Imaging Specific Cell Surface Protease Activity in Living Cells Using Reengineered Bacterial Cytotoxins

  • John P. Hobson
  • , Shihui Liu
  • Stephen H. Leppla
  • Thomas H. BuggeEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 539)


The scarcity of methods to visualize the activity of individual cell surface proteases in situ has hampered basic research and drug development efforts. In this chapter, we describe a simple, sensitive, and noninvasive assay that uses nontoxic reengineered bacterial cytotoxins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay takes advantage of the absolute requirement for site-specific endoproteolytic cleavage of cell surface-bound anthrax toxin protective antigen for its capacity to translocate an anthrax toxin lethal factor-β-lactamase fusion protein to the cytoplasm. A fluorogenic β-lactamase substrate is then used to visualize the cytoplasmically translocated anthrax toxin lethal factor-β-lactamase fusion protein. By using anthrax toxin protective antigen variants that are reengineered to be cleaved by furin, urokinase plasminogen activator, or metalloproteinases, the cell surface activities of each of these proteases can be specifically and quantitatively determined with single cell resolution. The imaging assay is excellently suited for fluorescence microscope, fluorescence plate reader, and flow cytometry formats, and it can be used for a variety of purposes.

Key words:

Anthrax toxin β-lactamase CCF2/AM Cell surface proteolysis Flow cytometry Fluorescence microscopy Fluorescence plate reader Furin Metalloproteinases Urokinase plasminogen activator. 



We thank Drs. Silvio Gutkind and Mary Jo Danton for critically reviewing this manuscript, and Drs. Kevin L. Holmes and David Stephany for expert assistance with flow cytometry. This work was supported by the NIH Intramural Research Program, by NIAID Support of Intramural Biodefense Research from ICs other than NIAID and by the Department of Defense (DAMD-17-02-1-0693) to Dr. Thomas H. Bugge. For imaging reagents, contact S. H. Leppla ( or T. H. Bugge (


