参见作者原研究论文

本实验方案简略版
Jun 2018

本文章节


 

In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
转谷氨酰胺酶-2蛋白底物的体外交联反应和底物掺入鉴定   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.

Keywords: Transglutaminase (谷氨酰胺转移酶), Transamidation (转酰胺基作用), Post-translational modification (翻译后修饰), High mobility group box 1 (高迁移率族蛋白), Isopeptide (异肽), In vitro protein crosslinking (体外蛋白交联)

Background

Transglutaminase (TG2) is a member of the transglutaminase enzyme family that catalyzes calcium-dependent transamidation reactions. The transglutaminase enzyme family includes TG1-7 and FXIIIa, as well as band 4.2, a non-catalytically active member that plays a role in protein scaffolding (Satchwell et al., 2009; Gundemir et al., 2012). TG2 is unique to the family in that it is ubiquitously expressed and pleiotropic in function; in addition to protein crosslinking TG2 functions as a protein disulfide isomerase (Hasegawa et al., 2003; Mastroberardino et al., 2006), G-protein (Nakaoka et al., 1994; Baek et al., 1996; Vezza et al., 1999), and kinase (Mishra and Murphy, 2004; Mishra et al., 2006 and 2007).

Protein crosslinking occurs between the γ-carboxamide group of a peptide-bound glutamyl residue (a glutamine donor substrate) and the ϵ-amino group of a lysyl residue (lysine acceptor substrate), resulting in the formation of ϵ-(γ-glutamyl) lysine isopeptide bonds (Folk and Finlayson, 1977). A schematic of the crosslink reaction is shown in Figure 1.


Figure 1. Schematic for the TG2 protein crosslink reaction. Protein crosslinking occurs by a transamidation reaction between the γ-carboxamide group of a peptide-bound glutamyl residue (shown in blue) and the ϵ-amino group of a lysyl residue (shown in red). Glutamine loses its amino group during the reaction, resulting in the formation of an ϵ-(γ-glutamyl) lysine isopeptide bond.

Although Tris-based buffers have been traditionally used for in vitro TG2 crosslink reactions, we found these buffers to be problematic for certain substrates, particularly those containing lysine donors only. Since Tris is a primary amine, it can potentially function as an acyl acceptor substrate in the transamidation reaction, competing with peptide-bound lysyl residues on protein substrates. To circumvent issues with Tris-based buffers, we instead used MOPS based buffer system for crosslink reactions. Since MOPSs is a tertiary amine, this avoids any potential issues with Tris- inhibition of the enzymatic reaction.

After confirmation that a protein is crosslinked by TG2, delineation of glutamyl vs. lysyl crosslink sites in the protein is an important next step to determine whether lysine and/or glutamine residues participate in the transamidation reaction.

The general scheme for in vitro crosslink reactions and substrate incorporation assays is described as follows:

1) Determine whether or not the protein of interest is a TG2 transamidation substrate.

Crosslink reactions between TG2 and purified recombinant forms of the putative substrate are performed in vitro and followed by Western blot analysis. This reaction can also be conducted in the context of endogenous proteins by using as substrates lysates from cell or tissue sources that contain the protein of interest instead of recombinant proteins. In either case, the appearance of a slower-migrating, size-shifted band by Western blot using antibodies against the putative substrate indicates a positive result. Regardless of whether purified recombinant proteins or endogenous sources from a cell or tissue lysate are included in the crosslink reaction, it is important to include the appropriate controls. The critical controls include reactions without TG2 enzyme, calcium, and/or using mutant TG2 enzyme lacking transamidation activity are for non-ambiguous data interpretation. Typical Western blot results for an in vitro crosslink reaction using purified recombinant HMGB1 are shown in Figure 2. Additionally, blocking peptide assays with the peptide antigen used to generate the antibody can be utilized to compete away the signals, confirming antibody specificity for TG2-modified substrates (Willis et al., 2018, Figure 3G).

2) Characterization of transamidation donor sites by substrate incorporation assays.

To determine whether the substrate in question undergoes crosslinking via glutamine or lysine donor residues or both, crosslink reactions between the purified recombinant proteins are supplemented with biotin-labeled glutamine- or lysine-donor probes. The crosslink reactions are then separated on SDS-PAGE and analyzed for biotin signal by Western blot with streptavidin-conjugated HRP. The utility of performing the substrate incorporation assay in this manner is that (in contrast to an ELISA-plate based format), additional qualitative information is gleaned about the size and relative abundance of probe-labeled reaction products on the gel. Additionally, specific bands of interest can be excised from the gel for protein identification or crosslink mapping by mass spectrometry (Willis et al., 2018).

