Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Polydom binds to Tie1 via CCP20. (A) Recombinant integrin α9β1, Tie1, Tie2, EphrinB2, and Podoplanin were coated on microtiter plates at 5 μg/ml and then incubated with full-length Polydom (5 μg/ml). Polydom was allowed to bind to integrin α9β1 in the absence or presence of 1 mM MnCl 2 . The data represent means ± SD of triplicate determinations. (B) Titration curves of Tie1 and Tie2 binding to Polydom. Tie1 (open squares) or Tie2 (closed squares) at the indicated concentrations was allowed to bind to microtiter plates coated with full-length Polydom (10 nM). The data represent means ± SD of triplicate determinations. (C) Schematic representation of Polydom and its recombinant fragments. Polydom consists of multiple domains including von Willebrand factor type A (vWFA), Ephrin 2-like (Eph-like), hyaline (HYR), similar to thyroglobulin type 2 repeat (STT2R), EGF, and pentraxin (PTX) domains, and an array of CCP domains. (D) Polydom and its fragments were coated at 2 μg/ml on microtiter plates and incubated with Tie1 (2 μg/ml). The data represent means ± SD of triplicate determinations. (E) GST fusion proteins of CCP20 and CCP22 were coated at 2 μg/ml on microtiter plates and incubated with Tie1 (2 μg/ml). Pol-C and GST alone were included as positive and negative controls, respectively. The data represent means ± SD of triplicate determinations.

Article Snippet: Recombinant Tie1 and Tie2, both containing their extracellular domains and expressed as Fc fusion proteins, recombinant human VEGF-C, and human Ang1 were obtained from R&D Systems.

Techniques: Recombinant, Incubation, Titration, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Glu2567 and Gly2568 in CCP20 are required for Tie1 binding. (A) Alignment of the amino acid sequences of CCP20 (white bars) and CCP22 (gray bars) by ClustalW. The asterisks denote identical amino acids, while the colons and periods denote strongly similar and weakly similar amino acids, respectively. The N-terminal half of CCP20/CCP22 is subdivided into Region-1 and Region-2. (B) Tie1 (2 μg/ml) was allowed to bind to microtiter plates coated with GST fusion proteins containing CCP20, CCP22, and their chimeras or with Pol-C or GST alone coated at 2 μg/ml. The data represent means ± SD of triplicate determinations. † P < 0.02 ( n = 3). (C and D) A series of alanine scanning mutants of CCP20/Region-2 (C; underlined) were expressed as GST fusion proteins and assessed for their binding activities toward Tie1 (D). The data represent means ± SD of triplicate determinations. The substituted alanine (A) and glycine (G) residues are shown in bold. (E and F) Full-length Polydom and its Glu2567Ala mutant (Polydom/E2567A; E) were assessed for their binding activities toward Tie1 (F). The data represent means ± SD of triplicate determinations.

Article Snippet: Recombinant Tie1 and Tie2, both containing their extracellular domains and expressed as Fc fusion proteins, recombinant human VEGF-C, and human Ang1 were obtained from R&D Systems.

Techniques: Binding Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Polydom promotes LEC migration via Tie1. (A) Photographs of LECs that migrated to the lower side of Transwell membranes in the presence of collagen, fibronectin, laminin, or Polydom (3 μg/ml) added to the lower chamber medium. The molar concentration of the proteins was collagen, 23 nM; fibronectin, 15 nM; laminin, 3.8 nM; and Polydom: 8.8 nM. Bar, 200 μm. (B) Cells that migrated to the lower side of the membranes were counted under a microscope. The data represent means ± SD of three independent experiments each assayed in triplicate. ***P < 0.001 ( n = 3). (C) Immunoprecipitates (IP) of Tie1 and Tie2 from lysates of LECs transfected with control, Tie1, or Tie2 siRNAs were analyzed by Western blotting under reducing conditions. The same lysates were probed with an antibody against α-tubulin as a control. The positions of molecular weight markers are shown on the left. (D) LECs transfected with control, Tie1, or Tie2 siRNAs were allowed to migrate in the presence of Polydom (1 μg/ml) for 16 h. Bar, 200 μm. (E) Cells that migrated to the lower side of the membranes were counted under a microscope in the absence (none) or presence of Polydom added to the lower chamber medium. The data represent means ± SD of three independent experiments each assayed in triplicate. ***P < 0.001 ( n = 3). (F) Total lysates of LECs transfected with control, Tie1, or Tie2 siRNAs were analyzed by Western blotting using anti-cleaved caspase-3 and anti-caspase-3 antibodies under reducing conditions. Lysates of LECs treated with staurosporine (0.2 μM, 4 h) were used as a positive control for cleaved caspase-3 detection. (G) Photographs of LECs that migrated to the lower side of the membranes in the presence of Polydom or Polydom/E2567A (1 μg/ml) in the lower chamber medium. Bar, 200 μm. (H) Cells that migrated to the lower side of the membranes were counted under a microscope. The data represent means ± SD of three independent experiments each assayed in triplicate. † P < 0.02 ( n = 3). Source data are available for this figure: .

