title: Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.
abstract: BACKGROUND
  Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.
  
  
  OBJECTIVE
  CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.
  
  
  RESULTS
  We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain.
  
  
  CONCLUSIONS
  We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: M Guy
authors: MG
authors: Roukens
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Josi
authors: J
authors: Peterson-Maduro
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Yvonne
authors: Y
authors: Padberg
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Michael
authors: M
authors: Jeltsch
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Veli-Matti
authors: VM
authors: Leppänen
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Frank L
authors: FL
authors: Bos
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Kari
authors: K
authors: Alitalo
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.).
authors: Stefan
authors: S
authors: Schulte-Merker
authors: From the Hubrecht Institute, KNAW-UMC Utrecht, Utrecht, The Netherlands (M.G.R., J.P.M., Y.P., F.L.B., S.S.-M., D.S.); Cardiovascular Research Institute, University of California San Francisco (F.L.B.); Translational Cancer Biology Program, University of Helsinki, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland (M.J., V.-M. L., K.A.); Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany (Y.P., S.S.M., D.S.); and Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Münster, Germany (Y.P., S.S.M., D.S.). d.schulte@uni-muenster.de.
authors: Dörte
authors: D
authors: Schulte
mesh_terms: D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D019465:Craniofacial Abnormalities / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D055879:Gene Knock-In Techniques; D005832:Genital Diseases, Male / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D005865:Gestational Age; D057809:HEK293 Cells; D006801:Humans; D008201:Lymphangiectasis, Intestinal / Q000235:genetics / Q000378:metabolism; D042601:Lymphatic Vessels / Q000196:embryology / Q000378:metabolism*; D008209:Lymphedema / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D015398:Signal Transduction; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C546964:CCBE1 protein, human; C000599480:CCBE1 protein, zebrafish; D002135:Calcium-Binding Proteins; C572375:Ccbe1 protein, mouse; D025521:Tumor Suppressor Proteins; C467486:VEGFC protein, human; D042582:Vascular Endothelial Growth Factor C; D029961:Zebrafish Proteins; C467483:vascular endothelial growth factor C, mouse; C493178:vascular endothelial growth factor C, zebrafish; D004815:Epidermal Growth Factor; D003094:Collagen
keywords: CCBE1 protein; Hennekam lymphangiectasia-lymphedema syndrome; endothelium, vascular; lymphangiogenesis; vascular endothelial growth factor