Materials and Reagents

  1. 10 cm tissue culture plates (Corning, Catalog number: 430293 )
  2. 1.5 ml microcentrifuge tubes (USA Scientific, catalog number: 1615-5510 )
  3. 15 ml conical tubes (Thermo Scientific, catalog number: 339651 )
  4. Transglutaminase from guinea pig liver (Sigma-Aldrich, catalog number: T5398 )
  5. MOPS [3-(N-morpholino)propanesulfonic acid] (Sigma-Aldrich, catalog number: M1254 )
  6. Biotin-pentylamine (EZ-LinkTM Pentylamine-Biotin) (Thermo Scientific, catalog number: 21345 )
  7. Biotin-TVQQEL (a.k.a. A25 peptide) (Zedira, catalog number: B001 )
  8. Roche Complete protease inhibitor cocktail (Sigma, catalog number: 11697498001 )
  9. Dithiothreitol (DTT) (Sigma, catalog number: D9163 )
  10. PMSF (phenylmethanesulfonyl fluoride) (Sigma, catalog number: 78830 )
  11. Western blotting materials
    1. 4-12% polyacrylamide gels (Thermo Scientific catalog number: NW04120BOX )
    2. Mini gel tank (Thermo Scientific catalog number: A25977 )
    3. Nitrocellulose or PVDF membranes
    4. Electrophoretic transfer apparatus (GE Biosciences TE-22 )
  12. Tris [tris(hydroxymethyl)aminomethane] base (Fisher, catalog number: BP152-500 )
  13. HMGB1(high mobility group box protein 1; Genscript, catalog number: Z02803 )
  14. PBS (phosphate-buffered saline) (Thermo Scientific, catalog number: 10010049 )
  15. NaCl (Sigma-Aldrich, catalog number: S7653 )
  16. Ethylenediaminetetraacetic acid (EDTA) (Sigma, catalog number: E9884 )
  17. Sodium deoxycholate (Sigma, catalog number: D6750 )
  18. Sodium dodecyl sulfate (SDS) (Sigma, catalog number: L3771 )
  19. NP-40 (a.k.a. Igepal CA-630) (Sigma, catalog number: I8896 )
  20. Glycerol (Sigma, catalog number: G6279 )
  21. Streptavidin-Horse-radish peroxidase (Streptavidin-HRP)
  22. Protease inhibitor 25x stock solution (see Recipes)
  23. Dithiothreitol (DTT) 1 M stock solution (see Recipes)
  24. Crosslink reaction buffer (see Recipes)
  25. TG2 reconstitution buffer (see Recipes)
  26. Modified RIPA buffer (see Recipes)
  27. 6x SDS-loading buffer (see Recipes)
  28. Tris/SDS pH 6.8 (see Recipes)

Equipment

  1. Incubator or heating block (Fisher Scientific, catalog number: 88-860-022 )
  2. Electrophoretic transfer apparatus (GE Healthcare, catalog number: TE-22 )
  3. Refrigerated benchtop centrifuge (Eppendorf, model: 5424R )
  4. Gel box for running gels (mini gel tank) (Thermo Scientific, catalog number: A25977 )
  5. Platform shaker (VWR, catalog number: 10127-876 )
  6. Tissue culture incubator (Thermo Scientific, model: Steri-CycleTM i160)
  7. Western blot imaging equipment (film + developer machine, Chemi-Doc, Licor, etc.)

Procedure

  1. Preparation of cell lysates
    1. Remove cells from the tissue culture incubator and place on ice. For adherent cells growing in a 10 cm plate, discard tissue culture media and rinse the cell monolayer twice with 5 ml ice-cold PBS. For suspension cells, proceed to Step A3.
    2. While keeping the plate(s) on ice, add 1ml of ice-cold PBS, scrape and transfer the detached cells to pre-chilled microcentrifuge tubes on ice.
    3. Harvest the cells by centrifugation at 2,000 x g for 5 min at 4 °C. Discard the PBS supernatant.
    4. Lyse the cells by gently resuspending cell pellets in the appropriate amount of ice-cold modified RIPA lysis buffer (generally 200-500 μl for one 10 cm plate of 50-70% confluent cells) and incubate for 10 min on ice.
    5. Optional: If the cells were lysed in a buffer that releases chromatin from nuclei, sonication will be necessary to reduce sample viscosity. Keeping the cell lysates on ice, sonicate with 3-5 1-second pulses at 50% output.
    6. Centrifuge the lysates at 15,000 x g for 5 min at 4°C to pellet any insoluble cell debris.
    7. Transfer the supernatants to new microcentrifuge tubes pre-chilled on ice and assess the protein concentration by bicinchoninic acid (BCA) assay per the manufacturer’s instructions.

  2. In vitro TG2 crosslinking reactions
    1. Reconstitute the lyophilized TG2 enzyme at 4 U/ml (= 4 mU/µl) in TG2 reconstitution buffer. For a 1 unit vial of TG2, reconstitute with 250 µl of buffer for a final concentration of 4 U/ml. Place on ice while setting up the crosslink reactions.
    2. Prepare the in vitro crosslink reactions by adding the reagents in the order listed below to microcentrifuge tubes. The TG2 enzyme concentration, total reaction volume, and substrate concentrations should be optimized for each application. Some typical final concentrations for the reagents in reaction buffer are noted below:
      1. Reaction buffer with protease inhibitors
      2. Crosslink substrates: cell lysate (0.25 µg/µl) or purified recombinant proteins (10-20 ng/µl)
      3. TG2 enzyme (0.05-1.0 mU/ml)
      4. CaCl2 (2-5 mM)
    3. Incubate the reactions for 60 min at 37 °C.
    4. Quench the crosslink reactions by adding SDS-PAGE loading buffer to 1x concentration, followed by heating at 95 °C for 5 min on a heating block.
    5. Assess samples for protein crosslinking by SDS-PAGE followed by Western blotting.
      When using in vitro crosslink reactions to test new potential TG2 substrates, positive and negative controls are also important. Using previously established crosslink substrates as a positive control will ensure the reactions are working as expected. Any commercially available protein with known TG2 substrate activity will work, with one caveat; the protein should have both glutamine and lysine crosslink sites. Otherwise a mixture of a protein with only glutamine donor sites and lysine acceptor sites would need to be included to ensure crosslinking occurs. Our lab typically uses recombinant Y-box 1 (YB-1) protein, which contains both glutamine and lysine crosslink sites and can be crosslinked to itself in vitro (Willis et al., 2013 and non-published data), or HMGB1, which contains only lysine acceptor sites but is crosslinked in vitro to Glutamine 636 of TG2 (Willis et al., 2018).