Article Snippet: Recombinant Tie1 and Tie2, both containing their extracellular domains and expressed as Fc fusion proteins, recombinant human VEGF-C, and human Ang1 were obtained from R&D Systems.

Techniques: Migration, Concentration Assay, Microscopy, Transfection, Control, Western Blot, Molecular Weight, Positive Control

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Polydom does not induce Tie1 phosphorylation. (A) Serum-starved LECs were treated with EBM-MV2 medium containing 0.5% FBS, 1 μg/ml Polydom, or 500 ng/ml Ang1 at 37°C for 15 min. Immunoprecipitates (IP) of Tie1 (left) and Tie2 (right) from cell lysates were immunoblotted under reducing conditions for phosphotyrosine residues (upper panels) followed by reimmunoblotting for total Tie1 or Tie2 (lower panels). (B) 293-F cells were transfected with the indicated expression plasmids for Tie1 and Tie2 and treated with 1 μg/ml Polydom or 500 ng/ml Ang1 at 37°C for 15 min. Immunoprecipitates of Tie1 and Tie2 from cell lysates were immunoblotted under reducing conditions for phosphotyrosine residues (upper panels), followed by reimmunoblotting for total Tie1 or Tie2 (lower panels). Co-transfection of Tie2 with Tie1 increased Tie1 phosphorylation irrespective of the presence or absence of Polydom (lanes 4, 8, and 12). No Tie1 phosphorylation was induced by Polydom without Tie2 co-transfection (lanes 2, 6, and 10). Signals for phosphorylated Tie2 (pTie2; closed stars) were detected in Tie1 immunoprecipitates from Tie2-transfected cells (uppermost panel; lanes 3, 4, 7, 8, 11, and 12) because the anti-Tie1 polyclonal antibody used for the immunoprecipitation crossreacts with Tie2. Weak signals (open stars) were detected at (or slightly above) the position of pTie2 in the Tie1 immunoprecipitates from cells that were either untransfected or only transfected with Tie1 (lanes 1, 2, 5, 6, 9, and 10). Because Tie2 was not transfected in these cells, the weak signals (open stars) at the pTie2 position should be derived from tyrosine-phosphorylated proteins that were endogenously expressed in 293-F cells and nonspecifically precipitated with the anti-Tie1 antibody used. Such bands were not detected in untransfected or Tie1-transfected cells after immunoprecipitation with an anti-Tie2 antibody (lower panels; lanes 1, 2, 5, 6, 9, and 10). Source data are available for this figure: .

Article Snippet: Recombinant Tie1 and Tie2, both containing their extracellular domains and expressed as Fc fusion proteins, recombinant human VEGF-C, and human Ang1 were obtained from R&D Systems.

Techniques: Phospho-proteomics, Transfection, Expressing, Cotransfection, Immunoprecipitation, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Multiple sequence alignment of CCP20 domains. The amino acid sequences of the CCP20 domains of mouse, human, rabbit, chicken, Xenopus , and zebrafish Polydom were aligned by ClustalW. The asterisks denote identical amino acids, while the colons and periods denote strongly similar and weakly similar amino acids, respectively. The glutamate (E) and glycine (G) residues corresponding to E2567 and G2568 in mouse Polydom (highlighted in pink), which are critically required for Tie1 binding, are conserved in these animals.

Article Snippet: Recombinant Tie1 and Tie2, both containing their extracellular domains and expressed as Fc fusion proteins, recombinant human VEGF-C, and human Ang1 were obtained from R&D Systems.

Techniques: Sequencing, Binding Assay