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Cell Mol Life Sci. 2015 Apr;72(7):1417-32. doi: 10.1007/s00018-014-1761-6. Epub 2014 Oct 24.

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

Author information

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

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 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). 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.

PMID:
25342221
DOI:
10.1007/s00018-014-1761-6
[Indexed for MEDLINE]

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