  3. Substrate Incorporation assays
    For substrate incorporation assays, purified recombinant proteins are incubated with TG2 enzyme as described above, but in the presence of biotin-pentylamine (BP), a TG2 lysine donor substrate, or biotin-TVQQEL (A25 peptide), a TG2 glutamine donor substrate. Reaction mixtures are then size-fractionated on SDS-PAGE and analyzed for substrate incorporation with streptavidin -HRP by Western blot.
    Reactions are set up in a similar manner to the TG2 crosslink reactions described above.
    1. Reconstitute the lyophilized TG2 enzyme at 4 U/ml in TG2 reconstitution buffer. Place on ice while setting up the substrate incorporation assays.
    2. Set up the substrate incorporation reactions by adding the reagents to microcentrifuge tubes in the order they are listed below:
      1. Reaction buffer with protease inhibitors
      2. Purified recombinant protein crosslink substrate (10-20 ng/µl)
      3. BP or A25 peptide (2 mM)
      4. TG2 enzyme (1.0 mU/ml)
      5. CaCl2 (2-5 mM)
    3. Incubate for 60 min at 37 °C.
    4. Stop the reactions by adding concentrated SDS-loading buffer to a final 1x concentration and heat for 5 min at 95 °C on a heating block. The samples are ready to be separated by SDS-PAGE.

Data analysis

  1. TG2 crosslink reactions:
    Run on SDS-PAGE and analyze by Western blot. TG2 substrate proteins will show a size-shift, often forming a slower migrating, higher molecular weight variant (or variants) near the top of the gel. This is often accompanied by a reduction in signal for the monomeric substrate (Willis et al., 2013 and 2018). Representative data for a crosslink reaction with recombinant HMGB1 as the substrate is shown in Figure 2.


    Figure 2. Representative Western blot results for an in vitro TG2 crosslink reaction. TG2 enzyme (0.2 mU/µl was incubated for 60 min at 37 °C in the presence of purified recombinant HMGB1 (rHMGB1) crosslink substrate (20 ng/µl) (Lane 3). TG2-only and recombinant HMGB1-only controls were loaded into lanes 1 and 2, respectively. Note that in lane 3, the ~29 kDa HMGB1 signal is diminished relative to lane 2, which coincides with the formation of the crosslinked HMGB1 oligomer near the top of the gel. Samples were separated on 4-12% polyacrylamide gels, transferred to nitrocellulose membranes and probed with HMGB1 antibody.

  2. Substrate incorporation assays:
    For the substrate incorporation assays, a reaction system using purified recombinant protein substrates facilitates straightforward analysis of the results by Western blot. Presence of a biotin-positive signal at the expected molecular weight for the substrate of interest indicates that biotinylated probes were incorporated into the substrate. Slower migrating/more retarded signals are indicative of the presence of multiple glutamyl and/or lysyl crosslink sites, since band migration is more retarded as the number of incorporated probe molecules increases (see Willis et al, 2018). It is also possible to excise proteins from the gel and map the specific crosslink sites by mass spectrometry, as noted in Willis et al., 2018.
      The assay can also be performed in situ, where live cells are incubated with membrane-permeable biotinylated probes (Willis et al., 2018). After cell lysis, the biotin-tagged candidate substrate can be isolated with streptavidin beads; identification of the substrate by Western blot can be interpreted (in the context of the proper controls) as a positive result. Alternatively, proteins can be eluted from the streptavidin beads and subjected to mass spec analysis to determine the identity of the isolated proteins.

Notes

  1. A range of concentrations for cell lysate, TG2 enzyme, and/or biotinylated probes may be used. Final concentrations should be determined empirically. Note that higher concentrations of the biotinylated probes have been shown to function as inhibitors (Lorand et al., 1992), so care should be taken not to use excessive concentrations of labeled probes.
    Depending on the abundance of transamidation substrate and other factors, a range of TG2 enzyme and protein substrate combinations should be tested with and without the biotin-labeled probes.
    Although reconstituted TG2 will retain enzyme activity when stored at 4 °C for at least a couple weeks, it is best to reconstitute and use fresh enzyme the same day.
    If the protein substrate of interest contains multiple transamidation donor sites, covalent addition of a number of probes will cause a size-shift on SDS-PAGE in proportion to the number of incorporated probe molecules.
  2. Important controls for in vitro crosslinking reactions:
    1. No TG2 enzyme: reaction input (cell lysate or recombinant proteins) only. [For cell or tissue lysates with high levels of endogenous transamidation activity, this should be suppressed by adding 2 mM EGTA (a calcium-chelator) or 10 mM Iodoacetamide (an irreversible TG2 transamidation inhibitor)].
    2. TG2 enzyme only.
    3. No substrate: include reactions lacking the purified recombinant substrate, or cell or tissue lysates from sources where there is no expression or expression of the putative substrate is knocked out.

