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Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization.

Gong Y, Renigunta V, Zhou Y, Sunq A, Wang J, Yang J, Renigunta A, Baker LA, Hou J - Mol. Biol. Cell (2015)

Bottom Line: The molecular nature of tight junction architecture and permeability is a long-standing mystery.Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist.These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine-Renal Division, Washington University Medical School, St. Louis, MO 63110 Center for Investigation of Membrane Excitability Diseases, Washington University Medical School, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus

Identifying loci in the claudin-16 transmembrane domain important for its interaction with claudin-19. (A) Helical-wheel view of the four TM domains in claudin-16. The positions of alanine insertion are labeled with arrows. (B, C) Effects of alanine insertion into claudin-16 TM domains on the claudin-16 and -19 interaction assayed with the Y2H β-gal reporter gene. The loci with β-gal reporter activity <20% of wild-type interaction level are labeled with asterisks.
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Figure 2: Identifying loci in the claudin-16 transmembrane domain important for its interaction with claudin-19. (A) Helical-wheel view of the four TM domains in claudin-16. The positions of alanine insertion are labeled with arrows. (B, C) Effects of alanine insertion into claudin-16 TM domains on the claudin-16 and -19 interaction assayed with the Y2H β-gal reporter gene. The loci with β-gal reporter activity <20% of wild-type interaction level are labeled with asterisks.

Mentions: To screen for amino acid loci in transmembrane domains important for claudin-16 and -19 interaction, we generated alanine insertion mutations (+A) along the four transmembrane helices of claudin-16 (Figure 2) and -19 (Figure 3) based on the published crystal structures of claudin-15 (Suzuki et al., 2014) and claudin-19 (Saitoh et al., 2015). The positions of alanine insertion were chosen periodically along each helix at two–amino acid intervals and marked with arrows for each amino acid locus, where insertion was placed to its C-terminal side (Figures 2 and 3A). We then subjected these mutant claudins to a previously established membrane Y2H assay with their wild-type claudin counterpart—for example, claudin-16 mutant with claudin-19 wild type (WT) or vice versa (Hou et al., 2008, 2009). None of the alanine insertion positions in TM1 or TM2 of claudin-16 affected its interaction with claudin-19 (Figure 2B), nor was any position found in TM1 or TM2 of claudin-19 important for its interaction with claudin-16 (Figure 3B). On the other hand, a number of loci in TM3 and TM4 of both claudins were critical for their interaction; insertions at these loci invariably abolished claudin interaction with β-gal reporter activity at only 20% or less of the wild-type interaction level (Figures 2 and 3C; positions labeled with asterisks). These positions appeared periodically at four–amino acid intervals (i → (i + 4)n) for claudin-16 and seven–amino acid intervals (i → (i + 7)n) for claudin-19; both arrangements aligned toward one side of the helix. Such structural arrangement was very similar to the interaction surface found in the GpA dimer, (i → (i + 4)n; MacKenzie et al., 1997) and in the leucine zipper coiled-coil protein interaction, (i → (i + 7)n; Zhou, 2011). From these loss-of-interaction loci, we were able to draw a favorable interaction surface for TM3 and TM4 in claudin-16 and -19. In claudin-16 TM3, the interfacial loci were 193, 200, and 204 (Figure 2A), because insertions at the 192 or 194 locus may both displaced the 193 residue, resulting in similar loss of interaction (Figure 2C). Of note, the 193-200-204 transition is mixed with i → (i + 7) and i → (i + 4). In claudin-16 TM4, the interfacial loci included 236, 240, and 244 in a typical i → (i + 4) format (Figure 2A). Insertion at the 246 locus also abolished interaction (Figure 2C), indicating an additional interfacial residue, 247 with i → (i + 7) transition from 240 (Figure 2A). In claudin-19 TM3, the interfacial loci were 128, 132, and 139 with i → (i + 4) followed by i → (i + 7) transition (Figure 3A), keeping in mind that 139 is the most likely locus because insertions at both 138 and 140 caused loss of interaction (Figure 3C). Two additional residues aligned in parallel—117 and 135—were also included at the interaction face (Figure 3A), based on the observation that insertion at a nearby locus, 118 or 134, abolished interaction (Figure 3C). In claudin-19 TM4, the interfacial loci were 164, 171, and 178 in a typical i → (i + 7) transition (Figure 3A). The 171 locus was deduced from the interaction data of insertion at 170 (Figure 3C). Because insertion at 174 or 176 either diminished or abolished interaction (Figure 3C), we included the 175 residue at the interaction face due to its proximity to these affected loci and continuity with other interfacial loci on the helical wheel (Figure 3A).


Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization.

Gong Y, Renigunta V, Zhou Y, Sunq A, Wang J, Yang J, Renigunta A, Baker LA, Hou J - Mol. Biol. Cell (2015)

Identifying loci in the claudin-16 transmembrane domain important for its interaction with claudin-19. (A) Helical-wheel view of the four TM domains in claudin-16. The positions of alanine insertion are labeled with arrows. (B, C) Effects of alanine insertion into claudin-16 TM domains on the claudin-16 and -19 interaction assayed with the Y2H β-gal reporter gene. The loci with β-gal reporter activity <20% of wild-type interaction level are labeled with asterisks.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Identifying loci in the claudin-16 transmembrane domain important for its interaction with claudin-19. (A) Helical-wheel view of the four TM domains in claudin-16. The positions of alanine insertion are labeled with arrows. (B, C) Effects of alanine insertion into claudin-16 TM domains on the claudin-16 and -19 interaction assayed with the Y2H β-gal reporter gene. The loci with β-gal reporter activity <20% of wild-type interaction level are labeled with asterisks.
Mentions: To screen for amino acid loci in transmembrane domains important for claudin-16 and -19 interaction, we generated alanine insertion mutations (+A) along the four transmembrane helices of claudin-16 (Figure 2) and -19 (Figure 3) based on the published crystal structures of claudin-15 (Suzuki et al., 2014) and claudin-19 (Saitoh et al., 2015). The positions of alanine insertion were chosen periodically along each helix at two–amino acid intervals and marked with arrows for each amino acid locus, where insertion was placed to its C-terminal side (Figures 2 and 3A). We then subjected these mutant claudins to a previously established membrane Y2H assay with their wild-type claudin counterpart—for example, claudin-16 mutant with claudin-19 wild type (WT) or vice versa (Hou et al., 2008, 2009). None of the alanine insertion positions in TM1 or TM2 of claudin-16 affected its interaction with claudin-19 (Figure 2B), nor was any position found in TM1 or TM2 of claudin-19 important for its interaction with claudin-16 (Figure 3B). On the other hand, a number of loci in TM3 and TM4 of both claudins were critical for their interaction; insertions at these loci invariably abolished claudin interaction with β-gal reporter activity at only 20% or less of the wild-type interaction level (Figures 2 and 3C; positions labeled with asterisks). These positions appeared periodically at four–amino acid intervals (i → (i + 4)n) for claudin-16 and seven–amino acid intervals (i → (i + 7)n) for claudin-19; both arrangements aligned toward one side of the helix. Such structural arrangement was very similar to the interaction surface found in the GpA dimer, (i → (i + 4)n; MacKenzie et al., 1997) and in the leucine zipper coiled-coil protein interaction, (i → (i + 7)n; Zhou, 2011). From these loss-of-interaction loci, we were able to draw a favorable interaction surface for TM3 and TM4 in claudin-16 and -19. In claudin-16 TM3, the interfacial loci were 193, 200, and 204 (Figure 2A), because insertions at the 192 or 194 locus may both displaced the 193 residue, resulting in similar loss of interaction (Figure 2C). Of note, the 193-200-204 transition is mixed with i → (i + 7) and i → (i + 4). In claudin-16 TM4, the interfacial loci included 236, 240, and 244 in a typical i → (i + 4) format (Figure 2A). Insertion at the 246 locus also abolished interaction (Figure 2C), indicating an additional interfacial residue, 247 with i → (i + 7) transition from 240 (Figure 2A). In claudin-19 TM3, the interfacial loci were 128, 132, and 139 with i → (i + 4) followed by i → (i + 7) transition (Figure 3A), keeping in mind that 139 is the most likely locus because insertions at both 138 and 140 caused loss of interaction (Figure 3C). Two additional residues aligned in parallel—117 and 135—were also included at the interaction face (Figure 3A), based on the observation that insertion at a nearby locus, 118 or 134, abolished interaction (Figure 3C). In claudin-19 TM4, the interfacial loci were 164, 171, and 178 in a typical i → (i + 7) transition (Figure 3A). The 171 locus was deduced from the interaction data of insertion at 170 (Figure 3C). Because insertion at 174 or 176 either diminished or abolished interaction (Figure 3C), we included the 175 residue at the interaction face due to its proximity to these affected loci and continuity with other interfacial loci on the helical wheel (Figure 3A).

Bottom Line: The molecular nature of tight junction architecture and permeability is a long-standing mystery.Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist.These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine-Renal Division, Washington University Medical School, St. Louis, MO 63110 Center for Investigation of Membrane Excitability Diseases, Washington University Medical School, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus