Cellular and Molecular Life Sciences

, Volume 72, Issue 7, pp 1417–1432 | Cite as

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5

  • Jonas Protze
  • Miriam Eichner
  • Anna Piontek
  • Stefan Dinter
  • Jan Rossa
  • Kinga Grażyna Blecharz
  • Peter Vajkoczy
  • Joerg Piontek
  • Gerd KrauseEmail author
Research Article


Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn150 and Thr153) and Cld5 (Asp149 and Thr151). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.


Tight junctions Protein–protein interactions Membrane proteins Drug delivery system Structure–function study Mutagenesis Molecular modeling 



Tight junctions


Clostridium perfringens Enterotoxin


C-terminal domain of Clostridium perfringens enterotoxin




Extracellular loop


Protein data bank


Root mean square deviation



This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants KR 1273/3-2, PI 837/2-1 and by the Sonnenfeld Stiftung (PhD-scholarship for Miriam Eichner).

Conflict of interest

The authors declare no conflict of interests.

Supplementary material

18_2014_1761_MOESM1_ESM.pdf (2.6 mb)
Supplementary material 1 (PDF 2632 kb)


  1. 1.
    Krause G, Winkler L, Mueller SL et al (2008) Structure and function of claudins. Biochim Biophys Acta 1778:631–645. doi: 10.1016/j.bbamem.2007.10.018 CrossRefPubMedGoogle Scholar
  2. 2.
    Angelow S, Ahlstrom R, Yu ASL (2008) Biology of claudins. Am J Physiol Ren Physiol 295:F867–F876. doi: 10.1152/ajprenal.90264.2008 CrossRefGoogle Scholar
  3. 3.
    Suzuki H, Nishizawa T, Tani K et al (2014) Crystal Structure of a claudin provides insight into the architecture of tight junctions. Science 344(80):304–307. doi: 10.1126/science.1248571 CrossRefPubMedGoogle Scholar
  4. 4.
    McClane BA (2001) The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions. Toxicon 39:1781–1791CrossRefPubMedGoogle Scholar
  5. 5.
    Katahira J, Inoue N, Horiguchi Y et al (1997) Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol 136:1239–1247CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Veshnyakova A, Protze J, Rossa J et al (2010) On the interaction of Clostridium perfringens enterotoxin with claudins. Toxins (Basel) 2:1336–1356. doi: 10.3390/toxins2061336 CrossRefGoogle Scholar
  7. 7.
    Sonoda N, Furuse M, Sasaki H et al (1999) Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: evidence for direct involvement of claudins in tight junction barrier. J Cell Biol 147:195–204CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Kimura J, Abe H, Kamitani S et al (2010) Clostridium perfringens enterotoxin interacts with claudins via electrostatic attraction. J Biol Chem 285:401–408. doi: 10.1074/jbc.M109.051417 CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Veshnyakova A, Piontek J, Protze J et al (2012) Mechanism of Clostridium perfringens enterotoxin interaction with claudin-3/-4 protein suggests structural modifications of the toxin to target specific claudins. J Biol Chem 287:1698–1708. doi: 10.1074/jbc.M111.312165 CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Kitadokoro K, Nishimura K, Kamitani S et al (2011) Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins. J Biol Chem 286:19549–19555. doi: 10.1074/jbc.M111.228478 CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Briggs DC, Naylor CE, Smedley JG 3rd et al (2011) Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins. J Mol Biol 413:138–149. doi: 10.1016/j.jmb.2011.07.066 CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Van Itallie CM, Betts L, Smedley JG 3rd et al (2008) Structure of the claudin-binding domain of Clostridium perfringens enterotoxin. J Biol Chem 283:268–274. doi: 10.1074/jbc.M708066200 CrossRefPubMedGoogle Scholar