Recipes

  1. Protease inhibitor 25x stock solution
    1. Dissolve 1 tablet Roche Complete Protease inhibitor cocktail tablets in 2 ml distilled water
    2. Store small aliquots at -20 °C
  2. Dithiothreitol (DTT) 1 M stock solution
    1. Weigh 1.54 g of DTT and add to a 15 ml conical tube
    2. Add distilled water to 10 ml and dissolve completely
    3. Store small aliquots at -20 °C
  3. Crosslink reaction buffer
    Note: Prepared fresh the day of the experiment; *add PMSF and DTT immediately prior to use.
    50 mM MOPS, pH 7.5
    0.2 mM PMSF*
    1 mM DTT*
    1x Protease inhibitor (Roche Complete Protease inhibitor cocktail)
  4. TG2 reconstitution buffer
    Note: Prepared fresh the day of the experiment; *add DTT immediately prior to use.
    50 mM MOPS, pH 7.5
    300 mM NaCl
    0.5 mM EDTA
    10 mM DTT*
  5. Modified RIPA buffer
    Note: Can be stored at 4°C after preparation; *add protease inhibitor, PMSF, and DTT immediately prior to use.
    50 mM MOPS, pH7.5
    150 mM NaCl
    0.5% sodium deoxycholate
    0.1% SDS
    1% NP-40
    0.2 mM PMSF*
    1 mM DTT*
    1x Protease inhibitor (Roche Complete Protease inhibitor cocktail)
  6. 6x SDS-loading buffer (recipe for 20 ml)
    Note: Prepare in advance and store at -20 °C in small aliquots.
    14 ml Tris/SDS pH 6.8
    6.0 ml glycerol
    2 g SDS
    1.86 g DTT
  7. Tris/SDS pH 6.8 (for making 6x SDS loading buffer)
    Note: Prepare in advance and store at room temperature.
    50 ml 1 M Tris, pH 6.8
    4 ml 10% SDS
    46 ml dH2O

References

  1. Baek, K. J., Kwon, N. S., Lee, H. S., Kim, M. S., Muralidhar, P. and Im, M. J. (1996). Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium. Biochem J 315 (Pt 3): 739-744.
  2. Folk, J. E. and Finlayson, J. S. (1977). The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases. Adv Protein Chem 31: 1-133. 
  3. Gundemir, S., Colak, G., Tucholski, J. and Johnson, G. V. (2012). Transglutaminase 2: a molecular Swiss army knife. Biochim Biophys Acta 1823(2): 406-419. 
  4. Hasegawa, G., Suwa, M., Ichikawa, Y., Ohtsuka, T., Kumagai, S., Kikuchi, M., Sato, Y. and Saito, Y. (2003). A novel function of tissue-type transglutaminase: protein disulphide isomerase. Biochem J 373(Pt 3): 793-803. 
  5. Lorand, L., Parameswaran, K. N., Velasco, P. T. and Murthy, S. N. (1992). Biotinylated peptides containing a factor XIIIa or a tissue transglutaminase-reactive glutaminyl residue that block protein cross-linking phenomena by becoming incorporated into amine donor sites. Bioconjug Chem 3(1): 37-41.
  6. Mastroberardino, P. G., Farrace, M. G., Viti, I., Pavone, F., Fimia, G. M., Melino, G., Rodolfo, C. and Piacentini, M. (2006). "Tissue" transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes. Biochim Biophys Acta 1757(9-10): 1357-1365. 
  7. Mishra, S., Melino, G. and Murphy, L. J. (2007). Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein. J Biol Chem 282(25): 18108-18115.
  8. Mishra, S. and Murphy, L. J. (2004). Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase. J Biol Chem 279(23): 23863-23868. 
  9. Mishra, S., Saleh, A., Espino, P. S., Davie, J. R. and Murphy, L. J. (2006). Phosphorylation of histones by tissue transglutaminase. J Biol Chem 281(9): 5532-5538.
  10. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M. J. and Graham, R. M. (1994). Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264(5165): 1593-1596. 
  11. Satchwell, T. J., Shoemark, D. K., Sessions, R. B. and Toye, A. M. (2009). Protein 4.2: a complex linker. Blood Cells Mol Dis 42(3): 201-210. 
  12. Vezza, R., Habib, A. and FitzGerald, G. A. (1999). Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, Gh. J Biol Chem 274(18): 12774-12779.
  13. Willis, W. L., Wang, L., Wada, T. T., Gardner, M., Abdouni, O., Hampton, J., Valiente, G., Young, N., Ardoin, S., Agarwal, S., Freitas, M. A., Wu, L. C. and Jarjour, W. N. (2018). The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens. J Biol Chem 293(22): 8394-8409.
  14. Willis, W. L., Hariharan, S., David, J. J. and Strauch, A. R. (2013). Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-box 1 (YB-1) translation-regulatory protein in TGFβ1-activated myofibroblasts. J Cell Biochem 114(12): 2753-2769.

简介

[摘要]转谷氨酰胺酶(TG2)催化谷氨酰和赖氨酰残基之间的蛋白质交联。催化活性通过转酰胺机制发生,导致异肽键的形成。由于TG2介导的transamidation是一些生物过程的机械重要性,检测,使快速和有效的识别和表征的候选底物是一个重要的第一步,以揭示交联蛋白的功能。在这里,我们描述了一个优化和灵活的协议,用于体外TG2交联反应和底物结合测定。我们以前曾采用这些技术在鉴定蛋白质高流动性基团盒1(HMGB1)作为TG2底物。然而,该协议可以适应任何候选转酰胺化底物的鉴定。

[背景]转谷氨酰胺酶(TG2)是转谷氨酰胺酶家族的成员,催化钙依赖性转酰胺化反应。转谷氨酰胺酶家族包括TG1-7和FXIIIa,以及带4.2的非催化活性成员,在蛋白质支架中发挥作用(Satchwell等,2009;Gundemir等,2012)。TG2在该家族中的独特之处在于,它是无处不在的表达和多向性功能;除了蛋白交联外,TG2还具有蛋白二硫异构酶(Hasegawa等,2003;Mastroberardino等,2006)、G蛋白(Nakaoka等,1994;Baek等,1996;Vezza等,1999)和激酶(Mishra和Murphy,2004;Mishra等,2006和2007)的功能。
蛋白质交联发生在与肽结合的谷氨酰残基(谷氨酰胺供体底物)的γ-羧酰胺基与赖氨酰残基(赖氨酸受体底物)的ϵ-氨基基之间,从而形成ϵ-(γ-谷氨酰)赖氨酸异肽键(Folk和Finlayson,1977)。交联反应的示意图如图1所示。


图1.TG2蛋白交联反应示意图。TG2蛋白交联反应的示意图。蛋白质交联是通过肽结合的谷氨酰残基的γ-羧酰胺基(蓝色所示)和赖氨酸残基的ϵ-氨基基团(红色所示)之间的转酰胺化反应发生的。谷氨酰胺在反应过程中失去其氨基基团,从而形成ϵ-(γ-谷氨酰)赖氨酸异肽键。

虽然基于Tris的缓冲液传统上被用于体外TG2交联反应,但我们发现这些缓冲液对于某些底物,特别是那些只含有赖氨酸供体的底物是有问题的。由于Tris是一种初级胺,它可以潜在地作为转酰胺化反应中的酰基受体底物,与蛋白质底物上的肽结合赖氨酸残基竞争。为了规避基于Tris的缓冲液的问题,我们反而使用基于MOPS的缓冲体系进行交联反应。由于MOPSs是一个三级胺,这避免了任何潜在的问题与Tris抑制的酶反应。
在确认蛋白质被TG2交联后,划定蛋白质中谷氨酰与赖氨酰交联位点是确定赖氨酸和/或谷氨酰胺残基是否参与转酰胺化反应的重要下一步。
体外交联反应和底物加入检测的一般方案描述如下。
1)确定感兴趣的蛋白质是否为TG2转酰胺化底物。
TG2和纯化的重组形式的假定底物之间的交联反应在体外进行,然后进行Western印迹分析。该反应也可以在内源性蛋白质的背景下进行,通过使用含有感兴趣的蛋白质的细胞或组织来源的裂解物作为底物,而不是重组蛋白质。在这两种情况下,使用针对底物的抗体进行Western印迹,出现一个迁移速度较慢的、大小移动的带子就表明结果是阳性的。无论纯化的重组蛋白还是来自细胞或组织裂解液的内源蛋白是否包括在交联反应中,都必须包括适当的对照。关键的控制包括没有TG2酶,钙,和/或使用缺乏转酰胺活性的突变体TG2酶的反应是为了非明确的数据解释。使用纯化的重组HMGB1的体外交联反应的典型Western印迹结果如图2所示。此外,可以利用用于产生抗体的多肽抗原的阻断肽检测来竞争掉信号,确认抗体对TG2修饰的底物的特异性(Willis等人 ,2018,图3G)。
2)通过底物加入试验来表征转酰胺化供体位点。
为了确定有关底物是否通过谷氨酰胺或赖氨酸供体残基或两者进行交联,用生物素标记的谷氨酰胺或赖氨酸供体探针补充纯化的重组蛋白之间的交联反应。然后将交联反应在SDS-PAGE上分离,并用链霉菌素结合的HRP进行Western印迹分析生物素信号。以这种方式进行底物结合检测的效用是(与基于ELISA板的格式相反),额外的定性信息是收集关于凝胶上探针标记的反应产物的大小和相对丰度。此外,感兴趣的特定条带可以从凝胶中切除,以便通过质谱法进行蛋白质鉴定或交联图谱(Willis等人,2018)。

关键字:谷氨酰胺转移酶, 转酰胺基作用, 翻译后修饰, 高迁移率族蛋白, 异肽, 体外蛋白交联

材料和试剂


1. 10厘米组织培养板(康宁,目录号:430293)。
2. 1.5毫升微量离心管(美国科学公司,目录号:1615-5510)。
3. 15毫升锥形管(Thermo Scientific,目录号:339651)。
4. 豚鼠肝脏转谷氨酰胺酶(Sigma-Aldrich,目录号:T5398)
5. MOPS [3-(N-吗啉基)丙磺酸] (Sigma-Aldrich,目录号:M1254)
6. 生物素-戊基胺(EZ-LinkTM戊基胺-生物素)(Thermo Scientific,目录号:21345)。
7. 生物素-TVQQEL(又名A25肽)(Zedira,目录号:B001)。
8. 罗氏全套蛋白酶抑制剂鸡尾酒(Sigma,目录号:11697498001)。
9. 二硫苏糖醇(DTT)(Sigma,目录号:D9163)。
10. PMSF(苯基甲烷磺酰氟)(Sigma,目录号:78830)。
11. Western印迹材料 
a. 4-12%聚丙烯酰胺凝胶(Thermo Scientific目录号:NW04120BOX)。
b. 迷你凝胶罐(Thermo Scientific目录号:A25977)。
c. 硝酸纤维素或PVDF膜
d. 电泳转移装置(GE Biosciences TE-22)。
12. 三(羟甲基氨基甲烷)碱(Fisher,目录号:BP152-500)
13. HMGB1(高流动性群盒蛋白1;Genscript,目录号:Z02803)
14. PBS(磷酸盐缓冲盐水)(Thermo Scientific,目录号:10010049)。
15. 氯化钠(Sigma-Aldrich,目录号:S7653)
16. 乙二胺四乙酸(EDTA)(Sigma,目录号:E9884)
17. 脱氧胆酸钠(Sigma,目录号:D6750)
18. 十二烷基硫酸钠(SDS)(Sigma,目录号:L3771)
19. NP-40(又名Igepal CA-630)(Sigma,目录号:I8896)。
20. 甘油(Sigma,目录号:G6279)
21. 链霉菌素-马-萝卜过氧化物酶(Streptavidin-HRP)
22. 蛋白酶抑制剂25倍液(见配方)。
23. 二硫苏糖醇(DTT)1M原液(见配方)
24. 交联反应缓冲液(见配方)
25. TG2重组缓冲液(见配方)。
26. 修改后的RIPA缓冲区(见配方)。
27. 6x SDS加载缓冲液(见配方)。
28. Tris/SDS pH 6.8(见配方)。


装备


1. 孵化器或加热块(Fisher Scientific,目录号:88-860-022)。
2. 电泳转移装置(GE Healthcare,目录号:TE-22)。
3. 冷藏台式离心机(Eppendorf,型号:5424R)。
4. 用于运行凝胶的凝胶盒(迷你凝胶罐)(Thermo Scientific,目录号:A25977)。
5. 平台振动器(VWR,目录号:10127-876)。
6. 组织培养箱(Thermo Scientific,型号:Steri-CycleTM i160)。
7. Western blot成像设备(胶片+显影机、Chemi-Doc、Licor等)。


流程


A. 细胞裂解液的制备
1. 从组织培养培养箱中取出细胞,并置于冰上。对于在10厘米板中生长的粘附细胞,丢弃组织培养基,用5毫升冰冷的PBS冲洗细胞单层两次。对于悬浮细胞,继续到步骤A3。
2. 虽然保持板(S)在冰上,加入1毫升冰冷的PBS,刮和转移分离的细胞在冰上预冷的微量离心管。
3. 收获细胞,在2,000 xg离心5分钟,在4℃下。丢弃PBS上清液。
4. 通过轻轻重悬细胞颗粒在适量的冰冷的改性RIPA裂解缓冲液(一般为一个10厘米板的50-70%汇合细胞200-500微升),并在冰上孵育10分钟。
5. 可选:如果细胞裂解在缓冲液中,释放染色质从核,超声将是必要的,以减少样品粘度。保持细胞裂解液在冰上,超声与3-5 1秒脉冲在50%的输出。
6. 离心裂解物在15,000 xg离心5分钟,在4℃下,以颗粒任何不溶性的细胞碎片。
7. 将上清液转移到新的微离心管中,在冰上预冷,并根据制造商的说明,通过双辛酸(BCA)检测评估蛋白质浓度。


B. 体外TG2的交联反应
1. 重组冻干的TG2酶在4 U/ml(= 4 mU/µl)在TG2重组缓冲液。对于1个单位小瓶的TG2,用250微升的缓冲液重组,最终浓度为4 U/ml。放在冰上,同时设置交联反应。
2. 准备体外交联反应,通过添加试剂在下面列出的顺序到微量离心管。TG2酶的浓度,总的反应体积,和底物浓度应优化为每个应用程序。一些典型的试剂在反应缓冲液中的最终浓度如下所述。
a. 含蛋白酶抑制剂的反应缓冲液
b. 交联底物:细胞裂解液(0.25 µg/µl)或纯化的重组蛋白(10-20 ng/µl)。
c. TG2酶(0.05-1.0 mU/ml)
d. 氯化钙(2-5毫摩尔)
3. 将反应在37℃下孵育60分钟。
4. 通过加入SDS-PAGE加载缓冲液至1倍浓度来淬灭交联反应,然后在95℃加热块上加热5分钟。
5. 通过SDS-PAGE评估样品的蛋白质交联情况,然后进行Western印迹。
当使用体外交联反应测试新的潜在TG2底物时,阳性和阴性对照也很重要。使用先前建立的交联底物作为阳性对照,将确保反应按预期工作。任何具有已知TG2底物活性的市售蛋白质都可以使用,但有一点需要注意;蛋白质应该同时具有谷氨酰胺和赖氨酸交联位点。否则就需要加入一种只有谷氨酰胺供体位点和赖氨酸受体位点的蛋白质的混合物,以确保交联的发生。我们的实验室通常使用重组Y-box 1 (YB-1)蛋白,它同时含有谷氨酰胺和赖氨酸交联位点,并且可以在体外与自身交联(Willis等人 ,2013年和未发表的数据),或者HMGB1,它只含有赖氨酸接受位点,但在体外与TG2的谷氨酰胺636交联(Willis等人 ,2018年)。


