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Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain
M Guy Roukens1, Josi Peterson-Maduro1, Yvonne Padberg1
1From 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.).
Insights
The collagen domains of CCBE1 are essential for activating VEGFC signaling and lymphangiogenesis, while EGF domains are less critical for these processes.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is vital for lymphangiogenesis and linked to Hennekam syndrome.
- CCBE1 regulates vascular endothelial growth factor-C (VEGFC) signaling.
Purpose of the Study:
- To investigate the functional roles of CCBE1's distinct protein domains (EGF and collagen repeats).
- To determine the in vivo and in vitro significance of CCBE1 domains in lymphangiogenesis and VEGFC signaling.
Main Methods:
- Generated CCBE1 deletion mutant knock-in mice.
- Assessed VEGFC signaling enhancement in zebrafish embryos.
- Tested VEGFC processing activation in vitro.
- Analyzed Hennekam syndrome mutations in CCBE1 domains.
Main Results:
- CCBE1 collagen domain deletion severely impaired lymphangiogenesis, mimicking CCBE1 knockout.
- CCBE1 EGF domain deletion allowed rudimentary lymphatic formation and partially rescued VEGFC signaling in zebrafish.
- CCBE1's EGF domains, not collagen domains, were necessary for in vitro VEGFC processing.
- Hennekam syndrome mutations in collagen domains had a more severe impact than those in EGF domains.
Conclusions:
- CCBE1's collagen domains are critical for VEGFC activation in vitro and in vivo.
- CCBE1's EGF domains are dispensable for VEGFC processing but necessary for full lymphangiogenic activity.
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.
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