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Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Morita K, Sasaki H, Furuse M, Tsukita S - J. Cell Biol. (1999)

Bottom Line: In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries.Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs.These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

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Specificity of pAbs. (A) Amino acid sequences of the cytoplasmic domains of claudin-5/TMVCF and claudin-6. The COOH-terminal KNYV is shared by these two claudin species. Two polypeptides corresponding to these sequences were used as antigens to produce pAbs in rabbits. (B) GST fusion proteins with the cytoplasmic domains of claudin-1 to -8 as well as the cytoplasmic domain of claudin-6 lacking its COOH-terminal KNYV (see claudin-6Δ in A) were produced in E. coli. The lysates of E. coli were separated by SDS-PAGE (C.B.B. staining), followed by immunoblotting with two distinct pAbs, which were raised against the cytoplasmic domain of claudin-5/TMVCF or claudin-6, respectively. The former recognized both GST–claudin-5/TMVCF and GST–claudin-6, but not GST–claudin-6Δ, indicating that this pAb (referred to as anti–claudin-5/6 pAb) specifically bound to the COOH-terminal KNYV sequence. The latter recognized GST–claudin-6 and GST–claudin-6Δ but not GST–claudin-5/TMVCF, indicating that this pAb (referred to as anti–claudin-6 pAb) was specific for the YSTSVPHSRGPSEYPT sequence of claudin-6 (see A). (C) Anti–claudin-5/6 pAb immunofluorescently stained L transfectants expressing claudin-5/TMVCF (C5L cells) and claudin-6 (C6L cells), whereas anti–claudin-6 pAb stained only C6L cells. Bar, 10 μm.
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Figure 1: Specificity of pAbs. (A) Amino acid sequences of the cytoplasmic domains of claudin-5/TMVCF and claudin-6. The COOH-terminal KNYV is shared by these two claudin species. Two polypeptides corresponding to these sequences were used as antigens to produce pAbs in rabbits. (B) GST fusion proteins with the cytoplasmic domains of claudin-1 to -8 as well as the cytoplasmic domain of claudin-6 lacking its COOH-terminal KNYV (see claudin-6Δ in A) were produced in E. coli. The lysates of E. coli were separated by SDS-PAGE (C.B.B. staining), followed by immunoblotting with two distinct pAbs, which were raised against the cytoplasmic domain of claudin-5/TMVCF or claudin-6, respectively. The former recognized both GST–claudin-5/TMVCF and GST–claudin-6, but not GST–claudin-6Δ, indicating that this pAb (referred to as anti–claudin-5/6 pAb) specifically bound to the COOH-terminal KNYV sequence. The latter recognized GST–claudin-6 and GST–claudin-6Δ but not GST–claudin-5/TMVCF, indicating that this pAb (referred to as anti–claudin-6 pAb) was specific for the YSTSVPHSRGPSEYPT sequence of claudin-6 (see A). (C) Anti–claudin-5/6 pAb immunofluorescently stained L transfectants expressing claudin-5/TMVCF (C5L cells) and claudin-6 (C6L cells), whereas anti–claudin-6 pAb stained only C6L cells. Bar, 10 μm.

Mentions: Two polypeptides, KYSAPRRPTANGDYDKKNYV and YSTSVPHSRGPSEYPTKNYV, which correspond to the COOH-terminal cytoplasmic domains of mouse claudin-5/TMVCF (aa 199–218) and claudin-6 (aa 200–219), respectively (a cysteine residue was added at their NH2 termini), were synthesized and coupled via the cysteine residue to keyhole limpet hemocyanin. These peptides were injected into rabbits as antigens. Rabbit antisera against the former and latter peptides were affinity-purified on nitrocellulose membranes with glutathione S-transferase (GST) fusion proteins with claudin-5/TMVCF and claudin-6Δ to obtain anti–claudin-5/6 pAb and anti–claudin-6 pAb, respectively (see Fig. 1 A).


Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Morita K, Sasaki H, Furuse M, Tsukita S - J. Cell Biol. (1999)

Specificity of pAbs. (A) Amino acid sequences of the cytoplasmic domains of claudin-5/TMVCF and claudin-6. The COOH-terminal KNYV is shared by these two claudin species. Two polypeptides corresponding to these sequences were used as antigens to produce pAbs in rabbits. (B) GST fusion proteins with the cytoplasmic domains of claudin-1 to -8 as well as the cytoplasmic domain of claudin-6 lacking its COOH-terminal KNYV (see claudin-6Δ in A) were produced in E. coli. The lysates of E. coli were separated by SDS-PAGE (C.B.B. staining), followed by immunoblotting with two distinct pAbs, which were raised against the cytoplasmic domain of claudin-5/TMVCF or claudin-6, respectively. The former recognized both GST–claudin-5/TMVCF and GST–claudin-6, but not GST–claudin-6Δ, indicating that this pAb (referred to as anti–claudin-5/6 pAb) specifically bound to the COOH-terminal KNYV sequence. The latter recognized GST–claudin-6 and GST–claudin-6Δ but not GST–claudin-5/TMVCF, indicating that this pAb (referred to as anti–claudin-6 pAb) was specific for the YSTSVPHSRGPSEYPT sequence of claudin-6 (see A). (C) Anti–claudin-5/6 pAb immunofluorescently stained L transfectants expressing claudin-5/TMVCF (C5L cells) and claudin-6 (C6L cells), whereas anti–claudin-6 pAb stained only C6L cells. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164984&req=5

Figure 1: Specificity of pAbs. (A) Amino acid sequences of the cytoplasmic domains of claudin-5/TMVCF and claudin-6. The COOH-terminal KNYV is shared by these two claudin species. Two polypeptides corresponding to these sequences were used as antigens to produce pAbs in rabbits. (B) GST fusion proteins with the cytoplasmic domains of claudin-1 to -8 as well as the cytoplasmic domain of claudin-6 lacking its COOH-terminal KNYV (see claudin-6Δ in A) were produced in E. coli. The lysates of E. coli were separated by SDS-PAGE (C.B.B. staining), followed by immunoblotting with two distinct pAbs, which were raised against the cytoplasmic domain of claudin-5/TMVCF or claudin-6, respectively. The former recognized both GST–claudin-5/TMVCF and GST–claudin-6, but not GST–claudin-6Δ, indicating that this pAb (referred to as anti–claudin-5/6 pAb) specifically bound to the COOH-terminal KNYV sequence. The latter recognized GST–claudin-6 and GST–claudin-6Δ but not GST–claudin-5/TMVCF, indicating that this pAb (referred to as anti–claudin-6 pAb) was specific for the YSTSVPHSRGPSEYPT sequence of claudin-6 (see A). (C) Anti–claudin-5/6 pAb immunofluorescently stained L transfectants expressing claudin-5/TMVCF (C5L cells) and claudin-6 (C6L cells), whereas anti–claudin-6 pAb stained only C6L cells. Bar, 10 μm.
Mentions: Two polypeptides, KYSAPRRPTANGDYDKKNYV and YSTSVPHSRGPSEYPTKNYV, which correspond to the COOH-terminal cytoplasmic domains of mouse claudin-5/TMVCF (aa 199–218) and claudin-6 (aa 200–219), respectively (a cysteine residue was added at their NH2 termini), were synthesized and coupled via the cysteine residue to keyhole limpet hemocyanin. These peptides were injected into rabbits as antigens. Rabbit antisera against the former and latter peptides were affinity-purified on nitrocellulose membranes with glutathione S-transferase (GST) fusion proteins with claudin-5/TMVCF and claudin-6Δ to obtain anti–claudin-5/6 pAb and anti–claudin-6 pAb, respectively (see Fig. 1 A).

Bottom Line: In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries.Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs.These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

Show MeSH
Related in: MedlinePlus