  1. 1.
    1. Werb Z. (1997) ECM and cell surface proteolysis: regulating cellular ecology. Cell 91(4), 439–42.CrossRefGoogle Scholar
  2. 2.
    2. Andreasen PA, Egelund R, Petersen HH.
(2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57(1), 25–40.CrossRefGoogle Scholar
  3. 3.
    3. McCawley LJ, Matrisian LM. (2000) Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol Med Today 6(4), 149–56.CrossRefGoogle Scholar
  4. 4.
    4. Turk B, Turk D, Turk V. (2000) Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477(1–2), 
98–111.CrossRefGoogle Scholar
  5. 5.
    5. Koblinski JE, Ahram M, Sloane BF. (2000) Unraveling the role of proteases in cancer. Clin Chim Acta 291(2), 113–35.CrossRefGoogle Scholar
  6. 6.
    6. Davie EW, Fujikawa K, Kisiel W. (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30(43), 10363–70.CrossRefGoogle Scholar
  7. 7.
    7. Bugge TH. Proteolysis in carcinogenesis. (2003) In: Ensley JF, Gutkind JS, Jacob JR, Lippman SM Head and Neck Cancer. San Diego: Academic Press, 137–49.CrossRefGoogle Scholar
  8. 8.
    8. Mohamed MM, Sloane BF. (2006) Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer 6(10), 764–75.CrossRefGoogle Scholar
  9. 9.
    9. Coussens LM, Fingleton B. (2002) Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295(5564), 2387–92.CrossRefGoogle Scholar
  10. 10.
    10. McIntyre JO, Matrisian LM. (2003) Molecular imaging of proteolytic activity in cancer. J Cell Biochem 290(6), 1087–97.CrossRefGoogle Scholar
  11. 11.
    11. Sloane BF, Sameni M, Podgorski I, Cavallo-Medved D, Moin K. (2006) Functional imaging of tumor proteolysis. Annu Rev Pharmacol Toxicol 46, 301–15.CrossRefGoogle Scholar
  12. 12.
    12. Hobson JP, Liu S, Rono B, Leppla SH, Bugge TH. (2006) Imaging specific cell-surface proteolytic activity in single living cells. Nat Meth 3(4), 259–61.CrossRefGoogle Scholar
  13. 13.
    13. Duesbery NS, Vande Woude GF. (1999) Anthrax toxins. Cell Mol Life Sci 55(12), 1599–609.CrossRefGoogle Scholar
  14. 14.
    14. Gordon VM, Klimpel KR, Arora N, Henderson MA, Leppla SH. (1995) Proteolytic 
activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular 
proteases. Infect Immun 63(1), 82–7.Google Scholar
  15. 15.
    15. Adachi M, Kitamura K, Miyoshi T, et al. (2001) Activation of epithelial sodium channels by prostasin in Xenopus oocytes. J Am Soc Nephrol 12(6), 1114–21.Google Scholar
  16. 16.
    16. Liu S, Netzel-Arnett S, Birkedal-Hansen H, Leppla SH. (2000) Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin. Cancer Res 60(21), 6061–7.Google Scholar
  17. 17.
    17. Liu S, Aaronson H, Mitola DJ, Leppla SH, Bugge TH. (2003) Potent antitumor activity of a urokinase-activated engineered anthrax toxin. Proc Natl Acad Sci USA 100(2), 657–62.CrossRefGoogle Scholar
  18. 18.
    18. Liu S, Schubert RL, Bugge TH, Leppla SH. (2003) Anthrax toxin: structures, functions and tumour targeting. Expert Opin Biol Ther 3(5), 843–53.CrossRefGoogle Scholar
  19. 19.
    19. Leppla SH, Arora N, Varughese M. (1999) Anthrax toxin fusion proteins for intracellular delivery of macromolecules. J Appl Microbiol 187(2), 284.CrossRefGoogle Scholar
  20. 20.
    20. Arora N, Leppla SH. (1993) Residues 1–254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides. 
J Biol Chem 268(5), 3334–41.Google Scholar
  21. 21.
    21. Zlokarnik G, Negulescu PA, Knapp TE, et al. (1998) Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279(5347), 84–8.CrossRefGoogle Scholar
  22. 22.
    22. Liu S, Bugge TH, Leppla SH. (2001) 
Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin. J Biol Chem 276(21), 17976–84.CrossRefGoogle Scholar
  23. 23.
    23. Ke SH, Coombs GS, Tachias K, Navre M, Corey DR, Madison EL. (1997) Distinguishing the specificities of closely related proteases. Role of P3 in substrate and inhibitor discrimination between tissue-type plasminogen activator and urokinase. J Biol Chem 272(26), 16603–9.CrossRefGoogle Scholar
  24. 24.
    24. Ke SH, Madison EL. (1997) Rapid and efficient site-directed mutagenesis by single-tube ‘megaprimer’ PCR method. Nucleic Acids Res 25(16), 3371–2.CrossRefGoogle Scholar
  25. 25.
    25. Coombs GS, Bergstrom RC, Pellequer JL, et al. (1998) Substrate specificity of prostate-specific antigen (PSA). Chem Biol 5(9), 475–88.CrossRefGoogle Scholar
  26. 26.
    26. Inglese J, Johnson RL, Simeonov A, et al. (2007) High-throughput screening assays for the identification of chemical probes. Nat Chem Biol 3(8), 466–79.CrossRefGoogle Scholar
  27. 27.
    27. Arora N, Leppla SH. (1994) Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells. Infect Immun 62(11), 4955–61.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • John P. Hobson
  • , Shihui Liu
  • Stephen H. Leppla
  • Thomas H. Bugge
    • 1
    Email author
  1. 1.Proteases and Tissue Remodeling Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial ResearchNational Institutes of HealthBethesda

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