核糖体
    1. 将冻结的兔网织红细胞溶解产物(储存于-80℃)和RIP(储存于-20℃)的冰冻等分试样在室温下解冻,并将其置于碎冰上。
    2. 反应在碎冰上制备成四个Eppendorf管,如下所示:

      1(控制)
      2(对照)
      3(+ RIP)
      4(+ RIP)
      裂解物
      40μl
      40μl
      40μl
      40μl
      RIP(1 mg/ml)
      -
      -
      1μl
      1μl

    3. 将40μl兔网织红细胞裂解液吸入四管
    4. 加入1μl1 mg/ml RIP到3号和4号管中
    5. 混合轻轻涡旋。
    6. 在水浴中30℃孵育1小时。
    7. 加入2μl0.5M EDTA(pH8.0)停止反应
    8. 混合轻轻涡旋。
    9. 加入500μl50 mM Tris/0.5%SDS。
    10. 旋转30秒(目的是在SDS的帮助下使蛋白质变性)。

  2. 脱蛋白
    注意:此过程应在通风橱下进行。
    1. 加入500微升苯酚
    2. 涡旋30秒。
    3. 在室温下以16,060 x g(13,000rpm)离心5分钟。
    4. 将水(上)相(400μl)回收到新的Eppendorf管中,而不会干扰蛋白质相间。

  3. RNA沉淀
    1. 为了在1.5ml管中用3体积乙醇进行RNA沉淀,将水相分成两个Eppendorf管(A和B),每个管分别为200μl。通过混合以下方法进行乙醇沉淀:

      1A(对照)
      1B(对照)
      2A(对照)
      2B(对照)
      水相
      200μl
      200μl
      200μl
      200μl
      3 M醋酸钠,pH 5.2
      20μl
      20μl
      20μl
      20μl
      绝对乙醇
      600μl
      600μl
      600μl
      600μl


      3A(+ RIP)
      3B(+ RIP)
      4A(+ RIP)
      4B(+ RIP)
      水相
      200μl
      200μl
      200μl
      200μl
      3 M醋酸钠,pH 5.2
      20μl
      20μl
      20μl
      20μl
      绝对乙醇
      600μl
      600μl
      600μl
      600μl

    2. 混合混合。
    3. 在-80°C(或至少3 h)保持过夜。
    4. 在4℃下以16,060 x g(13,000rpm)离心15分钟。定向转子中的每个管,盖铰链指向外部,这将指示颗粒状RNA的位置,因为颗粒几乎不可见。
    5. 拿出管子。小心地倒出上清液,弃去纸巾,不会看不见灰白色颗粒,避免其移位。
    6. 加入250μl70%乙醇
    7. 在4℃下以16,060 x g(13,000rpm)离心15分钟。定向转子中的每个管,盖铰接指向外
    8. 仔细倾倒上清液,弃去纸巾,避免颗粒移位。借助棉签去除管壁上的任何液滴。
    9. 将管子朝上放在干净的纸巾上,让小球风干15分钟。
    10. 将沉淀重悬于10μl无RNase的去离子水中。将管1A和1B以及3A和3B的内容物混合,分别获得20μl的管1和3。将这些样品储存在-80°C。管2A,2B,4A和4B仍然保持分离。

  4. 苯胺治疗RNA
    1. 向管2A,2B,4A和4B中加入一个体积(10μl)的2M苯胺(pH4.5)。
      注意:反应在碎冰上进行。
    2. 混合混合。
    3. 在冰上孵育10分钟。
    4. 通过加入200μl无RNase的去离子水停止反应,并通过涡旋混合
    5. 加入200μl饱和饱和水
    6. 涡旋20秒。
    7. 让样品放置20秒以允许相分离。
    8. 丢弃以太(上)相。
    9. 重复步骤D5至D8。
    10. 加入20μl3M乙酸钠(pH 5.2)和600μl无水乙醇。
    11. 混合混合。
    12. 在-80°C(或至少3 h)保持过夜。
    13. 在4℃下以16,060 x g(13,000rpm)离心15分钟。定向转子中的每个管,盖铰接指向外
    14. 拿出管子。小心地倒出上清液,弃去纸巾,不会看不见灰白色颗粒,避免其移位。
    15. 加入250μl70%乙醇
    16. 在4℃下以16,060 x g(13,000rpm)离心15分钟。定向转子中的每个管,盖铰接指向外
    17. 仔细倾倒上清液,弃去纸巾,避免颗粒移位。借助棉签去除管壁上的任何液滴。
    18. 将管子朝上放在干净的纸巾上,让小球风干15分钟。
    19. 将沉淀重悬于10μl无RNase的去离子水中。将管2A和2B以及4A和4B的内容物混合,分别得到20μl的管2和4。将这些样品储存在-80°C。