C. 基质结合检测
对于底物结合检测,纯化的重组蛋白与TG2酶孵育,如上所述,但在生物素-戊基胺(BP),TG2赖氨酸供体底物,或生物素-TVQQEL(A25肽),TG2谷氨酰胺供体底物的存在。然后将反应混合物在SDS-PAGE上进行尺寸分馏,并通过Western印迹分析底物与链霉菌素-HRP的结合情况。
反应的设置方式与上述TG2交联反应类似。
1. 重组冻干的TG2酶在4 U/ml的TG2重组缓冲液。放置在冰上,同时设置底物结合测定。
2. 设置底物加入反应,通过添加试剂到微量离心管中,他们在下面列出的顺序。
a. 含蛋白酶抑制剂的反应缓冲液
b. 纯化的重组蛋白交联底物(10-20 ng/µl)
c. BP或A25肽(2mM)
d. TG2酶(1.0 mU/ml)
e. 氯化钙(2-5毫摩尔)
3. 在37℃下孵育60分钟。
4. 停止反应,通过加入浓缩的SDS加载缓冲液至最终的1倍浓度,并在95℃下加热5分钟,在加热块上。样品准备通过SDS-PAGE分离。


数据分析


1. TG2交联反应。
在SDS-PAGE上运行并通过Western印迹分析。TG2底物蛋白将显示出一个大小转移,通常在凝胶顶部附近形成一个较慢的迁移,较高分子量的变体(或变体)。这通常伴随着单体底物的信号降低(Willis等人 ,2013和2018)。图2所示为以重组HMGB1为底物的交联反应的代表性数据。


 
图2.体外TG2交联反应的代表性Western blot结果。代表性的Western印迹结果为体外TG2交联反应。在纯化的重组HMGB1(rHMGB1)交联底物(20 ng/µl)的存在下,TG2酶(0.2 mU/µl)在37℃下孵育60分钟(车道3)。分别将仅含TG2和仅含重组HMGB1的对照品装入车道1和2中。请注意,在车道3中,约29 kDa HMGB1信号相对于车道2减弱,这与凝胶顶部附近的交联HMGB1低聚物的形成相吻合。在4-12%聚丙烯酰胺凝胶上分离样品,转移到硝酸纤维素膜上,用HMGB1抗体进行探针检测。


2. 基质结合试验。
对于底物整合检测,使用纯化的重组蛋白底物的反应系统有利于通过Western印迹直接分析结果。存在生物素阳性信号在预期的分子量为感兴趣的底物表示生物素化探针被纳入底物。较慢的迁移/更迟钝的信号表明存在多个谷氨酰和/或赖氨酰交联位点,因为随着纳入的探针分子数量的增加,带状迁移更迟钝(见Willis等人,2018)。也可以从凝胶中切除蛋白质,并通过质谱法绘制特定的交联位点,如Willis等人 ,2018年指出。
该检测也可以在原位进行,将活细胞与膜渗透性生物素化探针孵育(Willis等,2018)。在细胞裂解后,生物素标记的候选底物可以用链霉菌素珠分离;通过Western印迹鉴定底物可以被解释为(在适当的对照背景下)阳性结果。另外,蛋白质可以从链霉菌素珠中洗脱出来,并进行质谱分析以确定分离的蛋白质的身份。


笔记


1. 细胞裂解液、TG2酶和/或生物素化探针的浓度范围可以使用。最终浓度应根据经验确定。注意,较高浓度的生物素化探针已被证明具有抑制剂的功能(Lorand等,1992),因此应注意不要使用过高浓度的标记探针。
根据转酰胺化底物的丰度和其他因素,应使用和不使用生物素标记的探针测试一系列TG2酶和蛋白质底物组合。
虽然重组后的TG2在4℃下储存至少几周后仍能保持酶的活性,但最好在当天就重组并使用新鲜的酶。
如果感兴趣的蛋白质底物包含多个转酰胺供体位点,共价加入一些探针将导致SDS-PAGE上的大小移动,与加入的探针分子数量成正比。
2. 体外交联反应的重要对照。
a. 无TG2酶:仅反应输入(细胞裂解液或重组蛋白)。[对于内源性转酰胺活性较高的细胞或组织裂解液,应通过加入2mM EGTA(钙螯合剂)或10mM碘乙酰胺(不可逆的TG2转酰胺抑制剂)来抑制]。
b. TG2酶只。
c. 无底物:包括缺乏纯化的重组底物的反应,或细胞或组织裂解物来源的细胞或组织裂解物,其中没有表达或表达的putative substrate被敲除。


