Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain

Circ Res. 2015 May 8;116(10):1660-9. doi: 10.1161/CIRCRESAHA.116.304949. Epub 2015 Mar 26.


Rationale: 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.

Methods and 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.

Keywords: CCBE1 protein; Hennekam lymphangiectasia-lymphedema syndrome; endothelium, vascular; lymphangiogenesis; vascular endothelial growth factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / deficiency
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Collagen / metabolism
  • Craniofacial Abnormalities / genetics
  • Craniofacial Abnormalities / metabolism
  • Endothelial Cells / metabolism*
  • Epidermal Growth Factor / metabolism
  • Gene Expression Regulation, Developmental
  • Gene Knock-In Techniques
  • Genital Diseases, Male / genetics
  • Genital Diseases, Male / metabolism
  • Genotype
  • Gestational Age
  • HEK293 Cells
  • Humans
  • Lymphangiectasis, Intestinal / genetics
  • Lymphangiectasis, Intestinal / metabolism
  • Lymphatic Vessels / embryology
  • Lymphatic Vessels / metabolism*
  • Lymphedema / genetics
  • Lymphedema / metabolism
  • Mice
  • Mice, Transgenic
  • Mutation
  • Phenotype
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Signal Transduction
  • Transfection
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / deficiency
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Vascular Endothelial Growth Factor C / metabolism
  • Zebrafish / genetics
  • Zebrafish / metabolism
  • Zebrafish Proteins / chemistry
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism*


  • CCBE1 protein, human
  • CCBE1 protein, zebrafish
  • Calcium-Binding Proteins
  • Ccbe1 protein, mouse
  • Tumor Suppressor Proteins
  • VEGFC protein, human
  • Vascular Endothelial Growth Factor C
  • Zebrafish Proteins
  • vascular endothelial growth factor C, mouse
  • vascular endothelial growth factor C, zebrafish
  • Epidermal Growth Factor
  • Collagen

Supplementary concepts

  • Hennekam lymphangiectasia lymphedema syndrome