  5. RNA的分光光度定量
    1. 解冻冷冻样品1-4,每个20μl,放在冰上。
    2. 确定分光光度计中每个样品的RNA浓度为260 nm(石英比色管为10 mm路径长度,以水为空白)。如果A <260> = 1,则RNA浓度为40μgRNA/ml。通常该方法的产量为用苯胺处理的样品为45-50μgRNA,未处理样品为50-60μgRNA。

  6. 制备7M尿素/5%(w/v)聚丙烯酰胺凝胶
    在不含RNase的Corning管中制备25ml两种凝胶的混合物(10×10.5cm的玻璃板,0.1cm的间隔物):2.5ml 10×TBE; 3.1ml丙烯酰胺/双丙烯酰胺(40%); 10.5克尿素; 12.3毫升无RNase的去离子水; 0.2ml 10%过硫酸铵; 0.05 ml TEMED。

  7. 样品的制备
    将样品制成放在碎冰上的Eppendorf管
    1. 加入3μgRNA
    2. 用水完成体积至5μl。
    3. 加入5μl2x凝胶加载缓冲液
    4. 混合混合。
    5. 煮沸30秒(在100°C水浴中),立即将管放在碎冰上。

  8. 电泳
    1. 将凝胶板连接到凝胶装置上
    2. 将TBE电泳缓冲液(900 ml水和100 ml TBE)加入顶部和底部室
    3. 取出梳子并用缓冲液冲洗孔以除去残留的尿素(使用巴斯德吸管)。
    4. 使用微量移液器,装载样品。
    5. 运行凝胶在15毫安1小时50分钟
    6. 拆解凝胶。
    7. 用0.5μg/ml溴化乙锭或GelRed溶液在室温下将凝胶染色40分钟,轻轻摇匀(染色后不需脱色)。
    8. 将凝胶放在紫外线透射仪上。可视化和拍摄RNA带。
      注意:请注意,RNA会在凝胶内随时间扩散,因此检查和摄影应在电泳结束后不久进行。
      代表性数据如图3所示

      图3. BE27在兔网织红细胞裂解物中的rRNA N-糖基化酶活性。每个泳道含有从未处理(对照)或RIP(+ BE27)处理的来自兔的核糖体分离的3μgRNA。箭头表示28S,18S和5.8S rRNA,并且在苯胺处理(+)后作为RIP作用的结果释放RNA片段。指示A部分中对应的管的数量。将泳道5和6的样品分别作为泳道3和4的样品进行处理,但用3μg而不是1μg的BE27处理核糖体。也表示核苷酸中RNA标记的大小(M)。左侧的凝胶用溴化乙锭染色,凝胶右侧用GelRed染色。

数据分析

该方法对于鉴定rRNA N-糖基化酶活性非常特异,结果非常可靠。包括对照以排除通过苯胺处理随机产生的非特异性RNA断裂。另一方面,在每个实验中进行在不存在和存在苯胺的RIP处理的rRNA的比较分析,以排除由于可能的片段或降解RNA的可能污染物引起的非特异性效应。此外,可以通过用不同RIP浓度处理RNA来确认结果。 RNA分子量标记可用于确定释放的RNA片段的大小(其取决于测定中使用的核糖体的类型)。还建议在实验中包括以前发布的RIP(例如,蓖麻毒素),以明确地定位片段。

笔记

由于其敏感性和简便性,兔网织红细胞裂解物是检测rRNA N-糖基化酶活性的最佳系统。该方法也可用于测试特异性RNA酶活性(α类似刺激样活性),但不用苯胺处理。其他敏感核糖体也可以轻微修改使用,但由于在凝胶电泳后可以看到许多RNA条带,因此在检测到Endo的片段之前需要进行优化。已经使用这种修饰的方案成功地用于酵母,青霉菌,大肠埃希氏菌,根癌土壤杆菌,绿链球菌(Streptomyces lividans),乳酸发酵短杆菌,Vicia sativa,和Colo 320和HeLa细胞培养物。