  13. 13.
    Kokai-Kun JF, McClane BA (1997) Deletion analysis of the Clostridium perfringens enterotoxin. Infect Immun 65:1014–1022PubMedCentralPubMedGoogle Scholar
  14. 14.
    Smedley JG 3rd, Uzal FA, McClane BA (2007) Identification of a prepore large-complex stage in the mechanism of action of Clostridium perfringens enterotoxin. Infect Immun 75:2381–2390. doi: 10.1128/IAI.01737-06 CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Kondoh M, Takahashi A, Fujii M et al (2006) A novel strategy for a drug delivery system using a claudin modulator. Biol Pharm Bull 29:1783–1789CrossRefGoogle Scholar
  16. 16.
    Takahashi A, Kondoh M, Suzuki H, Yagi K (2011) Claudin as a target for drug development. Curr Med Chem 18:1861–1865CrossRefPubMedGoogle Scholar
  17. 17.
    Kondoh M, Masuyama A, Takahashi A et al (2005) A novel strategy for the enhancement of drug absorption using a claudin modulator. Mol Pharmacol 67:749–756. doi: 10.1124/mol.104.008375 CrossRefPubMedGoogle Scholar
  18. 18.
    Kakutani H, Kondoh M, Fukasaka M et al (2010) Mucosal vaccination using claudin-4-targeting. Biomaterials 31:5463–5471. doi: 10.1016/j.biomaterials.2010.03.047 CrossRefPubMedGoogle Scholar
  19. 19.
    Suzuki H, Kondoh M, Kakutani H et al (2012) The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice. Biomaterials 33:317–324. doi: 10.1016/j.biomaterials.2011.09.048 CrossRefPubMedGoogle Scholar
  20. 20.
    Turksen K, Troy T-C (2011) Junctions gone bad: claudins and loss of the barrier in cancer. Biochim Biophys Acta 1816:73–79. doi: 10.1016/j.bbcan.2011.04.001 PubMedGoogle Scholar
  21. 21.
    Saeki R, Kondoh M, Kakutani H et al (2009) A novel tumor-targeted therapy using a claudin-4-targeting molecule. Mol Pharmacol 76:918–926. doi: 10.1124/mol.109.058412 CrossRefGoogle Scholar
  22. 22.
    Kominsky SL, Tyler B, Sosnowski J et al (2007) Clostridium perfringens enterotoxin as a novel-targeted therapeutic for brain metastasis. Cancer Res 67:7977–7982. doi: 10.1158/0008-5472.CAN-07-1314 CrossRefPubMedGoogle Scholar
  23. 23.
    Casagrande F, Cocco E, Bellone S et al (2011) Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin. Cancer 117:5519–5528. doi: 10.1002/cncr.26215 CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Walther W, Petkov S, Kuvardina ON et al (2012) Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors. Gene Ther 19:494–503. doi: 10.1038/gt.2011.136 CrossRefPubMedGoogle Scholar
  25. 25.
    Neesse A, Hahnenkamp A, Griesmann H et al (2013) Claudin-4-targeted optical imaging detects pancreatic cancer and its precursor lesions. Gut 62:1034–1043. doi: 10.1136/gutjnl-2012-302577 CrossRefPubMedGoogle Scholar
  26. 26.
    Hsu L-W, Lee P-L, Chen C-T et al (2012) Elucidating the signaling mechanism of an epithelial tight-junction opening induced by chitosan. Biomaterials 33:6254–6263. doi: 10.1016/j.biomaterials.2012.05.013 CrossRefPubMedGoogle Scholar
  27. 27.
    Krug SM, Amasheh M, Dittmann I et al (2013) Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells. Biomaterials 34:275–282. doi: 10.1016/j.biomaterials.2012.09.051 CrossRefPubMedGoogle Scholar
  28. 28.
    Tscheik C, Blasig IE, Winkler L (2013) Trends in drug delivery through tissue barriers containing tight junctions. Tissue barriers 1:e24565. doi: 10.4161/tisb.24565 CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Takahashi A, Saito Y, Kondoh M et al (2012) Creation and biochemical analysis of a broad-specific claudin binder. Biomaterials 33:3464–3474. doi: 10.1016/j.biomaterials.2012.01.017 CrossRefPubMedGoogle Scholar
  30. 30.
