Claudin-3 and Claudin-5 Folding and Assembly into the Tight Junction are controlled by Non-conserved Residues in TM3 and ECL2 Segments [Membrane Biology]

January 29th, 2014 by Rossa, J., Ploeger, C., Vorreiter, F., Saleh, T., Protze, J., Guenzel, D., Wolburg, H., Krause, G., Piontek, J.

The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) which form the TJ strands are unclear. This limits 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 ultrastructure. To identify 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 (FFEM). A comprehensive screening was performed, based on the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment three, TM3 (A127/C128, S136/C137, S138/F139), the transition of TM3 to extracellular loop 2, ECL2 (T141/I142) and ECL2 (N148/D149, L150/T151, R157/Y158) 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 FFEM. 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 mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to formation of TJ strands with differing ultrastructure.
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