食谱

  1. 0.5 M EDTA(pH 8.0)
    1. 加入186.1g EDTA·2H 2 O二钠至800ml的ddH 2 O -/-
    2. 用磁力搅拌器剧烈搅拌
    3. 用NaOH(约20g NaOH颗粒)将pH调节至8.0
    4. 通过高压灭菌将其分配至等分试样并灭菌
    注意:通过加入NaOH将溶液的pH调节至〜8.0之前,EDTA的二钠盐不会进入溶液。在120°C高压灭菌15分钟。
  2. 50 mM Tris/0.5%SDS
    混合45ml ddH 2 O,2.5ml 1M Tris-HCl(用HCl调节至pH 7.8)和2.5ml 10%SDS。高压灭菌器120°C 15分钟
  3. 3 M醋酸钠(pH 5.2)
    20.412克醋酸钠三水合物
    加入约6毫升冰醋酸至pH 5.2,并加入高达50ml的ddH 2 O→/ 在120°C高压灭菌15分钟
  4. 70%乙醇
    70%乙醇绝对和30%去离子,RNase和无DNA酶水
  5. 2 M苯胺(pH 4.5)
    1. 将18.25毫升苯胺加入RNase和无DNA酶的水中
    2. 使用无RNA酶的电极用冰醋酸调节至pH 4.5,用RNase和不含DNA酶的水或用1%的焦碳酸二乙酯在水中洗涤。
    3. 加入双蒸水至100 ml
    4. 在Eppendorf管中分装,储存于-20°C
  6. 水饱和醚
    将一体积的去离子,RNase和无DNA酶的水加入一体积的乙醚中并混合。上层是水饱和醚
    注意:在通风橱下,室温下储存少量(10ml)。
  7. 10x TBE缓冲区
    1. 溶解(使用磁力搅拌器)将108g Tris碱和55g硼酸溶于800ml去离子,RNase和无DNA酶的水中。
    2. 加入40 ml 0.5 M EDTA(pH 8.0)
    3. 将音量调整为1 L
    4. 在120°C高压灭菌15分钟
    5. 在室温下存放
  8. 2x凝胶加载缓冲液
    TBE缓冲区
    100毫克/毫升蔗糖
    7 M尿素
    0.4μg/ml溴酚蓝
    储存于4°C直至使用

致谢

该协议是从以前的出版物(Endo和Tsurugi,1988; Sallustio和Stanley,1990)中修改的。这项工作得到了BIO39/VA39/14和BIO/VA17/15授权给L.C.的支持。

参考文献

  1. Barbieri,L.,Bolognesi,A.和Stirpe,F.(2001)。  1型核糖体失活蛋白的纯化和缀合。在Hall,WA(Ed。)。免疫毒素方法和方案 - 分子生物学中的系列方法。 Humana Press ,pp:71-85。
  2. Citores,L.,Iglesias,R.,Gay,C.and Ferreras,JM(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25976013"target ="_ blank">来自甜菜(Beta vulgaris L.)的核糖体灭活蛋白质BE27的抗真菌活性与绿霉霉青霉菌相比 Mol Plant Pathol 17(2):261-271。
  3. Endo,Y.和Tsurugi,K。(1988)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/3288622"target ="_ blank" >蓖麻毒素A链的RNA N-糖苷酶活性。具有核糖体和rRNA的蓖麻毒素A链的酶活性的特征.J Biol Chem 263(18):8735-8739。
  4. Iglesias,R.,Citores,L.,Di Maro,A.和Ferreras,JM(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25326773"target ="_ blank">来自甜菜的抗病毒蛋白质BE27的生物学活性( L. ):421-433。
  5. Iglesias,R.,Citores,L.,Ragucci,S.,Russo,R.,Di Maro,A.and Ferreras,JM(2016)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/26971856"target ="_ blank">来自Phytolacca dioica的核糖体失活蛋白的生物和抗病原活性。 Biochim Biophys Acta 1860(6):1256-1264。
  6. Pelham,HR和Jackson,RJ(1976)。  An来自网织红细胞裂解物的有效的mRNA依赖性翻译系统。 Eur J Biochem 67(1):247-256。
  7. Sallustio,S.和Stanley,P.(1990)。中国仓鼠卵巢核糖体突变体的分离对蓖麻毒素,相思豆毒素和新霉素具有差异性抗性。生物化学265(1):582-588。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Iglesias, R., Citores, L. and Ferreras, J. M. (2017). Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins. Bio-protocol 7(6): e2180. DOI: 10.21769/BioProtoc.2180.
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