    Winkler L, Gehring C, Wenzel A et al (2009) Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3. J Biol Chem 284:18863–18872. doi: 10.1074/jbc.M109.008623 CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Blasig IE, Winkler L, Lassowski B et al (2006) On the self-association potential of transmembrane tight junction proteins. Cell Mol Life Sci 63:505–514. doi: 10.1007/s00018-005-5472-x CrossRefPubMedGoogle Scholar
  32. 32.
    Piontek J, Winkler L, Wolburg H et al (2008) Formation of tight junction: determinants of homophilic interaction between classic claudins. FASEB J 22:146–158. doi: 10.1096/fj.07-8319com CrossRefPubMedGoogle Scholar
  33. 33.
    Piontek J, Fritzsche S, Cording J et al (2011) Elucidating the principles of the molecular organization of heteropolymeric tight junction strands. Cell Mol Life Sci 68:3903–3918. doi: 10.1007/s00018-011-0680-z CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Rossa J, Ploeger C, Vorreiter F et al (2014) Claudin-3 and Claudin-5 protein folding and assembly into the tight junction are controlled by non-conserved residues in the transmembrane 3 (TM3) and extracellular loop 2 (ECL2) segments. J Biol Chem 289:7641–7653. doi: 10.1074/jbc.M113.531012 CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Van den Ent F, Löwe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74. doi: 10.1016/j.jbbm.2005.12.008 CrossRefPubMedGoogle Scholar
  36. 36.
    Kleinschnitz C, Blecharz K, Kahles T et al (2011) Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome. Stroke 42:1081–1089. doi: 10.1161/STROKEAHA.110.592238 CrossRefPubMedGoogle Scholar
  37. 37.
    Baltzegar DA, Reading BJ, Brune ES, Borski RJ (2013) Phylogenetic revision of the claudin gene family. Mar Genomics 11:17–26. doi: 10.1016/j.margen.2013.05.001 CrossRefPubMedGoogle Scholar
  38. 38.
    Krause G, Winkler L, Piehl C et al (2009) Structure and function of extracellular claudin domains. Ann N Y Acad Sci 1165:34–43. doi: 10.1111/j.1749-6632.2009.04057.x CrossRefPubMedGoogle Scholar
  39. 39.
    Förster C, Silwedel C, Golenhofen N et al (2005) Occludin as direct target for glucocorticoid-induced improvement of blood-brain barrier properties in a murine in vitro system. J Physiol 565:475–486. doi: 10.1113/jphysiol.2005.084038 CrossRefPubMedCentralPubMedGoogle Scholar
  40. 40.
    Blecharz KG, Haghikia A, Stasiolek M et al (2010) Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis. Mult Scler 16:293–302. doi: 10.1177/1352458509358189 CrossRefPubMedGoogle Scholar
  41. 41.
    Fujita K, Katahira J, Horiguchi Y et al (2000) Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein. FEBS Lett 476:258–261CrossRefPubMedGoogle Scholar
  42. 42.
    Yelland TS, Naylor CE, Savva CG, et al. (2014) Structure of Clostridium perfringens enterotoxin with a peptide derived from a modified version of ECL-2 of Claudin 2. PDB ID: 3ZJ3, doi: 10.2210/pdb3zj3/pdb
  43. 43.
    Yelland TS, Naylor CE, Bagoban T et al (2014) Structure of a C. perfringens enterotoxin mutant in complex with a Modified Claudin-2 Extracellular Loop 2. J Mol Biol. doi: 10.1016/j.jmb.2014.07.001 PubMedGoogle Scholar

Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Jonas Protze
    • 1
  • Miriam Eichner
    • 1
  • Anna Piontek
    • 1
  • Stefan Dinter
    • 1
  • Jan Rossa
    • 1
  • Kinga Grażyna Blecharz
    • 2
  • Peter Vajkoczy
    • 2
  • Joerg Piontek
    • 3
  • Gerd Krause
    • 1
    Email author
  1. 1.Leibniz-Institut für Molekulare PharmakologieBerlinGermany
  2. 2.Department of NeurosurgeryCharité-Universitätsmedizin BerlinBerlinGermany
  3. 3.Institute of Clinical PhysiologyCharité-Universitätsmedizin BerlinBerlinGermany

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