Abstract
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.
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Abbreviations
- TJ:
-
Tight junctions
- CPE:
-
Clostridium perfringens Enterotoxin
- cCPE:
-
C-terminal domain of Clostridium perfringens enterotoxin
- Cld:
-
Claudin
- ECL:
-
Extracellular loop
- PDB:
-
Protein data bank
- RMSD:
-
Root mean square deviation
References
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
Angelow S, Ahlstrom R, Yu ASL (2008) Biology of claudins. Am J Physiol Ren Physiol 295:F867–F876. doi:10.1152/ajprenal.90264.2008
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
McClane BA (2001) The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions. Toxicon 39:1781–1791
Katahira J, Inoue N, Horiguchi Y et al (1997) Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol 136:1239–1247
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
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 147:195–204
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
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
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
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
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
Kokai-Kun JF, McClane BA (1997) Deletion analysis of the Clostridium perfringens enterotoxin. Infect Immun 65:1014–1022
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
Kondoh M, Takahashi A, Fujii M et al (2006) A novel strategy for a drug delivery system using a claudin modulator. Biol Pharm Bull 29:1783–1789
Takahashi A, Kondoh M, Suzuki H, Yagi K (2011) Claudin as a target for drug development. Curr Med Chem 18:1861–1865
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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 476:258–261
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
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
Acknowledgments
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).
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The authors declare no conflict of interests.
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J. Piontek and G. Krause contributed equally to this work.
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Protze, J., Eichner, M., Piontek, A. et al. Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5. Cell. Mol. Life Sci. 72, 1417–1432 (2015). https://doi.org/10.1007/s00018-014-1761-6
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DOI: https://doi.org/10.1007/s00018-014-1761-6