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.
The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.
From the Leibniz-Institut für Molekulare Pharmakologie, Department of Structural Biology, 13125 Berlin, Germany.
Jan
J
Rossa
Carolin
C
Ploeger
Fränze
F
Vorreiter
Tarek
T
Saleh
Jonas
J
Protze
Dorothee
D
Günzel
Hartwig
H
Wolburg
Gerd
G
Krause
Jörg
J
Piontek
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D062465:Claudin-3 / Q000737:chemistry*; D062507:Claudin-5 / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D005614:Freeze Fracturing; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010641:Phenotype; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019108:Tight Junctions / Q000648:ultrastructure*
C568122:CLDN3 protein, human; C107466:CLDN5 protein, human; D062465:Claudin-3; D062507:Claudin-5
Claudin; Confocal Microscopy; Electron Microscopy (EM); Freeze Fracture; Membrane Proteins; Mutagenesis; Protein Assembly; Protein-Protein Interaction; Tight Junctions