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

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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

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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).

Conflict of interest

The authors declare no conflict of interests.

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Authors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Jonas Protze, Miriam Eichner, Anna Piontek, Stefan Dinter, Jan Rossa & Gerd Krause

  2. Department of Neurosurgery, Charité-Universitätsmedizin Berlin, 13353, Berlin, Germany

    Kinga Grażyna Blecharz & Peter Vajkoczy

  3. Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, 12203, Berlin, Germany

    Joerg Piontek

Authors
  1. Jonas Protze
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  2. Miriam Eichner
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  4. Stefan Dinter
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  5. Jan Rossa
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  6. Kinga Grażyna Blecharz
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  8. Joerg Piontek
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Corresponding author

Correspondence to Gerd Krause.

Additional information

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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