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Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

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Identification of the active site of laminin γ2 chain using deletion mutants of domain V. (a) Three recombinant proteins, NE1/2, NE1/3, and NE2/3, were prepared by deleting each of three epidermal growth factor-like repeats in domain V. Right panel, SDS-PAGE profiles of purified NE1/2, NE1/3, and NE2/3 proteins; 1 μg protein was run in each lane. (b) Heparin-binding activity of three recombinant proteins. NE1/2, NE1/3, or NE2/3 (1 μg) was incubated with 100 μL heparin-Sepharose beads for 120 min, and bound proteins were analyzed by immunoblotting with anti-His-tag antibody. Relative intensity of the immunopositive bands was measured by ImageJ. (c) Effects of γ2 N-terminal domain V (NE) proteins on VE-cadherin localization. Confluent cultures of HUVECs were treated with PBS (Control) or 1 μg each recombinant protein. Through morphological changes of HUVECs, VE-cadherin at intercellular junctions moved to the cytoplasm. Right panel shows the average intensity of green fluorescence per cell in the cytoplasmic area, which was analyzed by ImageJ. Each value was obtained from 50 cells in 15 different images. *P < 0.05. N.S., not significant. Scale bars = 20 μm. (d) In vivo vascular permeability. Miles assay was carried out. Each mouse was injected with PBS (Control) and 1 μg each of γ2dV, NE1/2, NE1/3, and NE2/3 at separate points on the back skin (n = 5). One representative example of mouse skin is shown in the right panel.
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fig07: Identification of the active site of laminin γ2 chain using deletion mutants of domain V. (a) Three recombinant proteins, NE1/2, NE1/3, and NE2/3, were prepared by deleting each of three epidermal growth factor-like repeats in domain V. Right panel, SDS-PAGE profiles of purified NE1/2, NE1/3, and NE2/3 proteins; 1 μg protein was run in each lane. (b) Heparin-binding activity of three recombinant proteins. NE1/2, NE1/3, or NE2/3 (1 μg) was incubated with 100 μL heparin-Sepharose beads for 120 min, and bound proteins were analyzed by immunoblotting with anti-His-tag antibody. Relative intensity of the immunopositive bands was measured by ImageJ. (c) Effects of γ2 N-terminal domain V (NE) proteins on VE-cadherin localization. Confluent cultures of HUVECs were treated with PBS (Control) or 1 μg each recombinant protein. Through morphological changes of HUVECs, VE-cadherin at intercellular junctions moved to the cytoplasm. Right panel shows the average intensity of green fluorescence per cell in the cytoplasmic area, which was analyzed by ImageJ. Each value was obtained from 50 cells in 15 different images. *P < 0.05. N.S., not significant. Scale bars = 20 μm. (d) In vivo vascular permeability. Miles assay was carried out. Each mouse was injected with PBS (Control) and 1 μg each of γ2dV, NE1/2, NE1/3, and NE2/3 at separate points on the back skin (n = 5). One representative example of mouse skin is shown in the right panel.

Mentions: As shown in Figure 4(b), the most N-terminal domain of Lmγ2, domain V (γ2dV) had the highest activity to stimulate the endothelial cell permeability. It is composed of three N-terminal EGF-like-repeats, that is, disulfide bond-linked loop structures (NE1, 2, and 3). To localize the active site in domain V, we prepared three combinations of two repeats, NE1/2, NE1/3, and NE2/3 (Fig. 7a). In the pull-down assay with heparin-Sepharose, NE1/2 most strongly bound to heparin-Sepharose (Fig. 7b). NE1/3 weakly bound to heparin but not NE2/3 at all. Moreover, NE1/2 most evidently induced the delocalization of VE-cadherin from the cell–cell borders, but NE2/3 did not (Fig. 7c). Consistent with these results, NE1/2 increased vascular permeability in vivo more evidently than γ2dV (Fig. 7d). Neither NE1/3 nor NE2/3 showed significant activity. These data suggest that the first EGF-like repeat NE1 plays a critical role in the biological activities and heparin-binding activity of the Lmγ2 chain.


Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Identification of the active site of laminin γ2 chain using deletion mutants of domain V. (a) Three recombinant proteins, NE1/2, NE1/3, and NE2/3, were prepared by deleting each of three epidermal growth factor-like repeats in domain V. Right panel, SDS-PAGE profiles of purified NE1/2, NE1/3, and NE2/3 proteins; 1 μg protein was run in each lane. (b) Heparin-binding activity of three recombinant proteins. NE1/2, NE1/3, or NE2/3 (1 μg) was incubated with 100 μL heparin-Sepharose beads for 120 min, and bound proteins were analyzed by immunoblotting with anti-His-tag antibody. Relative intensity of the immunopositive bands was measured by ImageJ. (c) Effects of γ2 N-terminal domain V (NE) proteins on VE-cadherin localization. Confluent cultures of HUVECs were treated with PBS (Control) or 1 μg each recombinant protein. Through morphological changes of HUVECs, VE-cadherin at intercellular junctions moved to the cytoplasm. Right panel shows the average intensity of green fluorescence per cell in the cytoplasmic area, which was analyzed by ImageJ. Each value was obtained from 50 cells in 15 different images. *P < 0.05. N.S., not significant. Scale bars = 20 μm. (d) In vivo vascular permeability. Miles assay was carried out. Each mouse was injected with PBS (Control) and 1 μg each of γ2dV, NE1/2, NE1/3, and NE2/3 at separate points on the back skin (n = 5). One representative example of mouse skin is shown in the right panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317827&req=5

fig07: Identification of the active site of laminin γ2 chain using deletion mutants of domain V. (a) Three recombinant proteins, NE1/2, NE1/3, and NE2/3, were prepared by deleting each of three epidermal growth factor-like repeats in domain V. Right panel, SDS-PAGE profiles of purified NE1/2, NE1/3, and NE2/3 proteins; 1 μg protein was run in each lane. (b) Heparin-binding activity of three recombinant proteins. NE1/2, NE1/3, or NE2/3 (1 μg) was incubated with 100 μL heparin-Sepharose beads for 120 min, and bound proteins were analyzed by immunoblotting with anti-His-tag antibody. Relative intensity of the immunopositive bands was measured by ImageJ. (c) Effects of γ2 N-terminal domain V (NE) proteins on VE-cadherin localization. Confluent cultures of HUVECs were treated with PBS (Control) or 1 μg each recombinant protein. Through morphological changes of HUVECs, VE-cadherin at intercellular junctions moved to the cytoplasm. Right panel shows the average intensity of green fluorescence per cell in the cytoplasmic area, which was analyzed by ImageJ. Each value was obtained from 50 cells in 15 different images. *P < 0.05. N.S., not significant. Scale bars = 20 μm. (d) In vivo vascular permeability. Miles assay was carried out. Each mouse was injected with PBS (Control) and 1 μg each of γ2dV, NE1/2, NE1/3, and NE2/3 at separate points on the back skin (n = 5). One representative example of mouse skin is shown in the right panel.
Mentions: As shown in Figure 4(b), the most N-terminal domain of Lmγ2, domain V (γ2dV) had the highest activity to stimulate the endothelial cell permeability. It is composed of three N-terminal EGF-like-repeats, that is, disulfide bond-linked loop structures (NE1, 2, and 3). To localize the active site in domain V, we prepared three combinations of two repeats, NE1/2, NE1/3, and NE2/3 (Fig. 7a). In the pull-down assay with heparin-Sepharose, NE1/2 most strongly bound to heparin-Sepharose (Fig. 7b). NE1/3 weakly bound to heparin but not NE2/3 at all. Moreover, NE1/2 most evidently induced the delocalization of VE-cadherin from the cell–cell borders, but NE2/3 did not (Fig. 7c). Consistent with these results, NE1/2 increased vascular permeability in vivo more evidently than γ2dV (Fig. 7d). Neither NE1/3 nor NE2/3 showed significant activity. These data suggest that the first EGF-like repeat NE1 plays a critical role in the biological activities and heparin-binding activity of the Lmγ2 chain.

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

Show MeSH
Related in: MedlinePlus