title: Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.
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.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Protze
authors.forename: Miriam
authors.initials: M
authors.lastname: Eichner
authors.forename: Anna
authors.initials: A
authors.lastname: Piontek
authors.forename: Stefan
authors.initials: S
authors.lastname: Dinter
authors.forename: Jan
authors.initials: J
authors.lastname: Rossa
authors.forename: Kinga Grażyna
authors.initials: KG
authors.lastname: Blecharz
authors.forename: Peter
authors.initials: P
authors.lastname: Vajkoczy
authors.forename: Joerg
authors.initials: J
authors.lastname: Piontek
authors.forename: Gerd
authors.initials: G
authors.lastname: Krause
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D062445:Claudin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D062507:Claudin-5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003016:Clostridium perfringens / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D062445:Claudin-1; D062507:Claudin-5; D004768:Enterotoxins; D008164:Luminescent Proteins; D050505:Mutant Proteins; C035598:enterotoxin, Clostridium