食谱


1. 蛋白酶抑制剂25倍液
a. 将1片罗氏完全蛋白酶抑制剂鸡尾酒片溶于2毫升蒸馏水中。
b. 小量等分试样在-20℃下保存
2. 二硫苏糖醇(DTT)1M原液。
a. 称取1.54克DTT,加入到15毫升的锥形管中。
b. 加入蒸馏水10毫升,完全溶解。
c. 小量等分试样在-20℃下保存
3. 交联反应缓冲液
注:实验当天新鲜制备;*使用前立即添加PMSF和DTT。
50mM MOPS,pH值7.5。
0.2mM PMSF*
1mM DTT*
1x 蛋白酶抑制剂(罗氏完整蛋白酶抑制剂鸡尾酒)。
4. TG2重组缓冲液
注:实验当天新鲜制备;*使用前立即添加DTT。
50mM MOPS,pH值7.5。
300 mM氯化钠
0.5mM EDTA
10mM DTT*
5. 修改后的RIPA缓冲区
注:配制后可在4℃保存;*使用前立即加入蛋白酶抑制剂、PMSF和DTT。
50mM MOPS,pH7.5。
150mM氯化钠
0.5%脱氧胆酸钠
0.1% SDS
1% NP-40
0.2mM PMSF*
1mM DTT*
1x 蛋白酶抑制剂(罗氏完整蛋白酶抑制剂鸡尾酒)。
6. 6x SDS加载缓冲液(配方为20毫升)。
注:提前做好准备,在-20℃下分小等份保存。
14 毫升 Tris/SDS pH 6.8
6.0毫升甘油
2克 SDS
1.86克DTT
7. Tris/SDS pH值6.8(用于制作6倍的SDS装载缓冲液
注:提前准备,常温保存。
50毫升1M Tris,pH值6.8。
4 毫升 10% SDS
46毫升dH2O
参考文献


1. Baek, K. J., Kwon, N. S., Lee, H. S., Kim, M. S., Muralidhar, P.和Im, M. J.(1996年)。Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium.生物化学杂志315(Pt 3):739-744。
2. Folk, J. E. and Finlayson, J. S. (1977).( The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases.Adv Protein Chem 31: 1-133.
3. Gundemir,S.、Colak,G.、Tucholski,J.和Johnson,G.V.(2012年)。转谷氨酰胺酶2:一把分子瑞士军刀。Biochim Biophys Acta 1823(2): 406-419。
4. Hasegawa,G.、Suwa,M.、Ichikawa,Y.、Ohtsuka,T.、Kumagai,S.、Kikuchi,M.、Sato,Y.和Saito,Y.(2003)。A novel function of tissue-type transglutaminase: protein disulphide isomerase.Biochem J 373(Pt 3):793-803。
5. Lorand, L., Parameswaran, K. N., Velasco, P. T.和Murthy, S. N.(1992年)。含有因子XIIIa或组织转谷氨酰胺酶反应性谷氨酰胺残基的生物素化肽,通过融入胺供体位点来阻止蛋白质交联现象。Bioconjug Chem 3(1):37-41。
6. Mastroberardino, P. G.、Farrace, M. G.、Viti, I.、Pavone, F.、Fimia, G. M.、Melino, G.、Rodolfo, C.和Piacentini, M.(2006年)。"Tissue"transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes.Biochim Biophys Acta 1757(9-10):1357-1365。
7. Mishra, S., Melino, G. and Murphy, L. J. (2007).转谷氨酰胺酶2激酶活性促进蛋白激酶A诱导的视网膜母细胞瘤蛋白磷酸化。J Biol Chem 282(25): 18108-18115。
8. Mishra, S. and Murphy, L. J. (2004)。Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase. J Biol Chem 279(23): 23863-23868.J Biol Chem 279(23):23863-23868。
9. Mishra,S.,Saleh,A.,Espino,P.S.,Davie,J.R.和Murphy,L.J.(2006)。组织转谷氨酰胺酶对组蛋白的磷酸化。J Biol Chem 281(9): 5532-5538。
10. Nakaoka,H.,Perez,D.M.,Baek,K.J.,Das,T.,Husain,A.,Misono,K.,Im,M.J.和Graham,R.M.(1994)。Gh:一种具有转谷氨酰胺酶活性和受体信号功能的GTP结合蛋白。科学264(5165): 1593-1596。
11. Satchwell, T. J.、Shoemark, D. K.、Sessions, R. B.和Toye, A. M.(2009年)。蛋白4.2:一个复杂的链接器。Blood Cells Mol Dis 42(3): 201-210.
12. Vezza, R., Habib, A. and FitzGerald, G. A. (1999)。血栓素受体亚型通过新型GTP结合蛋白Gh进行的差异性信号传导。J Biol Chem 274(18): 12774-12779。
13. Willis, W. L., Wang, L., Wada, T. T., Gardner, M., Abdouni, O., Hampton, J., Valiente, G., Young, N., Ardoin, S., Agarwal, S., Freitas, M. A., Wu, L. C.和Jarjour, W. N.(2018).proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms highmolecular weight complexes with autoantigens.J Biol Chem 293(22):8394-8409.
14. Willis, W. L., Hariharan, S., David, J. J. and Strauch, A. R. (2013).Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-box 1 (YB-1) translation-regulatory protein in TGFβ1-activated myofibroblasts.J Cell Biochem 114(12): 2753-2769.

登录/注册账号可免费阅读全文
  • 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: © 2020 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. Willis, W. L., Foster, A., Henry, C., Wu, L. C. and Jarjour, W. (2020). In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates. Bio-protocol 10(12): e3657. DOI: 10.21769/BioProtoc.3657.
  2. Willis, W. L., Wang, L., Wada, T. T., Gardner, M., Abdouni, O., Hampton, J., Valiente, G., Young, N., Ardoin, S., Agarwal, S., Freitas, M. A., Wu, L. C. and Jarjour, W. N. (2018). The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens. J Biol Chem 293(22): 8394-8409.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。