Skip to main content
SpringerLink
Log in
Book cover

ADP-ribosylation and NAD+ Utilizing Enzymes pp 287–295Cite as

Detection of ADP-Ribosylating Bacterial Toxins

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1813))

Abstract

Many bacterial toxins catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to a host protein. Greater than 35 bacterial ADP-ribosyltransferase toxins (bARTTs) have been identified. ADP-ribosylation of host proteins may be specific or promiscuous. Despite this diversity, bARTTs share a common reaction mechanism, three-dimensional active site structure, and a conserved active site glutamic acid. Here, we describe how to measure the ADP-ribosylation of host proteins as purified proteins or within a cell lysate.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Aktories K, Barbieri JT (2005) Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol 3:397–410

    Article  CAS  Google Scholar 

  2. Simon NC, Aktories K, Barbieri JT (2014) Novel bacterial ADP-ribosylating toxins: structure and function. Nat Rev Microbiol 12:599–611

    Article  CAS  Google Scholar 

  3. Barbieri JT, Riese MJ, Aktories K (2002) Bacterial toxins that modify the actin cytoskeleton. Annu Rev Cell Dev Biol 18:315–344

    Article  CAS  Google Scholar 

  4. Oppenheimer NJ, Bodley JW (1981) Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem 256:8579–8581

    CAS  PubMed  Google Scholar 

  5. Liu S, Yahr TL, Frank DW, Barbieri JT (1997) Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa. J Bacteriol 179:1609–1613

    Article  CAS  Google Scholar 

  6. Wilson BA, Collier RJ (1992) Diphtheria toxin and Pseudomonas aeruginosa exotoxin A: active-site structure and enzymic mechanism. Curr Top Microbiol Immunol 175:27–41

    CAS  PubMed  Google Scholar 

  7. Koch-Nolte F, Haag F (1997) Mono(ADP-ribosyl)transferases and related enzymes in animal tissues. Emerging gene families. Adv Exp Med Biol 419:1–13

    Article  CAS  Google Scholar 

  8. Sixma TK, Pronk SE, Kalk KH, Wartna ES, van Zanten BA, Witholt B, Hol WG (1991) Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature 351:371–377

    Article  CAS  Google Scholar 

  9. Domenighini M, Magagnoli C, Pizza M, Rappuoli R (1994) Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol Microbiol 14:41–50

    Article  CAS  Google Scholar 

  10. Merritt EA, Hol WG (1995) AB5 toxins. Curr Opin Struct Biol 5:165–171

    Article  CAS  Google Scholar 

  11. Carroll SF, Lory S, Collier RJ (1980) Ligand interactions of diphtheria toxin. III Direct photochemical cross-linking of ATP and NAD to toxin. J Biol Chem 255:12020–12024

    CAS  PubMed  Google Scholar 

  12. Naglich JG, Metherall JE, Russell DW, Eidels L (1992) Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor. Cell 69:1051–1061

    Article  CAS  Google Scholar 

  13. O’Keefe DO, Cabiaux V, Choe S, Eisenberg D, Collier RJ (1992) pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys. Proc Natl Acad Sci U S A 89:6202–6206

    Article  Google Scholar 

  14. Kounnas MZ, Morris RE, Thompson MR, FitzGerald DJ, Strickland DK, Saelinger CB (1992) The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem 267:12420–12423

    CAS  PubMed  Google Scholar 

  15. Chaudhary VK, Jinno Y, FitzGerald D, Pastan I (1990) Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc Natl Acad Sci U S A 87:308–312

    Article  CAS  Google Scholar 

  16. Hessler JL, Kreitman RJ (1997) An early step in Pseudomonas exotoxin action is removal of the terminal lysine residue, which allows binding to the KDEL receptor. Biochemistry 36:14577–14582

    Article  CAS  Google Scholar 

  17. Teter K, Holmes RK (2002) Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin, Pseudomonas aeruginosa exotoxin A, and ricin. Infect Immun 70:6172–6179

    Article  CAS  Google Scholar 

  18. Taylor M, Burress H, Banerjee T, Ray S, Curtis D, Tatulian SA, Teter K (2014) Substrate-induced unfolding of protein disulfide isomerase displaces the cholera toxin A1 subunit from its holotoxin. PLoS Pathog 10:e1003925

    Article  Google Scholar 

  19. Teter K, Allyn RL, Jobling MG, Holmes RK (2002) Transfer of the cholera toxin A1 polypeptide from the endoplasmic reticulum to the cytosol is a rapid process facilitated by the endoplasmic reticulum-associated degradation pathway. Infect Immun 70:6166–6171

    Article  CAS  Google Scholar 

  20. Massey S, Banerjee T, Pande AH, Taylor M, Tatulian SA, Teter K (2009) Stabilization of the tertiary structure of the cholera toxin A1 subunit inhibits toxin dislocation and cellular intoxication. J Mol Biol 393:1083–1096

    Article  CAS  Google Scholar 

  21. Chinnapen DJ, Hsieh WT, te Welscher YM, Saslowsky DE, Kaoutzani L, Brandsma E, D'Auria L, Park H, Wagner JS, Drake KR, Kang M, Benjamin T, Ullman MD, Costello CE, Kenworthy AK, Baumgart T, Massol RH, Lencer WI (2012) Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1. Dev Cell 23:573–586

    Article  CAS  Google Scholar 

  22. Raghunathan K, Wong TH, Chinnapen DJ, Lencer WI, Jobling MG, Kenworthy AK (2016) Glycolipid crosslinking is required for cholera toxin to partition into and stabilize ordered domains. Biophys J 111:2547–2550

    Article  CAS  Google Scholar 

  23. Zhang J, Snyder SH (1993) Purification of a nitric oxide-stimulated ADP-ribosylated protein using biotinylated beta-nicotinamide adenine dinucleotide. Biochemistry 32:2228–2233

    Article  CAS  Google Scholar 

  24. Takei Y, Takahashi K, Kanaho Y, Katada T (1994) Pertussis toxin-catalyzed ADP-ribosylation of GTP-binding proteins with digoxigenin-conjugated NAD. Identification of the proteins in plasma membranes and nuclei. FEBS Lett 338:264–266

    Article  CAS  Google Scholar 

  25. Gulke I, Pfeifer G, Liese J, Fritz M, Hofmann F, Aktories K, Barth H (2001) Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from clostridium difficile. Infect Immun 69:6004–6011

    Article  CAS  Google Scholar 

  26. Simon NC, Barbieri JT (2014) Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion. J Biol Chem 289:10650–10659

    Article  CAS  Google Scholar 

  27. Simon NC, Vergis JM, Ebrahimi AV, Ventura CL, O'Brien AD, Barbieri JT (2013) Host cell cytotoxicity and cytoskeleton disruption by CerADPr, an ADP-ribosyltransferase of Bacillus cereus G9241. Biochemistry 52:2309–2318

    Article  CAS  Google Scholar 

  28. Carroll SF, Collier RJ (1984) NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc Natl Acad Sci U S A 81:3307–3311

    Article  CAS  Google Scholar 

Download references

Acknowledgments

JTB laboratory is supported by grants from the NIH: AI-030162 and AI-118389.

Author information

Authors and Affiliations

  1. Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, USA

    Chen Chen & Joseph T. Barbieri

Authors
  1. Chen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph T. Barbieri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph T. Barbieri .

Editor information

Editors and Affiliations

  1. Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Paul Chang

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chen, C., Barbieri, J.T. (2018). Detection of ADP-Ribosylating Bacterial Toxins. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8588-3_20

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8587-6

  • Online ISBN: 978-1-4939-8588-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation