Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4.
The tight junction tetraspan protein claudin-4 creates a charge-selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin-4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro-octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin-4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique.
Mitic
Laura L
LL
Department of Internal Medicine and Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Unger
Vinzenz M
VM
Anderson
James Melvin
JM
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D062506:Claudin-4; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D007834:Lasers; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012542:Scattering, Radiation; D012995:Solubility / Q000187:drug effects; D018411:Spodoptera; D019108:Tight Junctions / Q000737:chemistry*
C568157:CLDN4 protein, human; D062506:Claudin-4; D003902:Detergents; D008565:Membrane Proteins; D011994:Recombinant Proteins